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Chemico-Biological Interactions
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Parazoanthoxanthin A blocks Torpedo nicotinic acetylcholine receptors
Klara Bulc Rozman a,∗ , Romulo Araoz b , Kristina Sepčić c , Jordi Molgo b , Dušan Šuput a
a
Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Zaloška cesta 4, 1000 Ljubljana, Slovenia
CNRS, Institut de Neurobiologie Alfred Fessard – FRC2118, Laboratoire de Neurobiologie Cellulaire et Moléculaire – UPR9040, 91198 Gif-sur-Yvette cedex, France
c
Department of Biology, Biotechnical Faculty, University of Ljubljana, Večna pot 111, 1000 Ljubljana, Slovenia
b
a r t i c l e
i n f o
Article history:
Available online xxx
Keywords:
Parazoanthoxanthin A
Torpedo nicotinic acetylcholine receptor
Acetylcholinesterase inhibitor
Xenopus oocyte
a b s t r a c t
Nicotinic acetylcholine receptors are implicated in different nervous system-related disorders, and their
modulation could improve existing therapy of these diseases. Parazoanthoxanthin A (ParaA) is a fluorescent pigment of the group of zoanthoxanthins. Since it is a potent acetylcholinesterase inhibitor, it
may also bind to nicotinic acetylcholine receptors (nAChRs). For this reason its effect on Torpedo nAChR
(␣12 ␤␥␦) transplanted to Xenopus laevis oocytes was evaluated, using the voltage-clamp technique. ParaA
dose-dependently reduced the acetylcholine-induced currents. This effect was fully reversible only at
lower concentrations. ParaA also reduced the Hill coefficient and the time to peak current, indicating a
channel blocking mode of action. On the other hand, the combined effect of ParaA and d-tubocurarine
(d-TC) on acetylcholine-induced currents exhibited only partial additivity, assuming a competitive mode
of action of ParaA on nAChR. These results indicate a dual mode of action of ParaA on the Torpedo AChR.
© 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Nicotinic acetylcholine receptors (nAChRs) are ligand gated
receptors that mediate signal transduction in chemical synapses
in the central and peripheral nervous system [1,2]. Thus, they
play a role in various nervous system-related disorders, such
as Alzheimer’s disease, myasthenia gravis, schizophrenia, neuropathic pain, depression, attention deficit hyperactivity disorder,
tobacco addiction and drug abuse. Modulation of nAChR responses
could therefore improve the state of the disease [3–6]. The nAChR
is a complex protein consisting of five subunits, which form an
extracellular, transmembrane and cytoplasmic domain. A change
in any of the domains can alter the nAChR response, so the nAChR
offers a number of binding sites for different natural and synthetic
modulators convenient for drug design [1,3,6,7].
Zoanthoxanthins are a group of pigments isolated from Cnidarians. They are potential nAChR modulators, since their in vivo
action shows a systemic effect on the cholinergic system [8]. Parazoanthoxanthin A (ParaA) is a basic linear zoanthoxanthin, which
has been found to inhibit electric eel acetylcholinesterase (AChE)
in the micromolar range [9]. Many AChE inhibitors modulate
nAChR responses, including structurally similar tacrine [10–12].
The authors therefore decided to determine whether or not ParaA
has any effect on nAChR. For this purpose, Torpedo (␣12 ␤␥␦)
nAChR, a muscle-type of nAChR, was transplanted to Xenopus laevis
∗ Corresponding author. Tel.: +386 40 53 61 30; fax: +386 1 543 70 21.
E-mail address: [email protected] (K.B. Rozman).
oocytes, and the effects of ParaA were monitored using the voltageclamp technique.
2. Experimental
2.1. ParaA synthesis
Chemical synthesis of ParaA was carried out by means of a
biomimetic method [13]. The synthesis mixture, which also contained a small amount of pseudozoanthoxanthin A, was purified on
a HPLC system (Waters Corporation, Milford, MA, USA) according to
the previously described protocol [9]. In short, a semi-preparative
RP-column was eluted with a methanol and water gradient. The
fractions were detected with a diode array UV–vis detector at
290 nm, and fluorescence was monitored at 400 nm (excitation
295 nm). The solvents were evaporated and the ParaA was dissolved in methanol.
2.2. Oocyte collection and nAChR transplantation
Adult female X. laevis frogs were obtained live from the Centre
de Ressources Biologiques Xénopes (Rennes, France). Live animals
were maintained and treated according to the European standard
protocols approved by the Animal Ethics Committee of the CNRS.
The experiments were performed in accordance with the European
Community guidelines for laboratory animal handling, taking into
account the official edict of the French Ministry of Agriculture and
the recommendations of the Helsinski Declaration. X. laevis frogs
were anaesthetized by means of ethyl-3-aminobenzoate methane-
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sulfonate salt, and preparation of the oocytes was carried out as
described previously [14]. The stage V and VI oocytes without follicular coat were kept at 16 ◦ C in Barth’s medium (88 mM NaCl, 1 mM
KCl, 0.41 mM CaCl2 , 0.82 mM MgSO4 , 2.5 mM NaHCO3 , 0.33 mM
CaNO3 , 7.5 mM Hepes, pH 7.6) supplemented with kanamycin
(2 mg/ml). Viability was checked every day when changing the
Barth’s medium.
Torpedo marmorata were purchased live from the Station
Biologique de Roscoff (France). Membranes enriched in ␣12 ␤␥␦
nAChR T. marmorata nAChR were prepared from Torpedo’s electric
organ as has been described in Ref. [14]. Briefly, isolated Torpedo’s
electric organs were homogenized and centrifuged. The resulting
pellet was homogenized and nAChR-rich membranes were isolated by means of a sucrose gradient. Purified membranes enriched
with nAChR (3.5 mg/ml protein) were stored in 5 mM glycine at
−80 ◦ C until use. Viable oocytes were transferred to a clean Petri
dish with fresh Barth’s medium. A Nanoliter2000 Micro4 Controller
(World Precision Instruments Inc., Stevenage, Herts, U.K.) was used
to inject 50 nl of purified membranes enriched with nAChR into the
oocyte cytoplasm.
Sigma–Aldrich Co. (Saint Quentin Fallavier, France). All other drugs
were of analytical grade.
2.3. Electrophysiological recordings
All of the experiments were performed at 20 ◦ C, and the ionic
currents were measured with a two electrode voltage-clamp
amplifier (OC-725B, Warner Instruments, LLC, Hamden, USA). The
electrodes were filled with 3 M KCl and tested for their resistance. Only electrodes having a resistance between 2 and 5 M
were used. A single oocyte was placed in the recording chamber,
clamped to −60 mV, and perfused with modified Ringer solution,
where calcium chloride was replaced by barium chloride (100 mM
NaCl, 2.8 mM KCl, 1 mM MgCl2 , 0.3 mM BaCl2 , and 5 mM Hepes)
at 8 ml per min. Perfusion of the modified Ringer solution and the
drugs was controlled by computer-driven electromagnetic valves
(Warner Instruments, LLC, Hamden, USA). Data were acquired by a
pCLAMP-9/Digidata-1322A system (Molecular Devices, Union City,
CA, USA). Stock solutions of ACh and d-TC were prepared in distilled
water and ParaA in methanol. All stock solutions were diluted to the
final concentrations in the modified Ringer solution. On account of
ParaA solubility in methanol, the control ACh contained 0.05% of
methanol. The oocytes were challenged with 125 ␮M ACh in all the
experiments.
2.4. Data analysis
Amplitudes of the currents recorded in response to each drug
concentration were normalised to the maximum amplitude of the
current evoked by the control ACh. The dose–response curves were
fitted to the Hill equation:
Iinh
= [1 +
IACh
[inh] IC50
nH ]
Fig. 1. The dose–response curve of ParaA on ACh-induced currents on Xenopus
oocytes injected with Torpedo marmorata ␣12 ␤␥␦ nAChR-rich membranes. The
ParaA responses were normalized to control the responses and fitted to Eq. (1).
The calculated ParaA IC50 was 1.68 ␮M and the Hill coefficient 0.923. Each point
represents the mean ± S.D. (n = 18 oocytes). The structure of ParaA is shown in the
insert.
−1
(1)
where IACh is the peak current elicited by the control ACh; Iinh is
the current elicited in the presence of the inhibitor; [inh] is the
concentration of the inhibitor; IC50 is the antagonist concentration
required to halve the IACh ; and nH is the Hill coefficient.
The data were analyzed in SPSS 15.0. The average values were
calculated as the mean ± S.D. The statistical significances between
the control and drug elicited responses were analyzed using Student’s t-test or the Mann–Whitney–Wilcoxon test.
2.5. Drugs
Ethyl-3-aminobenzoate methanesulfonate salt, collagenase
type II, kanamycin and acetylcholine chloride were purchased from
3. Results
3.1. ParaA modified ACh-induced responses
The effect of ParaA on nAChR on Xenopus oocytes injected with
T. marmorata ␣12 ␤␥␦ nAChR-rich membranes was investigated.
The oocytes responded to the ACh application with stable currents
from the second day after the injection forward. Each oocyte was
first tested with ACh to ascertain consistent responses to ACh. The
interval between consecutive 5 s drug challenges was 150 s, which
ensured complete recovery of nAChR from desensitisation, and little or no run down of the ACh responses.
Application of ParaA alone produced no observable change in
the membrane conductance of the ACh responsive and unresponsive oocytes. If ParaA with ACh was applied to the ACh responsive
oocytes, the ParaA markedly changed the ACh-induced current.
This change was dose-dependent and fully reversible at lower
concentrations of ParaA. By fitting the normalized dose–response
currents to Eq. (1), an IC50 of 1.68 ␮M was obtained (Fig. 1). The
Hill coefficient of the dose–response curve was reduced to 0.923,
implying a stoichiometry of 1:1 in the interaction between ParaA
and Torpedo nAChR.
3.2. ParaA shortened time to peak current
The time course of ACh-induced currents was changed in the
presence of different ParaA concentrations (Fig. 2). Whereas the
control currents peaked at 4.43 ± 0.28 s, 3 ␮M ParaA shortened the
time to peak to 1.33 ± 0.19 s (p < 0.05). This change could not be
attributed to the effect of methanol, since the currents induced by
ACh alone peaked at 4.25 ± 0.38 s, which is not significantly different from the control currents. Neither ParaA nor methanol had
any residual effect on the time to peak in subsequent ACh-induced
currents (4.50 ± 0.21 and 4.30 ± 0.35, respectively).
3.3. Comparison of ParaA and d-TC effects on ACh-induced
currents
Shortened time to peak current, together with a Hill coefficient
value close to one led us to assume that ParaA acts as an open channel blocker. In order to confirm this assumption, the effect of ParaA
was compared with the effect of d-TC on the ACh-induced currents.
Please cite this article in press as: K.B. Rozman, et al., Parazoanthoxanthin A blocks Torpedo nicotinic acetylcholine receptors, Chem. Biol. Interact.
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Fig. 2. The effects of 3 ␮M ParaA on ACh-induced currents on Xenopus oocytes
with incorporated Torpedo nAChR. The representative superimposed currents were
elicited by challenging a single oocyte with 125 ␮M ACh with 0.05% methanol (control) and 125 ␮M ACh with 0.05% methanol and 3 ␮M ParaA (ParaA). ACh-induced
currents were measured at the beginning (ACh before) and at the end of the experiment (ACh after) in order to observe the reversibility of a ParaA induced blockage
of the ACh-induced currents. Reversibility was complete only at lower ParaA concentrations.
Since d-TC is a competitive antagonist of nAChR, perfusion of both
drugs and ACh should result in summation of the inhibiting effects.
But whereas 0.05 ␮M d-TC diminished the ACh-induced current
to 56.5 ± 1.1% and 2 ␮M ParaA to 72.2 ± 4.6%, the collective effect
was only 51.9 ± 2.2%. This reduction in the ACh-induced current
was significantly different (p < 0.05) from the ParaA and from the
d-TC-induced reduction, thus indicating only partial summation.
3
this property is independent of the presence of a nAChR antagonist
[21].
ParaA and tacrine are structurally similar, and both substances
are anticholinesterase agents. It is also possible that they could
similarly modify nAChR responses. This view is supported by the
observation that tacrine shortens the open time of nAChR, meaning
that it is an open channel blocker [20].
The results obtained in this study partially support the open
channel block by ParaA, since ParaA effects on ACh-induced currents are appropriate for this mode of action. However, the results
of co-application with d-TC raise the question of whether this is
the only mode of action ParaA has on nAChR. Since d-TC is a competitive antagonist of nAChR [2,7,10] the cumulative effect of the
co-application of both drugs should be more pronounced. Some
authors have proposed that d-TC acts also as open channel blocker
[22,23], but at higher concentrations than those used in our experiments. Further experiments using different agonist concentrations
and clamping voltages are needed to reveal all the possible mechanisms of the action of ParaA on nAChR.
Conflict of interest statement
The authors declare that there is no conflict of interest.
Acknowledgement
This work was supported by grant P-3-0019 from the Slovenian
Research Agency.
References
4. Discussion
Transplantation of Torpedo ␣12 ␤␥␦ nAChR into Xenopus oocytes
has proved to be an elegant method for the studying its function and of the pharmacological properties of various substances
[10,11,15,16]. It makes it possible to study native nAChRs without changes in their glycosylation and speculations about the
subunit stoichiometry, which are typical problems that occur in
heterologous expression systems and are known to alter the nAChR
responses [3,17]. In this study, the authors decided to use this
method to determine if the ParaA has any effect on nAChR. Our
results show that ParaA reduces ACh-induced currents on Torpedo
nAChR. This effect is likely to be caused by two modes of action of
ParaA, since the reduced time to peak and the Hill coefficient imply
an open channel blocking mode of action, whereas the partial summation with a d-TC effect indicates a possible competitive mode of
ParaA inhibition of nAChR. The actual mode of action of ParaA thus
needs to be further examined.
The effect of other AChE inhibitors has been studied on Torpedo nAChR. Galantamine and physostigmine potentiate agonist
effects at smaller doses, whereas higher concentrations produce
an open channel block [15,18,19]. The galantamine potentiating
effect is supposed to be due to the binding to an allosteric site,
since galantamine itself does not induce observable currents on
Torpedo nAChR [15] nor does it inhibit I-␣-bungarotoxin binding
to the agonist binding site [18]. On the other hand, physostigmine inhibited I-epibatidine binding to the agonist binding site,
and potentiating effect was agonist concentration-dependent, indicating a competitive interaction with nAChR agonists [19]. Zwart
et al. [19] observed similar potentiating by tacrine and proposed
the same mode of action for tacrine, too. However, other studies
have indicated that tacrine acts as open channel blocker since its
inhibition of ACh-induced currents is voltage dependent, agonist
concentration independent and is thought to bind into the open
nAChR-channel [11,20]. Tacrine is also a neuroprotective agent, and
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