Download Kinetics of metoclopramide effects on human 5

International Congress Series 1283 (2005) 49-54
ELSEVIER
Kinetics of metoclopramide effects on human
5-HT3* receptors*
J. Walkembach ", M. Bruss b, B.W. Urban ", M. Barann ab'*
" Klinik fur Anasthesiologie und opel-otive
Universitatskliniken Bonn, Gertnrrty
Institutju~,Pharmakologie und Toxikologie. Univer.sifafBonn, Germony
Abstract. The action of metoclopramide, a dnig which is used as an antiemetic and prokinetic, on
human (h)5-HT3, receptors, stably expressed in HEK-293 cells, was studied with patch clamp and
[3~]-radioligandbinding techniques. At clinical concentrations, metoclopramide inhibited peak and
integrated currents through h5-HT3, receptors concentration-dependently (ICs0=0.064 and 0.076
PM, respectively) when it was applied in equilibrium [6O s before and during 5-HT (30 FM)
exposure]. The onset and offset time constants of metoclopramide action were 1.3 s and 2.1 s,
respectively. The potency of metoclopramide when exclusively applied during the agonist pulse
decreased more than 200-fold (TCso= 19.0 PM, peak current suppression). Metoclopramide (0.10
PM) did not alter the ECso of 5-HT-induced peak currents. In contrast to the lack of competitive
interaction between metoclopramide and 5-HT in this functional assay, metoclopramide inhibited
specific ['H] GR65630 binding to human 5-HT3,, receptors in a surmountable inanner. This seeming
discrepancy between functional studies and radioligand binding experiments may be accounted for
by (1) the slow kinetics of inhibition of peak currents by metoclopramide compared with the fast
onset and offset kinetics of 5-HT-induced currents and (2) the low efficacy of metoclopramide in
inhibiting radioligand binding (e.g., only 20% binding inhibition compared with 79% peak current
suppression by 200 nM metoclopramide). O 2005 Elsevier B.V. All rights reserved.
Keyword.~:5-HTI receptor; Kinetics; Metoclopramide; Emesis
"' The results of this study have been published: J. Walkembach, M. Bniss, B.W. Urban, M. Barann, Interactions
of metoclopramide and ergotamine with human 5-HT3,, receptors and human 5-HT reuptake carriers, Bntish
Journal of Pharmacology, (2005). in press, [I].
* Corresponding author. Klinik fur Anasthesiologie und operative Intensivmedizin, Universitatskliniken Bonn,
Germany. Tel.: +49 228 287 4679; fax: +49 228 287 5157.
E-mail address: [email protected] (M. Barann)
0531-51311 0 2005 Elsevier B.V. All rights r e s e ~ e d
doi:10.1016lj.ics.2005.08.006
50
J. WnlA-emhach et nl. /International Congress Seria 1283 (2005) 49-54
1. Introduction
The 5-HT3 receptor is the only 5-HT receptor subtype which belongs to the superfamily
of ligand-gated ion channels [2]. The pentameric 5-HT3 receptor is a target for drugs,
including general anesthetics and cannabinoids [3-51. 5-HT3 receptors are located in
components that play a major role in the modulation of nausea and emesis [6,7].
Metoclopramide has been in use as an antiemetic and prokinetic agent for many years. The
drug is known for its D, (dopamine,) receptor antagonism but it also has some 5-HT3
antagonistic property [8]. We examined the actions of metoclopramide on human 5-HT3*
receptors using the patch clamp technique on excised outside out patches combined with a
fast solution exchange system. The mechanism of action of metoclopramide was hrther
evaluated in binding experiments.
2. Materials and methods
HEK-293 cells at 20% confluence were stably transfected by the modified calcium
phosphate method [9] with the hS-HT3* receptor cDNA subcloned into the mammalian
expression vector pCDNA3 (Invitrogen) under control of human cytomegalovirus
promoter. Cell culture was performed according b [1,5]. The condition for patch clamp
experiments on excised outside out patches was identical to those performed in [1,5]. Two
solution application systems were employed: (a) a two-tube system was used for
concentration-response curves of tested adjuvants. Patches were moved into position -0.5
mm from the outflow (0.3 mm in diameter) of a two-arm Teflon superfusion system [10].
(b) A multi-tube perfusion system (RSC 200, Biologie, France) was used for experiments
in which patches were pre-exposed to the respective drug for a variable amount of time
before agonist application [4]. The drug application systems were equipped with inert
materials such as Teflon tubing and glass to avoid loss of hydrophobic drugs [ I I]. Using
the two-tube fast solution exchange system, three different protocols of drug application
were used [1,3]: (1) equilibrium application: continuous exposure to the drug before and
during the application of 5-HT; (2) Open channel application: no drug application prior to
the 5-HT pulse, only simultaneous application of drug and 5-HT; (3) closed channel
application: pre-exposure to the drug before but not during the 5-HT application.
Radioligand binding to membranes from HEK-293 ~ e l l stably
s
transfected with the human
5-HT3* receptor cDNA was perfomed as described by us before [5]. Drugs and solutions:
5-hydroxytryptamine creatinine Sulfate, metoclopramide (monochloride) were obtained
from Sigma (Munchen, Gennany). [ 3 ~GR65630
]
(specific activity 64.8 Ci mmol- ' ) was
obtained from NEN DuPont (Dreieich, Germany). 5-HT and metoclopramide solutions
were prepared daily from aqueous stock solutions (containing 50 and 10 mM,
respectively). All stocks were stored at - 20 T.
3. Results
Application of 5-HT to excised patches for 2 s induced concentration-dependent inward currents
(EC5,= 10.3 FM; Hill coefficient= 1.5; Fig. 1). Metoclopramide inhibited 5-HT (30 FM)-induced
peak currents in a concentration-dependent manner. Following wash-periods of at least 60 s, the 5HT-induced currents were recovered to the respective control values at the beginning of the
J Walkemhach et al. / International Congtms Sertes 1283 (2005) 49-54
Effect of 200 nM rnetocloprarnide on
Iigand binding to 5-WaAreceptors
Effect of 100 nM rnetocloprarnide on
5-HTjA receptor function (patch clamp)
Fig. I . Left: concentration-response curves (- 100 mV) of 5-HT at h5-HT3. receptors stably transfected in HEK293 cells in the absence ( U ) and presence (A)of metoclopramide (100 nM: applied in eqiiilibrium mode, ++).
The inhibition was iinsurmountable by 5-HT. Shown are means I S.D. of 3-10 different patches. Right: effect of
metoclopramide (200 nM) on [ 3 ~GR65630
]
(3. 10 nM) binding. The moderate but significant ( p <0.01, paired
t-test) inhibition of 3 nM ['H] GR65630 binding was sutmountable by higher concentrations of ['H] GR65630.
Shown are means I S.D. of 3 different experiments.
experiment. The concentration-response curves of peak current suppression by metoclopramide
using the three application modes (methods) could be fitted to Hill equations [I]: when
metoclopramide was applied using closed channel application, the inhibitory potency was almost
identical to that obtained under equilibnum application ([Cio values: 0.060 FM and 0.064 pM,
respectively). In contrast, when metoclopramide was applied using the Open channel application, the
resulting inhibitory potency was lower (]Cjo approximately 19 PM). The effect of metoclopramide
(0.1 pM) on the concentration-response curve of 5-HT was detemlined to investigate whether the
mechanism of block was of competitive nature (Fig. 1): the extent of inhibition by metoclopramide
remained unchanged over a broad agonist concentration range (6-300 pM 5-HT), resulting in a
reduced maximum response to 5-HT. To examine the onset of the metoclopramide effects, we preexposed patches to metoclopramide (0.1 pM) using different durations (0.1, 0.3, 1. 3, 5 and 60 s) of
application time before the agonist pulse (Fig. 2). The time constant of drug onset was
1.3 s.
The offset (wash out) time constant was ~ , , , ~ = 2 s. 1(Fig. 2; mono-exponential fits). Calculating the
rate constants kOFF(0.48 s I) and koN (3.04 X 106 s M I ) from these time constants. a K D for
'
*
Illiie rourie
01 reC"Yely
250 ms
llom
Inhibition Dy I'icp.
"'4
Fig. 2. (A) The amplitude of 5-HT (30 PM; - 100 mV)-induced peak currents through h5-HT,,, receptor channels
as function of the duration of metoclopramide (100 nM) application prior to the 5-HT pulse (wash-in). (B) The
amplitude of 5-HT-induced peak currents through h5-HT,, receptor channels as function of the duration of
removal of metoclopramide (wash out). Means C S.D. of n 2 4 experiments. (C) Comparison of recovery (0)
from inhibition by metoclopramide (100 nM applied for 60 s, upper trace) and kinetics of the respective control
current induced by 5-HT (30 PM. lower trace).
J. ROlkemhach el al. / Inlernational Congress Series 1283 12005) 49-54
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metoclopramide of 155 nM was determined, which close to the ICso. In competition binding
experiments, metoclopramide (0.2 PM) significantly (p<0.01, paired t-test) reduced the specific
binding of 3 nM ['H] GR65630 by 21% but not that of 10 nM ['EI] GR65630, indicating that the
specific inhibition was surmountable (Fig. 1 , right panel).
4. Discussion
The major aim of the present study was to characterize pharmacologically the
interactions of metoclopramide with human 5-HT3Areceptors. As these receptors mediate
emesis and pain [12,13], their response to this drug may be relevant in addition to its wellcharacterized interaction with dopamine D2 receptors. Concentrations of metoclopramide
which inhibited 5-HT-induced currents in a concentration-dependent manner are similar to
those obtained during therapy (0.1-0.2 PM) [14]. High levels of metoclopramide are
reached within the area postrema, a region which contains 5-HT3 receptors at high density
[14]. The result that a pre-application of metoclopramide is necessary to obtain the full
inhibitory potency implies that the effect of metoclopramide is slower than the typical
opening and closing kinetics of 5-HT3 receptor currents. This is confinned by the
determination of the exact time Courses of onset and offset of inhibition by
metoclopramide. 5-HT concentration-response curves recorded in the absence and in
the presence of 0.1 FM metoclopramide had similar ECSo values (Fig. I), while the
maximum of the control curve was halved. This result is in contrast to a competitive
interaction of metoclopramide at the 5-HT binding site(s) suggested by radioligand
binding studies [15,16]. To rule out species differences, we performed, in addition to the
functional studies, radioligand binding experiments; we confirmed binding competition
between metoclopramide and the selective 5-HT3 receptor antagonist [ 3 ~GR65630
]
in our
receptor preparation. This seeming discrepancy between functional studies and radioligand
binding experiments may be accounted for by (1) slow kinetics of metoclopramide
compared with the fast onset and offset kinetics of 5-HT-induced currents (for example,
the wash out of pre-equilibrated metoclopramide is 150 times slower than the onset of 5HT-induced currents, see Fig. 2, right panel) and (2) the low efficacy of metoclopramide in
inhibiting radioligand binding (e.g., only 20% binding inhibition compared with 79% peak
current suppression by 200 nM metoclopramide, Fig. I).
Therefore, persuasive evidence that metoclopramide inhibits radioligand binding of
specific 5-HT3 receptor antagonists and contrasting reports of 5-HT3 antagonists acting via
non-competitive mechanisms (for review, See [2]) need not be a contradiction. As the
discussion above shows, it may simply reflect different functional endpoints being
examined by different experimental methods. Previously it had already been reported that
metoclopramide did not only show affinity for dopamine receptors [17] but also - to a
lesser extent for 5-HT3 receptors of different species [18]. The 1C50 values reported in
the present study are similar to results from functional studies on murine 5-HT3 receptors
[19], and human 5-HT3 receptors on whole cells [20-221. Comparisons of affinities of
metoclopramide to dopamine D2 and 5-HT3 receptors are inconclusive, some studies
report equal or even higher potencies at 5-HT3 receptors [15,16]. The low ICso value
reported here supports the conclusion that metoclopramide is at least as potent on human
5-HT3 receptors as on dopamine D2 receptors. The results indicate that metoclopramide at
-
J FFhlliembach et al / Intetnational Congres~Serres I283 (7005) 49-54
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clinical plasma concentrations is a potent antagonist of human 5-HT3 receptors, which is
compatible with its use as an antiemetic.
Acknowledgements
This study was supported by the Deutsche Forschungsgesellschaft (BA 1454).
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