TiPS - October 1992 [Vol. 131 36 Goldman, R. S., Finkbeiner, S. M. and 37 38 39 40
Smith, S. J. (1991) Nearosci.Left. 123,4-8 Calve. J. J. et al. (1990) Endocrinology 126,2288-2295 Shichiri, M. et al. (1989) Biockem. Biophys. Res. Commun. 163,l332-1337 StojilkoviCS. S., Iida, T., Merelli, F. and Catt, K. J. (1991) Mol. Pharmacol. 39, 762-770 Samson, W. K., Slcala,K. D., Alexander, 8. and Huang, F-L. S. (1991) Endocrinology 128,1465-1473
391 41 Kanyicska, B., Bunis, T. P. and Freeman, M. E. (1991) Biochem.Biopkys. Res. Commun.174,338-343 42 Hinson, J. P., Vinson, C. P., Kapas, S. and Teja, R. (1991) J. Endocrinol. 128, 275-280 43 Cow, E. N. and Gomez-Sanchez,C. E. (1990) Endocrinology l27,549-554 44 Boarder, M. R. and Marriott, D. B. (1991) Biockem. Pkarmacol.41,521-526 45 Yada, Y., Higuchi, K. and hnokawa, G. (1991) J. Biol. Chem.266,18352-18357
Recent advances in the electrophysiological characterization of 5HT3 receptors John A. Peters, Hilary M. Malone and Jeremy J. Lambert 5-HTs receptors are ligand-gated, cation-selective ion channels, mediating membrane depolarization and neuronal excitation. Established and potential therapeutic applications of selective 5-HT3 receptor antagonists, coupled with the localization of this receptor subtype within discrete areas of the CNS, have resulted in an intensification of research in this area. In this review, Jeremy Lambert and colleagues summarize recent developments in the electrophysiological characterization of 5-HTs receptors, and comment upon the unresolved issue of 5-HT3 receptor heterogeneity. The introduction of highly selective antagonists of the 5-HTs receptor in the mid-1980s was instrumental in generating widespread interest in a receptor subtype that, in its former guise as the ‘M’ receptor of Gaddum and Picarellil, was probably best known to pharmacologists for mediating an indirect contractile action of 5-I-IT upon the guineapig ileum. That 5-I-IT3 receptors occur upon some peripheral mediating neurons, transient depolarizing and neurotransmitter-releasing actions of 5-HT, has long been known’. Behavioural studies employing 5-I-IT3 receptor antagonists first suggested that this receptor may also be centrally located3. The subsequent use of radioli and-binding4, autoradiographk B and biochemical6 techniques amply confirmed this. 1. A. Peters is Lecturer, H. M. Maione is
a Postgraduate Student and 1. 1. Lambert is Professor of Neuropkarmacology at the Department of Pharmacology and Clinical Pkanuacology,Ninewells Hospitaland Medicnl School, The University, Dundee, UK DDl 9SY.
The established and potential therapeutic uses of 5-I-IT3 receptor antagonists have been the subject of recent reviews3,‘. Here, we summarize recent developments in the electrophysiological evaluation of 5-I-I& receptors, including the results of experiments examining the permeability and conductance of the channel integral to the receptor, the electrophysiological properties of a cloned 5-I-IT3 receptor subunits, and the demonstration that 5-I-IT3 receptors participate in fast s aptic transmission in the CNS Jm’ . Characteristics of b-HTs receptoroperated ion channels electrophysiological Recent studies support the contention that the 5-I-& receptor is a ligandgated, cation-selective ion channel, similar in many respects to the nicotinic acetylcholine receptorio. Table I summarizes some characteristics of the electrical response to 5-I-IT3 receptor activation determined in voltage-clamp
studies performed on peripheral
neurons and a variety of neuronal
46 Mazzoc&i, G. et al. (1998) Peptides 11,
767-772 47 Takuwa. N., Takuwa, Y., Yanagisawa, M., Yamashita, K. and Masaki,T. (1989) J, Biol. them. 264,785&7861 48 Gandhi, C. R, Stephenson, K. and Olson, M. S. (1998) J. Biol. Ckem. 265, 17432-17435 49 Delarue, C. et al. (1998) Emfocri~ofogy l27,2001-2Ml8 58 Gulati,A. and Rebello,S. (1991) tife 5ci. 48, uo7-1215
clonal cell lines. In v?titdy all studies, the current response has been found to reverse in polarity at a potential close to zero miIlivolts, consistent with the opening of cation-selective ion channels with essentially equal permeability to Na+ and K+ ions. There is also general agreement that receptor activation results from the binding of at least two agonist molecules** *-13, that cooperative interactions occur between such binding sites (see Ref. 11, for example), and that the resulting current response desensitizes rapidly, although the kinetics and voltage dependence of the latter process appear to be somewhat variableis-=. Ion substitution experiments performed on the neuronal clonal cell lines N18 (Ref. 16) and NGlo& 15 (Ref. 17), differentiated PC12 phaeochromocytoma cellsis, dissociated rat superior cervical ganglion cells14 and adult rabbit nodose ganglion neurons maintained in cell cuItu#, have established that the 5-I-IT3 receptor ion channel discriminates poorly between monovalent metal ions. In Nl8 cells and rabbit nodose ganglion neurons, the permeability of organic cations is inversely related to their geometric mean diameter; thus ammonium and its mono-methyl and mono-ethyl derivatives are highly permeant, whereas large molecules such as Tris and glucosamine permeate the channel poorly16J9. Significantly, in these cellsi4J6J9, the 5-HT3 receptor channel conducts Ca2+ and other divalent cations. These observations suggest the channel to be a large water-filled pore, analogous to the nicotinic acetylcholine receptor of the neuromuscular junctionzo. Indeed, a
theoretical treatment by Yang16, which modelled the channel as a simple cylinder, suggests a minimum pore size of 7.6& a value
TiPS - October 1992 CVol. 131 T-6
t. t&nmarv of eMrophysiologicalcharacteristics of 5HT3 receptorsdeterminedusingvariousrecordingtechniques Channel Recardlng cenductance (pS) References technique RbPNa EM&M
Cewpe NlE-115neumbtastomacetls NlE-115neumblastomacetls N18 neumbktoma c&s NG10915 hybridcells
RCB2Ohybrtdcetls RabtJitmdosegarlgEr.rncetts Rebbttnodosegargtfoncefts Ratsuperb cwvicatgangtii cells Guinea@ submucqusplexusneurons Gutnea& tii ganglionneumns
WCR/FA WCR WCRIFA WCR/FA FAISCR WCR vc WCFVSCR WCR/FA SCR WCR/SCR WCWSCR
-2.1 +20.0 -1.6 +3.5
1.09 0.42 1.10 0.89
-2.0 +7.0 -1.6 -4.7
1.09 0.43 1.06 1.24
+3.3 -1.0
0.44
0.31 0.59 3.614.4’ 7.2/12.0+
16.6 2.6 11.1 15.019.2 10
23 15 16 13,17 23 11 25,26 14 22 27
WCR, whobcetl recording;FA, fluctuationanalysis;SCR, singlechannelrecording;E$m, reversalpotenfal of the 5-HT-inducedcurrent;PK/ SI, fmmwab%t of K+ mtativetoNa’. *determinedindifferentiatedNG108-15 cells,+determinedin undifferentiated cells.
remarkably similar to that of 7.4A determined for the frog endplate nicotinic receptor using comparable methods and assumptions. Several investigations have revealed that the activation of the 5-HTs receptor-mediated current is extremely rapid, precluding a primary role for G proteins or second messengers in the activation of the ion channe111~“21. Furthermore, the internal application of G-protein activators, or inactivators, has little or no effect upon current responses recorded under whole-cell voltage clamp’6? Indeed, nucleotide-free intmAl~ar solutions support long-term recordings of 5-HTevoked macroscopic and singlechannel currents from whole cells and outside-out membrane patches, respectively1*~16~1-~. Recent studies suggest the conductance of the ion channel integral to the 5-HTa receptor to vary markedly between preparations (Table I). In undifferentiated cells of two neuronal clonal cell lines NlE-115 and NlB), singlechannel conductances below 1pS were inferred using fluctuation analysis techniques162s. However, in undifferentiated neuroblastoma X glioma hybrid NGlOB-15 cells, single-channel conductances of approximately 7 and l2pS were calculated from 5-HTinduced current fluctuations in whole cells and outside-out membrane patches, respectively24. Adding further complexity, the channel conductance in NG108-15 cells is apparently developmentally regulated**. On membrane patches excised from acutely dissociated guinea-pig submucous plexus neurons, 5-HT3 receptor-gated channels with
conductance states of 15 and 9pS, respectively, have been convincingly demonstrated by their susceptibility to antagonism by tropisetron (ICS205930) and ondansetron=. The former value is similar to that of 17pS found in membrane patches excised from rabbit nodose ganglion neurons25z6, whereas the latter might correspond to the predominant conductance state (lops) seen in guinea-pig coeliac ganglion neurons*‘. In rat superior cervical ganglion neurons14, the 5-I-IT3 receptor single-channel current-voltage relationship rectifies inwardly but, at the most negative potentials examined, yields conductances comparable to those observed in rabbit nodose ganglion and guinea-pig submucous plexus neurons (Table I). Interestingly, fluctuation analysis of whole-cell currents in rat superior cervical ganglion neurons gives an estimate of single-channel conductance that is approximately four times less than that directly observed in outside-out membrane patches. A plausible explanation for this discrepancy, suggested by the authors, might involve the coexistence within these cells of two populations of 5-I-ITS receptors; one of sufficient conductance to be directly resolved in singlechannel recordings, and a second of too low a conductance to stand out from background noise as a discrete evenP4. The conductance value obtained by fluctuation analysis would, therefore, be the weighted average of such events. Cloned 5-HT3 receptor subunit Recently, by expression cloning in Xenopus oocytes, David Julius
and his colleagues’ have isolated a cDNA clone encoding a 5-I-lTs receptor subunit (5-HT3R-A) from a cDNA library generated from poly(A)+ mRNA purified from cells of the neuronal hybrid cell line NCB20. These cells had previously been shown to express a robust 5-I-IT3 receptor-mediated electrophysiological response23a. In Xenopus oocytes injected with RNA transcripts made from the 5-HT&A clone, 5-I-IT and the 5-I-IT3 receptor-selective agonists 2-methyl&HT, 1-phenylbiguanide (PBG) and m-chlorophenylbiguanide, evoked rapidly activating inward current responses that demonstrated desensitization. Such currents were blocked by appropriate concentrations of the 5-HT3 receptor antagonists tropisetron and tropanserin (MDL72222), but were little affected by methysergide, an antagonist of 5-HTilike and 5-HT2 receptors. Interestingly, the response was also blocked by low concentrations of (+)-tubocurarine, a compound previously demonstrated to potently antagonize 5-HT3 receptormediated currents recorded from mouse hippocarnpal neurons*l and murine neuroblastoma cells29. Current-voltage relationships and ion substitution experiments suggest that the 5-HTsR-A subunits assemble, presumably as homopentamers, to form cationselective ion channels permeant even to large monovalent cation&?. As in previous studies of ‘native’ 5-I-& receptors’6,30, the presence of the divalent cations Ca*+ and Mg*+ in the extracellular medium at physiological concentrations suppressed the inward current response to 5-HT recorded from the oocytess. However, the
TiPS - October 1992 [Vol. 131 voltage-dependent apparently nature of the block exerted by Ca2+ and Mgr+ in the oocyte system contrasts with the voltageindependent mechanism of inhibition observed elsewhere1630. To draw any conclusion from this discrepancy would be premature, especially since the currentvoltage relationship in HER293 cells stably transfected with the 5-I-Ha-A does not, in the presence of similar concentrations of Ca2’ and Mg+, demonstrate the region of negative slope conductance observed in oocytes (Gill, C. et al. unpublished). Indeed, over a wide range of transmembrane potentials (-100 to +6OmV), the current-voltage relationship in HER293 cells exhibits the modest inward rectification that is often observed for 5-I-IT3 receptor-mediated current responses14.16.17,2~,23,30~
Hydrophobicity analysis suggests that the 5-I-H&A subunit has a topological organization typical of the ligand-gated ion channel superfamily. Four hydrophobic transmembrane regions (Ml-M4), with a long cytoplasmic loop between M3 and M4, and a large, extracellular N-terminal domain are envisaged. By analogy to the functionally similar nicotinic receptors, the 5-I-ITS receptor might be assembled from five such subunits, with the o-helix of the M2 region of each presumabl forming the lining of the channel2 B. Investigations of the single-channel properties of nicotinic channels modified by site-directed mutagenesis have established that negatively charged amino acids, which are thought to bracket the M2 o-helix, are crucial determinants of channel conductance20. It is of interest that similar amino acids occupy equivalent positions within the 5-I-I&R-A, particularly since native ~-I-ITS receptor-channel complexes vary greatly in their single-channel conductance (Table I and see above). Also of interest is a hydrophobic leucine (L286) residue located within the M2 transmembrane domain of the 5-HT&-A8. It is highly conserved in the ligandgated ion channel superfamily and occurs at a similar location in all nicotinic, GABA,, and glycine receptors cloned to date31. Mutation of L286 to phenylalanine (L286F) dramatically accelerates the rate of
393 onset of desensitization of the 5-HT&A expressed in Xenopus oocytes (J. L. Yakel, et al., pers. commun.). Conversely, the replacement of L286 by the more polar threonine (L286T) produces a mutant in which the kinetics of densensitization are greatly slowed relative to the wild-type subunit. These changes in desensitization kinetics parallel those previously reported to occur in nicotinic or-homooligomeric receptors subjected to similar mutations but, unlike the latter system31, they do not appear to be associated with a greatly altered affinity of the receptor for its agonist (J. L. Yakel et al., pers. commun.). Subtypes of 5-I-IT3 receptor It is now clear that marked interspecies variations exist in the pharmacological characteristics of 5-HTs receptors. In a comparative study of 5-I-IT, receptor-mediated responses recorded from guineapig isolated ileum, colon and vagus nerve, and rat isolated vagus nerve, little evidence for ~-I-ITS receptor heterogeneity was found within the guinea-pig tissues32. In contrast, the affinities of a range of antagonist compounds determined in the rat preparation were generally l& 188fold higher thzy ‘= -1. :& guinea-pig tissues3253. Additionally, although PBG is a potent agonist in rat vagus nerve33 , it proved to be inactive as either an agonist or antagonist in any of the guinea-pig tissues evaluated. Newberry et aZ.= examined the blockade by (+)-tubocurarine and tropisetron of 5-HT3 receptormediated depolarizing responses extracellularly from recorded superior cervical ganglia excised from the rat, mouse and guineapig. Their results indicate comparatively low antagonist potencies for both of these compounds in the guinea-pig, and additionally point to a pronounced discriminative effect of (+)-tubocurarine, which blocked 2-methylS-HTevoked depolarizations in mouse, rat and guinea-pig ganglia with pAP values of 8.1, 7.1 and 4.8, respectively. Results from single-cell studies, where 5-I-IT3 receptor-evoked inward currents were recorded under whole-cell voltage-clamp
conditions, support the above observations (Fig. 1). The I&, values of a range of antagonists determined in dissociated adult rabbit nodose ganglion neurons, were considerably lower than those found in the corresponding nearOIW isolated from the guineapie*. Additionally, the 5-I-IT3 receptor-evoked current in mouse nodose ganglion neurons is potently antagonized by (+)tubocurarine35. A high sensitivity to (+)-tubocurarine rn~v typify the ‘mouse 5-HT3 receptoi, this property now having been described in mouse hippocampa121, nodose ganglion35 and superior cervical ganglion34 neurons, several murine clonal cell linesUz1-29, and the cloned 5-I-IT&A8. The mechanism of the blockade is uncertain but, at Ieast in NlE115 neuroblastoma cells, it is voltage- and use-independent and unique to (+)-tubocurarine, other nicotinic receptor antagonists so far tested being ineffect&?. Additionally, in mouse and rat superior cervical ganglia=, antagonism by (+)-tubocurarine is surmountable and associated with a parallel rightward displacement of the dose-response curve to 2methyl5HT, suggestive of a competitive mechanism of action. However, in guinea-pig superior cervical ganglion neurons, where relatively high concentrations of (+)-tubocurarine are necessary to demonstrate blockade, a depression of the slope and maximum of the dos+response curve In the nodose is observedx. ganglion neurons of the rabbit, a transition from an apparently competitive to a noncompetitive blockade is reported to occur with increasing concentrations of (+)tubocurarine”. It is possible, by analogy with the nicotinic receptor of the motor endplate, that (+)tubocurarine exerts both receptorand ion channel-blocking actions. Whilst evidence for interspecies receptor 5-HT3 variation in properties is accumulating, intraspecies variation remains to be convincingly demonstrated. It seems likely, however, that differences in the characteristics of 5-I-lTs receptors within a species will eventually be found, in view of the diversity exemplified by other members of the ligandgated ion channel superfamily, such as the nicotinic, GABA* and
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glutamate receptors. Fragmentary evidence may already exist. The sensitivity of 5-Err3 receptors located on rabbit isolated preganglionic cervical sympathetic nerves36 and the terminals of cardiac sympathetic neNesjsr to blockade by tropanserin is considerably higher than that displayed by the 5-HTs receptors expressed by other neural elements in the same species. In mea-pig frontal cortical slices, PBG at low concentration (101~) is reported to activate a putative 5-HT,s receptor whose stimulation enhances the electcitally evoked release of [“I-I]5-HTss. Jn the FNS of this species, PBG is inactive at 5-H& receptoIs3354. Exceptionally low concentrations of 5-HT (EC& 0.4nM) and PBG @C, 0.64nna) are reported to augment a Ca*‘-dependent depolarization-evokedrelease of cholecystokinin-like immunoreactivity from rat brain synaptosomes, an effect insensitive to the 5-IiT1_like and S-I-IT2 receptor antagonist metitepine, but blocked by appropriate concentrations of tropisetron, ondansetron and tropanserin3*. Such EC& values for 5-HT. and PBG are at variance with the concentrations of these agonists required to activate S-I-IT, receptors in the rat peripheral nervouss~stem~~*.Finally, ~~orophenylbi~~de may discriminate between the 5-HTs receptors expressed by the closely related cell lines NlE-115 and NGlOg-15 acting as a full agonist in the former system, but as a partial agonist in the 1atterc’O. Clearly, these anomalous find-
r
control 200 pA 1
ings do not constitute proof of
intraspecies heterogeneity, but thev nonetheless require explanation. In this context, it is noteworthy that 5-HTsR-A sequences (amplified by the polymerase chain reaction) have been detected in mouse cortex, midbrain, spinal cord and heart, but not in mouse intestine, which might also be expected to express S-I-IT3 receptors. The possibility that the mouse intestine contains a S-I-IT3 subtype encoded by a separate gene deserves further investigation. 5-H’& receptors in the CNS It is only recently that electrophysiological techniques have been used to probe the characteristics of central 5-HT3 receptors. In embryonic mouse hippo~mp~ and striatal neurons in primary cell culture, 5-HT elicits three physiologically and pharmacologically distinct responses**. One of these, a rapidly activating and desensitizing inward current response, reversing in sign close to 0 mV and arising from an increase in membrane conductance, has been attributed to 5-HTs receptor activation. In hippocampal neurons, this response is mimicked by IL-methyl-5-HT, blocked completely by a low concentration (1nM) of tropisetron, and antagonized by metoclopramide (I& = 440nM) but not by ketanserin2*. Given the many extensive electrophysiological studies that have characterized the actions of 5-HT upon pyramidal neurons in hippocampal slices (Ref. 10 and refs therein) and the presence of
WIT3 recognition sites in this tissue*, it is at first sight surprising that this response had not been previously described. A possible reason for this has recently emerged. Recording intracellularly from Cl--loaded CA1 pyramidal neurons in rat hippocampal slices, Ropert and Guy”2 noted that, in addition to its welldocumented activation of an inwardly rectifying K+ channel (via G protein-coupled 5-HTr, receptor#O), 5-HT also increased the frequency of spontaneous synaptic events. The effect of 5-HT upon the frequency of postsynaptic potentials persisted in the presence of NMDA and AMPA/kainate receptor antagonists, but was abolished by the GABAA receptor antagonist bicuculline or by loading the neurons with poorly permeant anions. A facilitation of GABA* receptormediated inhibitory postsynaptic potentials (IPSPs) by 5-I-IT thus seemed likely_ In tetrodotoxin @IX)-treated slices, the (miniature) IPSP amplitude distribution was unimodal and 5-HT affected neither IPSP amplitude nor frequency. In control slices, the distribution of IPSP amplitudes was broadly similar with a mode of approximately 1mV. By contrast, in the presence of 5-HT the amplitude dis~bution was bimodal with peaks occurring at around 1 and 4mV. The interpretation of these data provided by the authors42 is that 5-HT elicits IPSPs of higher quantal content than in control, most probably as a result of the excitation of previously quiescent in-
TiPS - October 1992 Wol. 131 hibitory GABAergic interneurons. Significantly, the effect of 5-HT upon IPSPs (but not the postsynaptic conductance increase registered in the impaled CA1 neuron) was blocked by low concentrations (l-90nM) of tropisetron and mimicked by a-methyl5-HT, suggesting the involvement of 5-HT, receptors. A GABA-releasing action of 5-I-IT3 receptor activation could, in principle, explain the recently inhibitory reported effect of selective 5-HT, receptor activation upon the F-evoked release of [3H]acetylcholine and endogenous noradrenaline from slices of rat entorhinal cortex43 and respectively. It is hypothalamu&, significant that the inhibition of [3H]acetylcholine release . blocked by TTX (N. Barnes, per: commun.), consistent with the notion that 5-HT3 receptor stimulation excites inhibitory, perhaps GABAergic, intemeurons. In this context, it is noteworthy that 2methyl-5-HT inhibits the NMDAinduced firing of nociceptive spinal projection neurons located in the dorsal hom45. Such inhibition is blocked both by 5-HT3 (zacopride) and GABAA (bicuculline) receptor antagonists, again suggesting the excitation of inhibitory interneurons as a consequence of 5-HT3 receptor stimulation. Both pre- and postsynaptic effects attributed to the activation of 5-HT3 receptors have recently been reported to occur in brain slice preparations containing the nucleus tractus solitarius (NTS). In whole-cell recordings from NTS neurons, a rather high concentration (100 PM) of 2-methyl-5-HT evoked a TTX-insensitive depolarizing response associated with an increase in membrane conductance4’j. This effect was reduced by tropisetron (0.01-1.0 PM) or tropanserin (1 PM). An additional action, observed with lower cnn2-methyl-5-HT centrations of (0.25-~PM), was an increase in the frequency and amplitude of excitatory and inhibitory spontaneous postsynaptic potentials (sPSPS)~~. Unlike results obtained from the hippocampus, there was a modest increase in sPSP frequency in the presence of TTX (P. Brooks, pers. commun.). The further addition of Co*+, to reduce Ca’+-dependent transmitter re-
lease from presynaptic terminals, blocked this effect. However, the antagonism by Co2+ of 5-HT3 receptor-mediated currents in neuronal cell linesI’ may complicate the interpretation of the latter experiment. In contrast to its augmentation of sPSP amplitude, 2-methyl+ HT, over an identical concentration range, depressed the amplitude of EPSPs and IPSPs evoked by electrical stimulation of the afferent fibres of the solitary tract& (P. Brooks, pers. commun.). All of the effects of 2-methyl-5-I-IT upon postsynaptic potentials were antagonized by either tropisetron (10 nM) or tropanserin (10 PM), but were resistant to blockade by ritanserin (1ph.Q (P. Brooks, pers. commun.). One explanation of these data might be that the activation of presynaptically located 5-HT3 receptors produces a depolarization that facilitates spontaneous neurotransmitter release, but presynaptically inhibits evoked neurotransmitter release (Ref. 46 and P. Brooks, pers. commun.). Although the NTS is innervated by 5-HT-releasing neurons from the medial raphC nucleus, and ~-I-ITS receptors are located both pre- and postsynaptically in the NTS, 5-HT3 receptor-mediated postsynaptic potentials ii1 response to electrical stimulation were not detected (P. Brooks, pers. commun.). However, such events have been demonstrated to occur in rat lateral amygdala neurons in brain slices, by the use of conventional intracellular and whole-cell recording techniques’. In a proportion of neurons within this nucleus, electrical stimulation of presynaptic fibres caused the appearance of fast EPSPs resistant to blockade by NMDA, AMPAI kainate and, with the notable exception of (+)-tubocurarine, nicotinic receptor/channel blockers. Such EPSPs were of brief duration (tens of milliseconds) and insensitive to spipirone and ketanserin (at concentrations sufficient to abolish 5-HT2 receptor-mediated excitatory response:s in other brain regions), but were reduced in amplitude, in a concentrationdependent manner, by the ~-I-ITS receptor antagonists GR67330 (l300nM), tropisetron (3-300 nM), and ondansetron (30 nM-10 PM). Although the concentrations of these antagonists necessary to
achieve blockade are somewhat higher than might be anticipated from their Km, Ki or Kd values at 5-H’T3 recognition sites (determined from radioligand binding studies performed on rat cortical tissue’), selectivity of action was clearly demonstrated. GR67330 had no effect upon synaptic potentials mediated by excitatory amino acids or GABA, even at a concentration lOO-fold higher than that producing a 50% depression of the EPSP tiuoked by 5-HT3 receptor stimulation. Further evidence for the involvement of 5-I-IT includes the mimicry of the EPSP by ionophoretically applied 5-W, its augmentation by the 5-HT uptake inhibitor fluoxetine, and its suppression by superfused 5-H’P. The latter might occur via the rapid desensitization that typifies electrical responses mediated via 5-I-IT3 receptors1*,1621 and/or as a consequence of receptor occlusion. Like 5-HT3 receptor-evoked electrical responses recorded from neurons14,*9Z and peripheral neuronal clonal ceil line!?, the 5-I-ITS receptor-mediated excitatory postsynaptic current reversed polarity at a potential close to 0 mV, suggesting similarities in electrogenesis iYig. 2). these electroCollectively, physiological studies point to the existence of pre- and postsynaptic central 5-HT3 receptors regulating release and neurotransmitter rapid ionotropic mediating neumtransmission, respectively. Furthermore, they indicate substantial commonality in the functioning of the receptor-channel complex in the CNS, peripheral neurons, and neuronal clonal cell lines. This is to be contrasted with several recent reports by Wang, Ashby and their colleagues (summarized in Ref. 47) that have suggested the existence of a ‘5-I-ITS-like’ receptor, possibly coupled to phospholipase C, that mediates a non-desensitizing inhibition of rat medial prefrontal cortical neurons in vivo. While the pharmacology of both the suppressant action of 5-HT receptor-selective and 5-I-IT3 agonists upon the firing rate of such neurons, and the stimulation of phosphoinositide turnover in fronto-cingulate and entorhinal cortical slices in vitro, is entirely consistent with 5-I-& receptor
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GR67330
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T
wbv
[email protected], receptorsmetIMe fast excitatorypostsynapticpotentials (EPSPs) in rat .&era/ amyg&la neurwx? in vitro.a: Synapfic potentials evoketi by brief focal electrical s6muMon of the twsoiateml twa’eua are blocketi by GR67330. b: Depolarizing v to &wphorelkzaf& applied 5-HT mimic the EPSP and are blocked by tropMmn_ Reproduced, with permission,from Ref. 9. (Copyright,Cell Press.)
mediation, the purportedly direct nature of these responses requires further investigation.
Modulators of 5-HTs receptor function
Recent reports indicate that ethanol and the dissociative anaesthetic ketamine act to potentiate electrical responses elicited by 5-HTs receptor activation, but their mechanism(s) of action are unknown. in a subset of NCB20 cells and rat nodose ganglion neuronP, concentrations of ethanol within the range producing acute intoxication in man, reversibly enhance submaximal inward current responses evoked by 5-HT, receptor stimulation. This observation is intriguing in view of the reported ability of 5HT, receptor antagonists to reduce alcohol consumption in animals and their suppressant effect upon aversive behaviours triggered by ethanol withdrawa13. Ketamine (3-30~) produces a concentration-dependent enhancement of inward current responses mediated by 5-HT3 receptors in rabbit nodose ganglion neurons, via a mechanism distinct from suppression of 5-I-U uptake”. The generality of this effect and its physiological relevance remain to be explored. q
0
cl
The 5-HTJ receptor is now firmly established as a member of
the ligand-gated ion channel superfamily and, like others in this group, mediates rapid synaptic transmission in the mammalian CNS. The latter action is unique among the amine neurotransmitters, and contrasts with the relatively slow inhibitory synaptic potentials mediated by the G protein-linked 5-HTI, receptors”. Additionally, there is evidence that 5-I-IT3 receptor activation may modulate the release of a variety of CNS neurotransmitters includin~ acetylcholine43, cholecystokinin , dopamine6, GABA4’ and noradrenalineU. Assuming such actions to be physiologically re!evant, these observations may eventually provide a rational basis for the various purported psychoactive properties of 5-HT3 receptor antagonists3. The precise mechanism(s) by which 5-HT3 receptor activation modulates transmitter release is uncertain, but it is parsimonious to assume that enhanced release is the result of nerve terminal depolarization arising from either the stimulation of 5-HT3 receptors resident upon nerve terminals, or increased impulse flow generated by the activation of somatically located receptors. The blockade, or reduction, by TTX of 5-HT3 receptor-mediated release of GABAQ and dopamine6 suggests that impulse propagation plays a role in at least some circumstances. However, presynaptically located receptors must presumably
account for 5-HT3 receptor-mediated release of cholecystokininlike immunoreactivity from rat brain synaptosomes3’. Apart from providing a depolarizing stimulus to activate voltage-sensitive Ca2+ channels, 5-HT3 receptor stimulation might result in Ca” influx through the ion channel complex itselP4,16. Inhibition of evoked transmitter release, as in the case of noradrenaline@ and acetylcholinea, might best be explained as occurring secondary to enhanced release of inhibitory transmitter substances, or as the result of a depolarizing presynaptic block. While species differences in the pharmacological characteristics of 5-HT3 receptors now seem certain, intraspecies heterogeneity remains to be convincingly demonstrated. The cloning of a 5-HT3 receptor subunit seems certain not only to broaden our knowledge of 5-HT3 receptor function, but also to generate a new approach to the identification of receptor subtypes that will not be hampered by the possible lack of discriminatory ligands. Acknowledgements Work performed in the authors’ laboratory is supported by the Wellcome Trust. We thank Drs P. Brooks and J. Yakel and their colleagues for permission to quote their unpublished observations. References 1 Gaddum, J. H. and Picarelli, Z. P. (1957) Br. J. Phaimncol. 12.323-328 Fozard, J. R. (1984) Neuropharmacology 23,1473-1486 Costall, B., Navlor, R. 1. and Tvers, M. B. (1990) Pharm&ol. The;. 47,18i-202 Kilpatrick, G. J., Jones, B. J. and Tyers, M. 8. (1987) Nature 330, 746-748 Waeber. C., Dixon, K., Hover, D. and Palacios, J. M. (1988) Eur. J: Pharmacol. 151,351-352 Blandina, P., Goldfarb, J. and Green, J. P. (1988) Eur. 1. Pharmacol. 155. 349-350 ’ . Fozard, J. R. in Proceedings of Int. Acad. Drug Res. Workshop on Serotonin Receptor Subtypes (Langer, S. Z., ed.), Birkhauser (in press) Maricq, A. V., Peterson, A. S., Brake, A. J., Myers, R. M. and Julius, D. (1991) Science 254, 432-437 9 Sugita, S., Shen, K-Z. and North, R. A. (1992) Neuron 8,199-203 10 Bobker, D. H. and Williams, J. T. (1990) TrevrdsXeurosci. 13,169-173 11 Higashi, S. and Nishi, S. (1982) 1. Physiol. 323, 543-567 12 Neijt, H. C., Te Duits, I. J. and Vijverberg, H. P. M. (1988) Neuropharmncology 27,301-317
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13 ‘.‘akel, J. L., Shao, X. M. and Jackson, M. B. (1991) J. Pension. 436,29>308 14 Yang, J., Mathie, A. and Hille, B. (1992) J. Physiol. 448, 237-256 15 Neijt, H. C., Plomp, J. J. and Vijverberg, H. I’. M. (1989) J. Physiol. 411,257-269 16 Yang, J. (1990) J. Gen. Physiol. %, 1177-1198 17 Yakei, J. L., Shao, X. M. and Jackson, M. B. (1990) Brain RPS. 533,46-52 18 Furukawa, K., Akaike, N., Onodera, H. and Kogure, K. (1992) J. Neurophysiol. 67,812-819 19 Malone, H. M., Peters, J. A. and Lambert, J. J. (1991) SW. Neurosci. Abs. 17, 239.11 20 Hille, B. (1992) Ionic Channels of Excitable Membranes, 2nd edn, Sinauer 21 Yakel, J. L. and Jackson, M. B. (1988) Neuron 1,61!?-621 22 Derkach, V., Surprenant, A. and North, R. A. (1989) Nature 339,70&709
23 Lambert, J. J., Peters, J. A., Hales, T. G. and Dempster, J. (1989) Br. 1. Pharmacol. 97,27-40 24 Shao, X. M., Yakel, J. L. and Jackson, M. B. (1991) f. Neurophysjo~~ 65,630-638 25 Peters, J. A., Malone, H. M. and Lambert, J. J. (1991) in Serotonin: Molecular Biology, Receptors and Functional Effects (Fozard, J. R. and Saxena, P. R.,
eds), pp. 84-94, Birkhauser 26 Malone, H. M., Peters, J. A. and Lambert, J. J. (1991) Ne~ropeptides 19 (suppl.), 25-30 27 Surprenant, A., Matsumoto, S. and Gerzanich, V. (1991) Sot. Neurosci. Abs. 17,239.12 28 Peters, J. A. and Lambert, J. J. (1989) Trends Pku~aco~. Sci. 10, 172-175 29 Peters, J. A., Malone, H. M. and Lambert, J. J. (1990) Neurosci. Left. 110, 107-112 30 Peters, J. A., Hales, T. G. and Lambert, J. J. (1988) Eur. J. Pharmacol. 151,491-495 31 Revah, F. et al. (1991) Nature 353, 846-849 32 Butler, A. et al. (1990) Br. J. Pharrnacoi. 101,591-598
33 Ireland, S. J. and Tyers, M. B. (1987) Br. 1. Pharmacol. 90,229-238 34 Newberry, N. R., Cheshire, S. H. and Gilbert, M. J. (1991) BY. 1. Phar~aeo~.
102,61~20 35 Malone, H. M., Peters, J. A. and Lambert, J. J. (1991) Br. 1. PharmacoZ. 104, 68P 36 Elliot, I’. and Wallis, D. I. (1988) Naunyn-Schm~edebe~*s col. 338,608-615
Arch.
Pha~a-
37 Fozard, J. R. (1984) Naunyn-Schmiedeberg’s Arch. Pharmacol. 326, 36-44 38 Galzin, A. M., Poncet, V. and Langer,
S. 2. (1990) Br. J. F’harmacol. 100,307!’ 39 Paudice, I?. and Raiteri, M. (1991) Br. 1. Fharmacoi. 103,1790-1794 40 Boess, F. G., Septilveda, M. I., Lummis,
S. C. R. and Martin, I. L. (1992) Neuropharmacology 31,561-564 41 Yakel J. L., Trussed, L. c). and Jackson, M. B. (1988) J. Neurosci. 8, 1273-1285 42 Ropert, N. and Guy, N. (1991) J. Phys~o~. 441,121-136
43 Barnes, J. M., Barnes, N. M., Costall, B., Naylor, R. J. and Tyers, M. B. (1989) Nature 338, 762-763 44 Blandina, I’., Goldfarb,
J. and Green, J. P. (1991) in Serotonin: Mo~ecu~ur Biology, Receptors and Functio}la~ Effecfs (Fozard, J. R. and Saxena, P. R., eds), pp.
322-329, Birkhauser 45 Alhaider, A. A., Lei, S. 2. and Wilcox,
G. L. (1991) J. Neurosci. 11, 18Sl-1883 46 Glaum, S. R., Brooks, P. A., Murphy, S. M. and MilIer, R. J. (1990) J. Physiof.
438,253I’ 67 Wang, R. Y.. Ashby, C. R., Jr and E&:ards, E. (1991) in Serofonin: MO/ecutar Biology. Receptors and Functional Effects (Fozard, J- R. and Saxena, P. R.,
edsf, pp. 174-185, Birkhauser 48 Lovinger, D. M. and White, C. (1991) J. Pharmacol. Exp. Ther. 40,263-270 49 Peters, J. A., Malone, H. M. and
Toughs at the top The Billion-Dollar Battle:
Merck v Glaxo by Matthew Lynn, Heinemann, 1991. f 16.99 (244 paged 1SBN 0 434 43924 x Paris is ‘a city where gloomy, selfimportant waffle is treated as wisdom’. Base1 is ‘furtive and sly, as if it harboured some dark secret: a secret likely to corrupt and destroy all those who come close to it’. These rather oblique statements from the opening sections of this book, which deal successively with the events and personalities at the 1990 Meeting of the International Pharmaceutical Conference, the life of
Lambert, J. J- (1991) Br. J. Pka~acof. 103,1623-1625
GR67330: (+)1,2,3,9-tetrahydro-9-methyl3-[(5-methyl-lH-imidazol-4yI)methyl]4H-carbazol-4one tropisetron: [(1~,5ff~-S-me~yl-Sazabicyclo-(3.2.l.@ct-3ct-3cr-yljltf-indole-3carboqrlate tnqwse& lOrn,3a,5crH-tropan-3-yI-3,5dichlorobenzoate
Paracdsus, and the Valium-Roche saga of the 197Os, give notice of the idiosyncratic thesis the author subsequently develops, based on his analysis of the world’s two leading pharmaceutical companies, Merck and Glaxo. His message is simple: that the corporate attitudes of these giants are radically different and a reflection of the professional backgrounds and attitudes of their respective chief executives. In the red comer is Sir Paul Girolami of Glaxo, au accountant, ‘dedicated, passionate and ruthless’; in the green comer is Dr Roy Vagelos, a physician and ‘one of the most brilliant American s&ntists of his generation’. The battle is for ‘control of the world’s most
Smith, S. J. (1991) Nearosci.Left. 123,4-8 Calve. J. J. et al. (1990) Endocrinology 126,2288-2295 Shichiri, M. et al. (1989) Biockem. Biophys. Res. Commun. 163,l332-1337 StojilkoviCS. S., Iida, T., Merelli, F. and Catt, K. J. (1991) Mol. Pharmacol. 39, 762-770 Samson, W. K., Slcala,K. D., Alexander, 8. and Huang, F-L. S. (1991) Endocrinology 128,1465-1473
391 41 Kanyicska, B., Bunis, T. P. and Freeman, M. E. (1991) Biochem.Biopkys. Res. Commun.174,338-343 42 Hinson, J. P., Vinson, C. P., Kapas, S. and Teja, R. (1991) J. Endocrinol. 128, 275-280 43 Cow, E. N. and Gomez-Sanchez,C. E. (1990) Endocrinology l27,549-554 44 Boarder, M. R. and Marriott, D. B. (1991) Biockem. Pkarmacol.41,521-526 45 Yada, Y., Higuchi, K. and hnokawa, G. (1991) J. Biol. Chem.266,18352-18357
Recent advances in the electrophysiological characterization of 5HT3 receptors John A. Peters, Hilary M. Malone and Jeremy J. Lambert 5-HTs receptors are ligand-gated, cation-selective ion channels, mediating membrane depolarization and neuronal excitation. Established and potential therapeutic applications of selective 5-HT3 receptor antagonists, coupled with the localization of this receptor subtype within discrete areas of the CNS, have resulted in an intensification of research in this area. In this review, Jeremy Lambert and colleagues summarize recent developments in the electrophysiological characterization of 5-HTs receptors, and comment upon the unresolved issue of 5-HT3 receptor heterogeneity. The introduction of highly selective antagonists of the 5-HTs receptor in the mid-1980s was instrumental in generating widespread interest in a receptor subtype that, in its former guise as the ‘M’ receptor of Gaddum and Picarellil, was probably best known to pharmacologists for mediating an indirect contractile action of 5-I-IT upon the guineapig ileum. That 5-I-IT3 receptors occur upon some peripheral mediating neurons, transient depolarizing and neurotransmitter-releasing actions of 5-HT, has long been known’. Behavioural studies employing 5-I-IT3 receptor antagonists first suggested that this receptor may also be centrally located3. The subsequent use of radioli and-binding4, autoradiographk B and biochemical6 techniques amply confirmed this. 1. A. Peters is Lecturer, H. M. Maione is
a Postgraduate Student and 1. 1. Lambert is Professor of Neuropkarmacology at the Department of Pharmacology and Clinical Pkanuacology,Ninewells Hospitaland Medicnl School, The University, Dundee, UK DDl 9SY.
The established and potential therapeutic uses of 5-I-IT3 receptor antagonists have been the subject of recent reviews3,‘. Here, we summarize recent developments in the electrophysiological evaluation of 5-I-I& receptors, including the results of experiments examining the permeability and conductance of the channel integral to the receptor, the electrophysiological properties of a cloned 5-I-IT3 receptor subunits, and the demonstration that 5-I-IT3 receptors participate in fast s aptic transmission in the CNS Jm’ . Characteristics of b-HTs receptoroperated ion channels electrophysiological Recent studies support the contention that the 5-I-& receptor is a ligandgated, cation-selective ion channel, similar in many respects to the nicotinic acetylcholine receptorio. Table I summarizes some characteristics of the electrical response to 5-I-IT3 receptor activation determined in voltage-clamp
studies performed on peripheral
neurons and a variety of neuronal
46 Mazzoc&i, G. et al. (1998) Peptides 11,
767-772 47 Takuwa. N., Takuwa, Y., Yanagisawa, M., Yamashita, K. and Masaki,T. (1989) J, Biol. them. 264,785&7861 48 Gandhi, C. R, Stephenson, K. and Olson, M. S. (1998) J. Biol. Ckem. 265, 17432-17435 49 Delarue, C. et al. (1998) Emfocri~ofogy l27,2001-2Ml8 58 Gulati,A. and Rebello,S. (1991) tife 5ci. 48, uo7-1215
clonal cell lines. In v?titdy all studies, the current response has been found to reverse in polarity at a potential close to zero miIlivolts, consistent with the opening of cation-selective ion channels with essentially equal permeability to Na+ and K+ ions. There is also general agreement that receptor activation results from the binding of at least two agonist molecules** *-13, that cooperative interactions occur between such binding sites (see Ref. 11, for example), and that the resulting current response desensitizes rapidly, although the kinetics and voltage dependence of the latter process appear to be somewhat variableis-=. Ion substitution experiments performed on the neuronal clonal cell lines N18 (Ref. 16) and NGlo& 15 (Ref. 17), differentiated PC12 phaeochromocytoma cellsis, dissociated rat superior cervical ganglion cells14 and adult rabbit nodose ganglion neurons maintained in cell cuItu#, have established that the 5-I-IT3 receptor ion channel discriminates poorly between monovalent metal ions. In Nl8 cells and rabbit nodose ganglion neurons, the permeability of organic cations is inversely related to their geometric mean diameter; thus ammonium and its mono-methyl and mono-ethyl derivatives are highly permeant, whereas large molecules such as Tris and glucosamine permeate the channel poorly16J9. Significantly, in these cellsi4J6J9, the 5-HT3 receptor channel conducts Ca2+ and other divalent cations. These observations suggest the channel to be a large water-filled pore, analogous to the nicotinic acetylcholine receptor of the neuromuscular junctionzo. Indeed, a
theoretical treatment by Yang16, which modelled the channel as a simple cylinder, suggests a minimum pore size of 7.6& a value
TiPS - October 1992 CVol. 131 T-6
t. t&nmarv of eMrophysiologicalcharacteristics of 5HT3 receptorsdeterminedusingvariousrecordingtechniques Channel Recardlng cenductance (pS) References technique RbPNa EM&M
Cewpe NlE-115neumbtastomacetls NlE-115neumblastomacetls N18 neumbktoma c&s NG10915 hybridcells
RCB2Ohybrtdcetls RabtJitmdosegarlgEr.rncetts Rebbttnodosegargtfoncefts Ratsuperb cwvicatgangtii cells Guinea@ submucqusplexusneurons Gutnea& tii ganglionneumns
WCR/FA WCR WCRIFA WCR/FA FAISCR WCR vc WCFVSCR WCR/FA SCR WCR/SCR WCWSCR
-2.1 +20.0 -1.6 +3.5
1.09 0.42 1.10 0.89
-2.0 +7.0 -1.6 -4.7
1.09 0.43 1.06 1.24
+3.3 -1.0
0.44
0.31 0.59 3.614.4’ 7.2/12.0+
16.6 2.6 11.1 15.019.2 10
23 15 16 13,17 23 11 25,26 14 22 27
WCR, whobcetl recording;FA, fluctuationanalysis;SCR, singlechannelrecording;E$m, reversalpotenfal of the 5-HT-inducedcurrent;PK/ SI, fmmwab%t of K+ mtativetoNa’. *determinedindifferentiatedNG108-15 cells,+determinedin undifferentiated cells.
remarkably similar to that of 7.4A determined for the frog endplate nicotinic receptor using comparable methods and assumptions. Several investigations have revealed that the activation of the 5-HTs receptor-mediated current is extremely rapid, precluding a primary role for G proteins or second messengers in the activation of the ion channe111~“21. Furthermore, the internal application of G-protein activators, or inactivators, has little or no effect upon current responses recorded under whole-cell voltage clamp’6? Indeed, nucleotide-free intmAl~ar solutions support long-term recordings of 5-HTevoked macroscopic and singlechannel currents from whole cells and outside-out membrane patches, respectively1*~16~1-~. Recent studies suggest the conductance of the ion channel integral to the 5-HTa receptor to vary markedly between preparations (Table I). In undifferentiated cells of two neuronal clonal cell lines NlE-115 and NlB), singlechannel conductances below 1pS were inferred using fluctuation analysis techniques162s. However, in undifferentiated neuroblastoma X glioma hybrid NGlOB-15 cells, single-channel conductances of approximately 7 and l2pS were calculated from 5-HTinduced current fluctuations in whole cells and outside-out membrane patches, respectively24. Adding further complexity, the channel conductance in NG108-15 cells is apparently developmentally regulated**. On membrane patches excised from acutely dissociated guinea-pig submucous plexus neurons, 5-HT3 receptor-gated channels with
conductance states of 15 and 9pS, respectively, have been convincingly demonstrated by their susceptibility to antagonism by tropisetron (ICS205930) and ondansetron=. The former value is similar to that of 17pS found in membrane patches excised from rabbit nodose ganglion neurons25z6, whereas the latter might correspond to the predominant conductance state (lops) seen in guinea-pig coeliac ganglion neurons*‘. In rat superior cervical ganglion neurons14, the 5-I-IT3 receptor single-channel current-voltage relationship rectifies inwardly but, at the most negative potentials examined, yields conductances comparable to those observed in rabbit nodose ganglion and guinea-pig submucous plexus neurons (Table I). Interestingly, fluctuation analysis of whole-cell currents in rat superior cervical ganglion neurons gives an estimate of single-channel conductance that is approximately four times less than that directly observed in outside-out membrane patches. A plausible explanation for this discrepancy, suggested by the authors, might involve the coexistence within these cells of two populations of 5-I-ITS receptors; one of sufficient conductance to be directly resolved in singlechannel recordings, and a second of too low a conductance to stand out from background noise as a discrete evenP4. The conductance value obtained by fluctuation analysis would, therefore, be the weighted average of such events. Cloned 5-HT3 receptor subunit Recently, by expression cloning in Xenopus oocytes, David Julius
and his colleagues’ have isolated a cDNA clone encoding a 5-I-lTs receptor subunit (5-HT3R-A) from a cDNA library generated from poly(A)+ mRNA purified from cells of the neuronal hybrid cell line NCB20. These cells had previously been shown to express a robust 5-I-IT3 receptor-mediated electrophysiological response23a. In Xenopus oocytes injected with RNA transcripts made from the 5-HT&A clone, 5-I-IT and the 5-I-IT3 receptor-selective agonists 2-methyl&HT, 1-phenylbiguanide (PBG) and m-chlorophenylbiguanide, evoked rapidly activating inward current responses that demonstrated desensitization. Such currents were blocked by appropriate concentrations of the 5-HT3 receptor antagonists tropisetron and tropanserin (MDL72222), but were little affected by methysergide, an antagonist of 5-HTilike and 5-HT2 receptors. Interestingly, the response was also blocked by low concentrations of (+)-tubocurarine, a compound previously demonstrated to potently antagonize 5-HT3 receptormediated currents recorded from mouse hippocarnpal neurons*l and murine neuroblastoma cells29. Current-voltage relationships and ion substitution experiments suggest that the 5-HTsR-A subunits assemble, presumably as homopentamers, to form cationselective ion channels permeant even to large monovalent cation&?. As in previous studies of ‘native’ 5-I-& receptors’6,30, the presence of the divalent cations Ca*+ and Mg*+ in the extracellular medium at physiological concentrations suppressed the inward current response to 5-HT recorded from the oocytess. However, the
TiPS - October 1992 [Vol. 131 voltage-dependent apparently nature of the block exerted by Ca2+ and Mgr+ in the oocyte system contrasts with the voltageindependent mechanism of inhibition observed elsewhere1630. To draw any conclusion from this discrepancy would be premature, especially since the currentvoltage relationship in HER293 cells stably transfected with the 5-I-Ha-A does not, in the presence of similar concentrations of Ca2’ and Mg+, demonstrate the region of negative slope conductance observed in oocytes (Gill, C. et al. unpublished). Indeed, over a wide range of transmembrane potentials (-100 to +6OmV), the current-voltage relationship in HER293 cells exhibits the modest inward rectification that is often observed for 5-I-IT3 receptor-mediated current responses14.16.17,2~,23,30~
Hydrophobicity analysis suggests that the 5-I-H&A subunit has a topological organization typical of the ligand-gated ion channel superfamily. Four hydrophobic transmembrane regions (Ml-M4), with a long cytoplasmic loop between M3 and M4, and a large, extracellular N-terminal domain are envisaged. By analogy to the functionally similar nicotinic receptors, the 5-I-ITS receptor might be assembled from five such subunits, with the o-helix of the M2 region of each presumabl forming the lining of the channel2 B. Investigations of the single-channel properties of nicotinic channels modified by site-directed mutagenesis have established that negatively charged amino acids, which are thought to bracket the M2 o-helix, are crucial determinants of channel conductance20. It is of interest that similar amino acids occupy equivalent positions within the 5-I-I&R-A, particularly since native ~-I-ITS receptor-channel complexes vary greatly in their single-channel conductance (Table I and see above). Also of interest is a hydrophobic leucine (L286) residue located within the M2 transmembrane domain of the 5-HT&-A8. It is highly conserved in the ligandgated ion channel superfamily and occurs at a similar location in all nicotinic, GABA,, and glycine receptors cloned to date31. Mutation of L286 to phenylalanine (L286F) dramatically accelerates the rate of
393 onset of desensitization of the 5-HT&A expressed in Xenopus oocytes (J. L. Yakel, et al., pers. commun.). Conversely, the replacement of L286 by the more polar threonine (L286T) produces a mutant in which the kinetics of densensitization are greatly slowed relative to the wild-type subunit. These changes in desensitization kinetics parallel those previously reported to occur in nicotinic or-homooligomeric receptors subjected to similar mutations but, unlike the latter system31, they do not appear to be associated with a greatly altered affinity of the receptor for its agonist (J. L. Yakel et al., pers. commun.). Subtypes of 5-I-IT3 receptor It is now clear that marked interspecies variations exist in the pharmacological characteristics of 5-HTs receptors. In a comparative study of 5-I-IT, receptor-mediated responses recorded from guineapig isolated ileum, colon and vagus nerve, and rat isolated vagus nerve, little evidence for ~-I-ITS receptor heterogeneity was found within the guinea-pig tissues32. In contrast, the affinities of a range of antagonist compounds determined in the rat preparation were generally l& 188fold higher thzy ‘= -1. :& guinea-pig tissues3253. Additionally, although PBG is a potent agonist in rat vagus nerve33 , it proved to be inactive as either an agonist or antagonist in any of the guinea-pig tissues evaluated. Newberry et aZ.= examined the blockade by (+)-tubocurarine and tropisetron of 5-HT3 receptormediated depolarizing responses extracellularly from recorded superior cervical ganglia excised from the rat, mouse and guineapig. Their results indicate comparatively low antagonist potencies for both of these compounds in the guinea-pig, and additionally point to a pronounced discriminative effect of (+)-tubocurarine, which blocked 2-methylS-HTevoked depolarizations in mouse, rat and guinea-pig ganglia with pAP values of 8.1, 7.1 and 4.8, respectively. Results from single-cell studies, where 5-I-IT3 receptor-evoked inward currents were recorded under whole-cell voltage-clamp
conditions, support the above observations (Fig. 1). The I&, values of a range of antagonists determined in dissociated adult rabbit nodose ganglion neurons, were considerably lower than those found in the corresponding nearOIW isolated from the guineapie*. Additionally, the 5-I-IT3 receptor-evoked current in mouse nodose ganglion neurons is potently antagonized by (+)tubocurarine35. A high sensitivity to (+)-tubocurarine rn~v typify the ‘mouse 5-HT3 receptoi, this property now having been described in mouse hippocampa121, nodose ganglion35 and superior cervical ganglion34 neurons, several murine clonal cell linesUz1-29, and the cloned 5-I-IT&A8. The mechanism of the blockade is uncertain but, at Ieast in NlE115 neuroblastoma cells, it is voltage- and use-independent and unique to (+)-tubocurarine, other nicotinic receptor antagonists so far tested being ineffect&?. Additionally, in mouse and rat superior cervical ganglia=, antagonism by (+)-tubocurarine is surmountable and associated with a parallel rightward displacement of the dose-response curve to 2methyl5HT, suggestive of a competitive mechanism of action. However, in guinea-pig superior cervical ganglion neurons, where relatively high concentrations of (+)-tubocurarine are necessary to demonstrate blockade, a depression of the slope and maximum of the dos+response curve In the nodose is observedx. ganglion neurons of the rabbit, a transition from an apparently competitive to a noncompetitive blockade is reported to occur with increasing concentrations of (+)tubocurarine”. It is possible, by analogy with the nicotinic receptor of the motor endplate, that (+)tubocurarine exerts both receptorand ion channel-blocking actions. Whilst evidence for interspecies receptor 5-HT3 variation in properties is accumulating, intraspecies variation remains to be convincingly demonstrated. It seems likely, however, that differences in the characteristics of 5-I-lTs receptors within a species will eventually be found, in view of the diversity exemplified by other members of the ligandgated ion channel superfamily, such as the nicotinic, GABA* and
TiPS - October 1992 [Vol. 131
3%
I
-COtlWOl 2fWA/
glutamate receptors. Fragmentary evidence may already exist. The sensitivity of 5-Err3 receptors located on rabbit isolated preganglionic cervical sympathetic nerves36 and the terminals of cardiac sympathetic neNesjsr to blockade by tropanserin is considerably higher than that displayed by the 5-HTs receptors expressed by other neural elements in the same species. In mea-pig frontal cortical slices, PBG at low concentration (101~) is reported to activate a putative 5-HT,s receptor whose stimulation enhances the electcitally evoked release of [“I-I]5-HTss. Jn the FNS of this species, PBG is inactive at 5-H& receptoIs3354. Exceptionally low concentrations of 5-HT (EC& 0.4nM) and PBG @C, 0.64nna) are reported to augment a Ca*‘-dependent depolarization-evokedrelease of cholecystokinin-like immunoreactivity from rat brain synaptosomes, an effect insensitive to the 5-IiT1_like and S-I-IT2 receptor antagonist metitepine, but blocked by appropriate concentrations of tropisetron, ondansetron and tropanserin3*. Such EC& values for 5-HT. and PBG are at variance with the concentrations of these agonists required to activate S-I-IT, receptors in the rat peripheral nervouss~stem~~*.Finally, ~~orophenylbi~~de may discriminate between the 5-HTs receptors expressed by the closely related cell lines NlE-115 and NGlOg-15 acting as a full agonist in the former system, but as a partial agonist in the 1atterc’O. Clearly, these anomalous find-
r
control 200 pA 1
ings do not constitute proof of
intraspecies heterogeneity, but thev nonetheless require explanation. In this context, it is noteworthy that 5-HTsR-A sequences (amplified by the polymerase chain reaction) have been detected in mouse cortex, midbrain, spinal cord and heart, but not in mouse intestine, which might also be expected to express S-I-IT3 receptors. The possibility that the mouse intestine contains a S-I-IT3 subtype encoded by a separate gene deserves further investigation. 5-H’& receptors in the CNS It is only recently that electrophysiological techniques have been used to probe the characteristics of central 5-HT3 receptors. In embryonic mouse hippo~mp~ and striatal neurons in primary cell culture, 5-HT elicits three physiologically and pharmacologically distinct responses**. One of these, a rapidly activating and desensitizing inward current response, reversing in sign close to 0 mV and arising from an increase in membrane conductance, has been attributed to 5-HTs receptor activation. In hippocampal neurons, this response is mimicked by IL-methyl-5-HT, blocked completely by a low concentration (1nM) of tropisetron, and antagonized by metoclopramide (I& = 440nM) but not by ketanserin2*. Given the many extensive electrophysiological studies that have characterized the actions of 5-HT upon pyramidal neurons in hippocampal slices (Ref. 10 and refs therein) and the presence of
WIT3 recognition sites in this tissue*, it is at first sight surprising that this response had not been previously described. A possible reason for this has recently emerged. Recording intracellularly from Cl--loaded CA1 pyramidal neurons in rat hippocampal slices, Ropert and Guy”2 noted that, in addition to its welldocumented activation of an inwardly rectifying K+ channel (via G protein-coupled 5-HTr, receptor#O), 5-HT also increased the frequency of spontaneous synaptic events. The effect of 5-HT upon the frequency of postsynaptic potentials persisted in the presence of NMDA and AMPA/kainate receptor antagonists, but was abolished by the GABAA receptor antagonist bicuculline or by loading the neurons with poorly permeant anions. A facilitation of GABA* receptormediated inhibitory postsynaptic potentials (IPSPs) by 5-I-IT thus seemed likely_ In tetrodotoxin @IX)-treated slices, the (miniature) IPSP amplitude distribution was unimodal and 5-HT affected neither IPSP amplitude nor frequency. In control slices, the distribution of IPSP amplitudes was broadly similar with a mode of approximately 1mV. By contrast, in the presence of 5-HT the amplitude dis~bution was bimodal with peaks occurring at around 1 and 4mV. The interpretation of these data provided by the authors42 is that 5-HT elicits IPSPs of higher quantal content than in control, most probably as a result of the excitation of previously quiescent in-
TiPS - October 1992 Wol. 131 hibitory GABAergic interneurons. Significantly, the effect of 5-HT upon IPSPs (but not the postsynaptic conductance increase registered in the impaled CA1 neuron) was blocked by low concentrations (l-90nM) of tropisetron and mimicked by a-methyl5-HT, suggesting the involvement of 5-HT, receptors. A GABA-releasing action of 5-I-IT3 receptor activation could, in principle, explain the recently inhibitory reported effect of selective 5-HT, receptor activation upon the F-evoked release of [3H]acetylcholine and endogenous noradrenaline from slices of rat entorhinal cortex43 and respectively. It is hypothalamu&, significant that the inhibition of [3H]acetylcholine release . blocked by TTX (N. Barnes, per: commun.), consistent with the notion that 5-HT3 receptor stimulation excites inhibitory, perhaps GABAergic, intemeurons. In this context, it is noteworthy that 2methyl-5-HT inhibits the NMDAinduced firing of nociceptive spinal projection neurons located in the dorsal hom45. Such inhibition is blocked both by 5-HT3 (zacopride) and GABAA (bicuculline) receptor antagonists, again suggesting the excitation of inhibitory interneurons as a consequence of 5-HT3 receptor stimulation. Both pre- and postsynaptic effects attributed to the activation of 5-HT3 receptors have recently been reported to occur in brain slice preparations containing the nucleus tractus solitarius (NTS). In whole-cell recordings from NTS neurons, a rather high concentration (100 PM) of 2-methyl-5-HT evoked a TTX-insensitive depolarizing response associated with an increase in membrane conductance4’j. This effect was reduced by tropisetron (0.01-1.0 PM) or tropanserin (1 PM). An additional action, observed with lower cnn2-methyl-5-HT centrations of (0.25-~PM), was an increase in the frequency and amplitude of excitatory and inhibitory spontaneous postsynaptic potentials (sPSPS)~~. Unlike results obtained from the hippocampus, there was a modest increase in sPSP frequency in the presence of TTX (P. Brooks, pers. commun.). The further addition of Co*+, to reduce Ca’+-dependent transmitter re-
lease from presynaptic terminals, blocked this effect. However, the antagonism by Co2+ of 5-HT3 receptor-mediated currents in neuronal cell linesI’ may complicate the interpretation of the latter experiment. In contrast to its augmentation of sPSP amplitude, 2-methyl+ HT, over an identical concentration range, depressed the amplitude of EPSPs and IPSPs evoked by electrical stimulation of the afferent fibres of the solitary tract& (P. Brooks, pers. commun.). All of the effects of 2-methyl-5-I-IT upon postsynaptic potentials were antagonized by either tropisetron (10 nM) or tropanserin (10 PM), but were resistant to blockade by ritanserin (1ph.Q (P. Brooks, pers. commun.). One explanation of these data might be that the activation of presynaptically located 5-HT3 receptors produces a depolarization that facilitates spontaneous neurotransmitter release, but presynaptically inhibits evoked neurotransmitter release (Ref. 46 and P. Brooks, pers. commun.). Although the NTS is innervated by 5-HT-releasing neurons from the medial raphC nucleus, and ~-I-ITS receptors are located both pre- and postsynaptically in the NTS, 5-HT3 receptor-mediated postsynaptic potentials ii1 response to electrical stimulation were not detected (P. Brooks, pers. commun.). However, such events have been demonstrated to occur in rat lateral amygdala neurons in brain slices, by the use of conventional intracellular and whole-cell recording techniques’. In a proportion of neurons within this nucleus, electrical stimulation of presynaptic fibres caused the appearance of fast EPSPs resistant to blockade by NMDA, AMPAI kainate and, with the notable exception of (+)-tubocurarine, nicotinic receptor/channel blockers. Such EPSPs were of brief duration (tens of milliseconds) and insensitive to spipirone and ketanserin (at concentrations sufficient to abolish 5-HT2 receptor-mediated excitatory response:s in other brain regions), but were reduced in amplitude, in a concentrationdependent manner, by the ~-I-ITS receptor antagonists GR67330 (l300nM), tropisetron (3-300 nM), and ondansetron (30 nM-10 PM). Although the concentrations of these antagonists necessary to
achieve blockade are somewhat higher than might be anticipated from their Km, Ki or Kd values at 5-H’T3 recognition sites (determined from radioligand binding studies performed on rat cortical tissue’), selectivity of action was clearly demonstrated. GR67330 had no effect upon synaptic potentials mediated by excitatory amino acids or GABA, even at a concentration lOO-fold higher than that producing a 50% depression of the EPSP tiuoked by 5-HT3 receptor stimulation. Further evidence for the involvement of 5-I-IT includes the mimicry of the EPSP by ionophoretically applied 5-W, its augmentation by the 5-HT uptake inhibitor fluoxetine, and its suppression by superfused 5-H’P. The latter might occur via the rapid desensitization that typifies electrical responses mediated via 5-I-IT3 receptors1*,1621 and/or as a consequence of receptor occlusion. Like 5-HT3 receptor-evoked electrical responses recorded from neurons14,*9Z and peripheral neuronal clonal ceil line!?, the 5-I-ITS receptor-mediated excitatory postsynaptic current reversed polarity at a potential close to 0 mV, suggesting similarities in electrogenesis iYig. 2). these electroCollectively, physiological studies point to the existence of pre- and postsynaptic central 5-HT3 receptors regulating release and neurotransmitter rapid ionotropic mediating neumtransmission, respectively. Furthermore, they indicate substantial commonality in the functioning of the receptor-channel complex in the CNS, peripheral neurons, and neuronal clonal cell lines. This is to be contrasted with several recent reports by Wang, Ashby and their colleagues (summarized in Ref. 47) that have suggested the existence of a ‘5-I-ITS-like’ receptor, possibly coupled to phospholipase C, that mediates a non-desensitizing inhibition of rat medial prefrontal cortical neurons in vivo. While the pharmacology of both the suppressant action of 5-HT receptor-selective and 5-I-IT3 agonists upon the firing rate of such neurons, and the stimulation of phosphoinositide turnover in fronto-cingulate and entorhinal cortical slices in vitro, is entirely consistent with 5-I-& receptor
TiPS - October 2992 [Vol. 131
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[email protected], receptorsmetIMe fast excitatorypostsynapticpotentials (EPSPs) in rat .&era/ amyg&la neurwx? in vitro.a: Synapfic potentials evoketi by brief focal electrical s6muMon of the twsoiateml twa’eua are blocketi by GR67330. b: Depolarizing v to &wphorelkzaf& applied 5-HT mimic the EPSP and are blocked by tropMmn_ Reproduced, with permission,from Ref. 9. (Copyright,Cell Press.)
mediation, the purportedly direct nature of these responses requires further investigation.
Modulators of 5-HTs receptor function
Recent reports indicate that ethanol and the dissociative anaesthetic ketamine act to potentiate electrical responses elicited by 5-HTs receptor activation, but their mechanism(s) of action are unknown. in a subset of NCB20 cells and rat nodose ganglion neuronP, concentrations of ethanol within the range producing acute intoxication in man, reversibly enhance submaximal inward current responses evoked by 5-HT, receptor stimulation. This observation is intriguing in view of the reported ability of 5HT, receptor antagonists to reduce alcohol consumption in animals and their suppressant effect upon aversive behaviours triggered by ethanol withdrawa13. Ketamine (3-30~) produces a concentration-dependent enhancement of inward current responses mediated by 5-HT3 receptors in rabbit nodose ganglion neurons, via a mechanism distinct from suppression of 5-I-U uptake”. The generality of this effect and its physiological relevance remain to be explored. q
0
cl
The 5-HTJ receptor is now firmly established as a member of
the ligand-gated ion channel superfamily and, like others in this group, mediates rapid synaptic transmission in the mammalian CNS. The latter action is unique among the amine neurotransmitters, and contrasts with the relatively slow inhibitory synaptic potentials mediated by the G protein-linked 5-HTI, receptors”. Additionally, there is evidence that 5-I-IT3 receptor activation may modulate the release of a variety of CNS neurotransmitters includin~ acetylcholine43, cholecystokinin , dopamine6, GABA4’ and noradrenalineU. Assuming such actions to be physiologically re!evant, these observations may eventually provide a rational basis for the various purported psychoactive properties of 5-HT3 receptor antagonists3. The precise mechanism(s) by which 5-HT3 receptor activation modulates transmitter release is uncertain, but it is parsimonious to assume that enhanced release is the result of nerve terminal depolarization arising from either the stimulation of 5-HT3 receptors resident upon nerve terminals, or increased impulse flow generated by the activation of somatically located receptors. The blockade, or reduction, by TTX of 5-HT3 receptor-mediated release of GABAQ and dopamine6 suggests that impulse propagation plays a role in at least some circumstances. However, presynaptically located receptors must presumably
account for 5-HT3 receptor-mediated release of cholecystokininlike immunoreactivity from rat brain synaptosomes3’. Apart from providing a depolarizing stimulus to activate voltage-sensitive Ca2+ channels, 5-HT3 receptor stimulation might result in Ca” influx through the ion channel complex itselP4,16. Inhibition of evoked transmitter release, as in the case of noradrenaline@ and acetylcholinea, might best be explained as occurring secondary to enhanced release of inhibitory transmitter substances, or as the result of a depolarizing presynaptic block. While species differences in the pharmacological characteristics of 5-HT3 receptors now seem certain, intraspecies heterogeneity remains to be convincingly demonstrated. The cloning of a 5-HT3 receptor subunit seems certain not only to broaden our knowledge of 5-HT3 receptor function, but also to generate a new approach to the identification of receptor subtypes that will not be hampered by the possible lack of discriminatory ligands. Acknowledgements Work performed in the authors’ laboratory is supported by the Wellcome Trust. We thank Drs P. Brooks and J. Yakel and their colleagues for permission to quote their unpublished observations. References 1 Gaddum, J. H. and Picarelli, Z. P. (1957) Br. J. Phaimncol. 12.323-328 Fozard, J. R. (1984) Neuropharmacology 23,1473-1486 Costall, B., Navlor, R. 1. and Tvers, M. B. (1990) Pharm&ol. The;. 47,18i-202 Kilpatrick, G. J., Jones, B. J. and Tyers, M. 8. (1987) Nature 330, 746-748 Waeber. C., Dixon, K., Hover, D. and Palacios, J. M. (1988) Eur. J: Pharmacol. 151,351-352 Blandina, P., Goldfarb, J. and Green, J. P. (1988) Eur. 1. Pharmacol. 155. 349-350 ’ . Fozard, J. R. in Proceedings of Int. Acad. Drug Res. Workshop on Serotonin Receptor Subtypes (Langer, S. Z., ed.), Birkhauser (in press) Maricq, A. V., Peterson, A. S., Brake, A. J., Myers, R. M. and Julius, D. (1991) Science 254, 432-437 9 Sugita, S., Shen, K-Z. and North, R. A. (1992) Neuron 8,199-203 10 Bobker, D. H. and Williams, J. T. (1990) TrevrdsXeurosci. 13,169-173 11 Higashi, S. and Nishi, S. (1982) 1. Physiol. 323, 543-567 12 Neijt, H. C., Te Duits, I. J. and Vijverberg, H. P. M. (1988) Neuropharmncology 27,301-317
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13 ‘.‘akel, J. L., Shao, X. M. and Jackson, M. B. (1991) J. Pension. 436,29>308 14 Yang, J., Mathie, A. and Hille, B. (1992) J. Physiol. 448, 237-256 15 Neijt, H. C., Plomp, J. J. and Vijverberg, H. I’. M. (1989) J. Physiol. 411,257-269 16 Yang, J. (1990) J. Gen. Physiol. %, 1177-1198 17 Yakei, J. L., Shao, X. M. and Jackson, M. B. (1990) Brain RPS. 533,46-52 18 Furukawa, K., Akaike, N., Onodera, H. and Kogure, K. (1992) J. Neurophysiol. 67,812-819 19 Malone, H. M., Peters, J. A. and Lambert, J. J. (1991) SW. Neurosci. Abs. 17, 239.11 20 Hille, B. (1992) Ionic Channels of Excitable Membranes, 2nd edn, Sinauer 21 Yakel, J. L. and Jackson, M. B. (1988) Neuron 1,61!?-621 22 Derkach, V., Surprenant, A. and North, R. A. (1989) Nature 339,70&709
23 Lambert, J. J., Peters, J. A., Hales, T. G. and Dempster, J. (1989) Br. 1. Pharmacol. 97,27-40 24 Shao, X. M., Yakel, J. L. and Jackson, M. B. (1991) f. Neurophysjo~~ 65,630-638 25 Peters, J. A., Malone, H. M. and Lambert, J. J. (1991) in Serotonin: Molecular Biology, Receptors and Functional Effects (Fozard, J. R. and Saxena, P. R.,
eds), pp. 84-94, Birkhauser 26 Malone, H. M., Peters, J. A. and Lambert, J. J. (1991) Ne~ropeptides 19 (suppl.), 25-30 27 Surprenant, A., Matsumoto, S. and Gerzanich, V. (1991) Sot. Neurosci. Abs. 17,239.12 28 Peters, J. A. and Lambert, J. J. (1989) Trends Pku~aco~. Sci. 10, 172-175 29 Peters, J. A., Malone, H. M. and Lambert, J. J. (1990) Neurosci. Left. 110, 107-112 30 Peters, J. A., Hales, T. G. and Lambert, J. J. (1988) Eur. J. Pharmacol. 151,491-495 31 Revah, F. et al. (1991) Nature 353, 846-849 32 Butler, A. et al. (1990) Br. J. Pharrnacoi. 101,591-598
33 Ireland, S. J. and Tyers, M. B. (1987) Br. 1. Pharmacol. 90,229-238 34 Newberry, N. R., Cheshire, S. H. and Gilbert, M. J. (1991) BY. 1. Phar~aeo~.
102,61~20 35 Malone, H. M., Peters, J. A. and Lambert, J. J. (1991) Br. 1. PharmacoZ. 104, 68P 36 Elliot, I’. and Wallis, D. I. (1988) Naunyn-Schm~edebe~*s col. 338,608-615
Arch.
Pha~a-
37 Fozard, J. R. (1984) Naunyn-Schmiedeberg’s Arch. Pharmacol. 326, 36-44 38 Galzin, A. M., Poncet, V. and Langer,
S. 2. (1990) Br. J. F’harmacol. 100,307!’ 39 Paudice, I?. and Raiteri, M. (1991) Br. 1. Fharmacoi. 103,1790-1794 40 Boess, F. G., Septilveda, M. I., Lummis,
S. C. R. and Martin, I. L. (1992) Neuropharmacology 31,561-564 41 Yakel J. L., Trussed, L. c). and Jackson, M. B. (1988) J. Neurosci. 8, 1273-1285 42 Ropert, N. and Guy, N. (1991) J. Phys~o~. 441,121-136
43 Barnes, J. M., Barnes, N. M., Costall, B., Naylor, R. J. and Tyers, M. B. (1989) Nature 338, 762-763 44 Blandina, I’., Goldfarb,
J. and Green, J. P. (1991) in Serotonin: Mo~ecu~ur Biology, Receptors and Functio}la~ Effecfs (Fozard, J. R. and Saxena, P. R., eds), pp.
322-329, Birkhauser 45 Alhaider, A. A., Lei, S. 2. and Wilcox,
G. L. (1991) J. Neurosci. 11, 18Sl-1883 46 Glaum, S. R., Brooks, P. A., Murphy, S. M. and MilIer, R. J. (1990) J. Physiof.
438,253I’ 67 Wang, R. Y.. Ashby, C. R., Jr and E&:ards, E. (1991) in Serofonin: MO/ecutar Biology. Receptors and Functional Effects (Fozard, J- R. and Saxena, P. R.,
edsf, pp. 174-185, Birkhauser 48 Lovinger, D. M. and White, C. (1991) J. Pharmacol. Exp. Ther. 40,263-270 49 Peters, J. A., Malone, H. M. and
Toughs at the top The Billion-Dollar Battle:
Merck v Glaxo by Matthew Lynn, Heinemann, 1991. f 16.99 (244 paged 1SBN 0 434 43924 x Paris is ‘a city where gloomy, selfimportant waffle is treated as wisdom’. Base1 is ‘furtive and sly, as if it harboured some dark secret: a secret likely to corrupt and destroy all those who come close to it’. These rather oblique statements from the opening sections of this book, which deal successively with the events and personalities at the 1990 Meeting of the International Pharmaceutical Conference, the life of
Lambert, J. J- (1991) Br. J. Pka~acof. 103,1623-1625
GR67330: (+)1,2,3,9-tetrahydro-9-methyl3-[(5-methyl-lH-imidazol-4yI)methyl]4H-carbazol-4one tropisetron: [(1~,5ff~-S-me~yl-Sazabicyclo-(3.2.l.@ct-3ct-3cr-yljltf-indole-3carboqrlate tnqwse& lOrn,3a,5crH-tropan-3-yI-3,5dichlorobenzoate
Paracdsus, and the Valium-Roche saga of the 197Os, give notice of the idiosyncratic thesis the author subsequently develops, based on his analysis of the world’s two leading pharmaceutical companies, Merck and Glaxo. His message is simple: that the corporate attitudes of these giants are radically different and a reflection of the professional backgrounds and attitudes of their respective chief executives. In the red comer is Sir Paul Girolami of Glaxo, au accountant, ‘dedicated, passionate and ruthless’; in the green comer is Dr Roy Vagelos, a physician and ‘one of the most brilliant American s&ntists of his generation’. The battle is for ‘control of the world’s most
