Download Drugs of abuse modulate dopaminergic neurotransmission : effects

Drugs of abuse modulate
dopaminergic neurotransmission
Effects on exocytosis and neurotransmitter receptor function
Laura Hondebrink
Drugs of abuse modulate dopaminergic neurotransmission. Effects on exocytosis and
neurotransmitter receptor function. Laura Hondebrink, Utrecht University, Institute for
Risk Assessment Sciences (IRAS), 2011.
ISBN 978-90-393-5596-1
Copyright © Laura Hondebrink
Printing: Printservice Ede B.V., Ede, The Netherlands
Cover design: Anjolieke Dertien
Drugs of abuse modulate
dopaminergic neurotransmission
Effects on exocytosis and neurotransmitter receptor function
Drugs moduleren dopaminerge neurotransmissie
Effecten op exocytose en neurotransmitter receptor functie
(met een samenvatting in het Nederlands)
Proefschrift
ter verkrijging van de graad van doctor aan de Universiteit Utrecht op gezag van de
rector magnificus, prof.dr. G.J. van der Zwaan, ingevolge het besluit van het college
voor promoties in het openbaar te verdedigen op vrijdag 9 september 2011 des
middags te 2.30 uur
door
Laura Hondebrink
geboren op 10 december 1982 te Almelo
Promotors:
Prof. dr. J. Meulenbelt
Prof. dr. M. van den Berg
Co-promotor:
Dr. R.H.S. Westerink
This thesis was accomplished with financial support from the National Institute of
Public Health and the Environment (RIVM), grant number S/660001, Bilthoven, The
Netherlands.
Index
List of abbreviations
Chapter 1.
General Introduction
p. 1 - 12
Chapter 2.
Applied Methodologies
p. 13 - 22
Chapter 3.
Are high-throughput measurements of intracellular
p. 23 - 26
calcium using plate-readers sufficiently accurate
and reliable?
Chapter 4.
Amphetamine reduces vesicular dopamine content in
p. 27 - 44
dexamethasone-differentiated PC12 cells only following
L-DOPA exposure
Chapter 5.
High concentrations of MDMA (‘ecstasy’) and its
p.45 - 56
metabolite MDA inhibit calcium influx and depolarizationevoked vesicular dopamine release in PC12 cells
Chapter 6.
Methamphetamine, amphetamine, MDMA (‘ecstasy’), MDA p. 57 - 68
and mCPP modulate depolarization- and acetylcholineevoked increases in intracellular calcium levels in PC12 cells
Chapter 7.
Modulation of human GABAA receptor function: a novel
p. 69 - 78
mode of action of drugs of abuse
Chapter 8.
Summary and risk assessment
p. 79 - 90
Chapter 9.
Conclusions and recommendations
p. 91 - 94
References
p. 95 - 102
Dutch summary (Nederlandse samenvatting)
p. 103 - 110
Curriculum Vitae & List of publications
p. 111 - 113
Acknowledgements (Dankwoord)
p. 115 - 117
List of Abbreviations
5-HIAA
5-HT
5-HT-R
ACh
ACh-R
ADHD
AMPA-R
AMPH
BA
2+
Ca
2+
[Ca ]i
CAMKII
cAMP
cDNA
CYP
DA
DAT
DIMS
DRN
EC20
EC50
ECmax
ER
GABA
GABAA-R
GABAB-R
Glu
Glu-R
GPCR
GPe
GPi
HA
Hamph
hGABAA-R
HHA
HHMA
HMA
Hmeth
5-hydroxyindoleacetic acid, serotonin metabolite
serotonin
serotonin receptor
acetylcholine
acetylcholine receptor
attention-deficit hyperactivity disorder
α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor,
ionotropic glutamate receptor
amphetamine
benzoic acid
calcium
intracellular calcium concentration
2+
Ca -calmodulin-dependent protein kinase II
cyclic adenosine monophosphate
complementary DNA
cytochrome P450 superfamily; group of enzymes
dopamine
dopamine membrane transporter
drug information and monitoring system
dorsal raphe nuclei
effective concentration that evokes 20% of the maximum response
effective concentration that evokes 50% of the maximum response
effective concentration that evokes the maximum response
endoplasmic reticulum
γ -aminobutyric acid
ionotropic GABAA receptor
metabotropic GABAB receptor
glutamate
glutamate receptor
G-protein-coupled receptor
globus pallidus externa
globus pallidus interna
hippuric acid
4-hydroxyamphetamine
human GABAA receptor
3,4-dihydroxyamphetamine, MDMA metabolite
3,4-dihydroxymethamphetamine, MDMA metabolite
4-hydroxy-3-methoxyamphetamine, MDMA metabolite
4-hydroxymethamphetamine
HMMA
HNE
IP3
KO
LDCVs
L-DOPA
LDT
mACh-R
MAO
mCPP
MDA
MDMA
Meth
mGlu-R
MRN
MTT
NA
nACh-R
NCX
NE
NET
NMDA-R
NVIC
PA
PC12
PFC
pKa
PKA
PKC
PMCA
PPT
Q
SERCA
SERT
SNc
SNr
SSRI
SSVs
STN
TH
Thal
4-hydroxy-3-methoxymethamphetamine, MDMA metabolite
4-hydroxynorephedrine
inositol 1,4,5-trisphosphate
knockout
large dense-core vesicles
L-3,4-dihydroxyphenylalanine
laterodorsal tegmental nuclei
muscarinic acetylcholine receptor
monoamine oxidase
meta-chlorophenylpiperazine
3,4-methylenedioxyamphetamine, MDMA metabolite and active
substance in ecstasy tablets
3,4-methylenedioxymethamphetamine, active substance in ecstasy
tablets
methamphetamine
metabotropic glutamate receptor
medial raphe nuclei
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
nucleus accumbens
nicotinic acetylcholine receptor
+
2+
Na /Ca exchanger
norephedrine
noradrenaline membrane transporter
N-methyl-D-aspartate receptor, ionotropic glutamate receptor
national poisons information centre
phenylacetone
pheochromocytoma cell line
prefrontal cortex
logarithmic measure of the acid dissociation constant
protein kinase A
protein kinase C
2+
plasma membrane Ca -ATPase
pedunculopontine tegmental nuclei
vesicular dopamine content
2+
sarco/endoplasmic recticulum Ca -ATPase
serotonin membrane transporter
substantia nigra compacta
substantia nigra reticulata
selective serotonin reuptake inhibitors
small synaptic vesicles
subthalamic nucleus
tyrosine hydroxylase
thalamus
VGCC
VMAT
VP
VTA
WT
voltage-gated calcium channel
vesicular monoamine transporter
ventral pallidum
ventral tegmental area
wildtype
Chapter 1
General Introduction
General Introduction
Drugs of abuse
The term “drugs of abuse” refers to substances that are not taken for medical reasons, but
usually for mind-altering effects including feelings of emotional warmth, empathy toward
others, enhanced sensory perception, a general sense of well being and decreased anxiety.
Many of these drugs are only taken in recreational settings. However, problematic forms of
drug use may develop. Some drugs of abuse may cause dependency leading to addiction.
Furthermore, overdoses can easily lead to intoxications. In addition to mind-altering effects,
acute as well as long-term adverse effects have been described. For example, the main
constituent of ecstasy tablets, MDMA, is known to induce effects on the cardiovascular
system that include cardiac stimulation and tachycardia as well as vasoconstriction.
Moreover, MDMA affects thermoregulation, resulting in hyperthermia. The central nervous
system is an important target for drugs of abuse and behavioral changes as well as long-term
neurotoxicity have been reported (for review see Cole and Sumnall 2003). Apart from adverse
effects for the user, drugs of abuse have a much wider impact on society. Medical treatment
related to problematic forms of drug use, like addiction and intoxication, is a burden on the
public health care budget. Moreover, drugs of abuse are also believed to undermine social
development and feed corruption and organized crime (EMCDDA 2010).
The most recent data available on lifetime prevalence in the European adult
population (15-64 years old) indicate that almost a quarter of the population has used
cannabis, whereas around ~11 million people (3,3%) have used ecstasy and ~12 million (3.6%)
have used amphetamines (EMCDDA 2010). Although the prevalence of chemically
manufactured drug use appears low compared to cannabis use, the effects these chemically
manufactured drugs induce are usually more harmful. All of these chemically manufactured
drugs are considered to be hard drugs and are illicit drugs, whereas cannabis is considered to
be a soft drug that is prohibited in most countries (Opiumwet 1928; EMCDDA 2010).
European figures of hospital admittances are unavailable, but in the US, where a similar
prevalence for drug use is found, amphetamines and other stimulants (like substances in
ecstasy tablets) are responsible for ~100.000 emergency hospital admittances each year
(DAWN 2010). European member states report around ~8.000 deaths by a drug of abuse
overdose each year (EMCDDA 2010).
In the Netherlands, the annual report from the National Poisons Information Centre
(NVIC) reported approximately thousand information requests regarding drugs of abuse of
which one third involved amphetamine and ecstasy (van Velzen et al. 2010). Although
knowledge on mechanisms of action of drugs of abuse has increased extensively during the
last decades, the number of information requests at the NVIC suggests that current
knowledge on cellular and molecular mechanisms is insufficient. This is also reflected in the
treatment of patients with a drug intoxication, which consists primarily of stabilizing the
patient and monitoring vital functions. In addition, pharmacotherapy (e.g., benzodiazepines)
can be applied to reduce the stimulatory effects of certain drugs of abuse (e.g.,
amphetamines). Research into the cellular and molecular mechanisms of drugs of abuse
might contribute to the development of a more “evidence-based” treatment for intoxication
with drugs of abuse. In this thesis, cellular and molecular effects induced by
2
Chapter 1
methamphetamine, amphetamine, 3,4-methylenedioxymethamphetamine (MDMA), 3,4methylenedioxy-amphetamine (MDA) and meta-chlorophenylpiperazine (mCPP) are
described. These drugs were selected as they are the most commonly used illicit, chemically
manufactured drugs (EMCDDA 2010). Amphetamines are used as drugs of abuse in many
forms, whereas the other selected drugs are often found in ecstasy tablets; MDMA is the
main common constituent with MDA being an important metabolite. MDMA is an
amphetamine derivate that is recreationally used for its entactogenic, stimulating and
rewarding properties. The piperazine derivate mCPP is also commonly found in ecstasy
tablets and has been replacing MDMA as the main constituent in some countries (EMCDDA
2009). Some of these drugs of abuse are also used in regular medicine to treat patients. For
example, amphetamines are administrated to treat attention-deficit hyperactivity disorder
(ADHD) and mCPP has a legitimate use as the pharmacologically active metabolite of several
psychiatric pharmaceuticals (Rotzinger et al. 1998). A better understanding of the mechanism
of action of drugs of abuse is useful to improve the treatment of intoxications with drugs of
abuse.
This thesis focuses on the cellular mechanisms underlying the short-term toxic effects
of drugs of abuse on the central nervous system, more specifically on the dopaminergic
system. Therefore a short introduction in dopaminergic brain systems and presynaptic
processes is provided to enable a better understanding of the involved mechanisms.
Neurotransmission
In the brain, signals are transmitted via neurotransmission. Upon activation, a presynaptic
neuron will release chemical messenger molecules (neurotransmitters) that activate specific
receptors on neighboring postsynaptic neurons (see also Fig. 1.1). Provided that an adequate
number of postsynaptic receptors is activated, the signal is propagated from the postsynaptic
neuron to the following neuron.
Within the brain, regions can be distinguished that contain specific types of neurons,
e.g., some regions primarily contain dopaminergic neurons, whereas serotonergic neurons
dominate in other regions. In the following paragraph, the main dopaminergic pathways
present in the brain as well as neurotransmitter systems that may influence these pathways
will be discussed.
Presynaptic input and dopaminergic pathways
Dopamine (DA) is an important neurotransmitter, which is involved in a variety of human
behaviors, like executive function, working memory, motivation, mood regulation and
psychomotor function. Moreover, DA neurons also play an important role in many brain
pathologies, including schizophrenia, addiction, ADHD and Parkinson’s disease. Most DA
neurons are found in two nuclei; the ventral tegmental area (VTA) and the substantia nigra
compacta (SNc). However, DA neurons have also been found in other parts of the brain, such
as the striatum (see Fig. 1.2, Di Giovanni et al. 2010).
3
General Introduction
Figure 1.1. Neurotransmission. Pre- and postsynaptic neurons connect at the synapse where signal
transduction occurs. The presynaptic neuron contains vesicles filled with neurotransmitters, that can be
released upon activation of the presynaptic neuron. These neurotransmitters can diffuse across the
synaptic cleft and then bind to specific receptors on the postsynaptic neuron. Modified from:
http://www.neurevolution.net.
Three major DA pathways innervate the forebrain and basal ganglia; the nigrostriatal,
mesocortical and mesolimbic pathway. The nigrostriatal pathway arises from SNc and projects
to the striatum. DA release from this pathway modulates striatal output and is involved in
facilitating movement or inhibiting unwanted movement. The mesocortical pathway arises
from the VTA and projects to the prefrontal cortex (PFC) and is crucial in normal cognitive
processes. The mesolimbic pathway also arises from the VTA but projects to the nucleus
accumbens (NA; sometimes also referred to as the limbic striatum), hippocampus, amygdala
and PFC. This pathway is part of the reward circuitry and important in, e.g., addiction. Some
of the mentioned structures are part of larger structures, e.g., basal ganglia is a collective
term for the striatum, the globus pallidus externa and interna (GPe and GPi), the ventral
pallidum and the substantia nigra (SN). Furthermore, SN can be subdivided into the SNc and
substantia nigra reticulata (SNr) and the subthalamic nucleus (STN) (see Fig. 1.2, for review
see Andre et al. 2010; Lester et al. 2010).
DA that is released by neurons in these dopaminergic pathways can bind to DA
receptors expressed on pre- or postsynaptic neurons. These receptors are G-protein-coupled
receptors (GPCRs) that can be divided in two groups; D1-like receptors (D1 and D5) and D2like receptors (D2, D3 and D4). Activation of these receptors by DA results in modulation of
second messenger systems. Activation of D1-like receptors stimulates adenylyl cyclase and
phospholipase C activity, thereby increasing phosphorylation and activating calcium-binding
proteins.
4
Chapter 1
Activation of D1-like receptors increases the availability of vesicles for fusion with the cell
membrane and thus increases vesicular DA release. Activation of D2-like receptors, also
known as autoreceptors, inhibits adenylyl cyclase activity, resulting in less phosphorylation
and an inhibitory effect on dopaminergic neurotransmission (Neve et al. 2004).
Figure 1.2. Dopaminergic pathways present in the brain; the nigrostriatal, mesocortical and mesolimbic
pathway are indicated with grey lines. VTA; ventral tegmental area. Modified from the National Institute
on Drug Abuse (NIDA).
Apart from autoreceptors, dopaminergic neurons also receive many inputs from other
neurotransmitter systems, including the serotonergic, cholinergic, glutamatergic and GABAergic system. Depending on which type of receptor is activated, input can increase (excitatory
input) or decrease (inhibitory input) dopaminergic neurotransmission. In the following
paragraphs, several important inputs on presynaptic dopaminergic neurons will be discussed
to indicate how these can affect dopaminergic output.
The largest excitatory input on dopaminergic neurons originates from the
glutamatergic system. Glutamatergic neurons innervate the GPi/SNr, GPe and SNc, which in
turn innervate the thalamus. Furthermore, glutamatergic input from the thalamic nuclei
innervates the PFC, which in turn innervates the NAc, VTA, laterodorsal tegmental nuclei
(LDT), striatal cholinergic and GABA-ergic interneurons (Freed 1994; Floresco et al. 2001;
Andre et al. 2010). Glutamatergic input is mediated by activation of two types of glutamate
receptors (Glu-Rs) expressed on dopaminergic neurons: metabotropic, G-protein-coupled
glutamate receptors (mGlu-Rs) and ionotropic Glu-Rs. Ionotropic Glu-Rs consist of
combinations of 4 subunits that form an ion channel pore and include NMDA (GluN1-N3),
AMPA (GluA1-A4), kainate (GluK1-K5), and δ receptors (GluD1-D2). Binding of agonists to
ionotropic Glu-Rs opens the ion channel allowing sodium or calcium influx, depending on the
type of receptor, and potassium efflux. Binding of agonists to the mGlu-Rs activates second
messenger systems. Three groups of mGlu-Rs can be distinguished; group I mGlu-Rs (1 and 5),
which are linked to phospholipase C and activation of these receptors results in calcium
release from intracellular stores. Activation of mGlu-Rs of group II (mGlu-R2 and 3) and group
III (mGlu-R4 and 6-8) catalyzes cyclic adenosine monophosphate (cAMP) production.
5
General Introduction
cAMP can activate protein kinase A (PKA), which could increase the availability of releasable
vesicles (Simeone et al. 2004; Traynelis et al. 2010).
The mainly excitatory serotonergic input on the dopaminergic system arises from
serotonergic neurons within two serotonergic nuclei, the dorsal raphe nuclei (DRN) and the
medial raphe nuclei (MRN). Serotonergic neurons within the raphe nuclei innervate
dopaminergic neurons in the VTA, SN, NA, PFC and striatum (Di Giovanni et al. 2010). The
serotonergic system can influence the dopaminergic system via release of serotonin (5-HT),
which can bind to metabotropic and ionotropic serotonin receptors (5-HT-Rs) expressed on
dopaminergic neurons. They can be divided into seven classes (5-HT1-7-R), most of which are
metabotropic, GPCRs. The 5-HT3-R is the only 5-HT-R that forms a cation-selective ion
channel. All other 5-HT-Rs act on second messenger systems when activated. Activation of
most 5-HT-R subtypes, including activation of the 5-HT1A-R, 5-HT1B-R, 5-HT2A-R, 5-HT3-R, and 5HT4-R facilitates DA release, thereby increasing neurotransmission. However, activation of the
5-HT2C-R mediates an inhibitory effect on DA release (Di Giovanni et al. 2008; Di Giovanni et
al. 2010).
The cholinergic system has an excitatory as well as inhibitory input on the
dopaminergic system and input arises from cholinergic neurons within two nuclei located in
the pons region of the hindbrain; LDT and the pedunculopontine tegmental nuclei (PPT).
Processes from the PPT project to the SNc and modulate dopaminergic output. Furthermore,
cholinergic interneurons in the striatum, which account for 2% of the total number of neurons
in the striatum, also contribute to cholinergic input. The cholinergic system can influence the
dopaminergic system via release of acetylcholine (ACh) that can bind to two types of
receptors; the ionotropic nicotinic acetylcholine receptor (nACh-R) and the metabotropic
muscarinic acetylcholine receptor (mACh-R). At least five different types of nACh-Rs are
expressed on dopaminergic neurons, which consist of different combinations of five α and β
subunits, but also include a homomeric α7 subtype. Many nACh-Rs have a relatively high
permeability to calcium, with the α7 homomers having the highest permeability. Binding of
agonists, like ACh or nicotine, to these nACh-Rs results in a rapid increase in conductance for
sodium and/or calcium. This might initiate depolarization and calcium entry, which will
increase DA release. Five subtypes of mACh-Rs have been identified; M1-like receptors (M1, 3
and 5) and M2-like receptors (M2 and 4). Activation of M1-like receptors could increase
dopaminergic neurotransmission via formation of inositol 1,4,5-trisphosphate (IP3) and 1,22+
diacylglycerol, which increases the intracellular calcium concentration ([Ca ]i). Activation of
M2-like receptors results in inhibitory effects through inhibition of adenylyl cyclase and
calcium channels as well as through activation of potassium channels and mitogen-activated
kinases. Consequently, activation of mACh-Rs has been shown to result in both excitation and
inhibition of DA activity (MacDermott et al. 1999; Engelman and MacDermott 2004; Lester et
al. 2010).
The most important inhibitory input on the dopaminergic system originates from
GABA-ergic neurons. GABA-ergic interneurons in the striatum project to the GPe, GPi and SNr,
whereas GABA-ergic neurons in the GPi and SNr project to the thalamus. GABA-ergic neurons
in the ventral pallidium (VP) also project to the thalamus and to the VTA. The GABA-ergic
system can influence the dopaminergic system via release of GABA that can bind to two types
6
Chapter 1
of GABA-Rs; the ionotropic GABAA-R and the metabotropic GABAB-R. The GABAA-R consists of
different combinations of α, β, γ, δ, ε, ρ, θ and π subunits. In the brain, the α1β2γ2 subtype
combination is the most abundant and widely distributed form of the GABAA-R. The GABAA-R
contains two GABA binding sites and also allosteric modulatory sites like the benzodiazepine
binding site. Binding of GABA to GABAA-R induces an inward chloride current, resulting in
hyperpolarization of the membrane of the presynaptic dopaminergic neuron and thereby
decreasing neurotransmission (McKernan and Whiting 1996; Sigel 2002; D'Hulst et al. 2009).
The GABAB-R is a heterodimer consisting of B1 and B2 subunits, with the B1 subunit
containing the GABA binding domain and the B2 subunit the G-protein-coupling mechanism
as well as an allosteric modulatory site. The GABAB-R modulates calcium and potassium
channels and therefore a variety of effects can be expected upon activation of the GABAB-R
(Bowery et al. 2002; Andre et al. 2010; Lester et al. 2010).
In summary, dopaminergic output is affected by multiple inhibitory and excitatory inputs from
non-dopaminergic neurotransmitter systems, like the glutamatergic, serotonergic, cholinergic
and GABA-ergic system. Neurotransmitters that are released by neurons belonging to these
systems can activate receptors on dopaminergic neurons, thereby affecting dopaminergic
output. In addition to inputs, several intracellular presynaptic processes in dopaminergic
neurons play an essential role in neurotransmission. These will be discussed in the following
paragraph.
Presynaptic processes
A large part of this thesis deals with presynaptic dopaminergic transmission and therefore the
neurotransmitter DA will be used to describe presynaptic neurotransmission. For other
neurotransmitters, similar processes are involved, although different enzymes and
transporters are involved. DA is synthesized in the cytosol of the dopaminergic neuron by
conversion of L-tyrosine to L-dihydroxyphenylalanine (L-DOPA) by the enzyme tyrosine
hydroxylase (TH) and stored in vesicles. DA that is not stored in vesicles is slowly metabolized
by monoamine oxidase (MAO). DA is transported from the cytosol into the vesicles via the
vesicular monoamine transporter (VMAT) expressed on the vesicle membrane. Vesicles can
store large amounts of DA against the concentration gradient by binding DA to an
intravesicular matrix of storage proteins, such as chromogranins (Westerink 2006).
Upon activation of a neuron, its vesicles can fuse with the cell membrane and the
vesicular content will be released in the synaptic cleft. This process is referred to as exocytosis
(see Fig. 1.3). Prior to exocytosis, vesicles migrate towards the cell membrane. During this
trafficking, the cytoskeleton has an important function.
A critical component of the cytoskeleton is actin; vesicles are connected to actin
filaments via synapsins strands. These synapsins regulate the availability of vesicles;
phosphorylation of synapsins decreases the interaction with actin resulting in an increased
availability of vesicles. Therefore, enhanced synapsin phosphorylation can increase
neurotransmission. Once vesicles are trafficked towards the cell membrane, they can form a
tight SNARE complex at the cell membrane (docking).
7
General Introduction
Figure 1.3. Schematic representation of the intracellular vesicle cycle and DA cycle that are present in
dopaminergic neurons. Dopamine (DA) is synthesized in the cytosol after which it is stored in vesicles
that can translocate to the cell membrane. Vesicles that have docked and primed can fuse with the cell
2+
membrane (exocytosis) upon an increase in [Ca ]i. Vesicular DA content depends on DA synthesis
(tyrosine hydroxylase; TH), breakdown (monoamine oxidase; MAO) and uptake (dopamine membrane
transporter; DAT) as well as on vesicular uptake (vesicular monoamine transporter; VMAT) and
intravesicular storage capacity (granins). Modified from Westerink 2006.
Subsequent molecular arrangements are made to allow for exocytosis to occur (priming).
Vesicles are distributed in the presynaptic terminal in a distinctive manner, due to synapsin
regulation of the actin network. Consequently, different pools of available vesicles can be
distinguished. At least three vesicle pools can be distinguished: (1) an undocked, (2) a docked,
but unprimed and (3) a docked and primed pool, which is also referred to as the “readily
releasable pool”. This latter pool of vesicles can be directly released upon activation of the
neuron, whereas following depletion of this pool, a different vesicle pool is mobilized (for
review see Doussau and Augustine 2000; Sorensen 2004; Westerink 2006). Only the readily
releasable vesicle pool is able to fuse with the cell membrane and only following a strong
2+
increase in [Ca ]i. Following release into the synaptic cleft, DA can bind to DA receptors on
postsynaptic neurons or to DA autoreceptors present on the DA releasing neuron. To prevent
ongoing receptor activation and to recycle DA, DA is transported from the synapse into the
cytosol of the presynaptic neuron by the DA transporter (DAT) at the cell membrane or is
broken down by extracellular MAO (see Fig. 1.3, for review see Westerink 2006).
Modulation of any part of the vesicle cycle could affect the amount of DA secreted during
exocytosis. For example, the active substance in ecstasy tablets, MDMA, can increase TH
phosphorylation. This increases DA synthesis, which could contribute to increased DA levels
observed in vivo. Moreover, amphetamine causes reversal of DAT, causing it to transport DA
8
Chapter 1
from the cytosol to the extracellular space, thereby increasing extracellular DA levels
(Westerink 2006).
Calcium is involved in many cellular processes, such as gene expression, cell death,
differentiation and growth and, as mentioned, also in neurotransmission. As an increase in
2+
2+
[Ca ]i precedes exocytosis, [Ca ]i is a valuable readout to predict exocytosis. The calcium
2+
concentration in the extracellular space amounts up to 2 mM, whereas [Ca ]i in a resting
2+
state is low; ~100 nM. However, [Ca ]i can rapidly increase to µM levels upon stimulation. For
2+
neuronal survival, a stringent control of [Ca ]i is necessary and therefore a strict homeostasis
is maintained by specialized mechanisms, including calcium influx, calcium efflux, calcium
buffering and internal calcium storage.
2+
[Ca ]i can increase via influx of extracellular calcium trough voltage-gated calcium
channels (VGCCs) or ligand-gated ion channels. Furthermore, calcium can be released from
intracellular stores, including mitochondria, vesicles and the endoplasmic reticulum (ER),
which is the major intracellular calcium store. The calcium signal is terminated by closure of
the VGCCs or ligand-gated ion channels and removal of cytosolic calcium via efflux through
+
2+
2+
the Na /Ca exchanger (NCX) and the ATP-driven Ca pump (PMCA) in the cell membrane as
2+
well as via uptake in mitochondria and ER through the sarco/endoplasmic recticulum Ca ATPase (SERCA) (for reviews see Carafoli et al. 2001; Duszynski et al. 2006; Westerink 2006;
Celsi et al. 2009).
9
General Introduction
Thesis outline
Most drugs of abuse are known to influence the dopaminergic system. However, the
mechanisms of action are still not fully understood. For example, are drug-induced increases
in DA levels due to direct effects on the dopaminergic system (as discussed in Presynaptic
processes) or is dopaminergic output altered by drug-induced changes in input from other
neurotransmitter systems on the dopaminergic system (as discussed in Presynaptic input and
dopaminergic pathways)? Although many macroscopic effects have been described, scientific
progress would benefit from an enhanced insight on a cellular level. Furthermore, druginduced effects on other neurotransmitter systems that provide input on the dopaminergic
system should also be investigated. Therefore, this research aimed to unravel the cellular
mechanisms through which drugs of abuse cause the observed in vivo effects on the
dopaminergic system. The dopaminergic system in the brain and the concept of
neurotransmission are introduced in chapter 1, whereas the applied models and techniques
are described in chapter 2. Calcium plays an important role in neurotransmission and can
therefore serve as a functional readout to determine drug-induced effects on
neurotransmission. In this doctoral research a calcium fluorescent dye was applied in
2+
2+
combination with microscopy to measure [Ca ]i. Although this technique enables [Ca ]i
recordings of multiple single cells in one experiment, it is a time-consuming method and
therefore considered to have a medium throughput of measurements. Therefore, some
scientists and pharmaceutical companies have switched to high-throughput measurements of
2+
[Ca ]i. In chapter 3 the accuracy and reliability of these high-throughput measurements of
2+
[Ca ]i is further discussed.
In chapter 4 amphetamine-induced effects were investigated, since amphetamine is
well known for its effects on the dopaminergic system. Initially, this drug was chosen to serve
2+
as a positive control since effects on [Ca ]i as well as on exocytosis have been described
2+
previously. Surprisingly, in our cell models amphetamine did not affect [Ca ]i or exocytosis at
relevant concentrations. However, following L-DOPA pre-treatment, thereby adding DA that is
not bound to the vesicular granin matrix (dense-core), an amphetamine-induced reduction of
vesicle content was observed. A refinement of the widely-accepted theory on the mechanism
of action of amphetamine is proposed, i.e., amphetamine can only exert its effect on free
vesicular DA, indicating that the dense-core present in large dense-core vesicles (LDCVs) can
protect these vesicles against AMPH-induced leakage.
Subsequently, a drug of abuse was investigated that is more known for inducing
effects on the serotonergic system; MDMA, the primary constituent of ecstasy tablets. To
investigate whether MDMA also affects the dopaminergic system, effects on calcium
homeostasis as well as on DA exocytosis were investigated. In chapter 5 it is shown that
MDMA and its main metabolite MDA, in addition to influencing the serotonergic system, also
affect dopaminergic neurotransmission. Both substances inhibit depolarization-evoked
vesicular DA release and calcium influx in PC12 cells. These results indicate that the observed
MDMA- and MDA-induced increase in DA brain levels in vivo is not due to an increase in
exocytosis, but rather due to reversal of DAT or indirect effects on DA exocytosis, e.g.,
through inputs from different neurotransmitter systems.
10
Chapter 1
The cholinergic system serves as an important excitatory input on the dopaminergic system
and therefore, drug-induced effects on the cholinergic system could contribute to observed
dopaminergic effects. The cholinergic system influences the dopaminergic system through
several ACh-Rs (see also Presynaptic input and dopaminergic pathways) and in chapter 6 the
effects of methamphetamine, amphetamine, MDMA, MDA and mCPP on nACh-Rs were
2+
investigated by measuring changes in [Ca ]i. At a low concentration (10 µM),
2+
methamphetamine and amphetamine potentiated the ACh-evoked increase in [Ca ]i,
indicating agonistic drug properties. This increase in cholinergic input might contribute to
drug-induced increases in DA levels observed in vivo. However, at higher drug concentrations
it is unlikely that increased DA levels are related to an increase in cholinergic input, as all
tested drugs antagonized nACh-Rs. Although at 1 mM all drugs also inhibited depolarization2+
evoked increases in [Ca ]i, stronger reductions were observed in ACh-evoked increases,
indicating some degree of specificity for nACh-Rs.
The GABA-ergic system is the most important inhibitory input on the dopaminergic
system. Although drug-induced inhibition of the GABA-ergic system could explain increases in
DA levels, little is known about possible drug-induced effects on the GABA-ergic system. In
chapter 7 a novel mode of action is shown for methamphetamine, amphetamine, MDMA,
MDA and mCPP; modulation of the human GABAA-R, which depends on the degree of
receptor occupancy. At low receptor occupancy, high concentrations of methamphetamine,
MDMA, MDA and mCPP inhibited GABA-evoked current, possibly through competitive binding
to one of the GABA binding sites. At high receptor occupancy, MDA and mCPP induced
potentiations, which could involve interaction with a positive allosteric binding site such as
the benzodiazepine binding site. These results indicate that at high drug concentrations, e.g.,
during intoxication, diminished GABA-ergic inhibitory input might contribute to
overstimulation of the dopaminergic system by drugs of abuse. Therefore, the GABA-ergic
system should be investigated more thoroughly to explore new treatment options for
intoxications with drugs of abuse.
In chapter 8 the observed effects that are described in the different chapters are
integrated and placed into context with other available data on mechanisms of action of
drugs of abuse. Although the available data only allows for a limited risk assessment,
exposure data on drugs of abuse is available and is used to correlate our data to relevant drug
concentrations. Additional information that is needed to perform a more thorough risk
assessment, e.g., drug-induced effects following repeated drug exposure, concurrent drug
exposure and drug-induced effects on important dopaminergic inputs, is discussed and
recommendations for future research are provided. The main conclusions of this thesis as
well as recommendations for future research are described in chapter 9.
11
General Introduction
12
Chapter 2
Applied methodologies
Applied methodologies
Several techniques can be used to investigate effects of drugs of abuse on the dopaminergic
system. As many of these drugs are known to increase DA brain levels, we investigated
whether DA exocytosis contributed to this increase by using carbon fiber microelectrode
2+
amperometry. We also studied drugs of abuse-induced effects on the [Ca ]i as an increase in
2+
[Ca ]i precedes exocytosis and can also serve as a readout for ACh-R activation. To investigate
drugs of abuse-induced effects on the GABAA-R we used the two-electrode voltage-clamp
technique. Cell viability tests were performed to exclude that observed effects were due to
cytotoxicity. These techniques and the model systems used as well as their advantages and
limitations will be shortly described.
Ex vivo and in vitro test systems
Primary chromaffin cells and PC12 cells
To investigate possible drug-induced effects on neurotransmitter release as well as on
calcium homeostasis two cell models were used; primary mouse adrenal chromaffin cells and
rat adrenal pheochromocytoma (PC12) cells.
Following the discovery of the secretory properties of chromaffin cells and their close
relation to neurons, chromaffin cells have been used for decades to investigate the molecular
mechanism of catecholamine secretion (Borges et al. 2008). Adrenal chromaffin cells were
isolated from decapitated C57BL6 mice of 2-3 weeks old by removal of the adrenal glands and
separation of the medulla and cortex. The medulla is predominantly composed of chromaffin
cells, which are derived from the neural crest and resemble modified sympathetic neurons.
These cells synthesize, store and release two types of cathecholamines; adrenaline (~20%)
and noradrenaline (~80%). Chromaffin cells have large dense-core vesicles (LDCVs) which
store catecholamines (Diaz-Flores et al. 2008), as well as small synaptic vesicles (SSVs, Koval et
al. 2001). Isolation of chromaffin cells from adrenal glands requires collagenase treatment
and mechanical separation of the tissue by titration. Subsequently, the tissue is exposed to an
enzyme solution containing papain to digest remaining connective tissue and to isolate single
cells. Following isolation, single cells can attach to dishes (glass or plastic) using appropriate
coating (see chapter 3 for more details), after which they can be used in experiments.
The PC12 cell line was isolated from a tumour in the adrenal medulla of the rat (Greene
and Tischler 1976) and PC12 cells are thus chromaffin-derived cells. These cells are often used
as a model to study the physiology of dopaminergic neurons. Like chromaffin cells, PC12 cells
also synthesize, store and release catecholamines in a calcium dependent manner. Moreover,
LDCVs are present that store DA and SSVs that store ACh (for review see Westerink and Ewing
2008). Although vesicles in PC12 cells are known to also contain noradrenaline (Chen et al.
1994), the vesicles in our PC12 cells contain 99% DA and <1% noradrenaline (Westerink et al.
2002). The number of releasable vesicles as well as the amount of catecholamines per vesicle
is limited in these undifferentiated cells (Westerink et al. 2000). As a result, the secretory
response decreases during multiple stimulations. Therefore, modifications of the model
system are required to reliably detect possible drug-induced changes in exocytosis.
Dexamethasone differentiation enhances TH activity and thus increases DA synthesis
(Schubert et al. 1980).
14
Chapter 2
Furthermore, the coupling between VGCCs and vesicle release sites is increased following
dexamethasone differentiation (Westerink and Ewing 2008). As a result, the amount of
catecholamines per vesicle and the number of releasable vesicles during successive
stimulations is increased (Westerink and Vijverberg 2002; Westerink and Ewing 2008).
Fortunately, dexamethasone-differentiated PC12 cells are comparable with undifferentiated
PC12 cells with respect to qualitative calcium channel expression patterns, which makes
dexamethasone-differentiation a valuable and valid tool allowing reliable measurements of
DA exocytosis.
Both cell types serve as a model for neurons and can be used to investigate effects on
neurotransmitter release as well as on calcium homeostasis. However, both have their
specific (dis)advantages and characteristics. An important feature of chromaffin cells is that
these cells are derived from laboratory animals, which gives rise to ethical issues but also
increases variation in the obtained data. PC12 cells are a popular model because of their ease
of use, the large amount of available background data on their proliferation and
differentiation and because these cells mimic many features of DA neurons (Westerink and
Ewing 2008). They resemble neurons more closely than chromaffin cells with their smaller
vesicles and quantal size and with respect to processes involved in calcium homeostasis
(Duman et al. 2008; Westerink and Ewing 2008). A comparison between PC12 cells,
chromaffin cells and DA terminals, revealed similar amounts of TH, whereas lower amounts of
DAT were found in PC12 cells compared to neurons. Chromaffin cells express an intermediate
amount of DAT. Moreover, VMAT (type 2) is well represented in DA terminals, whereas it was
not detected in PC12 cells and intermediate levels were found in chromaffin cells (Fornai et
al. 2007). These differences should be taken into account when comparing in vitro with in vivo
data as well as when performing risk assessment by extrapolating results obtained in these
two cell models.
Xenopus laevis oocytes
To investigate direct drug-induced effects on GABAA-Rs, Xenopus laevis oocytes were used as
a research tool, a model that was already introduced in the late 1950s. Several characteristics
enable the oocytes to efficiently translate injected complementary (cDNA) into RNA and RNA
into protein. For example, a single oocyte has a similar amount of mitochondria and
ribosomes as 100.000 somatic cells. Moreover, oocytes also have a higher level of RNA
polymerases; four times above that of somatic cells. Studies using labelled proteins have
shown that these synthesized proteins are transported to areas where they normally occur,
whereas neurophysiological studies have shown that such proteins have normal functionality
(Brown 2004). Furthermore, preparation of oocytes is relatively easy and their size (~1 mm)
makes them easy to handle. Furthermore, Xenopus laevis are laboratory animals that are easy
kept and bred in captivity and therefore available at low costs (Wagner et al. 2000).
Therefore, the Xenopus laevis oocyte is widely used for the expression of heterologous
proteins, like receptors.
To harvest oocytes, female Xenopus laevis were anaesthetized (MS-222) and ovarian
lobes were removed by surgical laparotomy. Xenopus laevis were put on ice and their skin
was moistured during the procedure to prevent dehydration and damaging of the skin.
15
Applied methodologies
Experiments were conducted in accordance with Dutch law and the European Community
directives regulating animal research (86/609/EEC). Collagenase treatment was applied to
isolate single oocytes from the collected ovarian lobes. cDNA was injected into the oocytes
and depending on the receptor, expression on the cell membrane is accomplished 2-5 days
following injection (Fig. 2.1).
A limitation of the oocyte as a research model is their endogenous expression of
several ion channels, transporters and receptors. Endogenously expressed ion channels
include at least four different classes of chloride channels and the main ion conductance in
oocytes is a chloride conductance through calcium-activated chloride channels. Furthermore,
the presence of potassium, sodium and calcium channels on the oocyte cell membrane has
been described. Endogenously expressed transporters include the sodium/potassium-ATPase
and glucose and amino acids transporters (Sobczak et al. 2010). In some studies, oocytes not
injected with cDNA have been described to respond to ACh, carbachol, angiotensin II,
prostaglandines and oxytocin, indicating potential expression of ACh-Rs, angiotensin-,
prostaglandins- and oxytocin- receptors (Oron et al. 1988; Miledi and Woodward 1989;
Brooker et al. 1990; Lupu-Meiri et al. 1990). As proteins can influence the function of other
proteins, possible interaction between these expressed endogenous proteins and exogenous
proteins should also be taken into account.
Furthermore, oocytes have different signalling pathways compared to somatic cells,
which might influence observed effects. For example, some receptors that couple to the
protein kinase C (PKC) pathway in human cells, do not couple to the PKC pathway in Xenopus
Laevis oocytes (Wagner et al. 2000).
Figure 2.1. Ovarian lobes are harvested from female Xenopus Laevis frogs, after which single oocytes are
isolated. Oocytes are injected with cDNA of the receptor of interest and receptors will be expressed on
the cell membrane following an incubation period. Modified from C.J.G.M. Smulders and E.C. Antunes
Fernandes.
Other models to investigate heterologous expressed proteins include human embryonic
kidney cells (HEK-293) and Chinese hamster ovary (CHO) cells. Compared to CHO cells, HEK
cells are preferable for many applications due to their human origin, higher and cheaper
transfectability and higher expression yield (Preuss et al. 2000, Suen et al. 2010).
16
Chapter 2
Although all these models can express proteins, drug-induced effects on the expressed
protein are usually investigated via binding studies. Used in this manner, these research
models do not provide a direct measurement of receptor functionality.
Using Xenopus Laevis oocytes will provide the most direct measurement of receptor
functionality compared to other available models and is therefore the most suitable model for
our study.
Cell viability assay
Drug-induced changes in cell viability were determined by measuring the capacity of
undifferentiated PC12 cells to take up and reduce soluble 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) into insoluble formazan with mitochondrial
succinic dehydrogenase (Mosmann 1983; Denizot and Lang 1986). PC12 cells were exposed to
drugs of abuse in multi-well plates, followed by incubation with MTT. Proteins present in the
culture medium can affect the free drug concentration, as test compounds can bind to these
proteins. Therefore, cells were exposed to drugs of abuse in serum free medium. Following
MTT incubation, cells were lysed and the quantity of formazan was measured
spectrophotometrically and used as a measure for cell viability.
The MTT assay is based on mitochondrial activity, which should be kept in mind when
interpreting data obtained with this assay as substances that act as respiratory chain
inhibitors could affect formazan formation. Furthermore, the obtained results can be
significantly influenced by medium pH (Jabbar et al. 1989) as well as the D-glucose
concentration in culture medium (Vistica et al. 1991). Consequently, these parameters should
be strictly controlled to have similar values in each experiment.
Passive assays that investigate effects on cell viability (e.g., neutral red uptake;
Borenfreund and Puerner 1985) can be influenced by weak bases (Ohkuma and Poole 1981).
As amphetamines, as well as MDMA and MDA are weak bases, passive assays were therefore
considered less suitable. Although other techniques could be applied to measure cell viability,
MTT was the most suitable method to investigate drugs of abuse-induced effects.
Calcium homeostasis, intracellular calcium imaging
2+
Drug-induced changes in [Ca ]i in undifferentiated PC12 and chromaffin cells were
2+
determined by measuring fluorescence of the high-affinity Ca -responsive fluorescent dye
Fura 2-AM (Molecular Probes; Invitrogen, Breda, The Netherlands). Fura 2-AM can cross cell
membranes because of its acetoxymethyl (AM) ester-group and is calcium sensitive following
intracellular cleavage of its AM group, after which it is unable to cross cell membranes.
Therefore, only intracellular calcium is measured when using Fura 2-AM to determine
2+
changes in [Ca ]i.
An inverted microscope was used to select a suitable region of the glass-bottom dish
with a sufficient amount of single cells. Fluorescence was evoked with 340 and 380 nm
excitation wavelengths (F340 and F380) using a polychromator and collected at 510 nm with a
2+
digital camera. At low [Ca ]i, the emission signal following excitation with 340 nM is low,
2+
whereas the emission signal is high at high [Ca ]i (Fig. 2.2D). On the other hand, excitation at
17
Applied methodologies
2+
380 nM at low [Ca ]i results in a higher emission signal compared to excitation during higher
2+
2+
[Ca ]i. Thus, an increase in the ratio F340/F380 is directly correlated to the [Ca ]i.
2+
Most experiments investigating the effects of drugs of abuse on [Ca ]i, described in
2+
this thesis, consisted of several minutes baseline recording to measure basal [Ca ]i, after
2+
+
which an increase in [Ca ]i was triggered by application of K or ACh for several seconds to
2+
measure the stimulus-evoked [Ca ]i (Fig. 2.2 A and B). Following this first depolarization and a
recovery time, a 15 min exposure period precedes a second stimulus.
To be able to correct for the non-linear decrease in Fura 2 sensitivity for calcium with
increasing calcium concentrations, specific experimental parameters were determined, like
the maximum and minimum fluorescence (Rmax and Rmin) and the dissociation constant (Kd).
2+
2+
Figure 2.2. A-C. Fluorescence microscopy [Ca ]i recordings of cells in a basal state of low [Ca ]i (A) and
cells displaying an increased [Ca2+]i due to depolarization with 100 mM K+ (B) or to ionomycin exposure
(C). D. Fluorescence excitation spectrum of Fura 2 in solutions containing 0-40 µM calcium (D modified
from Invitrogen).
18
Chapter 2
2+
Using these parameters allows for reliable calculations of [Ca ]i according to a modified
2+
Grynkiewicz’s equation [Ca ]i = Kd* *(R-Rmin) / (Rmax-R) (Deitmer and Schild 2000).
When using fluorescence microscopy in combination with Fura 2, it should be taken
into account that Fura 2 acts as an additional calcium buffer by binding calcium. Moreover,
Fura 2 can translocate to intracellular compartments, which will result in a loss of sensitivity
to cytosolic free calcium (Takahashi et al. 1999). Fura 2 is a ratio dye, which has several
advantages compared to a single wavelength dye for example like Fluo 4. By using a ratio dye,
measurements are relatively independent from the amount of available dye, loading efficacy,
cell volume, dye bleaching and dye leakage.
2+
In addition to fluorescence microscopy to determine [Ca ]i, plate-reader fluorescence
2+
measurements can also be used to determine [Ca ]i. Although this technique has a higher
throughput of measurements, several limitations could hamper accurate measurements,
including the observed sustained increase in fluorescence instead of transient increases in
fluorescence (for more detail see chapter 3).
Vesicular neurotransmitter release; carbon fiber microelectrode amperometry
Carbon fiber microelectrode amperometry was used to investigate drug-induced effects on
vesicular DA release (exocytosis) in dexamethasone-differentiated PC12 cells and isolated
chromaffin cells. A carbon fiber microelectrode was placed against the cell surface and set at
a sufficiently high voltage (700 mV) to oxidize oxidizable neurotransmitters, such as DA (see
Fig. 2.3).
Figure 2.3. Experimental design for amperometric recording of exocytotic DA release from a single cell
using a carbon fiber microelectrode. An amperometric trace is recorded during depolarization-evoked
+
exocytosis (triggered by 100 mM K ) indicated by the bar on top. A single exocytotic event representing
the release of the content of a single vesicle is displayed on an extended time scale (lower trace).
Modified from Westerink 2004.
19
Applied methodologies
When exocytosis occurs, DA is instantaneously oxidized on the electrode surface and the
current caused by the electron transfer during this oxidation is proportional to the amount of
DA released.
Experiments to investigate effects of drugs of abuse on exocytosis, described in this
thesis, usually consisted of several minutes baseline recording to measure basal exocytosis.
+
Subsequently, an increase in exocytosis was triggered by application of K for several seconds
to measure depolarization-evoked exocytosis. Following this first depolarization and a
recovery period, a 15 min exposure precedes a second stimulus. Several parameters were
determined from these recordings, like the frequency of exocytotic events and vesicular DA
content. Vesicular DA content (Q) can be determined from the time integral of the current
during the release event.
Disadvantages related to detection of neurotransmitters using amperometry include its
limitation to oxidizable neurotransmitters, like DA and 5-HT. Important neurotransmitters
including GABA, glutamate and ACh, can not be measured. Furthermore, electrochemical
detection only occurs near/under the electrode, which only covers a small part of the cell
surface. Finally, additional techniques, like high-performance liquid chromatography (HPLC),
may be required to identify the released neurotransmitters.
Big advantages of amperometric detection of DA release are its high temporal
resolution (ms) and its high sensitivity (~9000 catechol molecules, 15 zeptomol). Furthermore,
amperometry only measures presynaptic changes and can therefore separate pre- from
postsynaptic effects. In contrast, when detecting presynaptic DA release by measuring the
postsynaptic response, changes could be due to pre- as well as postsynaptic changes, such as
desensitizing receptors or an alteration in the number of receptors.
A different technique that can also provide a read-out for presynaptic changes is
measurement of presynaptic cell membrane capacitance (patch-clamp). However, with this
technique the net result of endo- and exocytosis is measured, whereas exocytosis is
separated from endocytosis using amperometry. Furthermore, intracellular conditions are
disturbed with membrane capacitance measurements, which might influence the obtained
results. In contrast, voltammetry could be used to measure neurotransmitter concentration
(instead of quantity like with amperometry) without affecting intracellular conditions and will
simultaneously provide a chemical profile. However, it measures the balance between uptake
and release of neurotransmitter and has lower temporal resolution compared to
amperometry (Westerink 2004). Therefore, amperometry is the preferred technique to
investigate presynaptically drug-induced effects.
Measurement of the GABA-mediated current, two-electrode voltage-clamp technique
The two-electrode voltage-clamp technique was used to investigate drug-induced effects on
ionotropic GABAA-Rs. Oocytes were harvested from Xenopus laevis and cDNA coding for the
human α1β2γ2 GABAA-R was injected into the nuclei of stage V or VI oocytes. After 2-5 days
incubation at 21°C, ion currents were evoked by application of GABA and measured using the
two-electrode voltage-clamp technique. The oocyte was placed in a custom-built Teflon
chamber and voltage-clamped at -60 mV using two microelectrodes.
20
Chapter 2
The voltage electrode recorded intracellular potential (Vm) and the current electrode injected
current (Ii) into the oocyte to maintain a membrane potential of -60 mV (Fig. 2.4). Ion current
through the membrane was measured by means of a "bath clamp" that converted current to
potential. Oocytes were continuously perfused with saline and upon application of the
endogenous agonist GABA ion channels open. This results in an inward current that decreases
the membrane potential (hyperpolarization) of the oocyte. As the voltage recording electrode
measures a change in membrane potential, a sufficient amount of current is injected to
maintain a potential of -60 mV. This injected current is proportional to the number of opened
ion channels and is measured by the virtual ground circuit. This technique allows for direct
measurement of receptor functionality and is therefore a suitable technique to investigate
drug-induced effects on the GABAA-R.
Figure 2.4. Schematic representation of the two-electrode voltage-clamp technique. An oocyte is placed
in a Teflon chamber and continuously perfused. The membrane potential is fixed at -60 mV using
electrodes. One electrode measures the membrane potential (Vm) and one injects current (Ii) upon
detection of a change in membrane potential in order to maintain a membrane potential of -60 mV (Vc).
Membrane current is measured by a "bath clamp". Ref, reference electrode; R, resistor; Im, membrane
current; Vc, voltage command (-60 mV); Vi, injected potential. Modified from H. P. M. Vijverberg.
21
Applied methodologies
22
Chapter 3
Are high-throughput measurements of intracellular calcium using
plate-readers sufficiently accurate and reliable?
Letter to the editor
Laura Hondebrink1,2, Remco H.S. Westerink1
1
Neurotoxicology Research Group, Institute for Risk Assessment Sciences (IRAS), Utrecht
University, P.O. Box 80.176, NL-3508 TD Utrecht, The Netherlands.
2
The National Poisons Information Centre (NVIC), The National Institute for Public Health and
the Environment (RIVM), Bilthoven, The Netherlands.
Toxicology and Applied Pharmacology 249 (2010) 247-8
High-throughput measurements
Dear Editor:
Garcia-Ratés et al (2010) recently reported effects of 3,4-methylenedioxy-methamphetamine
2+
(MDMA) on intracellular calcium levels ([Ca ]i) in PC12 cells as well as on α7 and α4β2
nicotinic acetylcholine receptors (nACh-Rs) expressed in Xenopus oocytes. Their data on
2+
[Ca ]i, obtained by determination of fluo-4 fluorescence in a plate-reader, indicate an
2+
apparent contradiction (Figs. 1-2). MDMA attenuated the increase in [Ca ]i evoked by ACh,
nicotine and the specific α7 nACh-R agonist PNU282987, i.e., MDMA acts as a nicotinic
2+
antagonist. On the other hand, MDMA concentration-dependently increased basal [Ca ]i in
PC12 cells, i.e., MDMA acts as a nicotinic agonist, mainly on the α7 nACh-R (Fig. 4).
Interestingly, the authors noted that MDMA, as well as ACh, induced a rapid but
2+
sustained (apparently up to 5 min) increase in basal [Ca ]i (Figs. 1B, 2A). This is rather
2+
surprising since their data also indicated that the observed MDMA-induced increase in [Ca ]i
was largely due to activation of α7 nACh-Rs (Fig. 4), which are well-known to undergo rapid
(within seconds) desensitization, even in the presence of a positive allosteric modulator (e.g.,
Bale et al. 2005; Hurst et al. 2005; Couturier et al. 1990, see also Fig. 3B). As a result and due
to stringent calcium homeostasis (for review see e.g., Garcia et al. 2006; Verkhratsky et al.
2+
1998), the increase in [Ca ]i must be transient (e.g., Wu et al. 2009; Sekiguchi-Tonosaki et al.
2009; Hruska and Nishi 2007; Zaika et al. 2004) rather than sustained.
2+
Further, according to Garcia-Ratés et al (2010), the EC50 of ACh to increase [Ca ]i,
determined using fluo-4 fluorescence in a plate-reader, amounted to only 0.5 μM (maximum
effect is observed already at 1 μM; Fig. 2B). However, this is two orders of magnitude below
EC50 values for α7 nACh-Rs activation determined using electrophysiology (Bale et al. 2005;
Hurst et al. 2005; Papke and Porter Papke 2002; see also Fig. 3). Considering the strong
2+
correlation between activation/inactivation of α7 nACh-Rs and an increase/decrease in [Ca ]i
(e.g., Fayuk and Yakel, 2007), it appears unlikely that these low ACh concentrations (even in
2+
the presence of a positive allosteric modulator) already exert a maximal effect on [Ca ]i.
It is thus possible that the conclusions and interpretations of the authors, though in
itself sound, are based on an inaccurate methodology. Whereas single cell fluorescence
+
microscopy irrespective of the type of agonist (e.g., ACh, K , ATP, 5-HT) generally reveals
2+
transient increases in [Ca ]i, in line with electrophysiological data, results obtained with
2+
plate-readers are much more variable. There are some reports of transient increases in [Ca ]i
using a plate-reader (e.g., El Kouhen et al. 2009; Liu et al. 2006; Hurst et al. 2005). However,
in addition to Garcia-Ratés et al (2010), several previous studies using a plate-reader have
2+
shown sustained increases in [Ca ]i (e.g., Dickinson et al. 2006; even several factsheets from
producers of plate-readers). It is not unlikely that this step-wise increase in fluorescence is
actually an artifact due to the injection of buffer, agonist or test compound (see e.g., Kassack
et al. 2002).
24
Chapter 3
Considering the central role of calcium in virtually all cellular processes including
2+
neurotransmission and cell death, the apparent sustained increase in [Ca ]i would indicate a
continuous activation of cellular processes and an imminent death of the cell following any
2+
kind of stimulation. In our view, the apparent sustained increases in [Ca ]i observed in these
studies are thus not compatible with the widely accepted stringent calcium homeostasis that
is required for normal cell function. Hence, until the suitability of plate-readers to accurately
2+
and reliably determine the dynamic changes in [Ca ]i is unequivocally demonstrated, the
2+
rapidly increasing amount of data on [Ca ]i obtained with plate-readers should be interpreted
with great care.
25
High-throughput measurements
26
Chapter 4
Amphetamine reduces vesicular dopamine content in PC12 and
chromaffin cells only following L-DOPA exposure
Laura Hondebrink1,2,*, Jan Meulenbelt1,2,3, Johan G. Timmerman1,
Martin van den Berg1, Remco H.S. Westerink1
1
Neurotoxicology Research Group, Institute for Risk Assessment Sciences (IRAS), Utrecht
University, P.O. Box 80.176, NL-3508 TD Utrecht, The Netherlands.
2
The National Poisons Information Centre (NVIC), The National Institute for Public Health and
the Environment (RIVM), Bilthoven, The Netherlands.
3
Division Intensive Care Center, University Medical Center Utrecht, Utrecht, The Netherlands.
Journal of Neurochemistry 111 (2009) 624-633
Amphetamine-induced vesicular leakage
Abstract
Amphetamine increases brain dopamine levels via reversal of the membrane dopamine
transporter. Additional mechanisms have been suggested, including inhibition of vesicular
2+
monoamine transporters and vesicular leakage of dopamine and Ca . According to the
widely-accepted weak base theory, amphetamine disrupts the proton gradient required for
2+
filling vesicles with dopamine. As a result, dopamine and Ca will leak from vesicles, giving
rise to exocytosis of less-filled vesicles. As several contradictions have been described, the
aim of the present study was to re-examine this theory using amperometry and Fura 2
2+
imaging to measure amphetamine-induced changes in exocytosis and intracellular Ca levels,
respectively, in PC12 and chromaffin cells.
Unexpectedly, 15 min exposure to amphetamine (20-200 µM) does not affect the
2+
amount of dopamine released per vesicle, the frequency of exocytosis or intracellular Ca
levels in PC12 cells or chromaffin cells. Comparable results were found following prolonged
exposure to amphetamine (45 min) or at 37°C.
When cells were pre-treated with the dopamine precursor L-DOPA, vesicle content
increased to ~150%. When these pre-treated cells are exposed to amphetamine, vesicle
content is strongly reduced. These results indicate that amphetamine-induced vesicle leakage
occurs only under specific conditions, therefore arguing for re-evaluation of the theory of
amphetamine-induced vesicular dopamine leakage.
28
Chapter 4
Introduction
Dopamine (DA) brain levels are mainly determined by release and uptake of DA. DA is
2+
released via Ca -dependent exocytosis of DA-filled vesicles that fuse with the cell membrane,
thereby spreading their content into the extracellular space (Westerink 2006). In the brain,
two types of DA-containing vesicles exist; large dense-core vesicles (LDCVs) and small synaptic
vesicles (SSVs). In LDCVs, most DA is bound to intravesicular storage molecules, whereas in
SSVs DA is unbound (De Camilli and Jahn 1990). Vesicle filling depends mainly on the
intracellular concentration of DA, the activity of the vesicular dopamine transporter (VMAT)
and, in case of LDCVs, on the presence of intravesicular storage molecules, including
chromogranins and secretogranins (Westerink 2006). Following exocytosis, the released DA
molecules are taken up by the membrane dopamine transporter (DAT) (Kahlig and Galli 2003).
Theoretically, the above-mentioned processes are all potential targets for medicines and
drugs of abuse, like amphetamine.
The main target of amphetamine, however, appears to be DAT. Amphetamine
exposure causes DAT to operate in reverse mode, leading to DAT-mediated DA efflux instead
of uptake. This results in higher extracellular levels of DA (Kahlig and Galli 2003). This was
elegantly demonstrated ex vivo in brain striatal slices obtained from wildtype (WT) and DAT
knockout (KO) mice (Jones et al. 1998). In this study it was shown that basal, i.e.,
unstimulated, extracellular DA levels markedly increased in brain slices from WT mice
following amphetamine exposure, whereas this increase was absent in striatal slices from
DAT-KO mice.
Jones et al (1998) indicated a second effect of amphetamine, i.e., a reduction of
depolarization-evoked DA release. This reduction could be the result of a reduced frequency
of DA exocytosis or of a reduction in the amount of DA released per vesicle, e.g., caused by
reduced vesicle filling or by leakage of DA from already filled vesicles.
Indeed, several studies suggested effects of amphetamine on vesicle contents in
addition to the reversal of DAT. Biochemical studies have shown that amphetamine is capable
of inhibiting VMAT (Erickson et al. 1996, Partilla et al. 2006), thereby potentially reducing
vesicle filling. Although direct effects of amphetamine on storage molecules (chromogranins
and secretogranins) have not been reported, amphetamine has been reported to increase
mRNA coding for the secretogranin II precursor (chromogranin C) 3 hours after a single dose
exposure in rat striatum (Gonzalez-Nicolini and McGinty 2002).
So far, only very few studies indicated that amphetamine may also induce vesicular
leakage of DA. Almost 15 years ago, following the introduction of carbon fiber microelectrode
amperometry at single cells to measure the amount of DA released per vesicle (Leszczyszyn et
al. 1991), Sulzer et al (1995) found a significant reduction in vesicle content during
depolarization-evoked exocytosis in rat pheochromocytoma (PC12) cells exposed to
amphetamine. It has been argued that amphetamine acts as a weak base, thereby disrupting
the proton-gradient required for vesicle filling (Sulzer and Rayport 1990). Consequently,
amphetamine could cause redistribution of DA from vesicles to the cytosol, resulting in a
2+
reduced vesicle content (Sulzer et al. 1995). As vesicles are also an important source for Ca
(Winkler and Fischer-Colbrie 1992), amphetamine-induced disruption of the proton-gradient
29
Amphetamine-induced vesicular leakage
2+
could also induce leakage of vesicular Ca , resulting in an increased intracellular calcium
2+
2+
concentration ([Ca ]i). The increase in [Ca ]i could trigger exocytosis, though Sulzer et al
(1995) did not report an increased exocytosis during amphetamine exposure. A few years
later, Mundorf et al (1999) showed that in chromaffin cells amphetamine exposure induced
2+
an extremely fast (within seconds) increase in [Ca ]i as well as exocytosis, supposedly due to
calcium leakage from vesicles. Nonetheless, no significant reduction in vesicular DA content
was reported.
Surprisingly, these somewhat contradictory results (Sulzer et al. 1995, Mundorf et al.
1999) and the weak base theory (Sulzer and Rayport 1990) were combined, resulting in the
2+
theory that on a cellular level amphetamine induces vesicular leakage of DA and Ca ,
2+
resulting in an increase in [Ca ]i, and thus exocytosis of vesicles with less DA per vesicle
(Sulzer et al. 2005). The proof for this theory can be debated and the ex vivo DAT-KO study
(Jones et al. 1998) already indicated that depletion of the number of vesicles, reduced vesicle
filling and a reduction in the frequency of exocytosis could all also account for the observed
effects of amphetamine. Therefore the aim of the present study was to re-examine the
cellular effects of amphetamine using both amperometry and calcium imaging in PC12 as well
as chromaffin cells. The results indicate that amphetamine can reduce vesicular DA content,
but only under conditions in which the vesicles are maximally filled. The action of
amphetamine under physiological conditions may thus be largely restricted to SSVs, whereas
LDCVs are hardly affected.
These findings therefore argue for refinement of the widely-accepted theory that
amphetamine redistributes vesicular DA.
Materials and methods
Chemicals
D- and DL-Amphetamine were obtained from Spruyt Hillen (IJsselstein, The Netherlands).
Stock-solutions of D- and DL- amphetamine (200 mM), prepared in saline and stored at 4°C,
remained stable for at least 1 month as determined using HPLC. Amphetamine solutions (20
μM - 200 mM) were prepared immediately before experiments. All other chemicals, unless
otherwise noted, were obtained from Sigma-Aldrich (Zwijndrecht, the Netherlands).
PC12 Cell culture
Undifferentiated rat pheochromocytoma (PC12) cells (Greene and Tischler 1976, ATCC) were
2
maintained in monolayers in 25 cm tissue culture flasks (Nunc, Rochester, NY, USA) in
5% CO2/95% air at 37 °C in RPMI 1640 medium (Invitrogen, Breda, the Netherlands) until they
reached confluence. Medium was supplemented with 5% fetal calf serum, 10% horse serum
(ICN Biomedicals, Zoetermeer, The Netherlands), 100 U/ml penicillin and 100 µg/ml
streptomycin (Invitrogen, Breda, the Netherlands). Cells were cultured for up to 10 passages
and medium was refreshed every 2-3 days. For amperometric recordings, the cells were
subcultured in 35 mm dishes (Nunc) and differentiated for 5-7 days with 5 μM
dexamethasone to enhance exocytosis, as described previously (Westerink and Vijverberg
2002).
30
Chapter 4
2+
For [Ca ]i imaging experiments, undifferentiated PC12 cells were subcultured in glass-bottom
dishes (MatTek, Ashland, MA, USA) as described previously (Dingemans et al. 2007). All
plastics were coated with poly-L-lysine (50 µg/ml).
Isolation and culturing of mouse chromaffin cells
Male and female C57BL6 mice of (2-3 weeks old) were kindly provided by Dr. G.M.J. Ramakers
(Rudolf Magnus Institute, Utrecht, The Netherlands). Mice were housed with food and water
available ad libitum. Isolation of the adrenal glands was performed as described previously
(Westerink et al. 2006). For amperometric recordings cells were plated on 35 mm dishes
2+
(Nunc) coated with 33 µg/ml calf skin collagen. For Ca imaging experiments, chromaffin cells
were plated on glass coverslips (Menzel Glaser, Braunschweig, Germany) coated with 10
µg/ml laminin (Roche, Woerden, the Netherlands) and 100 µg/ml poly-L-lysine. Experiments
were performed at room temperature 1-2 days after plating. All experiments were approved
by the local committee on Animal Bioethics of Utrecht University.
Cell viability assay
Effects of amphetamine and L-DOPA on cell viability were determined by measuring the
capacity of undifferentiated PC12 cells to reduce 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan (Denizot and Lang 1986). PC12 cells
4
were seeded in a 96-wells plate two days prior to the cell viability test at a density of 8 * 10
cells per well. Cells were exposed for 60 min to different concentrations and isomers of
amphetamine (0.02-200 mM D-amphetamine or 0.2 mM DL-amphetamine) or 100 µM L-DOPA
in serum-free RPMI medium, after which the cells were incubated for 30 min in 100 µl MTT (1
mg/ml) in serum-free medium. Following MTT incubation, cells were lysed using lysisbuffer
containing 0.25 M sodium dodecyl sulfate dissolved in equal amounts of milliQ-filtered water
and 99% dimethylformamide. Lysates were measured spectrophotometrically at 595 nm
(FLUOstar Galaxy, BMG Labtechnologies, Offenburg, Germany) to determine the quantity of
the blue formazan, which is used as a measure for cell viability.
Intracellular calcium imaging
Cells were washed before experiments and maintained in saline containing (in mM) 125 NaCl,
5.5 KCl, 2 CaCl2, 0.8 MgCl2, 20 Hepes, 24 glucose and 36.5 sucrose. Experiments were
2+
performed at room temperature. Changes in [Ca ]i were measured in undifferentiated PC12
2+
cells and isolated chromaffin cells using the high-affinity Ca -responsive fluorescent dye Fura
2-AM (Molecular Probes; Invitrogen, Breda, The Netherlands) as described previously
(Dingemans et al. 2007). The F340/F380 ratio was measured every 5 s. Each experiment
2+
consisted of a 2.5 min baseline recording to measure basal [Ca ]i, after which an increase in
2+
+
2+
[Ca ]i was triggered with 125 mM K for 15 s to measure depolarization-evoked [Ca ]i.
Following this first depolarization and a 3 min recovery period, cells were exposed to saline
(control) or 200 µM DL-amphetamine for 15 min prior to a second depolarization. For specific
experiments, cells were incubated with 100 µM of the DA precursor L-DOPA for 15 min prior
to experiments.
31
Amphetamine-induced vesicular leakage
2+
Free cytosolic [Ca ]i was calculated according to a modified Grynkiewicz’s equation
2+
[Ca ]i = Kd* *(R-Rmin) / (Rmax-R) where Kd* is the dissociation constant of Fura 2 determined in
the experimental set-up (Deitmer and Schild 2000).
Carbon fiber microelectrode amperometry
Cells were washed before experiments and maintained in saline. Experiments were
+
performed at room temperature. Spontaneous and K -evoked (NaCl reduced to 5.5 mM and
KCl increased to 125 mM) vesicular catecholamine release from dexamethasonedifferentiated PC12 cells and isolated chromaffin cells was measured using carbon fiber
microelectrode amperometry as described previously (Westerink and Vijverberg 2002).
Amperometric current (sampled at 4 kHz, filtered at 2 kHz) was recorded using an EPC-7 patch
clamp (List Electronic, Darmstadt, Germany), and stored on disk for off-line analysis. Low
noise carbon fiber microelectrodes (5 µm) were purchased from Dagan Corporation
(Minnesota, USA).
Each single cell measurement consisted of a 2.5 min baseline recording to measure the
spontaneous release frequency, after which exocytotic capacity was recorded by a first
+
depolarization with 125 mM K for 15 s. Following this first depolarization and a 3 min
recovery period, the cell is exposed to saline (control) or to 20 or 200 µM D- or
DL-amphetamine for 15 min prior to a second depolarization (see Fig. 4.2 for an example
recording).
For specific experiments, the first depolarization is followed by a 15 min exposure to
100 µM of the DA precursor L-DOPA, after which the increase in content was measured during
a second depolarization. Subsequently, the original protocol was followed, i.e., 15 min
exposure to saline (control) or to 200 µM D-amphetamine, after which the cell was
depolarized again (see Fig. 4.3 for an example recording).
A custom-designed routine in Labview (National Instruments, Austin, Texas, USA) was
used for identification and analysis of currents associated with vesicular catecholamine
nd
release. The original signal was digitally filtered (low-pass 100 Hz, 2 order Bessel filter) for
integration of the detected events. For each cell, frequency of events was determined and
vesicle content was calculated from the total charge (Q) transferred during the event
according to Q/nF, where n=2 electrons for the oxidation of one catecholamine molecule and
F is Faraday’s constant (96,485 C/mole). Cells were excluded for data analysis if basal release
was high (>5 events/min), evoked release was low (<40 events/min) or when the release
frequency following depolarization did not return to basal values in the last minute before
exposure (>5 events/min).
Statistical analysis
All data are presented as mean ± SEM of n cells or N experiments. Data was averaged per cell
instead of pooling data from several cells (Westerink et al. 2000, Colliver et al. 2000b).
Consequently, all data were compared using a Student’s paired t-test, unless otherwise
noted.
32
Chapter 4
Results
Cell viability is not affected by 60 minutes exposure to ≤ 2 mM amphetamine
To determine the effects of amphetamine and the DA precursor L-DOPA on cell viability, PC12
cells were exposed to different concentrations and isomers of amphetamine and/or L-DOPA.
Cell viability, measured with MTT, was not affected by a 60 min exposure to 0.02-2 mM
D-amphetamine. Increasing D-amphetamine concentration to 20 and 200 mM resulted in a
dose-dependent reduction of cell viability to 77 ± 9 % and 52 ± 2%, respectively, of control
(N=2, 12 wells/condition; p < 0.001; Fig. 4.1).
Figure 4.1. MTT assay with PC12 cells exposed to different concentrations of D-amphetamine (D-AMPH)
for 60 min, demonstrating that ≥ 20 mM amphetamine reduces cell viability. Control values were set at
100%. *** p < 0.001 (ANOVA followed by Bonferroni’s post hoc test).
Exposure to 0.2 mM DL-amphetamine, 0.1 mM L-DOPA or to 0.1 mM L-DOPA co-applied with
0.2 mM D-amphetamine had no effect on cell viability (data not shown). These results thus
indicate that PC12 cells can be exposed to amphetamine for at least 60 min at concentrations
up to 2 mM without inducing overt cytotoxicity.
2+
Amphetamine exposure does not induce changes in [Ca ]i
2+
As exocytosis is triggered by an increase in [Ca ]i and a previous report suggested that
2+
2+
amphetamine induced changes in [Ca ]i in chromaffin cells (Mundorf et al. 1999), [Ca ]i was
2+
measured in PC12 cells and chromaffin cells using the Ca -responsive fluorescent dye
2+
Fura 2-AM. In control cells (n=79, N=11), basal [Ca ]i levels were low, i.e., 100 ± 2 nM,
2+
whereas depolarization evoked a strong, transient increase in [Ca ]i up to 2.2 ± 0.1 µM.
2+
2+
During the 3 min recovery period, [Ca ]i dropped to basal levels. This low [Ca ]i was
maintained during a subsequent 15 min exposure to saline. A second depolarization evoked
2+
an increase in [Ca ]i up to 2.0 ± 0.1 µM, i.e., up to ~90% of the first depolarization. When
PC12 cells were exposed for 15 min to 200 µM DL-amphetamine (n=43; N=7), results did not
2+
differ from control cells exposed to saline, i.e., [Ca ]i did not increase during 15 min exposure
2+
and the increase in [Ca ]i during the second depolarization again amounted to ~90% of the
first depolarization.
33
Amphetamine-induced vesicular leakage
2+
To rule out that amphetamine induced a very rapid and transient increase in [Ca ]i, which
could remain undetected at a low sampling frequency, the sampling frequency was increased
to 1 Hz. These experiments (n=32; N=4) also did not reveal any amphetamine-induced
2+
changes in basal [Ca ]i in PC12 cells (data not shown).
In additional experiments chromaffin cells were also exposed to saline (n=63; N=6) or
to 200 µM D-amphetamine (n=80; N=7) to test for cell-type specific effects of amphetamine
2+
2+
on [Ca ]i. As for PC12 cells, amphetamine did not affect basal [Ca ]i in chromaffin cells.
2+
These results thus indicate the absence of amphetamine-induced changes in [Ca ]i in
both PC12 and chromaffin cells. Consequently, it is unlikely that amphetamine exposure
induces exocytosis.
Amphetamine exposure does not affect release frequency and vesicle DA content
To further investigate whether amphetamine exposure induces any functional changes in
exocytosis, basal and depolarization-evoked vesicular release frequencies were determined in
the presence and absence of amphetamine (Fig. 4.2) in PC12 cells using amperometry. In
control cells (n=20), basal release frequencies were low, amounting to 0.5 ± 0.1 events/min,
whereas depolarization evoked a strong, transient increase in the release frequency (274 ± 33
events/min). During the 3 min recovery period, release frequency dropped to ~1 event/min.
This low level of exocytosis was maintained during a subsequent 15 min exposure to saline. A
second depolarization, again, evoked massive exocytosis (197 ± 27 events/min), though the
release frequency is ~20% lower than during the first depolarization, consistent with previous
results (Westerink and Vijverberg 2002).
When PC12 cells were exposed for 15 min to 20 µM (n=8) or 200 µM (n=7) of
D-amphetamine or to 20 µM (n=9) or 200 µM (n=14) of DL-amphetamine, results were similar
to control cells exposed to saline, i.e., basal release frequency did not increase during 15 min
exposure and the release frequency during the second depolarization amounted to 80% of
the first depolarization (Fig 4.2B; Table 4.1).
Figure 4.2. Amperometric recordings of a control cell (A) and an amphetamine exposed cell (B). Each cell
is depolarized with 125 mM K+ for 15 s (as indicated by the arrows on top of the recording) to evoke
exocytosis. In between depolarizations the cell is exposed to either saline (A) or 200 µM
DL-amphetamine (DL-AMPH, B) for 15 minutes (as indicated by the dashed line on top on the recording).
As illustrated in the recordings, amphetamine did not change the frequency of basal and
depolarization-evoked exocytosis.
34
Chapter 4
2+
In accordance with the observed absence of amphetamine-induced changes in [Ca ]i, these
results clearly indicate that exposure to amphetamine does not affect the frequency of
exocytosis, neither under basal conditions, nor during depolarization.
2+
As amphetamine exposure apparently does not liberate Ca from vesicles, vesicular DA
content was measured to determine whether amphetamine induces vesicular leakage of DA
in PC12 cells. In control cells (n=20), the average median content amounted to 507 ± 40 zmol
during the first depolarization. During the second depolarization, average median vesicle
content was not significantly different, amounting to 553 ± 49 zmol. Unexpectedly, vesicle
content during the second depolarization was also unaffected in cells exposed to 20 µM (n=8)
or 200 µM (n=7) of D-amphetamine or to 20 µM (n=9) or 200 µM (n=14) of DL-amphetamine
(Table 4.1).
Table 4.1. Release frequencies and vesicle contents (zmol) in control cells or in cells exposed to 20 or
200 µM D-amphetamine (D-AMPH 20 µM or D-AMPH 200 µM) or cells exposed to 20 or 200 µM
DL-amphetamine (DL-AMPH 20 µM or DL-AMPH 200 µM). Values are represented as average ± SEM.
“Recovery” represents the last minute prior to a 15 min “exposure” to saline or amphetamine and
st
“1 dep” represents the exocytotic frequency or vesicle content during a first depolarization with 125
+
mM K for 15 s. Frequencies and content values of the second depolarization are expressed as
percentage of the first. Frequency during exposure was tested against recovery and frequencies and
content during first and second depolarization were compared. Compared to the first depolarization,
control cells displayed a modest, but significant decrease in release frequency during the second
depolarization. There were no other significant differences in frequency or content. ** p< 0.01.
Even though previous reports (Jones et al. 1998, Kahlig and Galli 2003) indicated that effects
of amphetamine occur rapidly, even at room temperature, additional experiments were
performed in which the exposure duration to saline (n=6) or amphetamine (200
µM D-amphetamine, n=3) was increased to 45 min. These experiments also did not reveal any
differences in vesicle content (data not shown).
Since several processes, including vesicle filling, are temperature-dependent, additional
experiments were performed at 37 °C. PC12 cells were incubated with 200 µM
DL-amphetamine at 37 °C for 15 min. Subsequent measurements of vesicle content again did
not reveal any differences between control (n=8) and amphetamine-exposed cells (n=3; data
not shown).
35
Amphetamine-induced vesicular leakage
Two types of VMAT exist and amphetamine is a substrate for both, though it has been shown
that VMAT2 has a higher affinity for amphetamine than VMAT1 (Peter et al. 1994, Erickson et
al. 1996). Though PC12 cells have been used previously to investigate effects of
amphetamine, undifferentiated PC12 cells only express VMAT1 (Fornai et al. 2007). The
absence of VMAT2 in PC12 cells could theoretically obscure effects of amphetamine.
Therefore, additional experiments were performed using chromaffin cells, which are known
to express both VMAT1 and VMAT2 (Fornai et al. 2007). Nonetheless, using chromaffin cells
instead of PC12 cells still did not reveal any effects of amphetamine on exocytosis (data not
shown). When chromaffin cells exposed to 200 µM DL-amphetamine (n=4) were compared
with saline exposed chromaffin cells (n=6), no difference in release frequency or vesicle
content could be detected.
Though rather unexpected, these results clearly indicate that exposure to
amphetamine does not induce exocyosis and does not reduce vesicle content in PC12 or
chromaffin cells.
Amphetamine exposure affects vesicle content only in cells pretreated with L-DOPA
In PC12 and chromaffin cells, DA is retained in LDCVs in which DA is bound to storage
molecules. It would therefore be possible that amphetamine cannot induce vesicular leakage
of DA simply by the fact that DA is efficiently bound and is not freely available. When PC12
cells are incubated with the DA precursor L-DOPA, vesicle content increases to ~150% (Pothos
et al. 1996, Westerink et al. 2000). Importantly, loading PC12 cells with L-DOPA also increases
the size of LDCVs. The increase in size is mediated by an increase of the clear halo, in which
DA is freely available, surrounding the large dense-core of storage molecules (Sombers et al.
2005). Loading cells with L-DOPA would thus increase the amount of unbound DA in vesicles,
making the model system more sensitive to the possible effects of amphetamine.
When control cells (n=11) are exposed to 100 µM L-DOPA for 15 min in between two
depolarizations, vesicle content increased from 538 ± 178 zmol during the first depolarization
to 734 ± 202 zmol during the second depolarization, i.e., 141% of the original content (Fig.
4.3A).
When these L-DOPA-treated cells are subsequently exposed for 15 min to saline prior
to a third depolarization, vesicle content still modestly increased to 807 ± 298 zmol, i.e., 157%
of the original content and 110% compared to the second depolarization (Fig. 4.3A, B1).
Another group of cells received the same initial treatment, i.e., 15 min incubation with
100 µM L-DOPA in between two depolarizations. However, these cells (n=11) were exposed to
200 µM D-amphetamine prior to the third depolarization (Fig. 4.3B2). In these cells, L-DOPA
exposure increased vesicle content to 156% compared to the first depolarization. However,
following exposure to 200 µM D-amphetamine, vesicle content during the third depolarization
significantly decreased to 77% (p<0.001) compared to the second depolarization (i.e., 121% of
the first depolarization). It should be noted that also in these L-DOPA-incubated cells,
2+
amphetamine exposure did not induce any changes in [Ca ]i or in basal and depolarizationevoked release frequency.
36
Chapter 4
So, while vesicle content continues to increase in control cells following incubation with
L-DOPA, amphetamine-exposed cells show a marked reduction in vesicle content under these
2+
conditions (Table 4.2), without any changes in [Ca ]i or release frequency.
Figure 4.3. Amperometric recordings of L-DOPA and saline or amphetamine-exposed cells. A cell is
+
depolarized with 125 mM K for 15 s (as indicated by the arrows on top of the recordings) followed by
exposure to 100 µM L-DOPA for 15 min (as indicated by the brackets) and a second depolarization (A).
L-DOPA increased vesicle content up to ~150 %. Subsequently, the cell is exposed to either saline (B1) or
200 µM D-amphetamine ( D-AMPH, B2) for 15 min (as indicated by the dashed line) prior to a third
depolarization. Amphetamine exposure decreased vesicle content, returning to initial values, whereas
vesicle content further increased during saline exposure. Amphetamine did not affect release frequency.
Table 4.2. Quantification of the average median content of DA (zmol) in vesicles during first, second and
third depolarization. Values are represented as average ± SEM. Following the first depolarization, cells
were exposed to 100 µM L-DOPA for 15 min and afterwards depolarized for the second time. The second
depolarization was followed by a 15 min exposure to either saline or 200 µM D-amphetamine (D-AMPH)
after which the cell was depolarized for the third time. For each cell, the content during the second and
third depolarization was also expressed as a percentage of the first depolarization. The median content
of the third depolarization of each cell was also expressed as a percentage of the second depolarization;
the table depicts averages of all cells. Vesicle content increased significantly following L-DOPA exposure
(2nd compared to 1st) and is still significantly increased after saline exposure (3rd compared to 1st).
However, vesicle content was significantly reduced if L-DOPA exposure was followed by amphetamine
rd
nd
exposure (3 compared to 2 ). * p < 0.05, ** p < 0.01, *** p < 0.005.
37
Amphetamine-induced vesicular leakage
This is further illustrated in Fig. 4.4, which shows the cumulative distribution of pooled vesicle
content during the second depolarization, i.e., after incubation with L-DOPA, and third
depolarization, i.e., after exposure to saline (Fig. 4.4A) or 200 µM D-amphetamine (Fig. 4.4B).
As can be clearly seen in Fig. 4.4, the cumulative distribution of vesicle content in control cells
during the second and third depolarization is very similar, whereas in amphetamine-exposed
cells there is a clear left-shift in the cumulative distribution for the third depolarization.
Figure 4.4. Cumulative distribution of vesicle content pooled in binnings of 0.1 attomol. Events were
+
obtained during second depolarization with 125 mM K for 15 s, i.e., following L-DOPA exposure (solid
+
line) and third depolarization with 125 mM K for 15 s (dotted line), i.e., following saline (A) or 200 µM
D-amphetamine (AMPH, B) exposure. For cells exposed to saline both distributions are rather similar
whereas there is a clear left-shift following amphetamine exposure. The combined results clearly
indicate that exposure to amphetamine does not affect basal and depolarization-evoked release
2+
frequency, in accordance with an absence of effects on basal and depolarization-evoked [Ca ]i.
Amphetamine exposure reduces vesicle content only in L-DOPA incubated cells, likely by inducing
leakage of vesicular DA.
38
Chapter 4
Discussion
The present results demonstrate that amphetamine reduces vesicular DA content only in cells
pre-treated with the DA-precursor L-DOPA (Figs. 4.3 and 4.4). Rather unexpectedly, 15 min
exposure of PC12 cells to 20-200 μM D- or DL-amphetamine does not affect the number of
fused vesicles or the amount of DA released per vesicle under control conditions (Fig. 4.2;
Table 4.1). This is supported by the calcium data, which demonstrate the absence of
2+
amphetamine-induced changes in basal and depolarization-evoked [Ca ]i.
It can be ruled out that the absence of amphetamine-induced effects on exocytosis and
2+
basal [Ca ]i is due to amphetamine-induced cytotoxicity, as the concentrations (20-200 μM)
used in the present study are well below the concentrations that induce overt cytotoxicity
following 60 min exposure (20 mM; Fig. 4.1). Nonetheless, the concentrations of
amphetamine used in this study (20-200 μM) are comparable with other studies (Sulzer et al.
1993 and 1995, Floor and Meng 1996, Mundorf et al. 1999, Liu et al. 2003). Clinically,
amphetamine concentrations above 2 µM are considered toxicologically relevant, according
to the Dutch National Poisons Information Centre. Tolerant individuals, who consume over 1 g
of amphetamines daily, maintain steady-state plasma levels of ~20 µM (Ellenhorn and
Barceloux 1988). In Dutch post-mortem blood samples the concentration ranged up to
~80 µM (Verschraagen et al. 2007) and up to ~20 µM in blood samples from people arrested
for driving under the influence of drugs (Holmgren and Holmgren 2008).
Amphetamine is available on the illegal drug market as a racemic mixture
(D- and L-isomers) and as the optically pure D-enantiomer. Though the D-enantiomer is
reported to be ~five times more potent than the L-isoform in inhibiting VMAT1 and VMAT2
(Erickson et al. 1996), both enantiomers are found in equal amounts in blood samples from
people arrested for driving under the influence of drugs (Peters et al. 2003). Therefore, both
2+
D- and DL-amphetamine were used in the present study to investigate effects on [Ca ]i and
exocytosis. However, D- and DL-amphetamine were both unable to induce any effects on DA
exocytosis in PC12 and chromaffin cells.
Chromaffin cells differ from PC12 cells in several aspects; a.o. the expression of
different types of VMAT. Chromaffin cells express both VMAT1 and VMAT2, whereas PC12
cells express VMAT1 only (Yao et al. 2004, Fornai et al. 2007). Although amphetamine is a
substrate for both types (Peter et al. 1994, Erickson et al. 1996), it is known that
amphetamine has a higher specificity for VMAT2 as indicated by Ki values 20-fold higher for
VMAT1 then VMAT2 (Erickson et al. 1996). To exclude any cell-type specific (VMAT-related)
effects, chromaffin cells were also included in this study. However, as for PC12 cells, no
differences were found in the frequency of exocytosis or in vesicular DA content between
amphetamine- and saline-exposed mouse chromaffin cells.
Amphetamine enters the cell and vesicles mainly via DAT and VMAT, respectively. As
previous studies indicated that DA transporters function more efficiently at 37 °C compared
to room temperature (Vizi 1998, Vizi et al. 2004), effects of amphetamine exposure could be
obscured at room temperature. However, additional experiments with amphetamine
exposure at 37 °C also did not reveal any differences between control and
amphetamine-exposed cells.
39
Amphetamine-induced vesicular leakage
Similarly, prolonging the exposure of PC12 cells to amphetamine to 45 min did not reveal any
effects of amphetamine on exocytotic frequency or vesicle content.
The present findings, obtained using PC12 and mouse chromaffin cells, differ from previous
results obtained with PC12 and bovine chromaffin cells. The only report that demonstrated a
decrease in vesicle content (Sulzer et al. 1995), was based on unpaired comparison of pooled
data from a limited number of PC12 cells, instead of paired comparison of the average
median content per cell. Previous studies (Westerink et al. 2000, Colliver et al. 2000b) already
demonstrated that the variability both within as well as between cells argues for comparing
data averaged per cell, instead of using pooled data from several cells. The difference
between the present study and previous findings might therefore be explained by differences
in analysis and interpretation. Though less likely, it is also possible that differences in culture
conditions, allowing for more efficient vesicle filling (and thereby a larger fraction of unbound
DA in LDCVs) in the previous study, account for the observed differences.
2+
With respect to previously observed changes in [Ca ]i and exocytosis, Mundorf et al
2+
(1999) demonstrated that 25 µM amphetamine within seconds induced a rise in [Ca ]i as well
as an increase in basal exocytosis in bovine chromaffin cells. It was suggested that the
2+
2+
increase in [Ca ]i was due to amphetamine-induced leakage of vesicular Ca , which
subsequently triggered vesicle fusion. Considering the suggested intravesicular mechanism of
action, these amphetamine effects occurred surprisingly fast. More recently, Liu et al (2003)
showed, also in bovine chromaffin cells, that amphetamine acts as an α7 nicotinic ACh
2+
receptor (nACh-R) agonist, thereby rapidly increasing [Ca ]i. Activation of α7 nACh-Rs not
2+
2+
only induces depolarization, thereby triggering Ca influx through voltage-gated Ca
2+
2+
channels, but also results directly in Ca influx due to its Ca permeability (Seguela et al.
1993). The amphetamine-induced increase in DA release was diminished when α7 nACh-Rs
2+
were blocked (Liu et al. 2003). Additionally, the amphetamine-induced increase in [Ca ]i was
2+
attenuated when extracellular Ca was removed (Liu et al. 2003), as was also observed for
amphetamine-induced DA exocytosis (Mundorf et al. 1999). As Mundorf et al (1999) did not
report a significant decrease in vesicle content following amphetamine exposure, it is not
unlikely that the effects observed in bovine chromaffin cells are not due to
amphetamine-induced vesicle leakage, but are rather an indirect result of the agonistic
effects of amphetamine on α7 nACh-Rs.
In bovine chromaffin cells, α3, α5, α7, and β4 subunits of nAChRs have been cloned
and characterized (Campos-Caro et al. 1997) and numerous reports indicated the presence of
functional, but rapidly inactivating, α7 nACh-Rs in bovine chromaffin cells (Lopez et al. 1998,
Liu et al. 2003, El-Hajj et al. 2007). Moreover, Lopez et al (1998) showed that α7 and α3β4
2+
receptors were the main nAChR in triggering Ca entry and catecholamine release. However,
functional α7 nACh-Rs have not been demonstrated in mouse chromaffin cells and cell type
specific differences in the expression of functional α7 nACh-R may thus underlie the observed
differences in amphetamine effects. It is in this respect noteworthy that another study, using
2+
PC12 cells, did observe amphetamine effects on [Ca ]i only following repeated pretreatment
2+
with amphetamine, whereas acute effects of amphetamine exposure on [Ca ]i could not be
detected (Kantor et al. 2004).
40
Chapter 4
Additionally, Sulzer et al (1995) also did not report an increase in the frequency of exocytosis
in amphetamine-exposed PC12 cells. The absence of an amphetamine-induced increase in
2+
exocytosis, likely reflecting the absence of an amphetamine-induced increase in [Ca ]i, is also
evident from measurements of DA levels in ex vivo brain striatal slices obtained from DAT KO
mice (Jones et al. 1998).
Following pre-treatment with L-DOPA, vesicle content increases to ~150%, in
agreement with previous studies (Pothos et al. 1996, Westerink et al. 2000). Subsequent
exposure to amphetamine reduces vesicle content to original proportions (Fig. 4.3, Table 4.2).
2+
Following pre-treatment with L-DOPA, frequency of exocytosis or the [Ca ]i during
amphetamine exposure is not different from saline exposure. As amphetamine treatment
does not affect already filled LDCVs under control conditions (not L-DOPA treated, Fig. 4.2,
Table 4.1), these data indicate that only freely available, unbound DA can be liberated via
vesicular leakage.
There is evidence for a constant leak of DA from vesicles (Schonn et al. 2003) and
therefore ongoing uptake is required to keep vesicles filled. VMAT transporters can be
inhibited by amphetamine at concentrations used in this study (Erickson et al. 1996) and
could, theoretically, cause the vesicular content to run down. The present data argue against
this under conditions of low freely available DA (not L-DOPA loaded) since no decrease in
vesicle content was observed following amphetamine exposure (Table 4.1 and Fig. 4.2).
Possibly, the time frame of the experiments is too short compared with the magnitude of the
leak, though it should be noted that a decrease in vesicular content was also not found
following 45 min exposure. The constant leak of DA from vesicles, also in the absence of
amphetamine, is possibly also higher for unbound DA (which is increased following L-DOPA
pretreatment), and blockade of the vesicular transporter could play a greater role in that
case. The amphetamine-induced decrease in content following L-DOPA pre-treatment can be
explained by amphetamine’s effect on VMAT, possibly causing unbound DA efflux, or through
DA efflux caused by disruption of the proton gradient as the weak-base theory suggests.
It should be noted that the current data do not exclude a disruption of the
proton-gradient by amphetamine acting as a weak base, though indicate that this disruption
mainly affects extremely filled LDCVs which also contain unbound DA. It is therefore likely
that disruption of the proton-gradient, in addition to attenuating VMAT-mediated filling of
empty vesicles, will mainly affect SSVs, in which DA is freely available due to the absence of
storage molecules.
Noteworthy, the weak base theory (Sulzer and Rayport 1990) is to a large extent based
on serotonin-loaded chromaffin granules, thus possibly resembling our L-DOPA loaded LDCVs.
Additionally, it has been argued that the chromaffin granules may not retain their dense-core
during the procedure of vesicle isolation (Sulzer and Rayport 1990), and thus already
resemble SSVs.
At first glance, these findings contradict with in vivo findings, which clearly demonstrate a
reduction in stimulation-evoked DA release (Jones et al. 1998). However, PC12 cells only
contain DA-filled LDCVs in which most DA is bound to matrix molecules (Fig. 4.5A1), whereas
in the in vivo situation (Fig. 4.5B5), neurons contain both DA-filled LDCVs and SSVs (De Camilli
41
Amphetamine-induced vesicular leakage
and Jahn 1990). Transmission electron micrographs of PC12 cells loaded with L-DOPA
demonstrate that upon treatment with L-DOPA, the volume of the core remains rather stable,
while halo volumes are increased 3-fold (Colliver et al. 2000a, Sombers et al. 2005), indicating
that saturating the dense-core results in increased transmitter storage in the surrounding
halo (Colliver et al. 2000a). This DA is not bound to matrix molecules, but freely available (Fig.
4.5A2).
Figure 4.5. Proposed modification of the cellular mechanism of action of amphetamine. A. Vesicle filling
is determined a.o. by DA synthesis and uptake from extracellular DA. DA is synthesized from tyrosine
with L-DOPA as an intermediate. Extracellular DA is taken up by the DA membrane transporter (DAT).
Subsequently, cytosolic DA can be transported into the vesicle by vesicular transporters (VMAT). PC12
cells only contain DA-filled LDCVs, in which most DA is bound to matrix molecules (1). Transmission
electron micrographs of PC12 cells have shown that L-DOPA exposure mainly increases the volume of
the clear halo (2). Our experiments did not show a decrease in vesicle content following saline exposure.
Other studies have shown that amphetamine causes DAT-reversal as indicated by the outward arrow
(3). However, a decrease in vesicle content was measured when L-DOPA exposure was followed by
exposure to 200 µM D-amphetamine (4). B. In vivo, neurons contain both DA-filled LDCVs and SSVs. In
these SSVs, DA is not bound to matrix molecules but freely available (5). As DA in SSVs is freely available,
the amphetamine-induced reduction in stimulation-evoked DA release observed in vivo is likely
mediated by amphetamine-induced DA leakage from SSVs, but not from LDCVs (6).
42
Chapter 4
As demonstrated in our experiments, amphetamine can only reduce vesicle content following
L-DOPA pre-treatment. We therefore argue for a re-evaluation of the widely-accepted theory
on the mechanism of action of amphetamine. Based on our results, we suggest a refinement
of the theory, which is illustrated in Fig 4.5. Possibly amphetamine can only exerts its effect
on free DA which, in vitro, becomes more abundant following L-DOPA pre-treatment (Fig.
4.5A). However, in vivo dopaminergic neurons contain (Fig. 4.5B), in addition to LDCVs also
SSVs (De Camilli and Jahn 1990). As DA in SSVs is freely available, the amphetamine-induced
reduction in stimulation-evoked DA release observed in vivo (Jones et al. 1998) is likely
mediated by amphetamine-induced DA leakage from SSVs, but not from LDCVs (Fig. 4.5B6).
Our results indicate that the dense-core could protect a vesicle against amphetamine-induced
leakage.
Acknowledgements
We gratefully acknowledge T. Sinnige, R.G.D.M. van Kleef and Ing. A. de Groot for technical
assistance; G.Th.H. van Kempen, Dr. R.J. van den Berg, S. Rietjens, C.M. Waaijenberg, M. Kops
and the members of the neurotoxicology research group for thoughtful discussions and Dr.
G.J.M. Ramakers for providing mouse adrenal glands.
43
Amphetamine-induced vesicular leakage
44
Chapter 5
High concentrations of MDMA (‘ecstasy’) and its metabolite
MDA inhibit calcium influx and depolarization-evoked
vesicular dopamine release in PC12 cells
Laura Hondebrink1,2,*, Jan Meulenbelt1,2,3, Marijke Meijer1,
Martin van den Berg1, Remco H.S. Westerink1
1
Neurotoxicology Research Group, Institute for Risk Assessment Sciences (IRAS), Utrecht
University, P.O. Box 80.176, NL-3508 TD Utrecht, The Netherlands.
2
The National Poisons Information Centre (NVIC), The National Institute for Public Health and
the Environment (RIVM), Bilthoven, The Netherlands.
3
Division Intensive Care Center, University Medical Center Utrecht, Utrecht, The Netherlands.
Neuropharmacology 61 (2011) 202-208
MDMA and MDA inhibit vesicular dopamine release
Abstract
The popular synthetic drug of abuse 3,4-methylenedioxymethampetamine (MDMA) and its
metabolite 3,4-methylenedioxyamphetamine (MDA) act mainly on the serotonergic system,
though they also increase the amount of extracellular dopamine (DA) in the brain, presumably
via reversal of the membrane dopamine transporter (DAT). As the involvement of exocytotic
DA release is debated, we investigated if these drugs alter the intracellular calcium
2+
concentration ([Ca ]i) and subsequent DA exocytosis in single PC12 cells using respectively
2+
Fura 2 imaging and amperometry. MDMA and MDA did not change basal [Ca ]i or exocytosis,
2+
but inhibited depolarization-evoked increases in [Ca ]i and exocytosis following 15 min
exposure to high concentrations of drugs (1 mM). Surprisingly, MDA was more potent in
inhibiting exocytosis than MDMA and already inhibited exocytosis at concentrations that did
2+
not inhibit depolarization-evoked Ca influx (10-100 µM). Without 15 min pre-exposure, both
2+
drugs failed to inhibit depolarization-evoked Ca influx. These results indicate that at high
2+
concentrations both MDMA and MDA inhibit exocytosis via indirect inhibition of Ca influx,
whereas at lower concentrations MDA may also reduce vesicle cycling. Our data suggest that
the DAT-independent increase in extracellular DA in vivo is not due to direct stimulation of
exocytosis, but rather to effects of these drugs on other neurotransmitter systems that
innervate the dopaminergic system.
46
Chapter 5
Introduction
3,4-Methylenedioxymethamphetamine (MDMA) is an amphetamine derivate that is
recreationally used for its stimulating and rewarding properties. When used in excess, MDMA
can cause severe intoxications, resulting in ~13.000 hospital admittances each year in the US
(DAWN, 2010) and even deaths (EMCDDA, 2007). Although several mechanisms of actions
have been described for MDMA and its primary metabolite 3,4-methylenedioxyamphetamine
(MDA), the cellular and molecular mechanisms remain debated.
MDMA is mainly known for its effects on the serotonergic system. MDMA has been
shown to cause inhibition as well as reversal of the serotonin reuptake transporter (SERT),
acutely leading to higher extracellular serotonin (5-HT) levels in the brain (Crespi et al. 1997;
Rothman et al. 2001; Han and Gu 2006). This mechanism of action is thought to cause the
‘positive’ effects desired by the user, such as feelings of emotional warmth, empathy toward
others, enhanced sensory perception, a general sense of well being and decreased anxiety
(for review see Cole and Sumnall 2003; Green et al. 2003). On the other hand, feelings of
depression following use of MDMA have also been described. This may be attributed to
depletion of 5-HT in the brain observed one week after a single MDMA dose (Shankaran and
Gudelsky, 1998) or to a reduction in SERT density and structural damage to 5-HT terminals
observed following repeated doses (Wilson et al. 1989; Shankaran and Gudelsky 1998;
McCann et al. 2005).
Besides effects on the serotonergic system, MDMA also affects the dopaminergic
system. It was shown already in 1986 that exposure to MDMA caused dopamine (DA) release
in rat brain slices, in addition to 5-HT release. Exposure to the MDMA metabolite MDA
resulted in an even larger amount of released DA (Johnson et al. 1986). Numerous other
investigators have confirmed the DA releasing actions of MDMA using in vivo microdialysis in
rats (Hiramatsu and Cho, 1990; Gudelsky et al. 1994; Esteban et al. 2001). MDMA-induced DA
release is at least partially caused by reversal of the membrane DA transporter (DAT, Schmidt
et al. 1987; Nash and Brodkin, 1991). However, several studies indicate that MDMA-induced
DA release is still observed when this transporter is blocked. The reduction in MDMA-induced
DA release by blocking DAT is highly variable amongst different in vivo studies, ranging from a
30 to 100% decrease in DA release compared to not blocking DAT (Schmidt et al. 1987; Nash
and Brodkin, 1991; Kanthasamy et al. 2002; Pifl et al. 2005). Though this could be due to
different degrees of DAT blockade, it could also indicate involvement of a DAT-independent
2+
DA-releasing mechanism during MDMA exposure, which may be Ca -dependent vesicular DA
release (exocytosis). Similarly, exocytosis might also play a role in MDA-induced DA release,
which was reported to be two times higher than MDMA-induced DA release (Johnson et al.
1986) and could be inhibited by DAT blockers only partly (Rothman et al. 2009). Yet, Fitzgerald
and Reid (1993) showed in rat striatal slices that MDMA-induced DA release was unaltered in
2+
Ca free medium, suggesting a limited role for exocytosis, at least for MDMA-induced
increases in DA levels.
47
MDMA and MDA inhibit vesicular dopamine release
In addition to reversal of DAT and SERT, MDMA may also affect brain transmitter levels by
acting as a substrate (IC50 ~20 µM) for and an inhibitor (IC50 > 100 µM) of the vesicular
monoamine transporter. Moreover, it has also been suggested that MDMA causes vesicular
leakage by disrupting the vesicular proton gradient (Partilla et al. 2006). This would reduce
the amount of DA secreted per vesicle, as was previously proposed for amphetamine (Sulzer
et al. 2005), though a recent study indicated that this may apply only to small synaptic
vesicles rather than large dense-core vesicles (Hondebrink et al. 2009).
MDMA- and MDA-induced DA release thus appears largely, but not exclusively, due to
DAT-reversal. The aim of this study was therefore to investigate whether exocytosis of DA
contributes to the MDMA- and MDA-induced increase in extracellular DA levels. In addition,
drug-induced leakage of vesicular DA was also determined since this can contribute to higher
extracellular DA levels via subsequent DAT-mediated reverse transport.
Materials and Methods
Chemicals
DL-MDMA and DL-MDA were obtained from Duchefa (Haarlem, The Netherlands). Fresh stocksolutions of 1 M were prepared in saline and stored at 4°C for no more than 2 weeks. MDMA
and MDA solutions (1-1000 µM) were prepared immediately before experiments. CaCl2,
MgCl2, MgSO4, NaHCO3, NaOH, Ca(NO3)2, KCl, and HEPES were purchased from Merck
(Darmstadt, Germany). All other chemicals, unless otherwise noted, were obtained from
Sigma-Aldrich (Zwijndrecht, the Netherlands).
PC12 Cell culture
Undifferentiated rat pheochromocytoma (PC12) cells (Greene and Tischler, 1976, ATCC) were
2
maintained in monolayers in 25 cm tissue culture flasks (Nunc, Rochester, NY, USA) in a
humidified 5% CO2 / 95% air atmosphere at 37 °C in RPMI 1640 medium (Invitrogen, Breda,
the Netherlands) supplemented with 5% fetal calf serum, 10% horse serum (ICN Biomedicals,
Zoetermeer, The Netherlands), 100 U/ml penicillin and 100 µg/ml streptomycin (Invitrogen,
Breda, the Netherlands). Medium was refreshed every 2-3 days and cells were cultured for up
4
to 10 passages. Undifferentiated PC12 cells were subcultured in glass-bottom dishes (23 * 10
2+
cells/dish, MatTek, Ashland, MA, USA) for [Ca ]i imaging experiments. For amperometric
4
recordings, the cells were subcultured in 35 mm dishes (14 * 10 cells/dish, Nunc) and
differentiated for 5-7 days with 5 μM dexamethasone to enhance exocytosis, as described
previously (Westerink and Vijverberg, 2002). For cell viability assays, cells were seeded in a
4
96-wells plate (8 * 10 cells/well). All culture flasks, dishes and plates were coated with poly-Llysine (50 µg/ml).
Cell viability assay
Effects of MDMA and MDA on cell viability were determined by measuring the capacity of
undifferentiated PC12 cells to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) to formazan as described before (Hondebrink et al. 2009). Briefly, PC12 cells
were seeded in 96-wells plates (N=4) one day prior to a 60 min or 24 h exposure to different
48
Chapter 5
concentrations of MDMA or MDA (1-1000 µM, 20 wells/concentration). Following exposure,
cells were incubated with MTT (1 mg/ml) for 30 min and subsequently lysed using lysisbuffer.
Lysates were measured spectrophotometrically at 595 nm (FLUOstar Galaxy, BMG
Labtechnologies, Offenburg, Germany) to determine the quantity of the blue formazan, which
is used as a measure for cell viability.
Intracellular calcium imaging
Undifferentiated PC12 cells were washed before experiments and maintained in saline
containing (in mM) 125 NaCl, 5.5 KCl, 2 CaCl2, 0.8 MgCl2, 10 HEPES, 24 glucose and 36.5
2+
sucrose. The high-affinity Ca -responsive fluorescent dye Fura 2-AM (Molecular Probes;
2+
Invitrogen, Breda, The Netherlands) was used to measure changes in the intracellular Ca
2+
concentration ([Ca ]i) as described previously (Dingemans et al. 2007) with minor
modifications. Cells were continuously superfused with saline using a valvelink 8.2 (Automate
Scientific, California, USA). During experiments the F340/F380 ratio (R) was measured every
2+
3 s. Each experiment consisted of a 5 min baseline recording to measure basal [Ca ]i, after
2+
which an increase in [Ca ]i was triggered by depolarization of the cells by changing
+
superfusion to 100 mM K -containing saline for 20 s. Following this first depolarization and a
subsequent 8 min recovery period, during which cells were superfused with saline, cells were
exposed to saline (control), MDMA- or MDA-containing saline for 15 min (“pre-exposed”)
+
+
prior to a second depolarization with 100 mM K -containing saline (control) or 100 mM K and
MDMA- or MDA-containing saline. A separate set of experiments was performed to
investigate direct drug effects on voltage-gated calcium channels (VGCC). In these
experiments, MDMA or MDA was only present during the second depolarization (“non pre2+
exposed”). Experiments were performed at room temperature (20-22°C). Free cytosolic [Ca ]i
2+
was calculated according to a modified Grynkiewicz’s equation [Ca ]i = Kd* *(R-Rmin) / (Rmax-R),
where Kd* is the dissociation constant of Fura 2 determined in the experimental set-up
2+
(Deitmer and Schild, 2000). Cells that showed [Ca ]i 2x standard deviation (SD) above or
below average, either during basal recording or during depolarization, were excluded from
further analysis (~ 17%).
Carbon fiber microelectrode amperometry
As the number of releasable vesicles as well as the amount of catecholamines per vesicle is
limited in undifferentiated PC12 cells (see e.g., Westerink et al. 2000), cells were
differentiated using dexamethasone (see PC12 cell culture). This increases the amount of
catecholamines per vesicle and, importantly, increases the number of releasable vesicles
during successive stimulations without affecting the basal processes underlying exocytosis
(Westerink and Vijverberg, 2002). Cells were washed before experiments and continuously
+
superfused with saline. Spontaneous and K -evoked vesicular catecholamine release from
single dexamethasone-differentiated PC12 cells was measured using carbon fiber
microelectrode amperometry as described previously (Westerink and Vijverberg, 2002).
Amperometric current (sampled at 4 kHz, filtered at 2 kHz) was recorded using an EPC-7 patch
clamp (List Electronic, Darmstadt, Germany), and stored on disk for off-line analysis.
49
MDMA and MDA inhibit vesicular dopamine release
Low noise carbon fiber microelectrodes (5 µm) were purchased from Dagan Corporation
(Minneapolis, MN, USA).
Each single cell measurement consisted of a 5 min baseline recording to measure the
spontaneous release frequency, after which exocytotic capacity was recorded by a first
+
depolarization with 100 mM K for 20 s. Following this first depolarization and an 8 min
recovery period, the cell is exposed to saline (control), MDMA- or MDA-containing saline for
+
15 min prior to a second depolarization with 100 mM K for 20 s. Initially, experiments were
performed with 1 mM MDMA or MDA. Lower concentrations were tested only if the ‘no
observed effect level’ (NOEL) was not yet reached. Experiments were performed at room
temperature (20-22°C).
A custom-designed routine in Labview (National Instruments, Austin, Texas, USA) was
used for identification and analysis of currents associated with vesicular catecholamine
release, as described previously (Hondebrink et al. 2009). For each cell, the frequency of
events was determined and vesicle content was calculated from the total charge (Q)
transferred during the event according to Q/nF, where n = 2 electrons for the oxidation of one
catecholamine molecule and F is Faraday’s constant (96 485 C/mole). Cells were excluded for
data analysis (11% for frequency and 28% for content calculations) if they were outliers, basal
release was high (>5 events/min), evoked release was low (<40 events/min) or when the
release frequency following depolarization did not return to basal values in the last minute
before exposure (>5 events/min).
Statistical analysis
All data are presented as mean ± SEM of n cells or N experiments. Data was averaged per cell
instead of pooling data from several cells (Colliver et al. 2000; Westerink et al. 2000).
Consequently, all data were compared using a Student’s t-test (significant effects p < 0.05),
2+
unless otherwise noted. The second depolarization-evoked increase in [Ca ]i or exocytosis
2+
was expressed as a percentage of the first depolarization-evoked increase in [Ca ]i or
2+
exocytosis per cell to derive a treatment ratio. As the standard deviation for the [Ca ]i
2+
treatment ratio in control cells amounted to 22%, effects on [Ca ]i <25% were considered
irrelevant. An ANOVA followed by Bonferroni’s post-hoc test was used to determine the
concentration dependency of effects.
Results
MDMA and MDA do not induce overt cytotoxicity
PC12 cells were exposed to 1-1000 µM MDMA or MDA to determine effects on cell viability
using MTT. Cell viability was not affected by a 60 min exposure to 1-1000 µM MDMA or MDA.
Following a 24 h drug exposure, only 1000 µM MDA decreased cell viability to 80 ± 14% (p <
0.005). This indicates that MDA is more potent than MDMA in reducing cell viability, though
2+
drug-induced effects observed during Ca imaging or amperometric measurements are thus
not confounded by acute cytotoxicity (Fig. 5.1).
50
Chapter 5
Figure 5.1. Effects of MDMA or MDA exposure on cell viability. MTT assays were performed using
undifferentiated PC12 cells exposed to 1 mM MDMA or MDA for 60 min or 24 h. Only following a 24 h
exposure to 1 mM MDA a decrease in cell viability was observed. Control values were set at 100%. **
p < 0.005 vs control, # p < 0.001 vs 1 mM MDMA.
2+
MDMA and MDA decrease the depolarization-evoked increase in [Ca ]i without affecting
2+
basal [Ca ]i
2+
2+
As exocytosis is triggered by an increase in [Ca ]i, drug-induced effects on [Ca ]i in single
PC12 cells were investigated. In control cells (n=191, N=22), basal calcium levels were low (94
+
nM ± 19 nM), whereas depolarization of the cells with 100 mM K induced a transient
2+
2+
increase in [Ca ]i up to 2.04 ± 0.06 µM. Cells returned to near basal [Ca ]i during the 8 min
recovery period and these low calcium levels were maintained during a subsequent 15 min
2+
exposure to saline. A second depolarization evoked an increase in [Ca ]i up to
1.79 ± 0.06 µM, i.e., up to ~90% of the first depolarization (Fig. 5.2A). When cells were
+
exposed for 15 min to different concentrations (1-1000 µM) of MDMA or MDA in between K
2+
depolarizations, [Ca ]i remained stable. A comparison of treatment ratios in control cells and
drug-exposed cells revealed a relevant decrease only following 1 mM MDMA or 1 mM MDA
exposure (see Fig. 5.2), with 1 mM MDA being more potent in reducing the treatment ratio
than 1 mM MDMA (p < 0.005).
To investigate if the decrease in treatment ratio is due to an acute direct inhibition of
VGCCs, a separate set of experiments was performed in which MDMA or MDA was only
present during the second depolarization (“non pre-exposed”), i.e., without 15 min drug
exposure in between the two depolarizations (“pre-exposed”). However, 1 mM MDMA or
MDA did not exert a relevant decrease in treatment ratio (Fig. 5.2C), indicating that the
inhibition of VGCCs is indirect and that other mechanisms are involved in inhibiting calcium
influx (see discussion).
MDMA and MDA reduce depolarization-evoked exocytosis
2+
To reveal whether the observed changes in [Ca ]i have functional consequences for
exocytosis, basal and depolarization-evoked vesicular release frequencies were determined
using amperometry. In control cells (n=22), basal release frequencies were low
(0.3 ± 0.1 events/min), whereas depolarization increased the release frequency to 152 ± 18
events/min.
51
MDMA and MDA inhibit vesicular dopamine release
Release frequency dropped to 0.3 ± 0.1 events/min during the 8 min recovery period and
remained this low during a subsequent 15 min exposure to saline. A second depolarization
again evoked exocytosis with a frequency of 128 ± 15 events/min, i.e., 93 ± 9% compared to
the first depolarization (Fig. 5.3A).
2+
2+
Figure 5.2. Drug-induced effects on [Ca ]i. Representative recordings of [Ca ]i from individual PC12
cells exposed for 15 min (dotted line on top of the recording) to saline (control, grey line) or 1 mM
MDMA (black line) in between two 20 s depolarizations (100 mM K+ indicated by the arrows) illustrating
the MDMA-induced inhibition of the depolarization-induced increase in [Ca2+]i (A). No relevant decrease
in treatment ratio was observed following exposure to 1, 10, 100 µM MDMA (n=99, 77 and 69
respectively) or to 1, 10, 100 µM MDA (n= 61, 57 and 58 respectively). A relevant decrease in treatment
ratio (see methods) was observed only following exposure to 1 mM MDMA (n=89) and 1 mM MDA
(n=46). ** p < 0.005 vs control. # p < 0.001 vs 1 mM MDMA (B). Bar graph illustrating that the treatment
ratio is reduced only following 15 min drug exposure (“pre-exposed”), but not when drug exposure is
limited to the second depolarization (“non pre-exposed”). ° p < 0.001 vs non pre-exposed (C). The
treatment ratio of MDMA and MDA exposed cells is expressed as a percentage of the treatment ratio in
saline exposed cells.
Basal release frequencies during a 15 min exposure to MDMA or MDA remained unaltered
compared to control, which is consistent with the absence of drug-induced increases in basal
2+
[Ca ]i. However, exposure to high concentrations of MDMA or MDA decreased the release
frequency during the second depolarization (see Fig. 5.3B for example recording). Compared
to control cells, MDMA reduced the frequency of exocytosis only at 1 mM (47 ± 7% of the first
2+
depolarization), which is in accordance with the MDMA-induced reduction in [Ca ]i. Although
2+
MDA also reduced the depolarization-evoked increase in [Ca ]i only at 1 mM, effects on
52
Chapter 5
release frequency were observed at lower concentrations. 10 µM MDA already decreased the
frequency of exocytosis during the second depolarization to 67 ± 10% of the first
depolarization (p = 0.06), whereas the maximum inhibition was reached at 1 mM MDA
(40 ± 7%; see also Fig. 5.3). These results indicate that although MDMA and MDA do not
induce exocytotic DA release under basal conditions, they do reduce the frequency of
exocytosis during depolarization. Furthermore, MDA is more potent than MDMA in reducing
the frequency of exocytosis.
Figure 5.3. Drug-induced effects on exocytosis. Representative amperometric recordings of a saline (A)
+
and a MDMA-exposed cell (B) during two depolarizations with 100 mM K for 20 s, illustrating the
MDMA-induced reduction in the frequency of depolarization-evoked exocytosis. C. Each cell is
depolarized followed by exposure to MDMA or MDA for 15 min (indicated by // ) and a subsequent
second depolarization. Frequency of exocytosis was determined during both depolarizations and the
treatment ratio (see methods) was calculated. No relevant decrease in treatment ratio was observed
following exposure to 100 µM MDMA (n=11) or to 1 or 10 µM MDA (n=12 and 12). A relevant decrease
in treatment ratio was found following exposure to 1 mM MDMA (n=15, solid line) as well as 100 µM
and 1 mM MDA (n=13 and 10, dashed line) compared to control (n=22, c). * p < 0.05, ** p < 0.005 vs
control.
MDMA and MDA do not affect vesicle content
Amperometric events, representing exocytosis of single large dense-core vesicles, were
analyzed to determine vesicular DA content. Vesicular DA content of MDMA and MDA
exposed cells was compared to saline exposed cells to determine possible drug-induced
vesicular DA leakage. During the first depolarization the average median content in control
cells amounted to 697 ± 66 zmol. Vesicle content was unaffected during the second
depolarization, amounting to 749 ± 77 zmol. In cells exposed to MDMA or MDA the vesicle
content during the second depolarization was also not significantly different, indicating that a
15 min exposure to MDMA or MDA (up to 1 mM) does not induce acute vesicular leakage of
DA.
53
MDMA and MDA inhibit vesicular dopamine release
Discussion
It is well known that MDMA causes a rise in extracellular DA, but whether exocytosis
contributes to this rise was not yet investigated. We used PC12 cells to investigate drug2+
induced effects on DA exocytosis since they synthesize, store and release DA in a Ca dependent manner (Westerink and Ewing, 2008). Although PC12 cells are known to also
contain noradrenaline (Chen et al. 1994), our PC12 cells contain 99% DA and <1%
noradrenaline (Westerink et al. 2002). Our single cell amperometric experiments were
performed using a perfusion system, which enables measurement of exocytosis, without
being confounded by transporter-mediated DA release. In our study, the frequency of
exocytosis during MDMA or MDA exposure was not altered (Fig. 5.3), indicating that direct
effects on exocytosis are not contributing to the observed increase in extracellular DA levels
2+
following MDMA exposure. In support of this, the [Ca ]i also remained unaltered during
exposure (Fig. 5.2A).
MDMA- or MDA induced increases in extracellular DA levels observed in vivo could also
be explained by increases in stimulation-evoked exocytosis. However, our findings suggest
that MDMA and MDA do not exert any stimulatory effect on DA exocytosis, but rather an
inhibitory effect (40-60% decrease in release frequency, Fig. 5.3). This is comparable to an
in vivo microdialysis study in rats, which showed an MDMA- and MDA-induced decrease in
the depolarization-evoked DA increase, although they found a larger effect and also at lower
concentrations (75% reduction at 100 µM, Romero et al. 2006). Furthermore, previous
experiments in neuroblastoma cells using a DAT blocker (Pifl et al. 2005) as well as in
antisense-mediated DAT knock down rats in which the MDMA-induced release was almost
completely abolished (Kanthasamy et al. 2002) support our findings. Variable results were
obtained using brain slices, ranging from no effect to an inhibition or even potentiation of
electrically stimulated DA release, depending on which dopaminergic brain area was
investigated (Fitzgerald and Reid 1993; Iravani et al. 2000).
In addition to changes in the frequency of exocytosis, an increase in extracellular DA
levels can also be attributed to leakage of vesicular DA into the cytosol followed by reverse
DAT transport. To the best of our knowledge, our study is the first to use a whole cell model
to determine MDMA- and MDA-induced effects on vesicular DA content. Previously,
MDMA-induced monoamine efflux has been shown in chromaffin granules and vesicular
fractions that were preloaded with monoamines (EC50 values 30-200 µM, Rudnick and Wall,
1992; Partilla et al. 2006). The ability of a substance to dissipate the intravesicular pH is
thought to be predictive for its ability to induce vesicular leakage (Sulzer and Rayport, 1990).
However, the ability of MDMA to dissipate intravesicular pH is limited with a half maximal
effect of 1 mM (Rudnick and Wall, 1992). In line with this, we did not observe an effect of
MDMA or MDA on the amount of intravesicular DA in large dense-core vesicles, which appear
rather protected from leakage due to their dense-core (Hondebrink et al. 2009).
2+
Both MDA and MDMA inhibited depolarization-evoked Ca influx and exocytosis at
concentrations of 1 mM (Figs. 5.2 and 5.3). This was probably not due to direct inhibition of
nd
VGCCs (Fig. 5.2C), but rather to modulation of a 2 messenger pathway. It has been shown
previously that MDMA can activate protein kinase C (PKC; Nair and Gudelsky, 2004; Lin et al.
54
Chapter 5
2010). Since PKC activation has been reported to inhibit VGCCs (Sena et al. 1999) and
subsequently depolarization-evoked exocytosis (Westerink and Vijverberg, 2002), activation
2+
of PKC could thus explain the observed inhibition of Ca influx and exocytosis at high drug
concentrations (1 mM).
Surprisingly, we found only a weak correlation between the observed MDA-induced
2+
effects on the treatment ratio for [Ca ]i and its effects on the frequency of exocytosis during
2+
depolarization. At 10 and 100 µM MDA, the treatment ratio for [Ca ]i was unaffected,
whereas exocytosis was already inhibited (Figs. 5.2 and 5.3). It is therefore unlikely that the
specific inhibition of exocytosis by moderate concentrations of MDA can be explained by
2+
activation of PKC. Another possible cause for a decrease in exocytosis, without effects on Ca
influx, could be a decrease in vesicle cycling resulting in less releasable vesicles. Several
2+
proteins involved in vesicle cycling require phosphorylation by protein kinase A (PKA) or Ca calmodulin-dependent protein kinase II (CAMKII) to increase the amount of releasable
vesicles and thus exocytosis (for review see Westerink, 2006). It is also known that inhibition
of CAMKII activity in PC12 cells reduces evoked catecholamine release by as much as 50%,
2+
without affecting Ca influx (Schweitzer et al. 1995). As both PKA and CaMKII are inhibited by
MDMA (Moyano et al. 2004; Crawford et al. 2006), it is not unlikely that these pathways are
also inhibited by MDA. However, one must also assume that MDA is more potent than MDMA
in doing so, to explain the observed specificity. Noteworthy in this respect, MDA is also more
2+
potent in reducing cell viability (Fig. 5.1) and depolarization-evoked [Ca ]i. However, the
involvement of other mechanism(s) cannot be ruled out and future experiments using
nd
pharmacological modulation of 2 messenger pathways will have to shed light on this issue.
Metabolism of MDMA results in several metabolites, such as MDA, 3,4-dihydroxymethamphetamine (HHMA) and 4-hydroxy-3-methoxymethamphetamine (HMMA), which can be
found in plasma in vivo. The neurological effects observed following MDMA exposure can be
caused by MDMA itself or any of its metabolites. Of these metabolites (MDA, HHMA and
HMMA), only MDA was found in rat brain (Chu et al. 1996; Mueller et al. 2009) and therefore
we chose to investigate MDMA and MDA-induced effects on a dopaminergic in vitro model.
2+
MDMA and MDA both reduced depolarization-evoked [Ca ]i at 1 mM, although MDA showed
a significant larger inhibition than MDMA. MDA was also more potent in reducing the
frequency of exocytosis (10 µM vs 1 mM).
Concentrations of MDMA used in our study were based on relevant human
concentrations. In human volunteers, an oral intake of 75-150 mg MDMA (approximately
equivalent to one ecstasy tablet) resulted in a maximal blood concentration of 0.4 - 2 µM (de
la Torre et al. 2000; Samyn et al. 2002; Kolbrich et al. 2008). In cases in which serious toxicity
or even fatality occurred, blood levels of 2 - 44 µM were found. However, interindividual
differences are large since serious toxicity has also been reported at doses as low as 0.1 µM
(Kalant, 2001). In rats, the concentration of MDMA in brain was found to be 10 times higher
compared to blood (Chu et al. 1996; Mueller et al. 2009). Assuming a similar disposition in
humans, oral intake of one ecstasy tablet would result in a brain concentration of ~20 - 440
2+
µM. Though the concentration at which MDMA decreases [Ca ]i as well as the frequency of
exocytosis during depolarization (1 mM) is rather high, it should be noted that people often
consume more than one tablet.
55
MDMA and MDA inhibit vesicular dopamine release
Moreover, in human volunteers it has been shown that MDMA pharmacokinetics lacked
linearity, i.e., increasing the dose resulted in a more than proportional increase in blood and
thus also brain concentration (de la Torre et al. 2000). Thus, due to drug accumulation in the
brain, interindividual differences and multiple dosing causing a non-linear increase in blood
levels, high µM levels of MDMA may well be reached in the human brain.
Human relevant MDA concentrations are lower compared to MDMA. MDA was found
in blood at concentrations of 0.2 µM in human volunteers (de la Torre et al. 2000) and brain
concentrations in vivo (~1 µM) were only 3-5 times higher compared to blood (Escobedo et al.
2005; Mueller et al. 2009). We observed a strong but not significant inhibition of the
frequency of exocytosis already at 10 µM MDA, indicating that this mechanism of action could
play a role in the effects observed in humans during recreational MDMA intake.
Although MDMA and MDA did not affect basal and even decreased depolarizationevoked exocytosis in our study, it is possible that these drugs exert effects on additional
neurotransmitter systems in vivo, thereby indirectly stimulating DA exocytosis. It is known, for
example, that pretreatment with a 5-HT2 antagonist attenuates MDMA-induced DA release
(Nash 1990; Schmidt et al. 1994). Several neurotransmitters are reported to mainly have a
stimulatory effect on DA release, such as glutamate, 5-HT and acetylcholine, whereas others
have a mainly inhibitory effect on DA release, e.g., GABA and glycine. The direction of
regulation is dependent on which specific receptor is expressed and where it is expressed (for
review see Adell and Artigas, 2004; David et al. 2005). More sophisticated experiments, e.g.,
using (spontaneously active) co-cultures of different types of neurons are required to resolve
whether induction of exocytosis via these neurotransmitter systems contributes to the
MDMA- and MDA-induced increase in DA release observed in vivo.
We have shown that the DAT-independent increase in DA levels observed in vivo following
2+
MDMA or MDA exposure is not due to a direct effect on [Ca ]i or exocytosis. On the contrary,
2+
in PC12 cells the depolarization-evoked increase in [Ca ]i and exocytosis was decreased by
MDMA and even stronger by MDA. Based on our results, it can be suggested that
bioactivation plays an important role in the observed physiological and toxicological effects.
Experiments lacking the 15 min pre-exposure did not reveal a direct effect on VGCCs.
2+
Moreover, since [Ca ]i is not affected at MDA concentrations that already affect the
nd
frequency of exocytosis, we hypothesize that exocytosis is inhibited indirectly via 2
2+
messenger systems involved in vesicle cycling and Ca homeostasis. Future experiments will
have to demonstrate if the DAT-independent increase in extracellular DA in vivo following
nd
MDMA exposure is due to other mechanisms, such as modulation of 2 messenger pathways,
reduction of inhibitory (GABA-ergic) input, enhancement of excitatory (5-HT, glutamate or
acetylcholine) input or nonspecific DA transport via SERT reversal.
Acknowledgements
We gratefully acknowledge S.J. Rietjens and the members of the neurotoxicology research
group for thoughtful discussions.
56
Chapter 6
Methamphetamine, amphetamine, MDMA (‘ecstasy’),
MDA and mCPP modulate electrical and
cholinergic input in PC12 cells
Laura Hondebrink1,2,*, Jan Meulenbelt1,2,3, Saskia J. Rietjens2,
Marijke Meijer1, Remco H.S. Westerink1
1
Neurotoxicology Research Group, Institute for Risk Assessment Sciences (IRAS), Utrecht
University, P.O. Box 80.176, NL-3508 TD Utrecht, The Netherlands.
2
The National Poisons Information Centre (NVIC), The National Institute for Public Health and
the Environment (RIVM), Bilthoven, The Netherlands.
3
Division Intensive Care Center, University Medical Center Utrecht, Utrecht, The Netherlands.
In revision, Neurotoxicology
Drugs of abuse modulate electrical and cholinergic input
Abstract
Although reversal of the dopamine (DA) membrane transporter is the main mechanism
through which many drugs of abuse increase DA levels, drug-induced modulation of
exocytotic DA release by electrical (depolarization) and neurochemical inputs (e.g.,
acetylcholine (ACh)) may also contribute. We therefore investigated effects of
methamphetamine, amphetamine, 3,4-methylenedioxymethamphetamine (MDMA), 3,4methylenedioxyamphetamine (MDA) and meta-chlorophenylpiperazine (mCPP) (1-1000 µM)
on these inputs by measuring drug-induced changes in basal, depolarization- and ACh-evoked
2+
intracellular calcium concentrations ([Ca ]i) using a dopaminergic model (PC12 cells) and
Fura 2 imaging.
The strongest drug-induced effects were observed on cholinergic input. At 0.1 mM all
2+
2+
drugs inhibited the ACh-evoked [Ca ]i increases by 40-75%, whereas ACh-evoked [Ca ]i
increases were nearly abolished following higher drug exposure (1 mM, 80-97% inhibition).
2+
Additionally, high MDMA and mCPP concentrations increased basal [Ca ]i, but only following
prior stimulation with ACh. Interestingly, low concentrations of methamphetamine or
2+
amphetamine (10 µM) potentiated ACh-evoked [Ca ]i increases. Depolarization-evoked
2+
[Ca ]i increases were also inhibited following exposure to high drug concentrations, although
drugs were less potent on this endpoint.
Our data suggest that at high drug concentrations all tested drugs reduce stimulationevoked dopaminergic output through inhibition of electrical and cholinergic input, though
2+
MDMA and mCPP may also increase dopaminergic output via an increase in basal [Ca ]i. Also,
this study shows that mCPP, which is regularly found in ecstasy tablets, is the most potent
drug regarding all investigated endpoints. Furthermore, at low drug concentrations,
amphetamines increase cholinergic input and possibly DA exocytosis, which may contribute to
the drug-induced dopamine increases observed in vivo.
58
Chapter 6
Introduction
Amphetamines as well as MDMA and MDA are known to increase brain dopamine (DA) levels
in vivo (for review see Sulzer et al. 2005, Capela et al. 2009). Drugs of abuse increase
dopaminergic output at least partly via reversal of the dopamine membrane transporter
(DAT), which is the primary mode of action through which e.g., amphetamine increases DA
levels. However, exposure to drugs of abuse may also directly or indirectly affect vesicular DA
secretion (exocytosis). The frequency of exocytosis can be influenced by multiple inputs, e.g.,
by changes in electrical input (depolarization) and subsequent opening of voltage-gated
calcium channels (VGCCs). Furthermore, the cholinergic system provides important excitatory
input on dopaminergic neurons, e.g., application of the acetylcholine (ACh) receptor agonist
nicotine causes DA release in dopaminergic brain areas (Romanelli et al. 2007). As cholinergic
input modulates dopaminergic output, drug-induced effects on the dopaminergic system may
also be mediated through the cholinergic system.
Cholinergic activation is mediated by two types of receptors; the ionotropic nicotinic
acetylcholine receptor (nACh-R) and the metabotropic muscarinic acetylcholine receptor
(mACh-R). Dopaminergic neurons express several types of nACh-Rs that consist of different
combinations of five α and β subunits, but also include a homomeric α7 subtype (Collins et al.
2009, Lester et al. 2010). PC12 cells, which have been widely used as a dopaminergic model to
investigate catecholamine release (Westerink and Ewing, 2008), also express both nACh-Rs
and mACh-Rs (Shafer and Atchison, 1991). Many nACh-Rs have a relatively high calcium
permeability, with the α7 homomers having the highest permeability. Activation of nACh-Rs
by agonist binding can therefore directly result in calcium entry. The subsequent increase in
2+
the intracellular calcium concentration ([Ca ]i) can also elicit calcium release from
intracellular stores, which further facilitates DA exocytosis (MacDermott et al. 1999,
Engelman and MacDermott 2004, Lester et al. 2010).
The hypothesis that, in addition to DAT reversal, the cholinergic system could also
contribute to drug-induced dopaminergic effects is supported by previous in vivo and in vitro
data showing that amphetamines and MDMA affect the dopaminergic system via the
cholinergic system (Drew et al. 2000, Camarasa et al. 2008, Chipana et al. 2008). Moreover, in
dopaminergic models (PC12 and chromaffin cells), direct effects on DA exocytosis and
2+
intracellular calcium concentration ([Ca ]i) could not be detected at concentrations of
amphetamine, MDMA or MDA reached during recreational use (Hondebrink et al. 2009,
Hondebrink et al. 2011). Consequently, drug-induced changes on the cholinergic system
should be explored, as these might contribute to drug-induced dopaminergic effects observed
in vivo. We therefore investigated the effects of (meth)amphetamine and the active
substances in ecstasy tablets (MDMA, MDA and mCPP) on electrical and cholinergic input by
2+
measuring drug-induced changes in basal, depolarization- and ACh-evoked [Ca ]i in
dopaminergic PC12 cells.
59
Drugs of abuse modulate electrical and cholinergic input
Materials and Methods
Chemicals
D-Methamphetamine, DL-MDMA, DL-MDA, and mCPP were obtained from Duchefa (Haarlem,
The Netherlands). D-Amphetamine was obtained from Spruyt Hillen (IJsselstein, The
Netherlands). Stock-solutions were prepared in saline and stored at 4°C for no more than 2
weeks. Working solutions (1-1000 µM) were prepared immediately before experiments.
CaCl2, MgCl2, MgSO4, NaHCO3, NaOH, Ca(NO3)2, KCl, and HEPES were purchased from Merck
(Darmstadt, Germany). All other chemicals, unless otherwise noted, were obtained from
Sigma-Aldrich (Zwijndrecht, the Netherlands).
PC12 Cell culture
Undifferentiated rat pheochromocytoma PC12 cells (Greene and Tischler 1976, ATCC) were
2+
used to study effects on basal as well as depolarization- and ACh-evoked [Ca ]i. Dopaminergic
PC12 cells are regarded as a suitable model to study these endpoints (Westerink and Ewing
2008), which are predictive for dopaminergic output. Cells were maintained in monolayers in
2
25 cm tissue culture flasks (Nunc, Rochester, NY, USA) in a humidified 5% CO2 / 95% air
atmosphere at 37 °C in RPMI 1640 medium (Invitrogen, Breda, the Netherlands)
supplemented with 5% fetal calf serum, 10% horse serum (ICN Biomedicals, Zoetermeer, The
Netherlands), 100 U/ml penicillin and 100 µg/ml streptomycin (Invitrogen, Breda, the
Netherlands). Medium was refreshed every 2-3 days and cells were cultured for up to 10
4
passages. Undifferentiated PC12 cells were subcultured in glass-bottom dishes (23 * 10
2+
cells/dish, MatTek, Ashland, MA, USA) for [Ca ]i imaging experiments as described previously
(Hondebrink et al. 2009, Hondebrink et al. 2011). All culture flasks and dishes were coated
with poly-L-lysine (50 µg/ml).
Intracellular calcium imaging
2+
As many nACh-Rs are calcium permeable, the [Ca ]i increases upon nACh-Rs activation.
2+
2+
Therefore, [Ca ]i imaging can be used to determine drug-induced effects on basal [Ca ]i as
2+
well as on depolarization- and ACh-evoked increases in [Ca ]i. Before experiments, cells were
washed and maintained in saline containing (in mM) 125 NaCl, 5.5 KCl, 2 CaCl2, 0.8 MgCl2, 10
2+
HEPES, 24 glucose and 36.5 sucrose. Changes in [Ca ]i were measured in undifferentiated
2+
PC12 cells using the high-affinity Ca -responsive fluorescent dye Fura 2-AM (Molecular
Probes; Invitrogen, Breda, The Netherlands) as described previously (Hondebrink et al. 2009,
Hondebrink et al. 2011) with minor modifications. Cells were continuously superfused with
saline using a valvelink 8.2 (Automate Scientific, California, USA). The F340/F380 ratio (R) was
measured every 3 s. Each experiment consisted of a 5 min baseline recording to measure
2+
2+
basal [Ca ]i, after which an increase in [Ca ]i was triggered by changing superfusion to 100
+
mM K or 1 mM ACh for 20 s. Following this first stimulation and an 8 min recovery period,
cells were exposed to saline (control) or to methamphetamine, amphetamine, MDMA, MDA
+
or mCPP for 15 min (“pre-exposed cells”) prior to a second stimulation with 100 mM K or 1
mM ACh in the absence (control) or presence of methamphetamine, amphetamine, MDMA,
MDA or mCPP. A separate set of experiments was performed to investigate direct drug effects
60
Chapter 6
on nACh-Rs and VGCCs. In these experiments, the drug of abuse was not present in between
stimulations, but only during the second stimulus (“non pre-exposed cells”). All experiments
were performed at room temperature (20-22°C).
Data analysis
2+
Free cytosolic [Ca ]i was calculated according to a modified Grynkiewicz’s equation
2+
[Ca ]i = Kd* *(R-Rmin) / (Rmax-R), where Kd* is the dissociation constant of Fura-2 determined in
the experimental set-up (Deitmer and Schild 2000). All data are presented as mean ± SEM of n
cells or N experiments. All data were compared using a Student’s unpaired t-test (significant
effects p<0.05), unless otherwise noted. The second depolarization- or ACh-evoked increase
2+
in [Ca ]i was expressed as a percentage of the first depolarization- or ACh-evoked increase in
2+
[Ca ]i to derive an ‘evoked treatment ratio’. Negative treatment ratios were set to zero. To
2+
2+
determine effects on basal [Ca ]i during drug exposure, [Ca ]i was expressed as a percentage
2+
of [Ca ]i 2 min before exposure to derive a ‘basal treatment ratio’. Data was averaged per cell
2+
instead of pooling data from several cells. Cells that showed [Ca ]i 2x standard deviation (SD)
above or below average, either during basal recording or during stimulation, were excluded
from further analysis (18%). Considering the large standard deviation for the stimulus-evoked
treatment ratio (28% for depolarization and 40% for ACh), effects < 25% were considered
irrelevant. An ANOVA followed by Bonferroni’s post hoc test was used for testing
concentration dependency of effects.
Results
High concentrations of methamphetamine, amphetamine, MDMA, MDA and mCPP decrease
2+
the depolarization-evoked increase in [Ca ]i
2+
As exocytosis is triggered by an increase in [Ca ]i, drug-induced effects on the depolarization2+
evoked increase in [Ca ]i were investigated in PC12 cells. Moreover, comparison of effects on
depolarization- or ACh-evoked effects could distinguish specific from non-specific effects, as
ACh-R activation can also result in depolarization (Lester et al. 2010). In control PC12 cells
2+
(n=307, N=33), basal [Ca ]i was low (97 nM ± 1 nM), whereas depolarization of the cells with
+
2+
2+
100 mM K induced a rapid but transient increase in [Ca ]i up to 2.1 ± 0.1 µM. [Ca ]i returned
to near basal levels during the 8 min recovery period and low calcium levels were maintained
during a subsequent 15 min exposure to saline. A second depolarization evoked an increase in
2+
[Ca ]i up to ~85% of the first depolarization (Fig. 6.1A).
When cells were exposed for 15 min to methamphetamine, amphetamine, MDMA,
2+
MDA or mCPP (1-1000 µM) in between the depolarizations, basal [Ca ]i remained stable.
However, a comparison of ‘evoked treatment ratios’ (see Materials & Methods for details)
obtained from saline- and drug-exposed cells revealed a relevant decrease in the
depolarization-evoked treatment ratio following exposure to methamphetamine,
amphetamine, MDMA or MDA, but only at 1 mM (p<0.001, Fig. 6.2 and Table 6.1). mCPP was
more potent and already decreased the ‘evoked treatment ratio’ to ~65% at 100 µM
(p<0.001).
61
Drugs of abuse modulate electrical and cholinergic input
2+
Figure 6.1. Representative [Ca ]i recordings. PC12 cells were exposed for 15 min (dotted line on top of
recording) to saline (control, grey line) or 1 mM MDA (black line) in between two depolarizations (A) or
+
ACh (B) stimuli. Note the difference in y-axis scaling. Arrows indicate application of 100 mM K (A) or 1
mM Ach (B).
To investigate if the decrease in ‘evoked treatment ratio’ is due to acute, direct inhibition of
VGCCs, a separate set of experiments was performed in which the drug of abuse (at 1 mM)
was present only during the second depolarization (non pre-exposed cells). A relevant
decrease in ‘evoked treatment ratio’ was observed in these non pre-exposed cells only
following exposure to methamphetamine (25%) or mCPP (37%).
This indicates that methamphetamine and mCPP directly inhibit VGCCs, whereas
indirect mechanisms are involved in the amphetamine-, MDMA- and MDA-induced inhibition.
However, indirect mechanisms are also involved in the mCPP-induced reduction as the
‘evoked’ treatment ratio was even further reduced following 15 min mCPP pre-exposure (95%
decrease, p<0.001, Fig. 6.3A).
Methamphetamine, amphetamine, MDMA, MDA and mCPP affect the ACh-evoked increase
2+
in [Ca ]i
2+
As many nACh-Rs are calcium permeable and thus increase [Ca ]i upon activation,
2+
drug-induced effects on the ACh-evoked increase in [Ca ]i were measured to determine
potential effects on nACh-Rs. In control PC12 cells (n=478, N=57), basal calcium levels were
low (104 nM ± 1 nM), whereas activation of nACh-Rs with 1 mM ACh induced a rapid but
2+
2+
transient increase in [Ca ]i up to 324 ± 7 nM. [Ca ]i returned to near basal levels during the 8
min recovery period and low calcium levels were maintained during a subsequent 15 min
2+
saline exposure. A second ACh stimulus evoked an [Ca ]i increase up to 260 ± 7 nM, i.e., up to
~80% of the first stimulation (Fig. 6.1B).
Experiments were performed to determine effects of different drugs of abuse on the
2+
ACh-evoked increase in [Ca ]i. A comparison of ‘evoked treatment ratios’ obtained from
saline- and drug-exposed cells revealed a relevant increase up to ~40% following exposure to
10 µM methamphetamine or amphetamine (p<0.05; Fig. 6.2A).
62
Chapter 6
Figure 6.2. Drug-induced effects on depolarization- and ACh-evoked [Ca2+]i. PC12 cells were exposed for
+
15 min to saline or drug in between two 20 s K (solid lines) or ACh (dashed lines) stimuli. The ‘evoked
treatment ratio’ of drug-exposed cells is compared to the ‘evoked treatment ratio’ of saline-exposed
cells. Methamphetamine (Meth), amphetamine (Amph) (A), MDMA, MDA (B) and mCPP Cc) modulate
the depolarization- and the ACh-evoked treatment ratio. Values represent mean ± SEM, for exact values
and degrees of significance see Tables 6.1 and 6.2.
However, exposure to higher concentrations of methamphetamine or amphetamine resulted
in a concentration-dependent decrease in ‘evoked treatment ratio’ up to 90% at 1 mM
(p<0.001). Similarly, exposure to MDMA, MDA or mCPP concentration-dependently
decreased the ‘evoked treatment ratio’ at concentrations >10 µM up to 97% at 1 mM
(p<0.001, see Fig. 6.2 and Table 6.2).
To investigate if the decrease in ‘evoked treatment ratio’ is due to acute and direct
inhibition of nACh-Rs, a separate set of experiments was performed in which 1 mM drug was
only present during the second ACh stimulus (non pre-exposed cells). The ‘evoked treatment
ratio’ in non pre-exposed cells was decreased, varying from 40-90%, which indicates all drugs
can directly inhibit nACh-Rs (p<0.001). However, except for MDMA, all drugs displayed a
stronger decrease in ‘evoked treatment ratio’ following a 15 min pre-exposure (90-97%
decrease, p<0.001).
63
Drugs of abuse modulate electrical and cholinergic input
This indicates that indirect mechanisms also contribute to the observed decrease in ‘evoked
treatment ratio’ following exposure to high concentrations of amphetamines, MDA and
mCPP. Although MDMA pre-exposure caused an additional 14% decrease in ‘evoked
treatment ratio’ compared to non pre-exposed cells, it did not result in a significant stronger
decrease (p=0.07), possibly indicating that MDMA directly inhibits nACh-Rs and modulation of
second messenger pathways plays a minor role (Fig. 6.3B).
Figure 6.3. Depolarization- and ACh-evoked [Ca2+]i in pre-exposed and non pre-exposed cells.
Normalized depolarization-evoked (A) and ACh-evoked (B) treatment ratios in cells pre-exposed (black
bars) and non pre-exposed (white bars) to the different drugs (at 1 mM). Values represent mean ± SEM,
** p<0.001 vs control, ° p<0.001 pre-exposed vs non pre-exposed cells. Meth, methamphetamine;
Amph, amphetamine.
2+
Besides drug-induced changes in ACh-evoked increases in [Ca ]i, we also observed changes in
2+
basal [Ca ]i during exposure to 0.1 or 1 mM of MDMA or 1 mM mCPP. Exposure to 1 mM
2+
MDMA evoked a rapid increase in [Ca ]i, resulting in a relevant increase in ‘basal treatment
ratio’ up to ~280% vs 130% in saline exposed cells (Fig. 6.4A, p<0.001). The maximum
2+
amplitude of the MDMA-induced increase in [Ca ]i amounted up to 270 ± 24 nM (compared
to 126 ± 2 nM in saline exposed cells) and was observed during the first 5 min (average tmax =
2+
3.1 min) of exposure to 1 mM MDMA. Smaller increases in [Ca ]i were observed during
exposure to 1 mM mCPP or 100 µM MDMA. Relevant increases in ‘basal treatment ratio’
(~160%) were observed only during the first 5 min exposure to 1 mM mCPP or 100 µM MDMA
2+
2+
(average tmax ~ 1.7 min), corresponding to [Ca ]i of 170 ± 15 nM for both drugs. [Ca ]i
returned to basal levels immediately upon switching superfusion from MDMA or mCPP to ACh
in the presence of MDMA or mCPP.
Additional experiments, in which superfusion was switched to calcium free medium
following the first ACh stimulus and the 8 min recovery period, were performed to determine
2+
the source of the drug-induced increases in [Ca ]i. Removal of extracellular calcium during
2+
MDMA or mCPP exposure (1 mM) completely prevented the increase in [Ca ]i (Fig. 6.4. n=20,
N=3 for MDMA and n=23, N=3 for mCPP), indicating that intracellular calcium stores are not
involved in the observed effect.
64
Chapter 6
Table 6.1. Drug-induced effects on depolarization-evoked [Ca2+]i. Depolarization-evoked treatment
ratios of [Ca2+]i, following 15 min exposure to methamphetamine (Meth), amphetamine (Amph), MDMA,
MDA or mCPP. Values represent mean ± SEM (number of cells), ** p<0.001 vs control.
Table 6.2. Drug-induced effects on ACh-evoked [Ca2+]i. ACh-evoked treatment ratios of [Ca2+]i, following
15 min exposure to methamphetamine (Meth), amphetamine (Amph), MDMA, MDA or mCPP. Values
represent mean ± SEM (number of cells), ** p<0.001, * p<0.05 vs control.
Figure 6.4. MDMA induced changes in [Ca2+]i. Representative recordings of [Ca2+]i from PC12 cells
exposed for 15 min to 1 mM MDMA (dotted line on top of recording) following an ACh stimulus.
2+
Following a prior ACh stimulation, MDMA induced an increase in [Ca ]i in calcium containing (2 mM)
medium (A). In calcium free medium, no MDMA-induced increase in [Ca2+]i was observed (B). Arrows
indicate application of 1 mM ACh.
65
Drugs of abuse modulate electrical and cholinergic input
Discussion
We demonstrated that methamphetamine, amphetamine, MDMA, MDA and mCPP are all
able to modulate both electrical and cholinergic input in a dopaminergic cell model (PC12
cells, Fig. 6.2). A low concentration of methamphetamine- and amphetamine (10 µM)
2+
increased the ACh-evoked [Ca ]i (~25-40% increase in treatment ratio). This study is the first
to show that methamphetamine and amphetamine display agonistic properties for ACh-Rs on
a functional level using calcium as a read-out. Thus, although reversal of the dopamine
membrane transporter is the main mechanism through which e.g., amphetamine increases
DA levels, drug-induced enhancement of cholinergic input may also contribute to druginduced increases in DA levels. For amphetamine, this is further supported by Liu et al (2003),
who showed in chromaffin cells that the amphetamine-induced (10 µM exposure) increase in
DA levels was diminished when α7 nACh-Rs were blocked. The drug-induced increases in
2+
[Ca ]i we observed upon nACh-Rs activation following exposure to 10 µM methamphetamine
or amphetamine is possibly due to binding to an allosteric activator site of nACh-Rs.
In addition to increases in cholinergic input that may increase dopaminergic output at
2+
low drug concentrations, increases in [Ca ]i were observed that might evoke exocytosis
2+
during exposure to high concentrations of MDMA and mCPP (Fig. 6.4). The increase in [Ca ]i
2+
was abolished in calcium free medium, indicating that the increase in [Ca ]i resulted from
2+
influx of extracellular calcium. Interestingly, drug-induced increases in basal [Ca ]i were not
observed following depolarization, indicating that MDMA and mCPP are not simply ACh-R
2+
agonists but also require prior ACh-R activation to exert this effect. As increases in [Ca ]i can
trigger DA exocytosis, this mechanism may contribute to higher DA levels in vivo.
At high concentrations of methamphetamine, amphetamine, MDMA, MDA and mCPP
electrical as well as cholinergic input was diminished. It is therefore unlikely that at high
concentrations drug-induced changes in electrical or cholinergic input contribute to increased
DA levels observed in vivo. Decreased responses upon nACh-R activation, as observed for all
drugs >10 µM, may result from drug binding to a ligand-binding site of nACh-Rs, thereby
preventing receptor activation by ACh. However, other mechanisms can also explain druginduced decreases, e.g., binding to a negative allosteric site, which inhibits ion channel
function but does not directly affect ACh binding (for review on binding sites see Paterson and
Nordberg 2000). The dual effects of amphetamines may be explained by high affinity binding
of the drugs to a positive allosteric site with a small maximum effect size and parallel low
affinity binding to a ligand-binding or negative allosteric site with a larger maximum effect
size.
The observed decreases in ACh-evoked treatment ratio are supported by other studies.
Amphetamine (100 µM) was previously shown to decrease the open state of nACh-Rs
(Spitzmaul et al. 1999). Moreover, MDMA inhibited the ACh-evoked current in α7 and α4β2
nACh-Rs (EC50 ~ 10 µM, Garcia-Rates et al. 2010). For mCPP, we are the first to describe
effects on ACh-Rs, although a structurally similar substance, dimethylphenyl piperazine
(DMPP), displayed agonistic properties for α3β4 and the α3β2 nACh-Rs (EC50 ~ 30 µM,
2+
Cachelin and Jaggi 1991). Although we observed an increase in ACh-evoked [Ca ]i following 1
µM mCPP, this effect was below the considered relevant effect of 25%. However, we also
66
Chapter 6
observed mCPP-induced decreases in ACh-evoked treatment ratio (at 0.1 and 1 mM),
indicating differential effects of mCPP may occur at different drug concentrations and at
different nACh-Rs.
Second messenger pathways could be involved in many of the drug-induced reductions
2+
in ACh-evoked [Ca ]i as 15 min pre-exposure resulted in stronger reductions (Fig. 6.3).
Kinases, like calcium- or calmodulin-dependent kinases and protein kinase C, are part of a
2+
second messenger pathway that can be activated by increases in [Ca ]i (for example due to
activation of nACh-Rs). Activation of kinases enhances phosphorylation, which in turn can
increase DA exocytosis (for review see Duman and Nestler 2000; Westerink 2006).
Methamphetamine, amphetamine, MDA and mCPP possibly decrease the ACh-evoked
treatment ratio by affecting one of these second messenger pathways.
We also investigated drug-induced changes in the depolarization-evoked treatment
ratio to examine whether observed effects were mediated directly via ACh-Rs or a result of
the drug effects on VGCCs. All tested drugs decreased the depolarization-evoked treatment
ratio at high drug concentrations (1 mM, p<0.001), although mCPP was more potent and
already induced a reduction at 100 µM. Modulation of second messenger pathways was
involved in amphetamine-, MDMA- and MDA-induced decreases, as no relevant change in
‘evoked treatment ratio’ was observed in non pre-exposed cells. Although methamphetamine
and mCPP directly inhibited VGCCs, modulation of second messenger pathways is also
involved in mCPP-induced decreases since pre-exposure induced a stronger decrease (58%
more inhibition).
These drugs of abuse thus inhibit VGCCs as well as nACh-Rs, suggesting that at high
drug concentrations the drug-induced effects are not very specific. However, all drugs
induced stronger inhibitions of the ACh-evoked treatment ratio compared to the
depolarization-evoked treatment ratio, indicating specificity for nACh-Rs. Of the selected set
of drugs, mCPP is the only drug that decreased both the depolarization- and the ACh-evoked
treatment ratio at a similar concentration (100 µM). Possibly, mCPP acts as an L-type VGCC
antagonist as this category of substances would reduce the depolarization-evoked treatment
ratio. Furthermore, L-type VGCC antagonists are also capable of blocking agonist-induced
activation of nACh-Rs (Lopez et al. 1993), which could explain the observed reduction in both
the depolarization- and ACh-evoked treatment ratio.
In summary, methamphetamine and amphetamine act as partial agonists of ACh-Rs,
but only at 10 µM. At this low concentration, methamphetamine- and amphetamine-induced
increases in cholinergic input may increase DA exocytosis. However, decreases in cholinergic
input were observed following exposure to >10 µM of methamphetamine, amphetamine,
MDMA, MDA and mCPP. Moreover, at high concentrations all drugs inhibited VGCCs. Thus, at
these high concentrations the selected drugs of abuse do not exert any stimulatory effect on
electrical or cholinergic input, but rather an inhibitory effect. It is therefore unlikely that at
these high drug concentrations effects on the investigated inputs contribute to drug-induced
increases in DA levels in vivo.
67
Drugs of abuse modulate electrical and cholinergic input
Acknowledgements
We gratefully acknowledge Martin van den Berg, Marten Kops and the members of the
neurotoxicology research group for thoughtful discussions and Aart de Groot for software
development, which simplified the extensive analysis.
68
Chapter 7
Modulation of human GABAA receptor function:
a novel mode of action of drugs of abuse
Laura Hondebrink1,2,*, Jan Meulenbelt1,2,3, Regina G.D.M. van Kleef1,
Martin van den Berg1, Remco H.S. Westerink1
1
Neurotoxicology Research Group, Institute for Risk Assessment Sciences (IRAS), Utrecht
University, P.O. Box 80.176, NL-3508 TD Utrecht, The Netherlands.
2
The National Poisons Information Centre (NVIC), The National Institute for Public Health and
the Environment (RIVM), Bilthoven, The Netherlands.
3
Division Intensive Care Center, University Medical Center Utrecht, Utrecht, The Netherlands.
Neurotoxicology (2011) DOI 10.1016/j.neuro.2011.05.016
Drugs of abuse modulate GABA-ergic input
Abstract
Drugs of abuse are known to mainly affect the dopaminergic and serotonergic system,
although behavioral studies indicated that the GABA-ergic system also plays a role. We
therefore investigated the acute effects of several commonly used drugs of abuse
(methamphetamine, amphetamine, 3,4-methylenedioxymethamphetamine (MDMA), 3,4methylenedioxyamphetamine (MDA) and meta-chlorophenylpiperazine (mCPP)) on the
function of the human α1β2γ2 GABAA receptor (hGABAA-R), expressed in Xenopus oocytes,
using the two-electrode voltage-clamp technique. Although none of the tested drugs acted as
full agonist on the hGABAA-R, some drugs induced differential modulation of hGABAA-R
function, depending on the degree of receptor occupancy. Methamphetamine did not affect
the GABA-evoked current at high receptor occupancy, but induced a minor inhibition at low
receptor occupancy. Its metabolite amphetamine slightly potentiated the GABA-evoked
current. MDMA and its metabolite MDA both inhibited the current at low receptor
occupancy. However, MDMA did not affect the current at high occupancy, whereas MDA
induced a potentiation. mCPP induced a strong inhibition (max. ~80%) at low receptor
occupancy, but ~25% potentiation at high receptor occupancy. Competitive binding to one of
the GABA-binding sites could explain the drug-induced inhibitions observed at low receptor
occupancy, whereas an additional interaction with a positive allosteric binding site may play a
role in the observed potentiations at high receptor occupancy. This is the first study to
identify direct modulation of hGABAA-Rs as a novel mode of action for several drugs of abuse.
Consequently, hGABAA-Rs should be considered as target for psychiatric pharmaceuticals and
in developing treatment for drug intoxications.
70
Chapter 7
Introduction
The term “drugs of abuse” refers to substances that are usually not taken for medicinal
reasons but for their mind-altering effects. The most recent data available on lifetime
prevalence in the European adult population (15-64 years old) indicate that ~11 million
people (3,3%) have used ecstasy and ~12 million (3,6%) have used amphetamines (EMCDDA,
2010). These drugs are often taken in recreational settings, though intoxications may occur
when taken in excess. Amphetamines and other stimulants, such as the active substances in
ecstasy tablets, are responsible for ~100.000 emergency hospital admittances per year in the
US (DAWN, 2010).
Drugs of abuse can cause acute as well as long-term toxicity, mainly in the central and
peripheral nervous system but also in other targets, including the liver. In humans, exposure
to methylenedioxymethamphetamine (MDMA, the main active substance in ecstasy tablets)
has been described to acutely induce heightened sensory awareness and symptoms of
sympatic arousal. Moreover, neuroendocrine changes, hyperthermia and suppression of the
immune system have also been described. In animal studies, long-term MDMA-induced
toxicity includes loss of serotonergic terminals, whereas in humans lower density and activity
of the serotonin membrane transporter, a reduction in 5-HT2A receptors as well as
impairments in memory and learning have been reported (for review see Capela et al. 2009).
On a cellular and molecular level, amphetamines and the active substances found in
ecstasy tablets primarily increase brain dopamine (DA) and serotonin (5-HT) levels within the
first hours following exposure. The increase in DA and 5-HT is thought to be at least partly due
to leakage of DA and 5-HT from vesicles into the cytosol and reversal of membrane
transporters (for review see Sulzer et al. 2005). Additionally, drugs of abuse may also directly
evoke exocytosis (but see e.g., Hondebrink et al. 2009). Alternatively, drugs of abuse could
affect dopaminergic and serotonergic output either by increasing excitatory input or by
decreasing inhibitory input on the dopaminergic and serotonergic system, e.g., via
activation/potentiation of glutamate and acetylcholine receptors or via inhibition of GABA
and glycine receptors, respectively.
The GABA-ergic system is the major inhibitory input in the brain, with the α1β2γ2
subtype combination being the most abundant and widely distributed form of the GABAA
receptor (GABAA-R, McKernan and Whiting, 1996; D'Hulst et al. 2009). Therefore, interactions
between drugs of abuse, dopaminergic and GABA-ergic systems may underlie the observed
drug-induced alterations in neuronal activity and brain neurotransmitter levels. Since GABA
decreases firing of dopaminergic neurons (Kiyatkin and Rebec, 1998), increases in DA levels
could be due to a drug-induced reduction in GABA-ergic input on dopaminergic neurons.
Drugs of abuse could achieve this by decreasing GABA-ergic inhibitory input, e.g., by reducing
the extracellular GABA concentration. However, increases in GABA release in dopaminergic
brain areas have been reported following exposure to e.g., amphetamine (Yin et al. 2010).
Consequently, it is more likely that drugs of abuse reduce the number or function of
GABAA-Rs.
71
Drugs of abuse modulate GABA-ergic input
In line with this, in vivo studies revealed that GABAA-Rs play an important role in many druginduced behavioral changes, such as amphetamine-induced place preference, stereotypy and
hyperlocomotion (Karler et al. 1995; Meririnne et al. 1999; Reynolds et al. 2003). Moreover, it
was demonstrated in humans that the discriminative stimulus effects of amphetamine were
significantly attenuated by pretreatment with the benzodiazepine alprazolam, which
potentiates GABAA-R function (Rush et al. 2004). Finally, patients with drug of abuse
intoxications are often treated with GABAA-R agonists, like benzodiazepines (Department of
Health, 2006; Merck, 2008), because of symptoms of agitation and/or seizures. Improvement
of these symptoms following benzodiazepine treatment indicates that a decreased GABAergic input contributes to symptoms often seen in these patients. However, up to now only
cocaine was reported to act as a GABAA-R antagonist, inhibiting the GABA-evoked ion current
up to ~80% (Ye et al. 1997).
Though research on drug of abuse-induced effects on GABAA-R function is scarce,
available literature indicates that the human GABAA-R could be an important target for some
drugs of abuse. We therefore investigated whether several popular drugs of abuse
(amphetamine, methamphetamine, MDMA, 3,4-methylenedioxyamphetamine (MDA) and
meta-chlorophenylpiperazine (mCPP)) can directly modulate GABAA-R function.
Materials and Methods
Animals
Xenopus laevis oocytes were used to investigate drug-induced effects on GABAA-Rs. Oocytes
efficiently translate injected complementary (cDNA) into RNA and RNA into protein.
Neurophysiological studies and studies using labelled proteins have shown that the expressed
proteins are functional and transported to areas where they normally occur (Brown 2004).
This model thus enables direct measurement of receptor function with relative ease and at
low costs. Consequently, the Xenopus laevis oocyte is widely used for the expression of
heterologous proteins and was considered the most suitable model for the present study.
Experiments were approved by the Ethical Committee for Animal Experiments of
Utrecht University and conducted in accordance with Dutch law and the European
Community directives regulating animal research (86/609/EEC). All efforts were made to
minimize the number of animals used and their suffering. Mature female Xenopus laevis frogs
(provided by Dr. Scheenen, Radboud University Nijmegen, The Netherlands) were kept in
standard aquaria (0.5 x 0.4 x 1 m; 1–10/aquarium) with copper-free water (pH 6.5, 23°C) and
a 12 h light/dark cycle. Animals were fed earthworms three times a week (Hagens,
Nijkerkerveen, The Netherlands).
Chemicals
DL-3,4-Methylenedioxymethamphetamine
(MDMA), DL-3,4-methylenedioxy-amphetamine
(MDA), 1-(3-chlorophenyl)-piperazine (mCPP) and D-methamphe-tamine were obtained from
Duchefa (Haarlem, The Netherlands). D-Amphetamine was obtained from Spruyt Hillen
(IJsselstein, The Netherlands). Stock-solutions (1 M) were prepared in saline and stored at 4°C
for no more than 2 weeks. CaCl2, MgCl2, MgSO4, NaHCO3, NaOH, Ca(NO3)2, KCl, and HEPES
72
Chapter 7
were purchased from Merck (Darmstadt, Germany). All other chemicals, unless otherwise
noted, were obtained from Sigma-Aldrich (Zwijndrecht, the Netherlands).
Expression of α1β2γ2 GABAA receptors in Xenopus laevis oocytes
All procedures have been described previously (Zwart and Vijverberg, 1997; Hendriks et al.
2010). Briefly, female Xenopus laevis were anesthetized by submersion in 0.1% MS-222, and
ovarian lobes were surgically removed. Oocytes were incubated with collagenase type I (1.5
2+
mg/ml Ca -free Barth’s solution) for 2 h at room temperature. Complementary DNA (cDNA)
coding for the human α1, β2 and γ2 subunits of human GABAA receptors (Origene, Rockville,
USA) was dissolved in distilled water at a 1:1:1 molar ratio and injected (23 nl/oocyte, ~1 ng of
each subunit) into the nuclei of stage V or VI oocytes using a Nanoject Automatic Oocyte
Injector (Drummond, Broomall, PA). Oocytes were incubated for 2-5 days at 21°C in modified
Barth’s solution containing (in mM) 88 NaCl, 1 KCl, 2.4 NaHCO3, 0.3 Ca(NO3)2, 0.41 CaCl2, 0.82
MgSO4, 15 HEPES and 50 µg/ml neomycin (pH 7.6 with NaOH) to allow for receptor
expression.
Electrophysiological recordings
Electrophysiological recordings were performed at room temperature, 2-5 days following
cDNA injection. Ion currents associated with GABAA-R activity were measured with the twoelectrode voltage-clamp technique using a Gene Clamp 500B amplifier (Axon Instruments)
with high-voltage output stage as described previously (Zwart and Vijverberg, 1997). Oocytes
were placed in a custom-built Teflon oocyte recording chamber and voltage-clamped at -60
mV using two microelectrodes (0.2-1 MΩ) filled with KCl (3 M). Oocytes were continuously
perfused (~6 ml/min) with saline containing (in mM) 115 NaCl, 2.5 KCl, 1 CaCl2, 10 HEPES (pH
7.2 with NaOH). Membrane currents were low-pass filtered (8-pole Bessel; 3 dB at 0.3 kHz),
digitized (12 bits; 1024 samples/record), and stored on disk for computer analysis. Oocytes
were exposed to saline or saline containing GABA and/or drugs by switching the perfusate
using a ValveLink 8.2 perfusion system (AutoMate Scientific, Berkeley, CA). Agonistic drug
properties were examined by exposing GABA-responsive oocytes to 1-1000 µM drug for 20 s
(see Fig. 7.1A for example recordings). In subsequent experiments, we investigated the ability
of drugs of abuse to modulate the GABA-evoked current using co-applications of drugs (11000 µM) and GABA (10 µM or 1 mM). GABA-responsive oocytes were exposed to GABA for
10 s, followed by GABA with drug for 20 s prior to another 10 s GABA exposure (see Fig. 7.1B
for example recordings). A washout period of 4 min between each GABA application was
introduced, allowing receptors to recover from desensitization. At least two different batches
of oocytes were used to collect data for each analysis.
Data analysis and statistics
Inhibition or potentiation of GABA-evoked currents by drugs was compared to the control
current evoked by the application of 10 µM or 1 mM GABA. The percentage of drug-induced
alteration of the GABA-evoked ion current was calculated from the quotient of the amplitude
of the GABA-drug co-application response (during 20 s) and the amplitude of the control part
73
Drugs of abuse modulate GABA-ergic input
of the response. Data represent mean ± SEM of n oocytes. Data were compared using an
ANOVA followed by Bonferroni’s post-hoc test (significant effects p < 0.05).
Results
Lack of agonistic effects on human α1β2γ2 GABAA-R
Oocytes expressing human α1β2γ2 GABAA-Rs were exposed to different GABA concentrations
(0.1 µM - 10 mM) to obtain a concentration-effect curve for GABA. From this, the EC20, i.e.,
the concentration producing 20% of the maximal response, and Hill slope were determined
using the Hill equation. The EC20 and slope amounted to respectively 14 ± 0.04 µM and 1.20 ±
0.16 (n=9), which is in line with previous results (Ye et al. 1997; Hendriks et al. 2010). The EC50
amounted to 40 µM, indicative of consistent expression of the α1β2γ2 subunits combination
(Walters et al. 2000). Also, successive GABA stimulations (prior to and following drug
exposure) resulted in similar GABA-induced currents, indicating that these brief (20 s) drugexposures do not induce acute toxicity or changes in expression levels of GABAA-Rs.
To investigate whether the various drugs of abuse used in this study are able to
activate the human GABAA receptor, GABA-responsive oocytes were superfused with saline
containing 1 - 1000 µM of MDMA, MDA, methamphetamine, amphetamine or mCPP (n=3-13),
see Fig. 7.1A for example recording. None of these drugs of abuse induced ion currents,
clearly demonstrating that these compounds cannot activate human GABAA receptors (data
not shown).
Figure 7.1. Experimental design for investigating agonistic and modulatory effects of drugs of abuse on
the α1β2γ2 GABAA-R. A. For testing agonistic drug properties, oocytes were exposed to 10 µM GABA,
followed by a 4 min recovery period (indicated by //) prior to a 20 s drug exposure (1-1000 µM). B. To
investigate drug effects in the presence of GABA, oocytes were exposed to 10 µM or 1 mM GABA,
followed by a 4 min recovery period. Subsequently, oocytes were exposed to 10 µM or 1 mM GABA,
prior to a co-application of 10 µM or 1 mM GABA with 1-1000 µM drug and a subsequent 10 µM or 1
mM GABA exposure. The arrow indicates the difference between the expected and observed current,
which was used in calculating the percentage of modulation.
74
Chapter 7
Modulation of human α1β2γ2 GABAA-R at low receptor occupancy by drugs of abuse
To investigate effects of drugs of abuse on GABAA-Rs at low receptor occupancy, GABAresponsive oocytes were superfused with saline containing 10 µM GABA (~EC20) and 1-1000
µM of the test compound, as illustrated in Fig. 7.1B. At this low receptor occupancy, low
concentrations of methamphetamine and amphetamine (1-100 µM) did not affect the GABAevoked current. However, at 1 mM, methamphetamine induced a small inhibition of the
GABA-evoked current, whereas its metabolite amphetamine slightly potentiated the current
(Fig. 7.2A; Table 7.1).
Figure 7.2. Drugs of abuse exert dual effects on the human α1β2γ2 GABAA-R. Graphs illustrate the
average (±SEM) percentage of inhibition (-) or potentiation (+) of the GABA-evoked current induced by
different drugs of abuse. Insets show the structure of the different drugs of abuse as well as example
recordings of acute modulation of GABA-evoked currents by co-application with different drugs of abuse
(1 mM). Note the difference in scaling between the graphs. See Table 7.1 for exact values and levels of
significance.
75
Drugs of abuse modulate GABA-ergic input
MDMA and its metabolite MDA also did not affect the GABA-evoked ion current at
concentrations up to 100 µM. At 1 mM, MDMA induced a clear inhibition of the GABA-evoked
current, amounting to ~30%, while MDA was evidently less potent (Fig. 7.2B; Table 7.1).
Notably, mCPP, a drug that is replacing MDMA in ecstasy tablets (EMCDDA 2009),
showed the strongest inhibition. The GABA-evoked current was significantly inhibited by
100 µM mCPP (~20%) and 1 mM mCPP (~80%; Fig. 7.2C; Table 7.1).
Modulation of human α1β2γ2 GABAA-R at high receptor occupancy by drugs of abuse
To investigate effects of drugs of abuse on GABAA-Rs at high receptor occupancy, GABAresponsive oocytes were superfused with saline containing 1 mM GABA (~ECmax) and 1-1000
µM of the test compound, as illustrated in Fig. 7.1B. At these high GABA concentrations,
methamphetamine did not exert any effect on the GABA-evoked current. However, 1 mM
amphetamine induced a slight potentiation of the GABA-evoked current, comparable to the
potentiation observed at low receptor occupancy (Fig. 7.2A; Table 7.1).
MDMA and MDA at concentrations up to 100 µM also did not affect the GABA-evoked
current at high receptor occupancy (Fig. 7.2B; Table 7.1). Under these conditions of high
receptor occupancy, MDMA also did not affect the GABA-evoked current when applied at 1
mM, which contrasts with the inhibition observed under conditions of low receptor
occupancy. Surprisingly, at high receptor occupancy, MDA (1 mM) induced a potentiation of
the GABA-evoked ion current of ~20%, contrasting with the slight inhibition observed during
co-exposure with 10 µM GABA.
Similar to the effects observed at low GABA concentrations, mCPP also showed the
strongest modulation of the GABA-evoked current at high receptor occupancy. However,
during co-exposure with 1 mM GABA, mCPP induced a potentiation rather than an inhibition.
At 100 µM and 1 mM, the mCPP-induced potentiation of the GABA-evoked current amounted
to respectively ~10% and ~25% (Fig. 7.2C; Table 7.1).
Table 7.1. Summary table of the modulatory effects of drugs of abuse on the α1β2γ2 GABAA-R under
conditions of low and high receptor occupancy (10 µM and 1 mM GABA). Values indicate the average
percentage ± SEM of inhibition (-) or potentiation (+) of the GABA-evoked currents of (n) experiments. *
p < 0.01, ** p < 0.005.
76
Chapter 7
Discussion
This study revealed that none of the tested drugs of abuse was able to activate the human
GABAA-R. However, all drugs modulated the GABA-evoked current and this modulation was
dependent on the occupancy of the GABAA-R. The fast onset (<1 s) and rapid reversibility of
the observed effects indicate a direct interaction between these drugs of abuse and the
GABAA-R. Though the in vivo relevance of direct modulation of human GABAA-R function
remains to be determined, it may represent a novel mode of action for some drugs of abuse.
Despite the wide application of these drugs, until now few (direct) effects of the tested
drugs of abuse on the GABA-R have been described. We are the first to show that these drugs
of abuse exert direct and functional effects on the GABAA-R, which depend on receptor
occupancy. Previous research supports these observations since several indirect drug-induced
effects can be linked to the GABA-ergic system. Many drugs of abuse induce behavioral
effects, like hyperlocomotion, discriminative stimulus or place preference, which are inhibited
by agonists or positive modulators of GABAA-Rs (Hsieh, 1982; Reynolds et al. 2003; Rush et al.
2004). This indicates that a reduction of GABA-ergic input contributes to drug-induced
changes in behavior. Similarly, many drugs of abuse increase DA levels, which could be
achieved by a reduction of GABA-ergic input. Both phenomena are in accordance with the
reduction in GABA-evoked current we observed during exposure to methamphetamine,
MDMA, MDA and mCPP at low GABAA-R occupancy (Fig. 7.2; table 7.1), indicating that drug
effects on the GABAA-R may contribute to behavioral effects.
At high GABAA-R occupancy however, opposite effects can be observed for mCPP, MDA
and to a lesser extent MDMA (Fig. 7.2; table 7.1). Dual actions on the GABAA-R at different
degrees of receptor occupancy have been described by others (Woodward et al. 1994; Chisari
et al. 2010). The origin of these observations may be related to the presence of multiple
GABA binding sites on the GABAA-R (D'Hulst et al. 2009). At a non-saturating, low GABA
concentration, GABA likely occupies only one of two binding sites. In this state, high
concentrations of methamphetamine, MDMA, MDA and mCPP induced inhibitions, which
may be explained by competitive binding to one of the GABA binding sites. This hypothesis is
supported by the observation that the inhibition is no longer observed at high GABA
concentrations. However, the MDA- and mCPP-induced potentiations observed at high
receptor occupancy possibly involve an additional interaction with a positive allosteric binding
site, such as the benzodiazepine binding site. The absence of suitable and truly specific
binding agents hampers identification of the specific binding site involved.
The drugs of abuse selected for this study are among those most often used for
recreational purposes (EMCDDA, 2010). Amphetamines are used as drugs of abuse in many
forms, although they are also commonly used to treat attention-deficit hyperactivity disorder
(ADHD). Steady-state plasma levels of ~20 µM are found in tolerant individuals who consume
~1 g daily (Ellenhorn and Barceloux, 1988), which is similar to blood concentrations in people
arrested for driving under the influence of amphetamine (Holmgren et al. 2008). The other
selected drugs are often found in ecstasy tablets; MDMA is the main common constituent
with MDA being an important metabolite. The piperazine derivate mCPP is also commonly
found in ecstasy tablets and is replacing MDMA as the main constituent in some countries
77
Drugs of abuse modulate GABA-ergic input
(EMCDDA, 2009). Importantly, mCPP also has a legitimate use as the pharmacologically active
metabolite of several psychiatric pharmaceuticals (Rotzinger et al. 1998). For MDMA,
exposure to a dose equivalent to one ecstasy tablet, led to plasma levels of ~2 µM in
volunteers (de la Torre et al. 2000; Kolbrich et al. 2008). In cases that included serious toxicity,
blood levels up to ~45 µM were found (Kalant, 2001). Due to expected drug accumulation in
the brain (Mueller et al. 2009), inter-individual differences and multiple dosing causing a nonlinear increase in blood levels, high µM levels of MDMA may well be reached in the human
brain.
Thus although normal user concentrations for most of the drugs tested in our study are
in the µM range, high µM levels are likely to occur in the brain following heavy use. The wide
application of these drugs combined with the observed dual effects on the GABAA-R function
urges for additional research to unravel the clinical relevance of GABAA-Rs as a target for
psychiatric pharmaceuticals as well as during multiple drug use and drug intoxications.
Acknowledgements
We gratefully acknowledge S. Rietjens and the members of the neurotoxicology research
group for thoughtful discussions.
78
Chapter 8
Summary and Risk Assessment
Summary and Risk Assessment
Summary
Drug-induced effects on presynaptic dopaminergic neurotransmission
As indicated in chapter 1, amphetamine-like drugs are known to increase basal levels of
extracellular dopamine (DA), primarily via reversal of the dopamine membrane transporter
(DAT, Fischer and Cho 1979). For example, amphetamine is unable to increase DA levels in
brain slices from mice that lack this transporter (Jones et al. 1998). However, varying degrees
of MDMA-induced DA release were still observed upon DAT blockage (Schmidt et al. 1987;
Nash and Nichols 1991; Kanthasamy et al. 2002; Pifl et al. 2005), indicating that both DATmediated and exocytotic DA release may contribute to increased basal DA levels in the brain.
Additionally, the weak base properties of amphetamine and its analogues presumably enable
them to disrupt vesicular catecholamines storage (Sulzer and Rayport 1990). This would
decrease vesicular DA content and thus increase cytosolic DA. Subsequently, cytosolic DA
could be transported outwards via DAT-reversal, contributing to increased extracellular basal
DA levels. Additionally, disruption of vesicular storage could induce vesicular calcium release,
possibly resulting in DA exocytosis. This hypothesis was supported by an amphetamine2+
induced reduction in vesicular DA content (Sulzer et al. 1995) and an increase in [Ca ]i and
exocytosis (Mundorf et al. 1999). Although amphetamine-like drugs can thus increase basal
DA levels, amphetamine, MDMA and MDA all decrease stimulation-evoked DA release in vivo
(Jones et al. 1998; Romero et al. 2006), possibly by decreasing vesicular DA content. This has
been observed in a dopaminergic cell model; PC12 cells in which amphetamine exposure
reduced vesicular DA content during depolarization-evoked exocytosis (Sulzer et al. 1995).
However, in chromaffin cells no significant reduction in vesicle DA content was reported
(Mundorf et al. 1999). It thus remains controversial whether amphetamine-like substances
induce exocytosis of dopamine and affect vesicular dopamine content.
2+
In this thesis it is shown that amphetamine exposure did not affect [Ca ]i or the
exocytotic release frequency in PC12 or chromaffin cells at concentrations up to 200 µM
(chapter 4). Similar to amphetamine, MDMA and MDA (up to 1 mM) did not induce
2+
exocytosis or changes in basal [Ca ]i (chapter 5). Consequently, we conclude that
amphetamine-, MDMA- or MDA-induced exocytosis does not contribute to drug-induced
increases in basal DA levels in the brain as observed in vivo.
Furthermore, we show that the weak base properties of amphetamine are insufficient
to induce DA leakage from large dense-core vesicles (LDCVs) in PC12 or chromaffin cells
(chapter 4). Weak base properties of a substance can be predicted with a logarithmic
measure of the acid dissociation constant (pKa). As MDMA and MDA have pKa values
comparable to amphetamine (~10), they might also be able to disrupt the proton-gradient
and decrease vesicular DA content. However, with respect to our results obtained with
amphetamine, a MDMA- or MDA-induced decrease in vesicular DA content is less plausible.
Similar to amphetamine, MDMA and MDA did not reduce vesicular DA content (chapter 5),
indicating that DA in LDCVs is effectively protected from leakage. Consequently, the in vivo
observed decrease in stimulation-evoked DA release following amphetamine, MDMA or MDA
exposure (Jones et al. 1998, Romero et al. 2006) is unlikely due to leakage from LDCVs.
However, we show that amphetamine did reduce vesicular DA content in L-DOPA pretreated
80
Chapter 8
PC12 cells, which contain LDCVs with unbound DA. Therefore, we argue for refinement of the
widely-accepted weak base hypothesis and propose that amphetamine-like substances can
only exert their effect on freely available DA. As small synaptic vesicles (SSVs) contain freely
available DA, the weak base properties of amphetamine could induce catecholamine leakage
from SSVs, explaining the decrease in stimulation-evoked DA release in vivo (chapter 4).
Additionally, our data suggests that inhibition of voltage-gated calcium channels
(VGCCs) could also contribute to this decrease in stimulation-evoked DA release. We found
that amphetamine, as well as all other tested drugs of abuse (1 mM) decreased the
2+
depolarization-evoked increases in [Ca ]i (chapter 6). This correlated well with the decrease
in depolarization-evoked exocytosis we observed following MDMA or MDA exposure (chapter
5). Most likely, (meth)amphetamine and mCPP will induce comparable reductions in
2+
exocytosis of DA as they decreased the depolarization-evoked increase in [Ca ]i and an
2+
increase in [Ca ]i is required for DA exocytosis. Noteworthy, 100 µM MDA did not reduce
2+
depolarization-evoked [Ca ]i, although we did find a decrease in depolarization-evoked
2+
exocytosis. The current findings indicate that microscopic measurement of [Ca ]i cannot
always predict drug-induced effects on exocytosis. This technique has many advantages
2+
compared to plate reader [Ca ]i measurements, e.g., measurement on a single cell level and
2+
the ability to measure transient increases in [Ca ]i (chapter 3). However, its limitations should
be considered when it is applied in screening for substance-induced changes in exocytosis of
DA.
From our data it can be concluded that increases in basal extracellular DA levels
following exposure to amphetamine-like substances are most likely caused by DAT-reversal,
while exocytotic DA release hardly contributes. However, DA exocytosis can be affected by
changes in input from different neurotransmitter systems on the dopaminergic system, such
as an increase in excitatory (e.g., cholinergic) input or a decrease in inhibitory (e.g., GABAergic) input. Our data demonstrated that excitatory input could be increased at a low
concentration of methamphetamine and amphetamine (10 µM) as both drugs enhanced the
2+
ACh-evoked increase in [Ca ]i (chapter 6). This might reflect a stronger cholinergic input on
the dopaminergic system, which can contribute to increased basal DA levels. However, at
higher drug concentrations (0.1 and 1 mM) all tested drugs of abuse inhibited cholinergic
input, likely decreasing dopaminergic output.
We have also shown that drugs of abuse can affect inhibitory input on the
dopaminergic system, more specifically GABA-ergic input (chapter 7). At low GABA receptor
occupancy, methamphetamine, MDMA, MDA and mCPP decreased the GABA-evoked ion
current, whereas amphetamine increased the ion current. Therefore, drug-induced decreases
in inhibitory input can contribute to DAT-independent increases in basal levels of extracellular
DA. The inhibition of the GABA-evoked ion current was most likely due to competitive drug
binding to a GABA-binding site, as no inhibitions were observed at high GABA receptor
occupancy when excess GABA is present and competition for the GABA-binding site is
increased. Furthermore, amphetamine, MDA and mCPP potentiated the GABA-evoked ion
current at high receptor occupancy, indicating a possible additional interaction with a positive
allosteric binding site.
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Summary and Risk Assessment
Although drug-induced effects are depending on GABA receptor occupancy, modulation of
the GABAA-R appears to be a common mode of action for methamphetamine, amphetamine,
MDMA, MDA and mCPP.
Intracellular messenger systems
Our data indicated the involvement of intracellular messenger systems in several drugs of
2+
abuse-induced effects, e.g., drug-induced decreases in depolarization- and ACh-evoked [Ca ]i.
The observed drug-induced effects can be mediated via their possible effects on G-proteincoupled receptors (GPCRs) or via effects on proteins that alter receptor function. GPCRs
expressed by PC12 cells include mACh-Rs and adenosine receptors (Greene and Tischler
1976). Furthermore, it is known that activation of A1 adenosine receptors inhibits VGCCs
(Satoh et al. 1997; Poli et al. 2001; Schwartz et al. 2003), which would inhibit
2+
neurotransmission. Therefore, drug-induced decreases in depolarization-evoked [Ca ]i can be
mediated via agonistic drug effects on the adenosine receptor.
Also, activation of GPCRs can induce cascades of intracellular messengers, which can
cause diverse physiological effects. For example, the activity of specific proteins can be
altered via addition (protein kinases) or removal (protein phosphatases) of phosphate groups
(Duman and Nestler 2000). Unfortunately, literature on the selected five drugs of abuse and
their possible effects on intracellular messenger systems is scarce. Thus, it is unclear whether
these systems contribute to the in vivo effects that can be observed following exposure to
these drugs of abuse.
Possibly, the tested drugs of abuse induce changes in the activity of kinases like protein
2+
kinase C (PKC), protein kinase A (PKA) or Ca -calmodulin-dependent protein kinase II
2+
(CAMKII), through which they decrease depolarization-evoked [Ca ]i. MDMA-induced PKC
activation has already been described (Lin et al. 2010) and could explain the MDMA-induced
2+
inhibition in depolarization-evoked [Ca ]i we observed, as well as the reduction in exocytosis
of DA. Additional mechanisms are probably involved in the MDA-induced inhibition, because
we found a stronger potency for reducing depolarization-evoked increases in exocytosis
2+
compared to [Ca ]i. Possibly, MDA inhibits PKA or CAMKII, which could decrease vesicle
cycling. In support of this, CAMKII inhibition has been shown to reduce depolarization-evoked
exocytosis without affecting calcium influx (Schweitzer et al. 1995). Furthermore, as it is
known that MDMA can inhibit PKA and CaMKII (Moyano et al. 2004; Crawford et al. 2006), it
is not unlikely that MDA induces effects comparable to MDMA (chapter 5 and 6). Kinases
might also be involved in amphetamine- and mCPP-induced reductions in depolarization2+
evoked [Ca ]i, as pre-exposure was necessary to reduce the depolarization-evoked increase
2+
in [Ca ]i (amphetamine) or pre-exposure induced a stronger reduction in the depolarization2+
evoked increase in [Ca ]i (mCPP). However, no data is available on the possible effects of
these drugs on intracellular messenger systems.
2+
Kinases can also mediate the drug-induced decreases in ACh-evoked [Ca ]i we
observed (chapter 6). nACh-Rs can be phosphorylated by PKA, PKC or CAMK, which will
increase the desensitization rate (Decker et al. 2000) and could decrease the ACh-evoked
2+
2+
increase in [Ca ]i. Therefore, drug-induced decreases in the ACh-evoked increase in [Ca ]i
can be mediated by kinases.
82
Chapter 8
2+
Furthermore, the drug-induced decreases in ACh-evoked [Ca ]i could also be mediated by
drug-induced activation of GPCRs that couple to intracellular messenger systems which inhibit
the nACh-Rs response. It is known that activation of the M1-like mACh-Rs can activate PKC,
2+
which consequently could phosphorylate nACh-Rs and thus decrease ACh-evoked [Ca ]i.
2+
Therefore, drug-induced decreases in ACh-evoked [Ca ]i might be the result of agonistic drug
effects on M1-like mACh-Rs. However, activation of M1-like mACh-Rs would also increase
2+
basal [Ca ]i (Richelson 2000), which we did not observe during drug exposure. It is therefore
unlikely that this pathway is involved in drug-induced decreases in ACh-evoked calcium influx.
As data on intracellular messenger systems is scarce, it is difficult to link drug-induced
2+
effects to, e.g., changes in the ACh-evoked increase in [Ca ]i. However, modulation of
intracellular messenger systems appears to be a common mechanism for drugs of abuse,
justifying further investigation.
Multiple mechanisms of action
Combining the drug-induced effects we observed in dopaminergic cells and their different
inputs will provide a more comprehensive mode of action for each drug. However, we found
contradicting effects for most tested drugs of abuse, e.g., a decrease in cholinergic input that
could decrease dopaminergic output combined with a decrease in GABA-ergic input that
could increase output. How these different inputs balance in vivo is difficult to predict and will
depend on the investigated brain area, the concentration of the drug as well as on other, not
investigated, inputs on the dopaminergic system.
2+
In our studies, amphetamine did not induce any changes in basal [Ca ]i or exocytosis at
any tested concentration. However, at a high amphetamine concentration, VGCCs were
inhibited, the important inhibitory GABA-ergic input was increased and the important
excitatory cholinergic input was decreased. All these mechanisms can contribute to a
reduction in DA release following amphetamine exposure. At a lower amphetamine
concentration, an increase in cholinergic input was the only observed effect and this could
increase dopaminergic output.
At a high concentration, we observed methamphetamine-induced effects comparable
to amphetamine-induced effects. Methamphetamine inhibited VGCCs and cholinergic input,
which could reduce DA release. However, GABA-ergic input was either decreased or
unaffected, which would respectively increase or not affect DA release. At a lower
methamphetamine concentration, the only observed effect was an increase in cholinergic
input, possibly contributing to higher DA levels.
Our data revealed MDMA- and MDA-induced inhibitions of VGCCs, depolarizationevoked exocytosis and cholinergic input at a high concentration, likely reducing evoked DA
release from dopaminergic neurons. Also, MDA potentiates GABA-ergic input (at high GABA
receptor occupancy), further contributing to a decrease in DA release. However, at low GABA
receptor occupancy, both MDMA and MDA inhibit GABA-ergic input. This mechanism possibly
contributes to drug-induced increases in extracellular DA levels.
mCPP has been suggested as a safe alternative to MDMA (Bossong et al. 2010) as mCPP
does not induce long-term 5-HT depletion, in contrast to MDMA. Furthermore, mCPP-induced
effects on the dopaminergic system are regarded as insignificant by some (Baumann et al.
83
Summary and Risk Assessment
2001; Gobbi et al. 2002). However, our studies clearly show that mCPP does affect
dopaminergic neurotransmission. Of all tested drugs of abuse, mCPP induced the strongest
effects on all measured endpoints in our studies. At a high concentration, mCPP almost
2+
completely blocked depolarization-evoked and ACh-evoked increases in [Ca ]i. Thus, mCPP
inhibits VGCCs and cholinergic input. Furthermore, mCPP increased GABA-ergic input (at high
receptor occupancy). All these mechanisms could contribute to a decrease in DA release.
However, mCPP decreased GABA-ergic input at low receptor occupancy, which could enhance
DA release. Also, in vivo microdialysis studies from other labs have shown mCPP-induced
increases in DA levels (~150% of baseline) as well as robust increases in 5-HT levels (400-800%
of baseline, Eriksson et al. 1999; Baumann et al. 2001). These released catecholamines can be
oxidized, thereby generating oxygen radicals and possibly causing toxicity. Many users
reported negative adverse effects like nausea and hallucinations (Bossong et al. 2010) and,
fortunately, the amount of ecstasy tablets containing mCPP is already declining in some
countries (EMCDDA 2010). However, drug users can still be exposed to mCPP, which should
not be considered a safe alternative to MDMA.
Overall, it is expected that the effects on the dopaminergic system can be enhanced
and decreased depending on the type and, importantly, the concentration of the drug of
abuse as well as the concentration of the endogenous receptor ligands. Therefore, a spectrum
of effects is expected, and reported, following in vivo exposure. Consequently, it is difficult to
predict the effects methamphetamine, amphetamine, MDMA, MDA and mCPP will induce in
the in vivo situation.
Risk assessment
When considering the possible risks of drugs of abuse, several aspects should be taken into
account. First of all, which levels of exposure are found in human blood or brain and how do
these relate to concentrations at which effects were observed in our studies? This will be
discussed in the following paragraph.
Levels of exposure
The research aim determines the “relevant concentration” of a drug of abuse. For example,
investigating drug-induced effects following ingestion of one ecstasy tablet requires different
testing concentrations compared to investigating which mechanisms are involved during
intoxications due to repeated ingestions within a short period of time. Drug blood levels may
indicate relevant exposure levels and are available from volunteer studies for some drugs of
abuse. However, brain concentrations are also important in determining relevant exposure
levels, as this research has focused on drug-induced effects on neurotransmission. The
blood/brain ratio of a drug, obtained in animal in vivo studies, was used to translate human
blood concentrations to brain concentrations. It should be kept in mind that this in vivo
obtained ratio might differ from the ratio in humans. Furthermore, drug blood levels during a
drug intoxication were obtained from case reports, which often involved fatality and in which
concurrent exposure to other drugs of abuse was regularly involved. Also, post-mortem drug
redistribution could have influenced the blood levels obtained in these studies.
84
Chapter 8
The only available data on “recreational” blood levels of methamphetamine and
amphetamine was obtained in volunteer studies. Although differences in blood drug
concentration were observed for different routes of exposure (via inhalation, injection or
oral), blood concentrations were generally in a similar range. Exposure to low doses of
methamphetamine (10-20 mg) revealed sub µM blood levels of methamphetamine and low
nM amphetamine blood levels (Cook et al. 1992). Exposure to low doses of amphetamine (1020 mg) resulted in sub µM amphetamine blood levels (Angrist et al. 1987). At these low drug
exposures, blood concentrations increased proportionally with the dose. Noteworthy, these
exposures are extremely low, considering that it is not unusual for drug users to consume 500
mg amphetamine. Assuming a proportional increase in blood levels, this would imply relevant
blood levels of 3-15 µM. In vivo, brain concentrations of amphetamine were comparable to
those in blood (Sood et al. 2009). Taken all these data into account, “recreational” use of
amphetamines results in low µM brain levels. However, repeated ingestions within a short
period of time or poor metabolism can result in much higher concentrations; up to ~80 µM
amphetamine was detected in post-mortem blood samples (Verschraagen et al. 2007).
MDMA exposure, equivalent to the amount found in one ecstasy tablet (~100 mg),
resulted in sub µM to low µM blood levels (de la Torre et al. 2000; Samyn et al. 2002; Kolbrich
et al. 2008). Noteworthy, pharmacokinetics of MDMA lacks linearity at exposure levels >125
mg (de la Torre et al. 2000). Human brain concentrations could reach 20 µM, when applying
the in vivo obtained MDMA blood/brain ratio (Chu et al. 1996; Mueller et al. 2009). However,
higher concentrations may well be reached, as ingestion of several tablets within a short
period of time is not an uncommon pattern in ecstasy use (Engels and ter Bogt 2004).
Furthermore, during a drug intoxication, high µM drug levels can be expected in the brain; up
to ~60 µM of MDMA was detected in post-mortem blood samples (De Letter et al. 2004),
possibly translating to 600 µM MDMA in the brain. Following MDMA exposure, its metabolite
MDA was also detected in blood at sub µM levels (de la Torre et al. 2000). When applying the
in vivo obtained blood/brain ratio for MDA (Escobedo et al. 2005; Mueller et al. 2009), this
would translate to a human brain concentration of ~1 µM. Concentrations following direct
oral MDA exposure are of importance as MDA is not only a MDMA metabolite, but also found
in ecstasy tablets. Unfortunately, data on blood concentrations following direct oral MDA
exposure is not available. Direct oral MDA exposure might result in a blood concentration
comparable to MDMA, as exposure to methamphetamine or its metabolite amphetamine also
resulted in similar blood concentrations.
mCPP has been replacing MDMA in ecstasy tablets and varying mCPP amounts were
found in ecstasy tablets. On average 30 mg was detected (Bossong et al. 2010), a dose also
used in the treatment of psychiatric disorders (Kovaleva et al. 2008). Exposure to a
comparable dose, revealed sub µM blood levels in humans (Gijsman et al. 1998). Corrected
for the in vivo obtained blood/brain ratio (Nacca et al. 1998), the mCPP brain concentration
in humans may amount up to ~4 µM. Furthermore, interindividual differences are large since
cases of serious toxicity have been described in psychiatric patients who had similar mCPP
blood concentrations as human volunteers who did not develop serious toxicity (Klaassen et
al. 1998). During an intoxication, brain concentrations of mCPP could amount up to 40 µM, as
blood levels of 1 µM mCPP were reported during an overdose (Kovaleva et al. 2008).
85
Summary and Risk Assessment
In our studies, concentrations from 1 µM to 1 mM were tested, covering expected levels of
drugs of abuse in the brain following “recreational” use and during intoxication. However, we
mostly observed drug-induced effects at 0.1 and 1 mM. For example, depolarization-evoked
2+
[Ca ]i was inhibited only by 1 mM methamphetamine, amphetamine, MDMA, MDA and
mCPP, whereas mCPP also induced an inhibition at 0.1 mM. Furthermore, the drug-induced
modulation of the GABA-evoked ion current also only occurred during exposure to 1 mM
methamphetamine, amphetamine, MDMA, MDA and mCPP, whereas mCPP also modulated
the response at 0.1 mM. Therefore, most of our observed effects represent mechanisms of
action that are involved only during a drug intoxication. The exceptions are the enhanced
2+
ACh-evoked increases in [Ca ]i induced by 10 µM methamphetamine and amphetamine. This
represents a new mechanism of action, which can contribute to increased DA levels during
“recreational” use.
Additional aspects for risk assessment
Many other factors could affect drug-induced effects and should be considered when
performing a risk assessment. For example, drug-induced effects on the dopaminergic system
can be affected by formation of metabolites, simultaneous exposure to several drugs of abuse
or repeated exposure to a single drug of abuse. Furthermore, drug-induced effects on inputs
on the dopaminergic system can change dopaminergic output. These elements will be
discussed in the following paragraphs.
Formation of metabolites
Drug metabolism might contribute to drug-induced effects via formation of more or less toxic
metabolites. In humans, MDMA is mainly metabolized via O-demethylenation into 3,4dihydroxymethamphetamine (HHMA) and via N-dealkylation into MDA (Fig. 8.1, de la Torre et
al. 2004).
Following direct MDMA injection into the brain, no long-term changes in 5-HT
concentration or its metabolite 5-hydroxyindoleacetic acid were observed. This indicates that
peripherally formed MDMA metabolites are involved in MDMA-induced long-term changes
(Esteban et al. 2001). Experiments in which quinone-thioethers metabolites of MDMA were
directly injected into the brain confirmed this, as they did induce long-term changes in 5-HT
concentration (Miller et al. 1997; Bai et al. 1999). Another important aspect of MDMA
metabolism is the variation between species in the MDMA metabolites present in blood.
Following MDMA exposure, a low blood concentration of MDA was detected in humans,
whereas a much higher concentration was detected in rats and mice (for review see Easton
and Marsden 2006). Furthermore, different CYPs mediate conversion to HHMA in humans
(primarily CYP2D6) compared to rats (CYP2D1).
Although high blood concentrations of HHMA were measured in humans (Capela et al.
2009), HHMA was not detected in the brains of MDMA exposed animals, whereas MDA was
detected (Chu et al. 1996; Mueller et al. 2009). Therefore, we included MDA in our selected
set of drugs of abuse.
86
Chapter 8
Figure 8.1. MDMA metabolism. MDMA, 3,4-methylenedioxymethamphetamine, HHMA, 3,4dihydroxymethamphetamine;
HMMA,
4-hydroxy-3-methoxymethamphetamine;
MDA,
3,4methylenedioxyamphetamine;
HHA,
3,4-dihydroxyamphetamine;
HMA,
4-hydroxy-3methoxyamphetamine. Modified from de la Torre et al 2004.
Methamphetamine is converted into amphetamine (Musshoff 2000), which can be
further metabolized via oxidative deamination to benzoic acid or via aromatic hydroxylation
to 4-hydroxy-amphetamine (Fig. 8.2). Similar to MDMA metabolism, CYP2D6 is also involved
in metabolism of amphetamines, primarily during aromatic hydroxylation and Ndemethylation. However, other CYP enzymes as well as MAO are involved in metabolism of
amphetamines (Kraemer and Maurer 2002).
Metabolism of a drug of abuse can result in exposure to the primary drug as well as to
its metabolites. Depending on the route of metabolism, more toxic or less toxic metabolites
can be formed. Therefore, the formation of metabolites and their effects should be included
when performing a risk assessment.
Repeated drug exposure
Repeated drug exposure can change the severity of drug-induced effects. Repeated exposure
can refer to repeated dosing due to several exposures within a few hours (binge use), but also
to several exposures with varying time windows in between. Changes in pharmacokinetics
and drug-induced effects have been described following repeated exposures compared to a
single exposure. For example, following thirteen daily methamphetamine exposures, a slight
increase (~10%) in maximum methamphetamine blood levels was found in humans compared
to the first challenge (Cook et al. 1992). Furthermore, methamphetamine-induced effects on
DAT function were also stronger ex vivo following repeated methamphetamine injections in
rats (Kokoshka et al. 1998).
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Summary and Risk Assessment
Figure 8.2. Methamphetamine metabolism. Meth, methamphetamine; Amph, amphetamine; Hmeth, 4hydroxymethamphetamine; Hamph, 4-hydroxyamphetamine; PA, phenylacetone; NE, norephedrine;
HNE, 4-hydroxynorephedrine, BA, benzoic acid; HA, hippuric acid. Modified from Musshoff, 2000.
Also, a substantial higher MDMA blood concentration was observed in humans following a
second MDMA dose (24 h interval), possibly due to CYP2D6 inhibition, which decreases
MDMA metabolism (Farre et al. 2004). However, drug-induced effects can also be unaffected
or decreased following repeated exposure.In vivo studies mimicking MDMA binge use,
displayed a reduction in the MDMA-induced increase in extracellular 5-HT levels, although a
stronger increase in body temperature and locomotor activity was observed following
repeated (high) MDMA exposure (Rodsiri et al. 2011). These examples indicate that repeated
dosing may alter pharmacokinetics and pharmacodynamics of drugs of abuse.
Concurrent drug exposure
The simultaneous use of several drugs of abuse can influence drug-induced effects.
Concurrent exposure can result from consuming various tablets or powders containing
different active substances. However, concurrent exposure can also result from consuming
only one tablet, as ecstasy tablets often contain more then one active substance. Besides
MDMA, other commonly found substances in ecstasy tablets are MDA, mCPP, ketamine,
caffeine and methamphetamine (Vogels et al. 2009). Around one third of ecstasy tablets that
were offered to and investigated by the Drug Information and Monitoring System (DIMS) in
the Netherlands contained both mCPP and MDMA (Bossong et al. 2010). Possibly, single drug
88
Chapter 8
effects, as observed during our studies, can result in additive effects or even drug synergy
during simultaneous exposure to multiple drugs of abuse. Insight in mechanisms of action
during concurrent drug use might contribute to a more tailored treatment for patients with a
drug of abuse intoxication. Unfortunately, data on induced effects following exposure to
multiple drugs is limited. However, several effects can be predicted. For example, CYP2D6 is
involved in MDMA metabolism as well as in amphetamine and mCPP metabolism (Maurer et
al. 2004). During concurrent drug exposure, competition for CYP2D6 is likely to occur, which
will reduce metabolism and probably increase drug blood levels. Whether this poses a higher
risk for neurotoxicity should be investigated. For example, when metabolism results in the
formation of more toxic metabolites (bioactivation), less efficient metabolism (e.g., because
of CYP2D6 inhibition) can also decrease toxicity.
Apart from concurrent exposure to several drugs of abuse, concurrent drug exposure
to a drug of abuse and a drug used for a medical purpose can also occur. Prevalence of drugs
of abuse use is much higher among psychiatric populations (el-Guebaly 1990). Interactions
between drugs of abuse and drugs commonly prescribed in psychiatric disorders, like
monoamine oxidase (MAO) inhibitors and selective serotonin reuptake inhibitors (SSRIs) have
been described. For example, the combination of amphetamines and MAO inhibitors is
contraindicated and several fatalities have been reported. Furthermore, SSRIs inhibit various
CYPs, including the ones involved in MDMA and amphetamine metabolism. Consequently,
concurrent use of a SSRI and MDMA or amphetamine will decrease metabolism of both drugs
(Markowitz and Patrick 2001).
Thus, exposure to several drugs of abuse or to a drug of abuse in combination with a
pharmaceutical could alter drug-induced effects. This is therefore an additional factor to
consider when performing a risk assessment.
Additional inputs
In this thesis, effects of drugs of abuse on important excitatory (cholinergic) and inhibitory
(GABA-ergic) pathways in relation to dopaminergic activity are described. Unfortunately, due
to the absence of suitable in vitro models we could not investigate the glutamatergic system,
a major excitatory input that could contribute significantly to drug-induced effects. Druginduced increases in glutamatergic input can have considerable effects on dopaminergic
output and might explain DAT-independent increases in DA levels. For example,
amphetamine exposure evoked an increase in glutamate levels in rats (Reid et al. 1997),
resulting in an increased glutamatergic input on the dopaminergic system that may increase
vesicular DA release. Furthermore, pretreatment with memantine, an NMDA receptor
(glutamate receptor) and α-7 nACh-R antagonist, was shown in vivo to prevent MDMAinduced serotonergic neurotoxicity (Chipana et al. 2008a; Chipana et al. 2008b). Recently,
stem cells have been differentiated towards dopaminergic neurons and displayed calcium
responses upon exposure to Glu-R agonists (Malmersjo et al. 2010). Models like these could
contribute to further unraveling the cellular mechanisms of action of drugs of abuse.
89
Summary and Risk Assessment
90
Chapter 9
Conclusions and Recommendations
Conclusions and Recommendations
Conclusions
Our studies provide important and novel insights in the cellular mechanisms of action of
regularly abused drugs. Stimulatory as well as inhibitory drug-induced effects are observed.
The effects are dependent on drug concentration and stronger or opposite effects were
observed at higher concentrations that represent intoxication levels. Based on the endpoints
we measured it can be concluded that low levels of MDMA, MDA and mCPP do not affect DA
exocytosis, whereas low levels of methamphetamine and amphetamine can increase DA
exocytosis via activation of ACh-Rs. In our studies, all tested drugs (methamphetamine,
amphetamine, MDMA, MDA and mCPP) primarily display inhibitory effects on dopaminergic
neurotransmission at intoxication levels, e.g., a decrease in depolarization- or ACh-evoked
2+
[Ca ]i and / or exocytosis as well as an increase in GABA-ergic input at high GABA receptor
occupancy. At these high drug levels, the only effect that we observed that could enhance DA
exocytosis is the decrease in GABA-evoked ion current at low GABA receptor occupancy, which
may represent a decreased GABA-ergic input.
At low µM levels the main findings of our studies are:
- Methamphetamine, amphetamine, MDMA, MDA and mCPP do not affect VGCCs
(chapter 5 and 6).
- Amphetamine, MDMA and MDA do not affect DA exocytosis (chapter 4 and 5)
2+
- Methamphetamine and amphetamine increase ACh-evoked [Ca ]i, thus increasing
cholinergic input and possibly increasing dopaminergic output (chapter 6).
- MDMA, MDA and mCPP do not affect cholinergic input (chapter 6).
- Methamphetamine, amphetamine, MDMA, MDA and mCPP do not affect GABA-ergic
input (chapter 7).
Thus, MDMA, MDA and mCPP do not affect DA exocytosis, whereas methamphetamine and
amphetamine may increase dopaminergic output.
At intoxication levels the main findings of our studies are:
2+
- At concentrations up to 0.2 mM amphetamine does not affect basal [Ca ]i or DA
2+
exocytosis, depolarization-evoked [Ca ]i or DA exocytosis nor does it affect vesicle DA
content. Amphetamine can only reduce vesicular DA content following pretreatment
with L-DOPA, inducing the availability of freely available DA in LDCVs (chapter 4).
- Methamphetamine, amphetamine, MDMA, MDA and mCPP inhibit VGCCs (chapter 6),
thus decreasing dopaminergic output.
- MDMA and MDA inhibit depolarization-evoked DA exocytosis (chapter 5).
- Methamphetamine, amphetamine, MDMA, MDA and mCPP decrease ACh-evoked
2+
[Ca ]i, thus decreasing cholinergic input and possibly decreasing dopaminergic output
(chapter 6).
- Amphetamine increases the GABA-evoked ion current, thus increasing GABA-ergic
input and thereby possibly decreasing dopaminergic output (chapter 7).
- Methamphetamine, MDMA, MDA and mCPP modulate GABA-ergic input, depending
on GABA receptor occupancy (chapter 7).
92
Chapter 9
Thus, although GABA-ergic input is differentially affected by the drugs of abuse we
investigated, all other endpoints are decreased. Therefore, methamphetamine, amphetamine,
MDMA, MDA and mCPP are expected to inhibit dopaminergic output.
Recommendations
To improve the quality of risk assessment of drugs of abuse, additional information on several
aspects should be collected. Main points of interest are:
- Investigating effects on neurotransmission induced by metabolites of drugs of abuse
that are detected in brain following drug exposure.
- Investigating drug-induced effects on neurotransmission following repeated
exposure to a single drug of abuse.
- Investigating drug-induced effects on neurotransmission during concurrent drug
exposure.
- Investigating drug-induced effects on important dopaminergic inputs, like the
glutamatergic input.
- Investigating the possibilities of translating in vitro observed effects to the in vivo
situation. Furthermore, the relevance of these effects for clinical treatment of
patients with a drug intoxication should be investigated.
93
Conclusions and Recommendations
94
References
References
Adell A and Artigas F. (2004). The somatodendritic release of dopamine in the ventral tegmental area and its regulation by afferent
transmitter systems. Neurosci Biobehav Rev 28, 415-431.
Andre VM, Cepeda C and Levine MS. (2010). Dopamine and glutamate in Huntington's disease: A balancing act. CNS Neurosci Ther 16,
163-178.
Angrist B, Corwin J, Bartlik B and Cooper T. (1987). Early pharmacokinetics and clinical effects of oral D-amphetamine in normal
subjects. Biol Psychiatry 22, 1357-1368.
Bai F, Lau SS and Monks TJ. (1999). Glutathione and N-acetylcysteine conjugates of alpha-methyldopamine produce serotonergic
neurotoxicity: possible role in methylenedioxyamphetamine-mediated neurotoxicity. Chem Res Toxicol 12, 1150-1157.
Bale AS, Meacham CA, Benignus VA, Bushnell PJ and Shafer TJ. (2005). Volatile organic compounds inhibit human and rat neuronal
nicotinic acetylcholine receptors expressed in Xenopus oocytes. Toxicol. Appl. Pharmacol. 205, 77-88.
Baumann MH, Ayestas MA, Dersch CM and Rothman RB. (2001). 1-(m-chlorophenyl)piperazine (mCPP) dissociates in vivo serotonin
release from long-term serotonin depletion in rat brain. Neuropsychopharmacology 24, 492-501.
Borenfreund E and Puerner JA. (1985). Toxicity determined in vitro by morphological alterations and neutral red absorption. Toxicol
Lett 24, 119-124.
Borges R, Carbone E, Nistri A and Verkhratsky A. (2008). Chromaffin cells at the beginning of the 21st century. Acta Physiol (Oxf) 192,
143-144.
Bossong MG, Brunt TM, Van Dijk JP, Rigter SM, Hoek J, Goldschmidt HM and Niesink RJ. (2010). mCPP: an undesired addition to the
ecstasy market. J Psychopharmacol 24, 1395-1401.
Bowery NG, Bettler B, Froestl W, Gallagher JP, Marshall F, Raiteri M, Bonner TI and Enna SJ. (2002). International Union of
Pharmacology. XXXIII. Mammalian gamma-aminobutyric acid(B) receptors: structure and function. Pharmacol Rev 54, 247-264.
Brooker G, Seki T, Croll D and Wahlestedt C. (1990). Calcium wave evoked by activation of endogenous or exogenously expressed
receptors in Xenopus oocytes. Proc Natl Acad Sci USA 87, 2813-2817.
Brown DD. (2004). A tribute to the Xenopus laevis oocyte and egg. J Biol Chem 279, 45291-45299.
Cachelin AB and Jaggi R. (1991). Beta subunits determine the time course of desensitization in rat alpha 3 neuronal nicotinic
acetylcholine receptors. Pflugers Arch 419, 579-582.
Camarasa J, Marimon JM, Rodrigo T, Escubedo E and Pubill D. (2008). Memantine prevents the cognitive impairment induced by 3,4methylenedioxymethamphetamine in rats. Eur J Pharmacol 589, 132-139.
Campos-Caro A, Smillie FI, Dominguez del Toro E, Rovira JC., Vicente-Agullo F, Chapuli J, Juiz JM, Sala S, Sala F, Ballesta JJ and Criado
M. (1997). Neuronal nicotinic acetylcholine receptors on bovine chromaffin cells: cloning, expression, and genomic organization of
receptor subunits. J Neurochem 68, 488-497.
Capela JP, Carmo H, Remiao F, Bastos ML, Meisel A and Carvalho F. (2009). Molecular and cellular mechanisms of ecstasy-induced
neurotoxicity: an overview. Mol Neurobiol 39, 210-271.
Carafoli E, Santella L, Branca D and Brini M. (2001). Generation, control, and processing of cellular calcium signals. Crit Rev Biochem
Mol Biol 36, 107-260.
Celsi F, Pizzo P, Brini M, Leo S, Fotino C, Pinton P and Rizzuto R. (2009). Mitochondria, calcium and cell death: a deadly triad in
neurodegeneration. Biochim Biophys Acta 1787, 335-344.
Chen TK, Luo G and Ewing AG. (1994). Amperometric monitoring of stimulated catecholamine release from rat pheochromocytoma
(PC12) cells at the zeptomole level. Anal Chem 66, 3031-3035.
Chipana C, Camarasa J, Pubill D and Escubedo E. (2008a). Memantine prevents MDMA-induced neurotoxicity. Neurotoxicology 29,
179-183.
Chipana C, Torres I, Camarasa J, Pubill D and Escubedo E. (2008b). Memantine protects against amphetamine derivatives-induced
neurotoxic damage in rodents. Neuropharmacology 54, 1254-1263.
Chisari M, Shu HJ, Taylor A, Steinbach JH, Zorumski CF and Mennerick S. (2010). Structurally diverse amphiphiles exhibit biphasic
modulation of GABAA receptors: similarities and differences with neurosteroid actions. Br J Pharmacol 160, 130-141.
Chu T, Kumagai Y, DiStefano EW and Cho AK. (1996). Disposition of methylenedioxymethamphetamine and three metabolites in the
brains of different rat strains and their possible roles in acute serotonin depletion. Biochem Pharmacol 51, 789-796.
Cole JC and Sumnall HR. (2003). The pre-clinical behavioural pharmacology of 3,4-methylenedioxymethamphetamine (MDMA).
Neurosci Biobehav Rev 27, 199-217.
Collins AC, Salminen O, Marks MJ, Whiteaker P and Grady SR. (2009). The road to discovery of neuronal nicotinic cholinergic receptor
subtypes. Handb Exp Pharmacol, 85-112.
Colliver TL, Pyott SJ, Achalabun M and Ewing AG. (2000a). VMAT-Mediated changes in quantal size and vesicular volume. J Neurosci
20, 5276-5282.
Colliver TL, Hess EJ, Pothos EN, Sulzer D and Ewing AG. (2000b). Quantitative and statistical analysis of the shape of amperometric
spikes recorded from two populations of cells. J Neurochem 74, 1086-1097.
Cook CE, Jeffcoat AR, Sadler BM, Hill JM, Voyksner RD, Pugh DE, White WR and Perez-Reyes M. (1992). Pharmacokinetics of oral
methamphetamine and effects of repeated daily dosing in humans. Drug Metab Dispos 20, 856-862.
Couturier S, Bertrand D, Matter JM, Hernandez MC, Bertrand S, Millar N, Valera S, Barkas T and Ballivet M. (1990). A neuronal nicotinic
acetylcholine receptor subunit (alpha 7) is developmentally regulated and forms a homo-oligomeric channel blocked by alpha-BTX.
Neuron 5, 847-856.
Crawford CA, Williams MT, Kohutek JL, Choi FY, Yoshida ST, McDougall SA and Vorhees CV. (2006). Neonatal 3,4methylenedioxymethamphetamine (MDMA) exposure alters neuronal protein kinase A activity, serotonin and dopamine content,
and [35S] GTPgammaS binding in adult rats. Brain Res 1077, 178-186.
2+
Crespi D, Mennini T and Gobbi M. (1997). Carrier-dependent and Ca -dependent 5-HT and dopamine release induced by (+)amphetamine, 3,4-methylendioxymethamphetamine, p-chloroamphetamine and (+)-fenfluramine. Br J Pharmacol 121, 17351743.
96
References
David HN, Ansseau M and Abraini JH. (2005). Dopamine-glutamate reciprocal modulation of release and motor responses in the rat
caudate-putamen and nucleus accumbens of "intact" animals. Brain Res Brain Res Rev 50, 336-360.
DAWN (2010). Substance Abuse and Mental Health Services Administration, Office of Applied Studies. Drug Abuse Warning Network,
2007: National Estimates of Drug-Related Emergency Department Visits. Rockville, MD, 2010.
De Camilli P and Jahn R. (1990). Pathways to regulated exocytosis in neurons. Annu Rev Physiol 52, 625-645.
De la Torre R, Farre M, Ortuno J, Mas M, Brenneisen R, Roset PN, Segura J and Cami J. (2000). Non-linear pharmacokinetics of MDMA
('ecstasy') in humans. Br J Clin Pharmacol 49, 104-109.
De la Torre R, Farre M, Roset PN, Pizarro N, Abanades S, Segura M, Segura J and Cami J. (2004). Human pharmacology of MDMA:
pharmacokinetics, metabolism, and disposition. Ther Drug Monit 26, 137-144.
De Letter EA, Bouche MP, Van Bocxlaer JF, Lambert WE and Piette MH. (2004). Interpretation of a 3,4-methylenedioxymethamphetamine (MDMA) blood level: discussion by means of a distribution study in two fatalities. Forensic Sci Int 141, 85-90.
Decker WM, Sullivan JP, Arneric SP and Williams MT. (2000). Neuronal Nicotinic Acetylcholine Receptors: Novel Targets for CNS
Therapeutics. Psychopharmacology - 4th Generation of Progress (eds. Bloom FE and Kupfer DJ), http://www.acnp.org/g4/
GN401000009/Default.htm.
D'Hulst C, Atack JR and Kooy RF. (2009). The complexity of the GABAA receptor shapes unique pharmacological profiles. Drug Discov
Today 14, 866-875.
2+
Deitmer J and Schild D. (2000). Calcium - Imaging, Protocolle und Ergebnisse, in Ca und pH, Ionenmessungen in Zellen und Geweben,
(eds. Wigger F and Mechler M), pp.77-97. Spektrum Akademischer Verleger, Heidelberg.
Denizot F and Lang R. (1986). Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure
giving improved sensitivity and reliability. J Immunol Methods 89, 271-277.
Department of Health. Psychostimulant Users - Clinical guidelines for assessment and management. (2006). Mental Health and Drug
and Alcohol Office document number GL2006_001. Published January 13, 2006. Accessed January 20, 2011.
ttp://www.health.nsw.gov.au/policies/gl/2006/pdf/GL2006_001.pdf.
Diaz-Flores L, Gutierrez R, Varela H, Valladares F, Alvarez-Arguelles H and Borges R. (2008). Histogenesis and morphofunctional
characteristics of chromaffin cells. Acta Physiol (Oxf) 192, 145-163.
Dickinson JA, Hanrott KE, Mok MH, Kew JN and Wonnacott S. (2007). Differential coupling of alpha7 and non-alpha7 nicotinic
acetylcholine receptors to calcium-induced calcium release and voltage-operated calcium channels in PC12 cells. J Neurochem
100, 1089-1096.
Di Giovanni G, Di Matteo V and Esposito E. (2008). Serotonin-dopamine interaction: experimental evidence and therapeutic relevance.
Preface. Prog Brain Res 172, ix.
Di Giovanni G, Esposito E and Di Matteo V. (2010). Role of serotonin in central dopamine dysfunction. CNS Neurosci Ther 16, 179-194.
Dingemans MM, Ramakers GM, Gardoni F, van Kleef RG, Bergman A, Di Luca M, van den Berg M, Westerink RH and Vijverberg HP.
(2007). Neonatal exposure to brominated flame retardant BDE-47 reduces long-term potentiation and postsynaptic protein levels
in mouse hippocampus. Environ Health Perspect 115, 865-870.
Doussau F and Augustine GJ. (2000). The actin cytoskeleton and neurotransmitter release: an overview. Biochimie 82, 353-363.
Drew AE, Derbez AE and Werling LL. (2000). Nicotinic receptor-mediated regulation of dopamine transporter activity in rat prefrontal
cortex. Synapse 38, 10-16.
Duman RS and Nestler EJ. (2000). Signal Transduction Pathways for Catecholamine Receptors in Back to Psychopharmacology - The
Fourth Generation of Progress, (eds. Bloom FE and Kupfer DJ), Part 1, Preclinical section, Transmitter Systems, Amines.
http://www.acnp.org/publications/ psycho4generation.aspx
Duman JG, Chen L and Hille B. (2008). Calcium transport mechanisms of PC12 cells. J Gen Physiol 131, 307-323.
Duszynski J, Koziel R, Brutkowski W, Szczepanowska J and Zablocki K. (2006). The regulatory role of mitochondria in capacitative
calcium entry. Biochim Biophys Acta 1757, 380-387.
El-Guebaly N. (1990). Substance abuse and mental disorders: the dual diagnoses concept. Can J Psychiatry 35, 261-267.
El-Hajj RA, McKay SB and McKay DB. (2007). Pharmacological and immunological identification of native alpha7 nicotinic receptors:
evidence for homomeric and heteromeric alpha7 receptors. Life Sci 81, 1317-1322.
El Kouhen R, Hu M, Anderson DJ, Li J and Gopalakrishnan M. (2009). Pharmacology of alpha7 nicotinic acetylcholine receptor
mediated extracellular signal-regulated kinase signalling in PC12 cells. Br J Pharmacol 156, 638-648.
Ellenhorn MJ and Barceloux DG. (1988). Medical Toxicology. Diagnosis and Treatment of Human Poisoning. Elseviers Science
Publishing Co, New York.
EMCDDA (2007). European Monitoring Centre for Drugs and Drug addiction. The state of the drugs problem in Europe. Annual report
2007. ISBN 978-92-9168-288-1
EMCDDA (2009). European Monitoring Centre for Drugs and Drug Addiction. Recent changes in the ecstasy market. Drugnet Europe
online 68. http://www.emcdda.europa.eu/publications/drugnet/online/ 2009/68/article6
EMCDDA (2010). European Monitoring Centre for Drugs and Drug Addiction. The state of the drugs problem in Europe. Annual report
2010, 50-57. ISBN 978-92-9168-432-8. http://www.emcdda.europa.eu/ publications/annual-report/2010
Engelman HS and MacDermott AB. (2004). Presynaptic ionotropic receptors and control of transmitter release. Nat Rev Neurosci 5,
135-145.
Engels RC and ter Bogt T. (2004). Outcome expectancies and ecstasy use in visitors of rave parties in The Netherlands. Eur Addict Res
10, 156-162.
Erickson JD, Schafer MK, Bonner TI, Eiden LE and Weihe E. (1996). Distinct pharmacological properties and distribution in neurons and
endocrine cells of two isoforms of the human vesicular monoamine transporter. Proc Natl Acad Sci USA 93, 5166-5171.
Eriksson E, Engberg G, Bing O and Nissbrandt H. (1999). Effects of mCPP on the extracellular concentrations of serotonin and
dopamine in rat brain. Neuropsychopharmacology 20, 287-296.
97
References
Escobedo I, O'Shea E, Orio L, Sanchez V, Segura M, de la Torre R, Farre M, Green AR and Colado MI. (2005). A comparative study on
the acute and long-term effects of MDMA and 3,4-dihydroxymethamphetamine (HHMA) on brain monoamine levels after i.p. or
striatal administration in mice. Br J Pharmacol 144, 231-241.
Esteban B, O'Shea E, Camarero J, Sanchez V, Green AR and Colado MI. (2001). 3,4-Methylenedioxymethamphetamine induces
monoamine release, but not toxicity, when administered centrally at a concentration occurring following a peripherally injected
neurotoxic dose. Psychopharmacology (Berl) 154, 251-260.
Farre M, de la Torre R, Mathuna BO, Roset PN, Peiro AM, Torrens M, Ortuno J, Pujadas M and Cami J. (2004). Repeated doses
administration of MDMA in humans: pharmacological effects and pharmacokinetics. Psychopharmacology (Berl) 173, 364-375.
2+
Fayuk D and Yakel JL. (2007). Dendritic Ca signalling due to activation of alpha7-containing nicotinic acetylcholine receptors in rat
hippocampal neurons. J Physiol 582, 597-611.
Fischer JF and Cho AK. (1979). Chemical release of dopamine from striatal homogenates: evidence for an exchange diffusion model.
J Pharmacol Exp Ther 208, 203-209.
Fitzgerald JL and Reid JJ. (1993). Interactions of methylenedioxymethamphetamine with monoamine transmitter release mechanisms
in rat brain slices. Naunyn Schmiedebergs Arch Pharmacol 347, 313-323.
Floor E and Meng L. (1996). Amphetamine releases dopamine from synaptic vesicles by dual mechanisms. Neurosci Lett 215, 53-56.
Floresco SB, Todd CL and Grace AA. (2001). Glutamatergic afferents from the hippocampus to the nucleus accumbens regulate activity
of ventral tegmental area dopamine neurons. J Neurosci 21, 4915-4922.
Fornai F, Lenzi P, Lazzeri G, Ferrucci M, Fulceri F, Giorgi FS, Falleni A, Ruggieri S and Paparelli A. (2007). Fine ultrastructure and
biochemistry of PC12 cells: a comparative approach to understand neurotoxicity. Brain Res 1129, 174-190.
Freed WJ. (1994). Glutamatergic mechanisms mediating stimulant and antipsychotic drug effects. Neurosci Biobehav Rev 18, 111-120.
García AG, García-De-Diego AM, Gandía L, Borges R and García-Sancho J. (2006). Calcium signaling and exocytosis in adrenal
chromaffin cells. Physiol Rev 86, 1093-1131.
Garcia-Ratés S, Camarasa J, Sánchez-García AI, Gandía L, Escubedo and Pubill D. (2010). The effects of 3,4-methylenedioxymethamphetamine (MDMA) on nicotinic receptors: intracellular calcium increase, calpain/caspase 3 activation, and
functional upregulation. Toxicol. Appl. Pharmacol. 244, 344-353.
Gijsman HJ, Van Gerven JM, Tieleman MC, Schoemaker RC, Pieters MS, Ferrari MD, Cohen AF and Van Kempen GM. (1998).
Pharmacokinetic and pharmacodynamic profile of oral and intravenous meta-chloro-phenylpiperazine in healthy volunteers. J Clin
Psychopharmacol 18, 289-295.
Gobbi M, Moia M, Pirona L, Ceglia I, Reyes-Parada M, Scorza C and Mennini T. (2002). p-Methylthio-amphetamine and 1-(mchlorophenyl)piperazine, two non-neurotoxic 5-HT releasers in vivo, differ from neurotoxic amphetamine derivatives in their
mode of action at 5-HT nerve endings in vitro. J Neurochem 82, 1435-1443.
Gonzalez-Nicolini V and McGinty JF. (2002). Gene expression profile from the striatum of amphetamine-treated rats: a cDNA array and
in situ hybridization histochemical study. Brain Res Gene Expr Patterns 1, 193-198.
Green A. Mechan AO, Elliott JM, O'Shea E and Colado MI. (2003). The pharmacology and clinical pharmacology of 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy"). Pharmacol Rev 55, 463-508.
Greene LA and Tischler AS. (1976). Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond
to nerve growth factor. Proc Natl Acad Sci USA 73, 2424-2428.
Gudelsky GA, Yamamoto BK and Nash JF. (1994). Potentiation of 3,4-methylenedioxymethamphetamine-induced dopamine release
and serotonin neurotoxicity by 5-HT2 receptor agonists. Eur J Pharmacol 264, 325-330.
Han DD and Gu HH. (2006). Comparison of the monoamine transporters from human and mouse in their sensitivities to
psychostimulant drugs. BMC Pharmacol 6, 6.
Hendriks HS, Antunes Fernandes EC, Bergman A, van den Berg M and Westerink RH. (2010). PCB-47, PBDE-47, and 6-OH-PBDE-47
differentially modulate human GABAA and alpha4beta2 nicotinic acetylcholine receptors. Toxicol Sci 118, 635-642.
Hiramatsu M and Cho AK. (1990). Enantiomeric differences in the effects of 3,4-methylenedioxymethamphetamine on extracellular
monoamines and metabolites in the striatum of freely-moving rats: an in vivo microdialysis study. Neuropharmacol 29, 269-275.
Holmgren A, Holmgren P, Kugelberg FC, Jones AW and Ahlner J. (2008). High re-arrest rates among drug-impaired drivers despite zerotolerance legislation. Accid Anal Prev 40, 534-540.
Hondebrink L, Meulenbelt J, Timmerman JG, van den Berg M and Westerink RH. (2009). Amphetamine reduces vesicular dopamine
content in dexamethasone-differentiated PC12 cells only following l-DOPA exposure. J Neurochem 111, 624-633.
Hondebrink L, Meulenbelt J, Meijer M, van den Berg M and Westerink RH. (2011). High concentrations of MDMA ('ecstasy') and its
metabolite MDA inhibit calcium influx and depolarization-evoked vesicular dopamine release in PC12 cells. Neuropharmacol 61,
202-208.
Hruska M and Nishi R. (2007). Cell-autonomous inhibition of alpha7-containing nicotinic acetylcholine receptors prevents death of
parasympathetic neurons during development. J Neurosci 27, 11501-11509.
Hsieh MT. (1982). The involvement of monoaminergic and GABAergic systems in locomotor inhibition produced by clobazam and
diazepam in rats. Int J Clin Pharmacol Ther Toxicol 20:227-235.
Hurst RS, Hajós M, Raggenbass M, Wall TM, Higdon NR, Lawson JA, Rutherford-Root KL, Berkenpas M.B, Hoffmann WE, Piotrowski
DW, Groppi VE, Allaman G, Ogier R, Bertrand S, Bertrand D and Arneric SP. (2005). A novel positive allosteric modulator of the
alpha7 neuronal nicotinic acetylcholine receptor: in vitro and in vivo characterization. J Neurosci 25, 4396-4405.
Iravani MM, Asari D, Patel J, Wieczorek WJ and Kruk ZL. (2000). Direct effects of 3,4-methylenedioxymethamphetamine (MDMA) on
serotonin or dopamine release and uptake in the caudate putamen, nucleus accumbens, substantia nigra pars reticulata, and the
dorsal raphe nucleus slices. Synapse 36, 275-285.
Jabbar SA, Twentyman PR and Watson JV. (1989). The MTT assay underestimates the growth inhibitory effects of interferons. Br J
Cancer 60, 523-528.
98
References
Johnson MP, Hoffman AJ and Nichols DE. (1986). Effects of the enantiomers of MDA, MDMA and related analogues on [3H] serotonin
and [3H] dopamine release from superfused rat brain slices. Eur J Pharmacol 132, 269-276.
Jones SR, Gainetdinov RR, Wightman RM and Caron MG. (1998). Mechanisms of amphetamine action revealed in mice lacking the
dopamine transporter. J Neurosci 18, 1979-1986.
Kahlig KM and Galli A. (2003). Regulation of dopamine transporter function and plasma membrane expression by dopamine,
amphetamine, and cocaine. Eur J Pharmacol 479, 153-158.
Kalant H. (2001). The pharmacology and toxicology of "ecstasy" (MDMA) and related drugs. CMAJ 165, 917-928.
Kanthasamy A, Sprague JE, Shotwell JR and Nichols DE. (2002). Unilateral infusion of a dopamine transporter antisense into the
substantia nigra protects against MDMA-induced serotonergic deficits in the ipsilateral striatum. Neuroscience 114, 917-924.
Kahlig KM and Galli A. (2003). Regulation of dopamine transporter function and plasma membrane expression by dopamine,
amphetamine, and cocaine. Eur J Pharmacol 479, 153-158.
Kantor L, Zhang M, Guptaroy B, Park YH and Gnegy ME. (2004). Repeated amphetamine couples norepinephrine transporter and
calcium channel activities in PC12 cells. J Pharmacol Exp Ther 311, 1044-1051.
Kassack MU, Höfgen B, Lehmann J, Eckstein N, Quillan JM and Sadée W. (2002). Functional screening of G-protein-coupled receptors
by measuring intracellular calcium with a fluorescence microplate reader. J Biomol Screen 7, 233-246.
Kiyatkin EA and Rebec GV. (1998). Heterogeneity of ventral tegmental area neurons: single-unit recording and iontophoresis in awake,
unrestrained rats. Neuroscience 85, 1285-1309.
Klaassen T, Ho Pian KL, Westenberg HG, den Boer JA and van Praag HM. (1998). Serotonin syndrome after challenge with the 5-HT
agonist meta-chlorophenylpiperazine. Psychiatry Res 79, 207-212.
Kokoshka JM, Vaughan RA, Hanson GR and Fleckenstein A E. (1998). Nature of methamphetamine-induced rapid and reversible
changes in dopamine transporters. Eur J Pharmacol 361, 269-275.
Kolbrich EA, Goodwin RS, Gorelick DA, Hayes RJ, Stein EA and Huestis MA. (2008). Plasma pharmacokinetics of 3,4-methylenedioxymethamphetamine after controlled oral administration to young adults. Ther Drug Monit 30, 320-332.
Koval LM, Yavorskaya EN and Lukyanetz EA. (2001). Electron microscopic evidence for multiple types of secretory vesicles in bovine
chromaffin cells. Gen Comp Endocrinol 121, 261-277.
Kovaleva J, Devuyst E, De Paepe P and Verstraete A. (2008). Acute chlorophenylpiperazine overdose: a case report and review of the
literature. Ther Drug Monit 30, 394-398.
Kraemer T and Maurer HH. (2002). Toxicokinetics of amphetamines: metabolism and toxicokinetic data of designer drugs,
amphetamine, methamphetamine, and their N-alkyl derivatives. Ther Drug Monit 24, 277-89.
Lester DB, Rogers TD and Blaha CD. (2010). Acetylcholine-dopamine interactions in the pathophysiology and treatment of CNS
disorders. CNS Neurosci Ther 16, 137-162.
Leszczyszyn DJ, Jankowski JA, Viveros OH, Diliberto EJ Jr, Near JA and Wightman RM. (1991). Secretion of catecholamines from
individual adrenal medullary chromaffin cells. J Neurochem 56, 1855-1863.
Lin PL, Tsai MC, Lu GL, Lu DY, Chuang CM, Yang H Y, Huang SS and Chen YH. (2010). Ecstasy and methamphetamine elicit action
potential bursts via different mechanisms in a central snail neuron. Neurotoxicology 31, 26-41.
Liu EC and Abell LM. (2006). Development and validation of a platelet calcium flux assay using a fluorescent imaging plate reader. Anal
Biochem 357, 216-224.
2+
Liu PS, Liaw CT, Lin MK, Shin SH, Kao LS. and Lin LF. (2003). Amphetamine enhances Ca entry and catecholamine release via nicotinic
receptor activation in bovine adrenal chromaffin cells. Eur J Pharmacol 460, 9-17.
Loffler M, Bubl B, Huethe F, Hubbe U, McIntosh JM, Jackisch R and Feuerstein TJ. (2006). Dopamine release in human neocortical
slices: characterization of inhibitory autoreceptors and of nicotinic acetylcholine receptor-evoked release. Brain Res Bull 68, 361373.
Lopez MG, Fonteriz RI, Gandia L, de la Fuente M, Villarroya M, Garcia-Sancho J and Garcia AG. (1993). The nicotinic acetylcholine
receptor of the bovine chromaffin cell, a new target for dihydropyridines. Eur J Pharmacol 247, 199-207.
Lopez MG, Montiel C, Herrero CJ, Garcia-Palomero E, Mayorgas I, Hernandez-Guijo JM, Villarroya M, Olivares R, Gandia L, McIntosh
JM, Olivera BM and Garcia AG. (1998). Unmasking the functions of the chromaffin cell alpha7 nicotinic receptor by using short
pulses of acetylcholine and selective blockers. Proc Natl Acad Sci USA 95, 14184-14189.
Lupu-Meiri M, Shapira H, Matus-Leibovitch N and Oron Y. (1990). Two types of intrinsic muscarinic responses in Xenopus oocytes. I.
Differences in latencies and 45Ca efflux kinetics. Pflugers Arch 417, 391-397.
MacDermott AB, Role LW and Siegelbaum SA. (1999). Presynaptic ionotropic receptors and the control of transmitter release. Annu
Rev Neurosci 22, 443-485.
2+
Malmersjo S, Liste I, Dyachok O, Tengholm A, Arenas E and Uhlen P. (2010). Ca and cAMP signaling in human embryonic stem cellderived dopamine neurons. Stem Cells Dev 19, 1355-1364.
Markowitz JS and Patrick KS. (2001). Pharmacokinetic and pharmacodynamic drug interactions in the treatment of attention-deficit
hyperactivity disorder. Clin Pharmacokinet 40, 753-772.
Maurer HH, Kraemer T, Springer D and Staack RF. (2004). Chemistry, pharmacology, toxicology, and hepatic metabolism of designer
drugs of the amphetamine (ecstasy), piperazine, and pyrrolidinophenone types: a synopsis. Ther Drug Monit 26, 127-131.
McCann UD, Szabo Z, Seckin E, Rosenblatt P, Mathews WB, Ravert HT, Dannals RF and Ricaurte GA. (2005). Quantitative PET studies of
the serotonin transporter in MDMA users and controls using [11C] McN5652 and [11C] DASB. Neuropsychopharmacology 30,
1741-1750.
McKernan RM and Whiting PJ. (1996). Which GABAA-receptor subtypes really occur in the brain? Trends Neurosci 19, 139-143.
Merck. Amphetamines. Merck Manual, Psychiatric disorders, Drug use and dependence, Amphetamines. http://www.merckmanuals.
com/professional/sec15/ch198/ ch198k.html. Published July 2008. Accessed January 20 2011.
Meririnne E, Kankaanpaa A, Lillsunde P and Seppala T. (1999). The effects of diazepam and zolpidem on cocaine- and amphetamineinduced place preference. Pharmacol Biochem Behav 62, 159-164.
99
References
Miledi R and Woodward RM. (1989). Membrane currents elicited by prostaglandins, atrial natriuretic factor and oxytocin in follicleenclosed Xenopus oocytes. J Physiol 416, 623-643.
Miller RT, Lau SS and Monks TJ. (1997). 2,5-Bis-(glutathion-S-yl)-alpha-methyldopamine, a putative metabolite of (+/-)-3,4-methylenedioxyamphetamine, decreases brain serotonin concentrations. Eur J Pharmacol 323, 173-180.
Mosmann T. (1983). Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J
Immunol Methods 65, 55-63.
Moyano S, Frechilla D and Del Rio J. (2004). NMDA receptor subunit and CaMKII changes in rat hippocampus induced by acute MDMA
treatment: a mechanism for learning impairment. Psychopharmacology (Berl) 173, 337-345.
Mueller M, Yuan J, Felim A, Neudorffer A, Peters FT Maurer HH, McCann UD, Largeron M and Ricaurte GA. (2009). Further studies on
the role of metabolites in (+/-)-3,4-methylenedioxymethamphetamine-induced serotonergic neurotoxicity. Drug Metab Dispos 37,
2079-2086.
Mundorf ML, Hochstetler SE and Wightman RM. (1999). Amine weak bases disrupt vesicular storage and promote exocytosis in
chromaffin cells. J Neurochem 73, 2397-2405.
Musshoff F. (2000). Illegal or legitimate use? Precursor compounds to amphetamine and methamphetamine. Drug Metab Rev 32, 1544.
Nacca A, Guiso G, Fracasso C, Cervo L and Caccia S. (1998). Brain-to-blood partition and in vivo inhibition of 5-hydroxytryptamine
reuptake and quipazine-mediated behaviour of nefazodone and its main active metabolites in rodents. Br J Pharmacol 125, 16171623.
Nair SG and Gudelsky GA. (2004). Protein kinase C inhibition differentially affects 3,4-methylenedioxymethamphetamine-induced
dopamine release in the striatum and prefrontal cortex of the rat. Brain Res 1013, 168-173.
Nash JF. (1990). Ketanserin pretreatment attenuates MDMA-induced dopamine release in the striatum as measured by in vivo
microdialysis. Life Sci 47, 2401-2408.
Nash JF and Brodkin J. (1991). Microdialysis studies on 3,4-methylenedioxymethamphetamine-induced dopamine release: effect of
dopamine uptake inhibitors. J Pharmacol Exp Ther 259, 820-825.
Nash JF and Nichols DE. (1991). Microdialysis studies on 3,4-methylenedioxyamphetamine and structurally related analogues. Eur J
Pharmacol 200, 53-58.
Neve KA, Seamans JK and Trantham-Davidson H. (2004). Dopamine receptor signaling. J Recept Signal Transduct Res 24, 165-205.
Ohkuma S and Poole B. (1981). Cytoplasmic vacuolation of mouse peritoneal macrophages and the uptake into lysosomes of weakly
basic substances. J Cell Biol 90, 656-664.
Opiumwet (1928). Dutch law regarding drugs. Published 1928, Accessed online February 2011. http://wetten.overheid.nl/
BWBR0001941/geldigheidsdatum_14-02-2011
Oron Y, Gillo B and Gershengorn MC. (1988). Differences in receptor-evoked membrane electrical responses in native and mRNAinjected Xenopus oocytes. Proc Natl Acad Sci USA 85, 3820-3824.
Papke RL and Porter Papke JK. (2002). Comparative pharmacology of rat and human alpha7 nAChR conducted with net charge
analysis. Br J Pharmaco. 137, 49-61.
Partilla JS, Dempsey AG, Nagpal AS, Blough BE, Baumann MH and Rothman RB. (2006). Interaction of amphetamines and related
compounds at the vesicular monoamine transporter. J Pharmacol Exp Ther 319, 237-246.
Paterson D and Nordberg A. (2000). Neuronal nicotinic receptors in the human brain. Prog Neurobiol 61, 75-111.
Peter D, Jimenez J, Liu Y, Kim J and Edwards RH. (1994). The chromaffin granule and synaptic vesicle amine transporters differ in
substrate recognition and sensitivity to inhibitors. J Biol Chem 269, 7231-7237.
Peters FT, Samyn N, Wahl M, Kraemer T, De Boeck G and Maurer HH. (2003). Concentrations and ratios of amphetamine,
methamphetamine, MDA, MDMA, and MDEA enantiomers determined in plasma samples from clinical toxicology and driving
under the influence of drugs cases by GC-NICI-MS. J Anal Toxicol 27, 552-559.
Pifl C, Nagy G, Berenyi S, Kattinger A, Reither H and Antus S. (2005). Pharmacological characterization of ecstasy synthesis byproducts
with recombinant human monoamine transporters. J Pharmacol Exp Ther 314, 346-354.
Poli A, Di Iorio P, Beraudi A, Notari S, Zaccanti F, Villani L and Traversa U. (2001). The calcium-dependent [3H] acetylcholine release
from synaptosomes of brown trout (Salmo trutta) optic tectum is inhibited by adenosine A1 receptors: effects of enucleation on
A1 receptor density and cholinergic markers. Brain Res 892, 78-85.
Pothos E, Desmond M and Sulzer D. (1996). L-3,4-dihydroxyphenylalanine increases the quantal size of exocytotic dopamine release in
vitro. J Neurochem 66, 629-636.
Preus AK, Connor JA and Vogel H. (2000). Transient transfection induces different intracellular calcium signaling in CHO K1 versus Hek
293 cells. Cytotechnology 33, 139-45.
Rao TS, Correa LD, Adams P, Santori EM and Sacaan AI. (2003). Pharmacological characterization of dopamine, norepinephrine and
serotonin release in the rat prefrontal cortex by neuronal nicotinic acetylcholine receptor agonists. Brain Res 990, 203-208.
Reid MS, Hsu K Jr and Berger SP. (1997). Cocaine and amphetamine preferentially stimulate glutamate release in the limbic system:
studies on the involvement of dopamine. Synapse 27, 95-105.
Reynolds DS, O'Meara GF, Newman RJ, Bromidge FA, Atack JR, Whiting PJ, Rosahl TW and Dawson GR. (2003). GABAA alpha1 subunit
knock-out mice do not show a hyperlocomotor response following amphetamine or cocaine treatment. Neuropharmacology 44,
190-198.
Richelson E. (2000). Cholinergic Transduction Psychopharmacology - 4th Generation of Progress (eds. Bloom FE and Kupfer DJ) http://
www.acnp.org/g4/GN401000011/Default.htm.
Rodsiri R, Spicer C, Green AR, Marsden CA and Fone KC. (2011). Acute concomitant effects of MDMA binge dosing on extracellular 5HT, locomotion and body temperature and the long-term effect on novel object discrimination in rats. Psychopharmacology (Berl)
213, 365-376.
100
References
Romanelli MN, Gratteri P, Guandalini L, Martini E, Bonaccini C and Gualtieri F. (2007). Central nicotinic receptors: structure, function,
ligands, and therapeutic potential. ChemMedChem 2, 746-767.
Romero CA, Bustamante DA, Zapata-Torres G, Goiny M, Cassels B and Herrera-Marschitz M. (2006). Neurochemical and behavioural
characterisation of alkoxyamphetamine derivatives in rats. Neurotox Res 10, 11-22.
Rothman RB, Baumann MH, Dersch CM, Romero DV, Rice KC, Carroll FI and Partilla JS. (2001). Amphetamine-type central nervous
system stimulants release norepinephrine more potently than they release dopamine and serotonin. Synapse 39, 32-41.
Rothman RB, Dersch CM, Ananthan S and Partilla JS. (2009). Studies of the biogenic amine transporters. 13. Identification of "agonist"
and "antagonist" allosteric modulators of amphetamine-induced dopamine release. J Pharmacol Exp Ther 329, 718-728.
Rotzinger S, Fang J, Coutts RT and Baker GB. (1998). Human CYP2D6 and metabolism of m-chlorophenyl-piperazine. Biol Psychiatry 44,
1185-1191.
Rudnick G and Wall SC. (1992). The molecular mechanism of "ecstasy" [3,4-methylenedioxymethamphetamine (MDMA)]: serotonin
transporters are targets for MDMA-induced serotonin release. Proc Natl Acad Sci USA 89, 1817-1821.
Rush CR, Stoops WW, Wagner FP, Hays LR and Glaser PE. (2004). Alprazolam attenuates the behavioral effects of d-amphetamine in
humans. J Clin Psychopharmacol 24:410-420.
Sigel E. (2002). Mapping of the benzodiazepine recognition site on GABAA receptors. Curr Top Med Chem 2, 833-839.
Samyn N, De Boeck G, Wood M, Lamers CT, De Waard D, Brookhuis KA, Verstraete AG and Riedel WJ. (2002). Plasma, oral fluid and
sweat wipe ecstasy concentrations in controlled and real life conditions. Forensic Sci Int 128, 90-97.
Satoh Y, Hirashima N, Tokumaru H and Kirino Y. (1997). Activation of adenosine A1 and A2 receptors differentially affects
acetylcholine release from electric organ synaptosomes by modulating calcium channels. Neurosci Res 29, 325-333.
Schmidt CJ, Levin JA and Lovenberg W. (1987). In vitro and in vivo neurochemical effects of methylenedioxymethamphetamine on
striatal monoaminergic systems in the rat brain. Biochem Pharmacol 36, 747-755.
Schmidt CJ, Sullivan CK and Fadayel GM. (1994). Blockade of striatal 5-hydroxytryptamine2 receptors reduces the increase in
extracellular concentrations of dopamine produced by the amphetamine analogue 3,4-methylendioxymeth-amphetamine. J
Neurochem 62, 1382-1389.
Schonn J, Desnos C, Henry J and Darchen F. (2003). Transmitter uptake and release in PC12 cells overexpressing plasma membrane
monoamine transporters. J Neurochem 84, 669-677.
Schubert D, LaCorbiere M, Klier FG and Steinbach JH. (1980). The modulation of neurotransmitter synthesis by steroid hormones and
insulin. Brain Res 190, 67–79.
Schwartz AD, Whitacre CL, Lin Y and Wilson DF. (2003). Adenosine inhibits N-type calcium channels at the rat neuromuscular junction.
Clin Exp Pharmacol Physiol 30, 174-177.
Schweitzer ES, Sanderson MJ and Wasterlain CG. (1995). Inhibition of regulated catecholamine secretion from PC12 cells by the
2+
Ca /calmodulin kinase II inhibitor KN-62. J Cell Sci 108 (Pt 7), 2619-2628.
Seguela P, Wadiche J, Dineley-Miller K, Dani JA and Patrick JW. (1993). Molecular cloning, functional properties, and distribution of rat
brain alpha7: a nicotinic cation channel highly permeable to calcium. J Neurosci 13, 596-604.
2+
Sekiguchi-Tonosaki M, Obata M, Haruki A, Himi T and Kosaka J. (2009). Acetylcholine induces Ca signaling in chicken retinal
pigmented epithelial cells during dedifferentiation. Am J Physiol Cell Physiol 296, C1195-1206.
2+
Sena CM, Santos RM, Boarder MR and Rosario LM. (1999). Regulation of Ca influx by a protein kinase C activator in chromaffin cells:
2+
differential role of P/Q- and L-type Ca channels. Eur J Pharmacol 366, 281-292.
Shankaran M and Gudelsky GA. (1998). Effect of 3,4-methylenedioxymethamphetamine (MDMA) on hippocampal dopamine and
serotonin. Pharmacol Biochem Behav 61, 361-366.
Simeone TA, Sanchez RM and Rho JM. (2004). Molecular biology and ontogeny of glutamate receptors in the mammalian central
nervous system. J Child Neurol 19, 343-360; discussion 361.
Sobczak K, Bangel-Ruland N, Leier G and Weber WM. (2010). Endogenous transport systems in the Xenopus laevis oocyte plasma
membrane. Methods 51, 183-189.
Sombers LA, Maxson MM and Ewing AG. (2005). Loaded dopamine is preferentially stored in the halo portion of PC12 cell dense core
vesicles. J Neurochem 93, 1122-1131.
Sood P, Cole S, Fraier D and Young AM. (2009). Evaluation of metaquant microdialysis for measurement of absolute concentrations of
amphetamine and dopamine in brain: a viable method for assessing pharmacokinetic profile of drugs in the brain. J Neurosci
Methods 185, 39-44.
Sorensen JB. (2004). Formation, stabilisation and fusion of the readily releasable pool of secretory vesicles. Pflugers Arch 448, 347362.
Spitzmaul GF, Esandi MC and Bouzat C. (1999). Amphetamine acts as a channel blocker of the acetylcholine receptor. Neuroreport 10,
2175-2181.
Suen KF, Turner MS, Gao F, Liu B, Althage A, Slavin A, Ou W, Zuo E, Eckart M, Ogawa T, Yamada M, Tuntland T, Harris JL and Trauger
JW. (2010). Transient expression of an IL-23R extracellular domain Fc fusion protein in CHO vs. HEK cells results in improves
plasma exposure. Protein Expr Purif 71, 96-102.
Sulzer D and Rayport S. (1990). Amphetamine and other psychostimulants reduce pH gradients in midbrain dopaminergic neurons and
chromaffin granules: a mechanism of action. Neuron 5, 797-808.
Sulzer D, Maidment NT and Rayport S. (1993). Amphetamine and other weak bases act to promote reverse transport of dopamine in
ventral midbrain neurons. J Neurochem 60, 527-535.
Sulzer D, Chen TK, Lau YY, Kristensen H, Rayport S and Ewing A. (1995). Amphetamine redistributes dopamine from synaptic vesicles
to the cytosol and promotes reverse transport. J Neurosci 15, 4102-4108.
Sulzer D, Sonders MS, Poulsen NW and Galli A. (2005). Mechanisms of neurotransmitter release by amphetamines: a review. Prog
Neurobiol 75, 406-433.
Takahashi A, Camacho P, Lechleiter JD and Herman B. (1999). Measurement of intracellular calcium. Physiol Rev 79, 1089-1125.
101
References
Traynelis SF, Wollmuth LP, McBain CJ, Menniti FS, Vance KM, Ogden KK, Hansen KB., Yuan H, Myers SJ and Dingledine R. (2010).
Glutamate receptor ion channels: structure, regulation, and function. Pharmacol Rev 62, 405-496.
van Velzen AG, van Gorcum TF, van Riel AJHP, Meulenbelt J and de Vries I. (2010). Acute intoxications among humans and animals:
Annual Report 2009 National Poisons Information Centre RIVM rapport 660100004, Published 2010, Accessed online February
2011. http://www.rivm.nl/bibliotheek/ rapporten/660100004.html
Verkhratsky A, Orkand RK and Kettenmann H. (1998). Glial calcium: homeostasis and signaling function. Physiol Rev 78, 99-141.
Verschraagen M, Maes A, Ruiter B, Bosman IJ Smink BE and Lusthof KJ. (2007). Post-mortem cases involving amphetamine-based
drugs in The Netherlands. Comparison with driving under the influence cases. Forensic Sci Int 170, 163-170.
Vistica DT, Skehan P, Scudiero D, Monks A, Pittman A and Boyd MR. (1991). Tetrazolium-based assays for cellular viability: a critical
examination of selected parameters affecting formazan production. Cancer Res 51, 2515-2520.
Vizi ES. (1998). Different temperature dependence of carrier-mediated (cytoplasmic) and stimulus-evoked (exocytotic) release of
transmitter: a simple method to separate the two types of release. Neurochem Int 33, 359-366.
Vizi ES, Palkovits M, Lendvai B, Baranyi M, Kovacs KJ and Zelles T. (2004). Distinct temperature-dependent dopamine-releasing effect
of drugs of abuse in the olfactory bulb. Neurochem Int 45, 63-71.
Vogels N, Brunt TM, Rigter S, van Dijk P, Vervaeke H and Niesink RJ. (2009). Content of ecstasy in the Netherlands: 1993-2008.
Addiction 104, 2057-2066.
Wagner CA, Friedrich B, Setiawan I, Lang F and Broer S. (2000). The use of Xenopus laevis oocytes for the functional characterization of
heterologously expressed membrane proteins. Cell Physiol Biochem 10, 1-12.
Walters RJ, Hadley SH, Morris KDW and Amin J. (2000). Benzodiazepines act on GABAA receptors via two distinct and separable
mechanisms. Nat Neurosci 3, 1274-81.
Westerink RH, de Groot A and Vijverberg HP. (2000). Heterogeneity of catecholamine-containing vesicles in PC12 cells. Biochem
Biophys Res Commun 270, 625-630.
2+
Westerink RH and Vijverberg HP. (2002). Ca -independent vesicular catecholamine release in PC12 cells by nanomolar concentrations
2+
of Pb . J Neurochem 80, 861-873.
2+
2+
Westerink RH, Klompmakers AA, Westenberg HG and Vijverberg HP. (2002). Signaling pathways involved in Ca - and Pb -induced
vesicular catecholamine release from rat PC12 cells. Brain Res 957, 25-36.
Westerink RH. (2004). Exocytosis: using amperometry to study presynaptic mechanisms of neurotoxicity. Neurotoxicology 25, 461470.
Westerink RH. (2006). Targeting exocytosis: ins and outs of the modulation of quantal dopamine release. CNS Neurol Disord Drug
Targets 5, 57-77.
Westerink RH, Rook MB, Beekwilder JP and Wadman WJ. (2006). Dual role of calbindin-D28K in vesicular catecholamine release from
mouse chromaffin cells. J Neurochem 99, 628-640.
Westerink RH and Ewing AG. (2008). The PC12 cell as model for neurosecretion. Acta Physiol (Oxf) 192, 273-285.
Wilson MA, Ricaurte GA and Molliver ME. (1989). Distinct morphologic classes of serotonergic axons in primates exhibit differential
vulnerability to the psychotropic drug 3,4-methylenedioxymethamphetamine. Neuroscience 28, 121-137.
Winkler H and Fischer-Colbrie R. (1992). The chromogranins A and B: the first 25 years and future perspectives. Neuroscience 49, 497528.
Woodward RM, Polenzani L and Miledi R. (1994). Effects of fenamates and other nonsteroidal anti-inflammatory drugs on rat brain
GABAA receptors expressed in Xenopus oocytes. J Pharmacol Exp Ther 268: 806-817.
Wu JC, Chruscinski A, De Jesus Perez VA, Singh H, Pitsiouni M, Rabinovitch M, Utz PJ and Cooke JP. (2009). Cholinergic modulation of
angiogenesis: role of the 7 nicotinic acetylcholine receptor. J Cell Biochem 108, 433-446.
Yao J, Erickson JD and Hersh LB. (2004). Protein kinase A affects trafficking of the vesicular monoamine transporters in PC12 cells.
Traffic 5, 1006-1016.
Ye JH, Liu PL, Wu WH and McArdle JJ. (1997). Cocaine depresses GABAA current of hippocampal neurons. Brain Res 770, 169-175.
Yin HS, Lai CC, Tien TW, Han SK and Pu XL. (2010). Differential changes in cerebellar transmitter content and expression of calcium
binding proteins and transcription factors in mouse administered with amphetamine. Neurochem Int 57, 288-296.
Zaika OL, Pochynyuk OM, Kostyuk PG, Yavorskaya EN and Lukyanetz EA. (2004). Acetylcholine-induced calcium signalling in adrenalineand noradrenaline-containing adrenal chromaffin cells. Arch Biochem Biophys 424, 23-32.
Zhou FM, Liang Y and Dani JA. (2001). Endogenous nicotinic cholinergic activity regulates dopamine release in the striatum. Nat
Neurosci 4, 1224-1229.
Zwart R and Vijverberg HP. (1997). Potentiation and inhibition of neuronal nicotinic receptors by atropine: competitive and noncompetitive effects. Mol Pharmacol 52, 886-895.
102
Nederlandse Samenvatting
Nederlandse samenvatting
Drugs of abuse
“Drugs of abuse”, of kortweg drugs, is een term die wordt gebruikt voor recreatief gebruikte
drugs zonder dat daarvoor een medische noodzaak is. Vaak worden ze gebruikt voor de
‘prettige’ effecten die ze veroorzaken, zoals warme gevoelens voor anderen, een toegenomen
waarnemingsvermogen, minder angstgevoelens en een algemeen gevoel van welbevinden.
Een aanzienlijk aantal mensen heeft ooit drugs gebruikt; 11 miljoen Europeanen hebben ooit
ecstasy tot zich genomen en ook amfetamines zijn populair met 12 miljoen Europeanen die
dit ooit hebben gebruikt. Hoewel veel mensen het gebruik van drugs uitsluitend als recreatief
zien, kan het ook problemen met zich mee brengen. Bij sommige gebruikers resulteert
druggebruik in een overdosis en ook het gevaar van verslaving dreigt. Europese cijfers over
ziekenhuisopnames door een overdosis zijn helaas niet beschikbaar, maar in de Verenigde
Staten leiden intoxicaties met drugs jaarlijks tot meer dan 100.000 ziekenhuisopnames.
Nederlandse cijfers laten zien dat het Nationaal Vergiftigingen Informatie Centrum (NVIC)
jaarlijks ongeveer duizend informatie verzoeken behandelt die drugs betreffen. Het grote
aantal informatieverzoeken over intoxicaties met drugs impliceert dat het kennisniveau
betreffende dit onderwerp beperkt is en geeft ook aan dat drug intoxicaties een relevant
maatschappelijk probleem vormen. Het is daarom van belang om meer inzicht te verkrijgen in
het werkingsmechanisme van deze middelen in het menselijk lichaam.
Er is veel literatuur beschikbaar over de veranderingen die drugs kunnen veroorzaken
in het menselijk lichaam. Gerapporteerd zijn bijvoorbeeld een versnelde hartslag, verstoring
van de thermoregulatie en een toename in de concentraties van neurotransmitters (zoals
dopamine) in de hersenen. Echter, er is slechts een geringe hoeveelheid informatie
beschikbaar over de cellulaire werkingsmechanismen die verantwoordelijk zijn voor deze
effecten. Het is bijvoorbeeld onbekend of de toename in de dopamine concentratie in de
hersenen het resultaat is van een direct of indirect effect van de drugs op het dopaminerge
systeem. Drugs kunnen bijvoorbeeld het dopaminerge systeem indirect beïnvloeden via
andere neurotransmitter systemen die een belangrijke invloed hebben op het dopaminerge
systeem.
Het ontbreken van kennis over de cellulaire werkingsmechanismen heeft zijn weerslag
op de behandeling van patiënten met een drug overdosis. Aangezien de specifieke cellulaire
mechanismen die de effecten in het menselijk lichaam veroorzaken onbekend zijn, richt
behandeling zich voornamelijk op de symptomen die door de intoxicatie worden veroorzaakt.
Zo worden de vitale functies van de patiënt tijdens de behandeling ondersteund en worden
veranderingen in het “milieu interieur” van het lichaam zo veel mogelijk gecorrigeerd om de
patiënt te stabiliseren. Daarnaast wordt soms farmacotherapie (bijvoorbeeld
benzodiazepines) toegepast om de stimulerende werking van bepaalde drugs te dempen.
Voor de behandeling van patiënten met een drug intoxicatie is het van belang om de
werkingsmechanismen te ontrafelen, zodat een ‘evidence-based’ behandeling kan worden
ingesteld. Daarom heeft dit onderzoek zich gericht op het onderzoeken van mogelijke
veranderingen op cellulair niveau geïnduceerd door vijf geselecteerde relevante drugs.
Geselecteerd zijn methamfetamine, amfetamine, 3,4-methylenedioxymethamfetamine
(MDMA), 3,4-methylenedioxyamfetamine (MDA) en meta-chlorophenylpiperazine (mCPP).
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Nederlandse samenvatting
Methamfetamine en amfetamine worden gebruikt in vele verschillende vormen, terwijl
blootstelling aan MDMA, MDA en mCPP vooral voortkomt uit inname van ecstasy tabletten.
Presynaptische input en dopaminerge verbindingen
Veel drugs beïnvloeden voornamelijk het dopaminerge systeem. In de hersenen zijn
verschillende dopaminerge gebieden te onderscheiden die voornamelijk dopaminerge
neuronen bevatten. Deze neuronen zijn onderling verbonden waardoor verschillende
dopaminerge gebieden met elkaar in contact staan. Ook zijn er gebieden te onderscheiden
met neuronen die behoren tot andere neurotransmitter systemen, zoals het glutamaterge,
serotonerge, cholinerge of GABA-erge systeem, die deze dopaminerge neuronen innerveren.
De hoeveelheid dopamine die deze neuronen afgeven is afhankelijk van cellulaire processen
in het presynaptisch neuron, maar ook van de verschillende innervaties vanuit andere
neurotransmitter systemen. Voorbeelden van presynaptische processen die bijdragen aan de
dopamine afgifte zijn de aanmaak en afbraak van dopamine, de opslagcapaciteit voor
dopamine in de blaasjes (ook wel vesicles genoemd), de effectiviteit van specifieke dopamine
transporters en de calcium huishouding in het neuron. Daarnaast brengen dopaminerge
neuronen verschillende receptoren tot expressie op hun celmembraan. Deze kunnen een
stimulerend of remmend effect hebben op de vesiculaire dopamine afgifte wanneer deze
worden geactiveerd. Deze receptoren kunnen dopamine afgifte vergroten wanneer zij
behoren tot het glutamaterge, cholinerge of serotonerge system. Wanneer zij behoren tot het
dopaminerge systeem (autoreceptoren) of het GABA-erge systeem kunnen zij dopamine
afgifte juist verlagen. Dit proefschrift beschrijft de effecten van vaak gebruikte drugs op
presynaptische processen zoals vesiculaire dopamine afgifte (exocytose). Aangezien een
calcium toename voorafgaat aan exocytose, zijn ook drugs-geïnduceerde effecten op de
calcium huishouding bepaald. Bovendien zijn ook de drugs-geïnduceerde effecten onderzocht
op twee typen receptoren die vesiculaire dopamine afgifte kunnen reguleren: de
acetylcholine en GABA receptoren (hoofdstuk 1).
Onderzoeksmodellen en technieken
Om cellulaire drugs-geïnduceerde effecten te onderzoeken is gebruik gemaakt van
verschillende celsystemen en technieken. In dit onderzoek zijn zowel in vitro als ex vivo
modellen gebruikt. Presynaptische effecten zijn onderzocht met behulp van PC12 cellen en
chromafiene cellen. Beide celtypen zijn veel gebruikte celsystemen die model staan voor
dopaminerge neuronen. Deze modellen zijn toegepast om drugs-geïnduceerde effecten op de
calcium huishouding te meten met behulp van fluorescentie microscopie. Ook zijn
amperometrische metingen uitgevoerd om de effecten van drugs op vesiculaire dopamine
afgifte direct te meten. Drugs-geïnduceerde effecten op het cholinerge systeem zijn bepaald
door effecten op acetylcholine receptoren in PC12 cellen te meten. Aangezien veel van deze
receptoren calcium permeabel zijn, is een verandering van de acetylcholine-geïnduceerde
2+
toename in de intracellulaire calcium concentratie ([Ca ]i) door blootstelling aan drugs
indicatief voor effecten op acetylcholine receptoren.
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Nederlandse samenvatting
Drugs-geïnduceerde effecten op GABA-erge input zijn gemeten door GABAA receptoren tot
expressie te brengen in Xenopus Laevis oocyten. Deze receptoren behoren tot de ionotrope
receptoren en activatie van deze specifieke receptoren geeft een instroom van chloor ionen.
Deze ionstroom en drugs-geïnduceerde veranderingen hierin zijn gemeten met behulp van de
voltage-clamp techniek (hoofdstuk 2).
Amfetamine heeft alleen een effect op ongebonden dopamine
Amfetamine induceert een toename in de dopamine concentratie in de hersenen.
Verschillende presynaptische amfetamine-geïnduceerde effecten zijn beschreven die deze
toename kunnen verklaren. Het huidige werkingmechanisme van amfetamine wordt in de
literatuur beschreven als een verstoring van het vesiculaire milieu, waardoor calcium en
2+
dopamine uit de vesicles lekken. Deze toename in [Ca ]i zou vervolgens leiden tot vesiculaire
dopamine afgifte. Echter, in onze celsystemen induceerde een relevante amfetamine
2+
concentratie geen verandering in vesiculaire dopamine afgifte of in [Ca ]i. Nadere
bestudering van de al aanwezige literatuur liet tegengestelde amfetamine-geïnduceerde
effecten zien. Voor ons reden om dieper in te gaan op amfetamine-geïnduceerde effecten.
Door de cellen te laden met een dopamine precursor, L-DOPA, bevatten de vesicles in de
cellen dopamine dat niet is gebonden aan de granine matrix (dense-core) in deze vesicles. In
deze opgeladen cellen induceerde blootstelling aan amfetamine een afname in de
hoeveelheid dopamine per vesicle. Daarom stellen we een verfijning voor van het in de
literatuur veronderstelde werkingsmechanisme van amfetamine. Amfetamine heeft
waarschijnlijk alleen invloed op de hoeveelheid dopamine per vesicle wanneer er
ongebonden dopamine aanwezig is. Mogelijk beschermt de aanwezigheid van een dense-core
in de vesicles tegen amfetamine-geïnduceerde dopamine lekkage uit de vesicles (hoofdstuk
4).
MDMA en MDA induceren geen exocytose
MDMA is het primaire actieve bestanddeel in ecstasy tabletten en staat vooral bekend om
zijn effecten op het serotonerge systeem. Echter, toenames in dopamine concentraties in de
hersenen na MDMA blootstelling zijn ook beschreven. Wij hebben gevonden dat het
onwaarschijnlijk is dat directe effecten op de vesiculaire afgifte van dopamine bijdragen aan
2+
deze toename, omdat MDMA geen toenames induceert in [Ca ]i of vesiculaire dopamine
afgifte in PC12 cellen. Hetzelfde geldt voor MDA, een belangrijke metaboliet van MDMA,
maar ook een stof die regelmatig als bestanddeel in ecstasy tabletten wordt aangetroffen.
MDMA en MDA remmen zelfs de depolarisatie-geïnduceerde toename in vesiculaire
2+
dopamine afgifte en [Ca ]i, indicatief voor een inhibitie van spanningsafhankelijke calcium
kanalen. Een toename in de dopamine concentratie in de hersenen kan worden veroorzaakt
door vesiculaire dopamine afgifte, maar ook door omkering van de dopamine membraan
transporter. Tijdens normale omstandigheden neemt deze transporter extracellulair
dopamine op in het dopaminerge neuron. Bij omkering van de werking van de transporter
wordt dopamine uit het cytosol getransporteerd naar de extracellulaire ruimte, waardoor de
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Nederlandse samenvatting
extracellulaire dopamine concentratie toeneemt. De toename in de extracellulaire dopamine
concentratie die MDMA en MDA in vivo induceren, wordt dus waarschijnlijk veroorzaakt door
omkering van de dopamine membraan transporter. Andere verklaringen voor een
toegenomen dopamine concentratie in de hersenen zijn indirecte drugs-geïnduceerde
effecten op vesiculaire dopamine afgifte, bijvoorbeeld via drugs-geïnduceerde effecten op
andere neurotransmitter systemen die een belangrijke invloed uitoefenen op het
dopaminerge systeem (hoofdstuk 5).
Methamfetamine, amfetamine, MDMA, MDA en mCPP moduleren acetylcholine receptoren
Het cholinerge systeem heeft een belangrijke stimulerende invloed op het dopaminerge
systeem, waardoor drugs-geïnduceerde effecten op het cholinerge systeem ook de afgifte van
dopamine kunnen beïnvloeden. Deze stimulerende invloed verloopt via acetylcholine
receptoren en daarom hebben we de effecten van methamfetamine, amfetamine, MDMA,
2+
MDA en mCPP hierop onderzocht. De acetylcholine-geïnduceerde toename in [Ca ]i is
verhoogd na blootstelling aan een lage concentratie methamfetamine en amfetamine (10
µM). Dit is indicatief voor agonistische effecten op acetylcholine receptoren en deze
toegenomen cholinerge input op het dopaminerge systeem kan leiden tot een verhoogde
vesiculaire dopamine afgifte. Echter, bij hogere concentraties methamfetamine, amfetamine,
MDMA, MDA en mCPP (0.1 en 1 mM) neemt de acetylcholine-geïnduceerde toename in
2+
[Ca ]i juist af, indicatief voor antagonistische effecten op acetylcholine receptoren. Deze
afgenomen cholinerge input op het dopaminerge systeem kan leiden tot een verlaagde
2+
vesiculaire dopamine afgifte. Ook de depolarisatie-geïnduceerde toename in [Ca ]i neemt af
na blootstelling aan deze drugs. Echter, dit is een minder gevoelige uitkomstmaat, aangezien
uitsluitend hoge drug concentraties (1 mM) een afname veroorzaken, met uitzondering van
mCPP dat ook een reductie induceert bij 0.1 mM (hoofdstuk 6).
Methamfetamine, amfetamine, MDMA, MDA en mCPP moduleren de α1β2γ2 GABAA
receptor
Hoewel het GABA-erge systeem de belangrijkste remmende invloed op het dopaminerge
systeem verzorgt, zijn er weinig drugs-geïnduceerde effecten beschreven op het GABA-erge
systeem. Deze remmende invloed op het dopaminerge systeem verloopt via GABA
receptoren. Daarom hebben we de effecten van methamfetamine, amfetamine, MDMA, MDA
en mCPP op de α1β2γ2 GABAA receptor onderzocht. Deze combinatie van subunits (α1β2γ2) is
de meest voorkomende vorm van de GABAA receptor. Hierdoor hebben we een nieuw
werkingsmechanisme voor de geteste drugs kunnen onthullen. Methamfetamine,
amfetamine, MDMA, MDA en mCPP hebben een direct functioneel effect op de GABAA
receptor. Bij een lage bezettingsgraad van de GABAA receptor, remmen hoge concentraties
methamfetamine, MDMA, MDA en mCPP de GABA-geïnduceerde ionstroom. Bij een hoge
bezettingsgraad van de GABAA receptor, potentiëren hoge concentraties amfetamine, MDA
en mCPP de GABA-geïnduceerde ionstroom.
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Nederlandse samenvatting
Hoewel de effecten afhankelijk zijn van de bezettingsgraad van de GABAA receptor, kan een
afgenomen GABA-erge input bijdragen aan overstimulatie van het dopaminerge systeem
(hoofdstuk 7).
Risicobeoordeling
Een belangrijk aspect van risicobeoordeling is de mate van blootstelling en hoe deze
blootstelling zich verhoudt tot de concentraties waarbij effecten zijn gevonden. Wij hebben
een reeks van concentraties gebruikt om zowel de cellulaire effecten tijdens recreatief
gebruik als tijdens een overdosis te kunnen onderzoeken. De meeste effecten die we hebben
gevonden treden pas op bij hoge concentraties van 0.1 en 1 mM. Methamfetamine,
amfetamine, MDMA, MDA en mCPP remmen bijvoorbeeld de depolarisatie- en acetylcholine2+
geïnduceerde toenames in [Ca ]i alleen bij 0.1 of 1 mM. Ook de veranderingen op de GABAA
receptor treden alleen op tijdens blootstelling aan hoge concentraties drugs. Deze
werkingsmechanismen zijn dus waarschijnlijk alleen betrokken tijdens een drug overdosis. De
2+
uitzondering hierop is de stijging in de acetylcholine-geïnduceerde toename in [Ca ]i na
blootstelling aan lage concentraties methamfetamine of amfetamine. Een toegenomen
cholinerge invloed op het dopaminerge systeem is daarom een mechanisme dat bij zou
kunnen dragen aan de toegenomen dopamine concentratie in de hersenen tijdens recreatief
gebruik.
Naast het bepalen van relevante blootstellingconcentraties en de verhouding hiervan
tot effectconcentraties, zijn vele andere factoren van belang om een goede risicobeoordeling
van drugs te kunnen maken. Een belangrijke factor in het beoordelen van mogelijke
schadelijke effecten van drugs is metabolisme. Metabolisme van een drug kan leiden tot
minder of juist meer schadelijke effecten, doordat de gevormde metabolieten minder
schadelijk of juist schadelijker zijn dan de oorspronkelijke drug. De vorming van metabolieten
en de effecten die deze metabolieten veroorzaken zou daarom moeten worden opgenomen
in de risicobeoordeling. Andere belangrijke aspecten zijn herhaalde drug blootstelling en
blootstelling aan meerdere drugs, aangezien deze de drugs-geïnduceerde effecten op
neurotransmissie kunnen veranderen en daardoor mogelijk ook de klinische symptomen.
Verder kunnen drugs ook effecten induceren op neurotransmitter systemen die een
belangrijke invloed uitoefenen op het dopaminerge systeem, zoals het glutamaterge systeem.
Helaas zijn er weinig gegevens beschikbaar over deze aspecten, waardoor het niet mogelijk is
om een volledige risicobeoordeling uit te voeren. Verder onderzoek moet hierover uitsluitsel
geven (hoofdstuk 8).
Conclusie
Het onderzoek beschreven in dit proefschrift geeft belangrijke nieuwe inzichten in de
cellulaire werkingsmechanismen van populaire drugs. De effecten zijn afhankelijk van de drug
concentratie en, vergeleken met een recreatieve dosis, worden sterkere en soms zelfs
tegenovergestelde effecten gezien bij concentraties die kunnen worden bereikt in de
hersenen na een overdosis. Gebaseerd op de eindpunten die wij hebben gemeten, kan
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Nederlandse samenvatting
worden gesteld dat lage concentraties van MDMA, MDA en mCPP geen effect hebben op
vesiculaire dopamine afgifte. Echter, blootstelling aan lage concentraties methamfetamine en
amfetamine kan leiden tot een toename in vesiculaire dopamine afgifte via een toegenomen
cholinerge invloed. In onze studies induceren drug concentraties op overdosis niveau
voornamelijk remmende effecten op dopaminerge neurotransmissie, bijvoorbeeld een drugs2+
geïnduceerde afname in de depolarisatie- of acetylcholine-geïnduceerde toename in [Ca ]i en
een toename in de GABA-geïnduceerde ionstroom bij een hoge GABAA receptor
bezettingsgraad. Tijdens deze hoge drugconcentraties is de afname in de GABA-geïnduceerde
ionstroom bij een lage bezettingsgraad van de GABAA receptor het enige gedetecteerde effect
dat de vesiculaire dopamine afgifte kan doen toenemen. Gebaseerd op onze gemeten
eindpunten zijn de belangrijkste conclusies (hoofdstuk 9):
Tijdens recreatieve blootstelling (lage µM):
- Methamfetamine, amfetamine, MDMA, MDA en mCPP hebben geen effect op
spanningsafhankelijke calcium kanalen (hoofdstuk 5 en 6).
- Amfetamine, MDMA en MDA beïnvloeden de vesiculaire afgifte van dopamine niet
(hoofdstuk 4 en 5).
- Methamfetamine en amfetamine potentiëren de acetylcholine-geïnduceerde
2+
toename in [Ca ]i, resulterend in een toegenomen cholinerge invloed en mogelijk in
een toename in vesiculaire dopamine afgifte (hoofdstuk 6).
- MDMA, MDA en mCPP hebben geen effect op de cholinerge invloed op het
dopaminerge systeem (hoofdstuk 6).
- Methamfetamine, amfetamine, MDMA, MDA en mCPP hebben geen effect op de
GABA-erge invloed op het dopaminerge systeem (hoofdstuk 7).
MDMA, MDA en mCPP hebben bij recreatief gebruik geen effect op vesiculaire dopamine
afgifte, terwijl methamfetamine en amfetamine vesiculaire dopamine afgifte mogelijk
vergroten door een toegenomen cholinerge invloed.
Tijdens overdosering (0.1 en 1 mM):
- Bij blootstellingen tot 0.2 mM heeft amfetamine geen effect op basale niveaus van
2+
[Ca ]i of vesiculaire dopamine afgifte. Ook op de depolarisatie-geïnduceerde
2+
toenames in [Ca ]i of vesiculaire dopamine afgifte heeft amfetamine geen effect. De
hoeveelheid dopamine per vesicle was ook onveranderd na amfetamine blootstelling.
Amfetamine kan de hoeveelheid dopamine per vesicle alleen verlagen wanneer er
ongebonden dopamine aanwezig is (hoofdstuk 4).
- Methamfetamine,
amfetamine,
MDMA,
MDA
en
mCPP
remmen
spanningsafhankelijke calcium kanalen en daardoor mogelijk vesiculaire dopamine
afgifte (hoofdstuk 6).
- MDMA en MDA remmen depolarisatie-geïnduceerde vesiculaire dopamine afgifte
(hoofdstuk 5).
- Methamfetamine, amfetamine, MDMA, MDA en mCPP reduceren de acetylcholine2+
geïnduceerde toename in [Ca ]i, resulterend in een verlaagde cholinerge invloed en
mogelijk een afname in vesiculaire dopamine afgifte (hoofdstuk 6).
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Nederlandse samenvatting
-
Amfetamine potentieert de GABA-geïnduceerde ionstroom, resulterend in een
toegenomen GABA-erge invloed en dus mogelijk in een afname in vesiculaire
dopamine afgifte (hoofdstuk 7).
- Methamfetamine, MDMA, MDA en mCPP moduleren GABA-erge invloed en
modulatie (inhibitie of potentiatie) is afhankelijk van de bezettingsgraad van de
GABAA receptor (hoofdstuk 7).
Hoewel drugs GABA-erge input zowel kunnen potentiëren als inhiberen, worden alle andere
2+
gemeten eindpunten, zoals de depolarisatie- en acetylcholine-geïnduceerde toename in [Ca ]i
en de vesiculaire dopamine afgifte, geremd. Daarom is het waarschijnlijk dat bij
overdoseringen met methamfetamine, amfetamine, MDMA, MDA en mCPP de vesiculaire
dopamine afgifte wordt verlaagd.
Aanbevelingen
Om de risicobeoordeling van drugs te verbeteren, is er aanvullende informatie op
verschillende vlakken nodig. Belangrijke gebieden die aanvulling behoeven zijn:
- Onderzoek naar effecten op neurotransmissie veroorzaakt door metabolieten van de
drugs die ook in de hersenen gedetecteerd worden na blootstelling aan een drug.
- Onderzoek naar drugs-geïnduceerde effecten op neurotransmissie na herhaalde
blootstelling aan één drug.
- Onderzoek naar drugs-geïnduceerde effecten op neurotransmissie tijdens
blootstelling aan verschillende drugs op hetzelfde tijdstip.
- Onderzoek naar drugs-geïnduceerde effecten op neurotransmittersystemen die een
belangrijke invloed uitoefenen op het dopaminerge systeem, zoals het glutamaterge
systeem.
- Onderzoek naar de translatie van in vitro geïnduceerde effecten naar de in vivo
situatie en uiteindelijk naar de klinische behandeling van patiënten die een
vergiftiging hebben opgelopen na blootstelling aan deze drugs.
110
Curriculum Vitae
&
List of publications
Curriculum Vitae
Curriculum Vitae
Laura Hondebrink was born in Almelo, the Netherlands, on December 10, 1982. She
graduated high school in 2001 at the OSG Erasmus in Almelo. In 2001 she started her study
Biomedical Sciences at Radboud University Nijmegen and in 2006 she obtained her Master of
Science degree with Toxicology as principal subject. During her Master she completed two
internships. She worked as an intern at the Rathanau Institute in The Hague on a project that
aimed at implementing scientific neuroscience results, through citizens, on a policy level.
After this she worked as an intern at The Netherlands Organization for Applied Scientific
Research (TNO) at the former department “Toxicology and Applied Pharmacology”. At TNO
she contributed to the development of an in vitro assay to detect the neurotoxic potential of
substances to reduce the use of laboratory animals. After completing her Master she started
her PhD-research in 2007 on the cellular mechanisms of action of popular drugs of abuse that
influence dopaminergic neurotransmission, under supervision of Dr. Remco Westerink and
Professors Martin van den Berg and Jan Meulenbelt at the Institute for Risk Assessment
Sciences (IRAS) at Utrecht University. This PhD-research has provided important and novel
insights in the cellular mechanisms of action of regularly abused drugs, which are described in
this thesis.
112
List of publications
List of publications
Hondebrink, L., Meulenbelt, J., Rietjens, S., Meijer, M., van den Berg, M., Westerink, R.H.S.
Methamphetamine, amphetamine, MDMA (‘ecstasy’), MDA and mCPP modulate
depolarization- and acetylcholine-evoked increases in intracellular calcium levels in PC12 cells.
In revision, Neurotoxicology.
Hondebrink, L., Meulenbelt, J., van Kleef, RG., van den Berg, M., Westerink, R.H.S. (2011)
Modulation of human GABAA receptor: a novel mode of action for drugs of abuse.
Neurotoxicology DOI 10.1016/j.neuro.2011.05.016
Hondebrink, L., Meulenbelt, J., Meijer, M., van den Berg, M., Westerink, R.H.S. (2011). MDMA
(‘ecstasy’) and its metabolite MDA inhibit depolarization-evoked vesicular dopamine release
and calcium influx in PC12 cells. Neuropharmacology 61, 202-208.
Hondebrink, L., Timmerman, J.T., van den Berg, M., Meulenbelt, J., Westerink, R. (2010).
Effects of amphetamine on neurotransmitter release in PC12 and chromaffin cells. Toxicol.
Lett. 196 (Suppl 1): S223.
Westerink, R.H.S., Hondebrink, L. (2010). Are high-throughput measurements of intracellular
calcium using plate-readers sufficiently accurate and reliable? Tox. App. Pharm 249 (3), 247248.
Hondebrink, L., Timmerman, J.G., van den Berg, M., Meulenbelt, J., Westerink, R.H.S. (2009).
Amphetamine reduces vesicular dopamine content in dexamethasone-differentiated PC12
cells only following L-DOPA exposure. J Neurochem 111(2), 624-33.
113
Curriculum Vitae
114
Dankwoord
Dankwoord
Dankwoord
Bij deze wil ik iedereen bedanken die op enige wijze een bijdrage heeft geleverd aan de
totstandkoming van dit proefschrift.
Beste promotoren, Jan en Martin, bedankt voor de fijne wetenschappelijke samenwerking,
jullie steun, advies en commentaren op wetenschappelijk gebied en voor de grote mate van
vrijheid bij dit onderzoek.
Remco, je hebt me er vaak door heen gesleept. Door mijn resultaatgerichtheid was het voor
mij soms lastig om het nut van experimenten in te blijven zien. Gelukkig was jij altijd
enthousiast over de data. Zonder jouw expertise, kritische noten en wetenschappelijke
feedback had er een heel ander proefschrift gelegen. Bedankt!
(Ex)collega’s van de “Neurotoxicology Research Group”: bedankt voor de fijne werksfeer, het
aanhoren van mijn twijfels en frustraties en, ook niet onbelangrijk, voor jullie feedback tijdens
werkbesprekingen. Milou, je bent een echte gedegen wetenschapster en daardoor een goed
voorbeeld geweest voor mij, dank daarvoor. Jakub, bedankt voor het relativeren tijdens
stressvolle periodes. Harm en Hester, bedankt voor alle koffiemomentjes en discussies. Elsa,
bedankt voor je vrolijkheid, dansjes en culinaire hoogstandjes. Aart, bedankt voor het
eindeloos maken van nieuwe macro’s en ook voor het oplossen van problemen bij de
amperometrie of oocyten opstelling. Het lijkt wel of je magische krachten hebt; wanneer jij
alleen al naar de opstelling kijkt, verdwijnt het probleem! Gina, bedankt voor al je hulp met
de cellen en de kikkers en de leuke gesprekken op het lab. Alfons, bedankt voor je
wetenschappelijke input en andere kijk op data.
My roomy for the longest time and good friend Irene; thank you for all the tips and
suggestions on a scientific as well as on a personal level. I will miss you and wish you good
luck with everything!
Johan, Marieke en Marten, ik wil jullie bedanken voor jullie inzet en gezelligheid. Jullie
hebben jezelf vrijwillig opgesloten in het donkere calcium imaging hok en daar heel wat data
bij elkaar gemeten. Bedankt! Marten, succes met je Master. Johan, veel succes met je
onderzoek als PhD student. Marieke, succes met het afronden van je studie en succes met het
vinden van een leuke baan.
Evelyn, bedankt voor je gezelligheid, je deur staat altijd open en je bent altijd erg attent!
Saskia, bedankt voor het meedenken over de lijn van het onderzoek en je bijdrage aan de
totstandkoming van dit proefschrift. Ik wens je veel succes in je carrière.
Rocio, thanks for helping me start up at IRAS! All the questions on printers, computers, coffee
machines etcetera must have been a pain in the ass. I wish you all the best!
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Dankwoord
Alle IRAS (ex)collega’s, bedankt voor de gezelligheid tijdens het wachten bij de
koffieautomaat, bij 1 van de vele traktaties, bij “cookie time” en op IRAS feestjes!
Op deze plaats wil ik ook vrienden en familie bedanken die zich in meer of mindere mate
konden voorstellen waar ik zo druk mee was. Marit, bedankt voor alle gezellige eetdates en
voor je peptalks waarin alles weer in perspectief werd geplaatst. Rachel, dank voor alle
gezellige avondjes uit en de broodnodige afleiding. Frieda, bedankt voor alle gezellige breaks
in het zonnetje en voor de pizza met rosé avonden!
Pap en mam, bedankt voor jullie steun tijdens deze periode. Jullie hebben me altijd
voorgehouden dat je iets moet doen waar je gelukkig van wordt. Promoveren heeft zijn ups
en zijn downs en het moet lastig voor jullie zijn geweest om daarmee om te gaan. Toch
hebben jullie me altijd gesteund in mijn keuze om hiermee verder te gaan en daarvoor wil ik
jullie bedanken.
Lievie, jouw gezelschap is essentieel geweest de afgelopen jaren. Onze wandeltochten,
duikvakanties en verre vakanties hebben voor een goede balans gezorgd en een mogelijkheid
om de batterijen weer op te laden. Jouw aandacht, steun en interesse zijn voor mij van
onschatbare waarde geweest. Bedankt!
117
Dankwoord
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