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5-HT4 receptors: general pharmacology and control of ionic channels
230 that this was yet a third subtype which was called the alpha-2C receptor. A fourth pharmacologic subtype, alpha-2D, has been identified in bovine pineal gland and the rat submaxillary gland. To date, no consistent and significant difference in the affinity of agonists for these subtype has been clearly shown. By contrast, there are several antagonists that are 10- to 100-fold selective for at least one of the subtypes. The classical mechanism of action for alpha-2 adrenergic receptor is the inhibition of adenylyl cyclase. The alpha-2A,-2b and -2C subtypes have all been shown to attenuate cyclic AMP production in intact cells. It is now clear that there are additional signal transduction mechanisms (e.g. activation of K * channels) which actually may be more directly responsible for the alpha-2 adrenergic receptor-mediated physiological effects of the catecholamines. To date no receptor subtype selectivity has been demonstrated for alpha-2 receptor signal transduction mechanisms. The genes encoding for the alpha-2 adrenergic receptor subtypes in the human have been cloned, sequenced, and expressed. They are designated according to the human chromosomal localization as C-10, C-2 and C-4. The homologous gene/cDNA for the subtypes have been cloned from the rat. We have recently shown that the cloned human alpha-2 adrenergic receptor subtypes designated as alpha-2-C10, alpha-2-C2 and alpha-2-C4 have identical or very similar pharmacological characteristics to the pharmacologically defined alpha-2A, alpha-2B, and alpha-2C receptor subtypes, respectively.
References Bylund, D.B. (1988) Subtypes of 0t2-adrenoceptors: pharmacologicaland molecular biologicalevidence converge. TIPS 9, 356-361. Bylund, D.B. (1992) Subtypes of alpha-1 and alpha-2 adrenergic receptors. FASEB J. 6, 832-839. Bylund,D.B., Blaxall,H.S., Iversen,L.J., Caron,M.G.,Lefkowitz,R.J. and Lomasney,J.W. (1992) Pharmacologicalcharacteristicsof alpha-2 adrenergic receptors: comparisonof pharmacologicallydefined subtypeswith subtypesidentifiedby molecularcloning.Mol. Pharmacol., in press. Harrison, J.K., Pearson, W.R. and Lynch, K.R. (1991) Molecularcharacterizationof ¢x1- and ct~-adrenoceptors.TIPS 12, 62-67. Lomasney, J.W., Cotecchia, S., Lefkowitz, R.J. and Caron, M.G. (1991) Molecular biology of 0t-adrenergic receptors: implications for receptor classificationand for structure-functionrelationships.Biochim. et. Biophys. Acta 1095, 127-139. McGrath, J.C., Brown, C.M. and Wilson, V.G. (1989) Alpha-adrenoceptors:A critical review. Med, Res. Rev. 9, 407-533.
5-HT4 receptors: general pharmacology and control of ionic channels
Bockaert, j . t Fagni, L. 1, Ouadid, H. 2, Nargeot, j.2 and Dumuis, A. 1 1Centre CNRS - INSERM de Pharmacologie-Endocronologie, rue de la Cardonille, 34094 Montpellier Cedex 5, France ZCRBM - UPR 8402, INSERM U. 249, B.P. 5051, 34033 Montpellier Cedex 1, France Key words: Serotonin receptors; Ca 2. and K ÷ channels; cAMP; Neuronal cells; Human cardiomyocytes
Summary 5-HT4 receptors have a unique pharmacology. In addition to indole derivatives, two agonist classes have been discovered, benzamides and benzimidazolones. Classical 5-HT receptor antagonists are generally inactive on this receptor. Three antagonists have been described and derive from each class of agonist (ICS 205 930, SDZ 205 557, DAU 6285). 5-HT4 receptors increase cAMP formation and, depending on the cell, inhibit K ÷ channels (neurons) or activate Ca 2* channels (human cardiomyocytes).
Introduction Four years ago we described [3] in colliculi neurons a 5-HT receptor which stimulates cAMP production. Since the
231 pharmacology of this receptor was clearly different from the 3 classes of 5-HT receptors previously described (5-HTI, 5-HT2, 5-HT3), we named this receptor 5-HT4. There have been many discussions [2] concerning the existence or the non-existence of this receptor. However, the discovery of specific classes of 5-HT4 agonists and antagonists and its unique tissular and cellular location have finally convinced colleagues that this receptor does exist [I]. Three main primary transduction mechanisms are used by 5-HT receptors to trigger their cellular functions: (i) coupling to adenyl cyclase (5-HT1A, 5-HTIB, 5-HT1D, 5-HT4), (ii) coupling to phospholipase C (5-HT2, 5-HT1C), and (iii) the direct ionic permeability, the receptor being a channel itself (5-HT3). 5-I-1"1"4receptors are the only ones which are positively coupled to adenylyl cyclase. Therefore, depending on the cells, they will trigger functions controlled by cAMP. Two examples will be given here. Results
General pharmacology and cloning The general agonist potencies at the 5-HT4 receptor in rat oesophagus and mouse colliculi neurons are given in Table 1. Concerning the indoles, 5-methoxytryptamine is a potent agonist, as well as ~x-methyl-5-HT. In contrast, 5-carboxamidotryptamine and 2-methyltryptamine are weak agonists. (The benzamides (e.g., cisapride, renzapride, zacopride, metoclopramide) and benzimidazolones (e.g., BIMU-1, BIMU-8) are key ligands for the identification of 5-HT4 receptors. However, these drugs are potent 5-HT3 antagonists. Benzamides are either full agonists (colliculi neurons) or partial agonists (human heart). Three antagonists have been described: ICS 205 930 (pA2=6.5 at 5-HT4 receptors) having low affinity; the benzimidazdone DAU 6285 (pA2=6.8 and 6.0 at 5-HT4 and 5-HT3 receptors, respectively). The absence of very potent agonists or antagonists of 5-HT4 receptors and its likely original structure has delayed its molecular cloning. We (L. Journot) are now, in collaboration with P. Seeburg, trying to clone this receptor using an original expression system. Table 1. Agonist potency"at the 5-HT4 receptor in rat <>esophagealtunica musculads mucosae Agonist class ECR*
Intrinsic activity relative to 5-HT
Indoles
Oesophagus
Benzamides
Benzimidazolones
5-HTt BIMU-I BIMU-8 5-Methoxytryptamine# ct-Methyl-5-HT
1 1.6 2 2 10 12 12 21
Colliculi 1 3.6 0.7 1.0
Colliculi 1 0.7 1.2 1.0
0.7 7.1 1.2
1 0.7 0.9 1.0 1.0 0.8 0.9 0.8
Cisapride# (S)-Zacopride Renzapride (RS)-Zacopride
86
11.8
0.9
1.4
95 128 130
31 36.7 32.7
1.0 0.8 0.9
1
Metoclopramide (R)-Zacopride
1.0 1.0
0.5 < 0.5
5-Carboxamidotryptamine
Tryptamine Methyl-5-HT
Oesophagus
906 1548
> I00 > 100
1.4
1 1.3
1
* ECR-equi-effective concentration ratio (ECs0 test + ECso 5-HT). r ECso 5-HT in oesophagus preparation-8.2 and in colliculi neurons-7.0. Some workers report 5-melhoxytryptamine or cisapride or both to be weak antagonists relative to 5-HT.
Control of IC and Ca2÷ channels In coUiculi neurons, 5-1-I"1"4receptors block the voltage activated K" current [4]. This effect is mimicked by 8-bromo-cAMP and blocked by the 1-17 and PKI non-specific and specific inhibitors, respectively. The pharmacology of this 5-HT receptor is identical to that of the 5-HT4 receptor which stimulates cAMP in the same cells. This inhibition of K ÷ channels by 5-HT4 receptors may be implicated to neurotransmitter release and synaptic plasticity. In human cardiomyocytes, the 5-HT4 receptor is as potent as isoprenaline in increasing the Ca 2÷ current of the L-type [5]. This effect is blocked by PKI. The effects of cAMP and 5-HT are not additive. The pharmacology is similar but not identical to the pharmacology of 5-HT receptors controlling K ÷ channels in colliculi neurons. In particular, benzamides are weak partial
232 agonists on 5-HT4 receptors in human heart. Interestingly, 5-HT4 receptors are not expressed in rat, rabbit, guinea pig or frog myocytes. In conclusion, 5-HT4 receptors are likely to control many physiological functions via cAMP dependent protein kinase activations and phosphorylations of ionic channels. This receptor is the only well described 5-HT receptor which has resisted from being cloned so far.
References I. Bockaen, J., Fozard, J.R., Dumuis, A. and Clarke, D.E. (1992) The 5-HT4 receptor: A place in the sun, Trends Pharmacol. Sci. 12, 141-145. 2. Clarke, D.E., Craig, D.A. and Fozard, J.R. (1989) The 5-HT4 receptor: Naughty but nice, Trends Pharmacol. Sci. 10, 385-386. 3. Dumuis, A., Bouhelai, R., Sebben, M., Cory, R. and Bockaert, J. (1988) A non-classical 5-hydroxytryptamine receptor positively coupled with adenylate cyclase in the central nervous system, Mol. Pharmacol. 34, 880-887. 4. Fagni, L., Dumuis, A., Sebben, M. and Bockaert, J. (1991) The 5-HT4 receptor subtype inhibits K ÷ current in coUiculi neurons via activation of a cAMP-dependent protein kinase, Br. J. Pharmacol. 105, 973-979. 5. Ouadid, H., Seguin, J., Dumuis, A., Bockaert, J. and Nargeot, J. (1992) Serotouin increases Ca2~ current in human atrial myocytes via the newly described 5-HT4 receptors, Mol. Pharmacol. 41,346-351.
Abstract not received
The selectivity of zolpidem and aipidem for the ~l-subunit of the G A B A A receptor
Langer, S.Z. 1, Faure-Halley, C. 1, Seeburg, p.2, Graham, D. 1 and Arbilla, S. 1 tSynthdlabo Recherche (L.E.KS.), 31, ave. Paul Vaillant Couturier, 92220 Bagneux, France 2Universita't Heidelberg, Feld 282, 6900 Heidelberg, Germany Key words: GABA A receptor, Omega modulatory sites, Zolpidem, Alpidem, Triazolam, Zopiclone
Summary The pharmacological profile of recombinant txl132),2 and ct5152~/2GABA A receptors was investigated using 3H-flumazenil as a probe and compared to that exhibited by native GABA A receptors present in rat cerebellum and spinal cord. Diazepam, zopiclone and triazolam did not differentiate between these various GABA a receptors. In contrast, the imidazopyridines zolpidem and alpidem showed high affinity for the cerebellar GABA a receptor, intermediate affinity for the spinal cord receptor, but no affinity for the 0t.5~2Y2 recombinant foma of the receptor. Two subpopulations of omega (t0) or benzodiazepine binding sites coI and co2 that are allosterically coupled to the GABA recognition site of the GABA x receptor were initially proposed on the basis of their pharmacological profile and distinctive distribution in the brain (Langer and Arbilla, 1988). The o~l modulatory site was distinguished by its high affinity for drugs such as CL 218,872, 0-CCE and the imidazopyridines, alpidem and zolpidem and its enrichment in the substantia nigra, cerebellum molecular layer and cortex layer IV. In contrast, the co2 modulatory site was defined as the binding site that did not exhibit high aff'mity for t~l selective compounds and which was notably enriched in the spinal cord. Available evidence now suggests more extensive GABA A receptor heterogeneity. The recent cloning of cDNAs encoding GABA A receptor
References Bylund, D.B. (1988) Subtypes of 0t2-adrenoceptors: pharmacologicaland molecular biologicalevidence converge. TIPS 9, 356-361. Bylund, D.B. (1992) Subtypes of alpha-1 and alpha-2 adrenergic receptors. FASEB J. 6, 832-839. Bylund,D.B., Blaxall,H.S., Iversen,L.J., Caron,M.G.,Lefkowitz,R.J. and Lomasney,J.W. (1992) Pharmacologicalcharacteristicsof alpha-2 adrenergic receptors: comparisonof pharmacologicallydefined subtypeswith subtypesidentifiedby molecularcloning.Mol. Pharmacol., in press. Harrison, J.K., Pearson, W.R. and Lynch, K.R. (1991) Molecularcharacterizationof ¢x1- and ct~-adrenoceptors.TIPS 12, 62-67. Lomasney, J.W., Cotecchia, S., Lefkowitz, R.J. and Caron, M.G. (1991) Molecular biology of 0t-adrenergic receptors: implications for receptor classificationand for structure-functionrelationships.Biochim. et. Biophys. Acta 1095, 127-139. McGrath, J.C., Brown, C.M. and Wilson, V.G. (1989) Alpha-adrenoceptors:A critical review. Med, Res. Rev. 9, 407-533.
5-HT4 receptors: general pharmacology and control of ionic channels
Bockaert, j . t Fagni, L. 1, Ouadid, H. 2, Nargeot, j.2 and Dumuis, A. 1 1Centre CNRS - INSERM de Pharmacologie-Endocronologie, rue de la Cardonille, 34094 Montpellier Cedex 5, France ZCRBM - UPR 8402, INSERM U. 249, B.P. 5051, 34033 Montpellier Cedex 1, France Key words: Serotonin receptors; Ca 2. and K ÷ channels; cAMP; Neuronal cells; Human cardiomyocytes
Summary 5-HT4 receptors have a unique pharmacology. In addition to indole derivatives, two agonist classes have been discovered, benzamides and benzimidazolones. Classical 5-HT receptor antagonists are generally inactive on this receptor. Three antagonists have been described and derive from each class of agonist (ICS 205 930, SDZ 205 557, DAU 6285). 5-HT4 receptors increase cAMP formation and, depending on the cell, inhibit K ÷ channels (neurons) or activate Ca 2* channels (human cardiomyocytes).
Introduction Four years ago we described [3] in colliculi neurons a 5-HT receptor which stimulates cAMP production. Since the
231 pharmacology of this receptor was clearly different from the 3 classes of 5-HT receptors previously described (5-HTI, 5-HT2, 5-HT3), we named this receptor 5-HT4. There have been many discussions [2] concerning the existence or the non-existence of this receptor. However, the discovery of specific classes of 5-HT4 agonists and antagonists and its unique tissular and cellular location have finally convinced colleagues that this receptor does exist [I]. Three main primary transduction mechanisms are used by 5-HT receptors to trigger their cellular functions: (i) coupling to adenyl cyclase (5-HT1A, 5-HTIB, 5-HT1D, 5-HT4), (ii) coupling to phospholipase C (5-HT2, 5-HT1C), and (iii) the direct ionic permeability, the receptor being a channel itself (5-HT3). 5-I-1"1"4receptors are the only ones which are positively coupled to adenylyl cyclase. Therefore, depending on the cells, they will trigger functions controlled by cAMP. Two examples will be given here. Results
General pharmacology and cloning The general agonist potencies at the 5-HT4 receptor in rat oesophagus and mouse colliculi neurons are given in Table 1. Concerning the indoles, 5-methoxytryptamine is a potent agonist, as well as ~x-methyl-5-HT. In contrast, 5-carboxamidotryptamine and 2-methyltryptamine are weak agonists. (The benzamides (e.g., cisapride, renzapride, zacopride, metoclopramide) and benzimidazolones (e.g., BIMU-1, BIMU-8) are key ligands for the identification of 5-HT4 receptors. However, these drugs are potent 5-HT3 antagonists. Benzamides are either full agonists (colliculi neurons) or partial agonists (human heart). Three antagonists have been described: ICS 205 930 (pA2=6.5 at 5-HT4 receptors) having low affinity; the benzimidazdone DAU 6285 (pA2=6.8 and 6.0 at 5-HT4 and 5-HT3 receptors, respectively). The absence of very potent agonists or antagonists of 5-HT4 receptors and its likely original structure has delayed its molecular cloning. We (L. Journot) are now, in collaboration with P. Seeburg, trying to clone this receptor using an original expression system. Table 1. Agonist potency"at the 5-HT4 receptor in rat <>esophagealtunica musculads mucosae Agonist class ECR*
Intrinsic activity relative to 5-HT
Indoles
Oesophagus
Benzamides
Benzimidazolones
5-HTt BIMU-I BIMU-8 5-Methoxytryptamine# ct-Methyl-5-HT
1 1.6 2 2 10 12 12 21
Colliculi 1 3.6 0.7 1.0
Colliculi 1 0.7 1.2 1.0
0.7 7.1 1.2
1 0.7 0.9 1.0 1.0 0.8 0.9 0.8
Cisapride# (S)-Zacopride Renzapride (RS)-Zacopride
86
11.8
0.9
1.4
95 128 130
31 36.7 32.7
1.0 0.8 0.9
1
Metoclopramide (R)-Zacopride
1.0 1.0
0.5 < 0.5
5-Carboxamidotryptamine
Tryptamine Methyl-5-HT
Oesophagus
906 1548
> I00 > 100
1.4
1 1.3
1
* ECR-equi-effective concentration ratio (ECs0 test + ECso 5-HT). r ECso 5-HT in oesophagus preparation-8.2 and in colliculi neurons-7.0. Some workers report 5-melhoxytryptamine or cisapride or both to be weak antagonists relative to 5-HT.
Control of IC and Ca2÷ channels In coUiculi neurons, 5-1-I"1"4receptors block the voltage activated K" current [4]. This effect is mimicked by 8-bromo-cAMP and blocked by the 1-17 and PKI non-specific and specific inhibitors, respectively. The pharmacology of this 5-HT receptor is identical to that of the 5-HT4 receptor which stimulates cAMP in the same cells. This inhibition of K ÷ channels by 5-HT4 receptors may be implicated to neurotransmitter release and synaptic plasticity. In human cardiomyocytes, the 5-HT4 receptor is as potent as isoprenaline in increasing the Ca 2÷ current of the L-type [5]. This effect is blocked by PKI. The effects of cAMP and 5-HT are not additive. The pharmacology is similar but not identical to the pharmacology of 5-HT receptors controlling K ÷ channels in colliculi neurons. In particular, benzamides are weak partial
232 agonists on 5-HT4 receptors in human heart. Interestingly, 5-HT4 receptors are not expressed in rat, rabbit, guinea pig or frog myocytes. In conclusion, 5-HT4 receptors are likely to control many physiological functions via cAMP dependent protein kinase activations and phosphorylations of ionic channels. This receptor is the only well described 5-HT receptor which has resisted from being cloned so far.
References I. Bockaen, J., Fozard, J.R., Dumuis, A. and Clarke, D.E. (1992) The 5-HT4 receptor: A place in the sun, Trends Pharmacol. Sci. 12, 141-145. 2. Clarke, D.E., Craig, D.A. and Fozard, J.R. (1989) The 5-HT4 receptor: Naughty but nice, Trends Pharmacol. Sci. 10, 385-386. 3. Dumuis, A., Bouhelai, R., Sebben, M., Cory, R. and Bockaert, J. (1988) A non-classical 5-hydroxytryptamine receptor positively coupled with adenylate cyclase in the central nervous system, Mol. Pharmacol. 34, 880-887. 4. Fagni, L., Dumuis, A., Sebben, M. and Bockaert, J. (1991) The 5-HT4 receptor subtype inhibits K ÷ current in coUiculi neurons via activation of a cAMP-dependent protein kinase, Br. J. Pharmacol. 105, 973-979. 5. Ouadid, H., Seguin, J., Dumuis, A., Bockaert, J. and Nargeot, J. (1992) Serotouin increases Ca2~ current in human atrial myocytes via the newly described 5-HT4 receptors, Mol. Pharmacol. 41,346-351.
Abstract not received
The selectivity of zolpidem and aipidem for the ~l-subunit of the G A B A A receptor
Langer, S.Z. 1, Faure-Halley, C. 1, Seeburg, p.2, Graham, D. 1 and Arbilla, S. 1 tSynthdlabo Recherche (L.E.KS.), 31, ave. Paul Vaillant Couturier, 92220 Bagneux, France 2Universita't Heidelberg, Feld 282, 6900 Heidelberg, Germany Key words: GABA A receptor, Omega modulatory sites, Zolpidem, Alpidem, Triazolam, Zopiclone
Summary The pharmacological profile of recombinant txl132),2 and ct5152~/2GABA A receptors was investigated using 3H-flumazenil as a probe and compared to that exhibited by native GABA A receptors present in rat cerebellum and spinal cord. Diazepam, zopiclone and triazolam did not differentiate between these various GABA a receptors. In contrast, the imidazopyridines zolpidem and alpidem showed high affinity for the cerebellar GABA a receptor, intermediate affinity for the spinal cord receptor, but no affinity for the 0t.5~2Y2 recombinant foma of the receptor. Two subpopulations of omega (t0) or benzodiazepine binding sites coI and co2 that are allosterically coupled to the GABA recognition site of the GABA x receptor were initially proposed on the basis of their pharmacological profile and distinctive distribution in the brain (Langer and Arbilla, 1988). The o~l modulatory site was distinguished by its high affinity for drugs such as CL 218,872, 0-CCE and the imidazopyridines, alpidem and zolpidem and its enrichment in the substantia nigra, cerebellum molecular layer and cortex layer IV. In contrast, the co2 modulatory site was defined as the binding site that did not exhibit high aff'mity for t~l selective compounds and which was notably enriched in the spinal cord. Available evidence now suggests more extensive GABA A receptor heterogeneity. The recent cloning of cDNAs encoding GABA A receptor