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The 5-HT7 receptor: orphan found
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The 5=HT,receptor: orphan found
Rilchard M. Eglen, Jeffrey R. Jasper, David J. Chang and Graeme R. Martin
Il. M. Eglen. Vice President and Director, J. R. Jasper, Research Scientist II, D. J. Chang. Research Manager, and 6. Il. Martin. Department Head, Center for Biological Research, Neurobiology Unit, Roche Bioscience. Palo Alto, CA 94304. USA.
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In the 40 or more years since serotonin was first isolated and identified as 5-hydroxytryptamine (5-HT)l, perceptive operational studies have shown that the monoamine elicits a complex array of pharmacological and physiological responses by acting at a diversity of 5-I-IT receptors. Many of these receptors are now widely recognized as important targets for therapeutics2. Whilst the operational characteristics of these 5-m receptors have served for many years as the primary basis for their differentiation, gene cloning techniques now enable the primary structure of the receptors to be determined; this information, together with the operational data, enables unambiguous classification3P. However, a number of functional receptors for 5-m have been described for which a corresponding gene product has yet to be identified. In the absence of complementary structural data these remain ‘orphans’ of the present IUPHAR (International Union of Pharmacology) scheme for classifying and naming 5-HT receptorsd. Conversely, gene products that encode 5-HT receptor proteins have been identified for which an endogenous physiological correlate has yet to be defined. These are identified by a lower case appellation, as in the case of 5-htlE,5-htlp 5-h&*,5-h&and, until recently, 5-ht, gene products. When evidence for endogenous expression and function of the proteins is obtained, they are allocated an upper case notation denoting that they are fully defined transmitter receptors. The most recent orphan 5-HT receptor to achieve ‘upper case’ status is the 5-I-IT, receptor. This adoption was based on the close identity between the operational profile of the 5-HT, gene product expressed in mammalian cells and a long recognized 5-HT receptor, previously designated ‘5-I-IT&e’, which is positively coupled to adenylate cyclase in a variety of neuronal and
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non-neuronal tissuess7. This article briefly reviews the characteristics of endogenous and recombinant 5-HT, receptor systems and highlights emerging new data on the molecular biology of this unique receptor class.
Molecular biology The predicted amino acid sequence of the 5-HT, receptor protein is unique, with low (<40%) overall homology with other 5-HT receptors&15.Like the 5-HT, and putative 5-h& receptors, it preferentially couples to adenylate cyclase via G,. The 5-HT, receptor represents a mammalian homologue of the 5-HT, Drosophila (5-HTdr,,l)receptor (overall homology = 57%), with divergence from a primordial 5-HT receptor predating the evolution of adrenoceptors, muscarinic and dopamine receptorsl6. The receptor gene, located on human chromosome lOq23.3q24.4 (Ref. 17), has two introns in the coding region, one located after the third transmembrane domain and the other close to the C-terminus. In the rat, a five base insertion (GTAAG) at the carboxy region intron introduces an in-frame stop codon, resulting in a 13 amino acid truncated splice variant9 (Fig. 1). Recent studies have identified a clone from a human placental cDNA library that contains the identical five base insertion at the carboxy intron site which results in a similar truncated gene productl5. Consistent with NC-IUPHAR nomenclature guidelines’s the human receptor variants can be named h 5-HT,,,, (long form) and h 5-HT,,, (short form), denoting the order of their discovery. Radioligand binding studies show that in terms of the available ligands, the pharmacology of the h 5-HT,, receptor stably expressed in HEK293 cells, resembles that of the h 5-HT,,,, receptor expressed in Cos7 cell@. Likewise, the pharmacology of the rat short and long variants of the receptor is
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similar (see Table 1). Interestingly, preliminary reverse transcription polymerase chain reaction (RT-PCR) studies in the rat have identified two further novel isoforms of the 5-HT, receptor arising from the insertion of an additional exon which is different in each case (Ref. 19). These novel isoforms also couple to Gs to stimulate adenylate cyclase and reveal no differences in their operational response to 5-HT (Ref. 19). Assuming that they have a physiological role, these isoforms would be named r 5-HT7,,, and r 5-HT,(,, receptor variants.
Pharmacology The recombinant 5-HT, receptor can be radiolabelled with [3H]5-HT, [3H]5_carboxamidotryptamine ([3H]5-CT) or [luI]D-lysergic acid diethylamide ([lEI]LSD). Competition binding studies demonstrate a unique pharmacological profile with good agreement of ligand affinities across several species (Table 1). Operationally, the recombinant receptor is defined by high affinities for 5-CT, LSD, methiothepin, mesulergine and methysergide, as well the antipsychotics clozapine and loxapi&J. Furthermore, (k)B-hydroxy-2dipropylaminotetralin @-OH-DPAT), generally considered a selective agonist for the 5-I-IT,, receptor, can act in some systems as a partial agonist at 5-HT, receptors8-15.In all recombinant systems studied to date, activation of the 5-I-IT,receptor augments intracellular adenylate cyclase activity, with no evidence for coupling of the receptor to other intracellular signalling mechanisms. Operational characterization of the 5-HT7 receptor in recombinant systems has provided an unambiguous profile of ligand affinities. A similar profile is obtained at the orphan 5-HT receptor, previously classified as ‘5-HT,-like’, that mediates smooth muscle relaxation in a variety of tissue@ (Table 2). First characterized in the guinea-pig isolated ileum and cat saphenous vei$*-23, this receptor has subsequently been found in the neonatal pig vena cava24,E,the rabbit jugular26
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I
1 Guin pig MOUSe Rat-long Rat-short Hum-long Hum-short
Guin pig Mouse
Pat-long Rat-short Hum-long Hum-short
Guin pig Mouse Rat-long Rat-short Hum-long Hum-short
MMGVNSSGRP MMDVNSSGRP bQ4DVNSSGRP MMDVNSSGRP MMDVNSSGRP MMDVNSSGRP
DLYGHLHSIL DLYGHLRSLI DLYGHLRSLI DLYGHLRSLI DLYGHLRSFL DLYGHLRSFL
LP..GRGLPD LPSVGRRLQD LPEVGRGLQD LPEVGRGLQD LPEVGRGLPD LPEVGRGLPD
WSPDGGADFG LSPDOOAHSV LSPDGGAHPV LSPXGAHPV LSPDGGADPV LSPDGGADPV
VSTWTPRLLS VSSWMPHLLS VSSWMPHLLS VSSmPHLLS AGSWAPHLLS AGSWAPHLLS
GVPBVAASPS GFPEVTASPA GFLEVTASPA GFLEVTASPA . ..EXl'ASPA . ..EVTASPA
PSWEGTNDNV PTWDAPPDNV PTWDAPPDNV PlWDAPPDNV PTWDAPPDNA PTWDAPPDNA
Ul GNCLWISVC GNCLWISVC GNCLWISVC GNCLWISVC GNCLWISVC GNCLWISVC
FVKKLRQPSN FVKNVRQPSN FVKKLRQPSN FVKKLRQPSN FVKKLRQPSN FVKKLRQPSN
YLIVSLALAD YLIVSLALAD YLIVSLALAD YLIVSLALAD YLIVSLALAD YLIVSLALAD
II LSVAVAVMPF LSVAVAVMPP LSVAVAVMPF LSVAVAVMPF LSVAVAVMPF LSVAVAVMPF
VSVTDLIGGK VSVTDLIGGK VSVTDLIGDK VSVTDLIGGK VSVTDLIGGK VSVTDLIGGK
WIFGHFPCNV WIFGHFFCNV WIFGHFFCNV
201 CMPKMILSVW CMAKMILSVW C!MaKMILSVW CMAKMILSVW CMAKMILSVW CNFdZMILSVW
IV LLSASITLPP PLSASITLPP LLSASITLPP LLSASITLPP LLSASITLPP LLSASITLPP
LFGWAQNVND LFGWAQNVND LFGWAQNVND LFGWAQNVND LFGWAQNVND LFGWAQNVND
DKVCLISQDF DKVCLISQDF DKVCLINQDF DKVCLISQDF DKVCLISQDF DKVCLISQDF
GYTIYSTAVA GYTIYSTAVA GYTIYSTAVA GYTIYSTAVA GYTIYSTAVA GYTIYSTAVA
FYIPNSVMLF FYIPMSVMLF FYIPMSVMLF
LLKHERKNIS LLKHERKNIS LLKH?XRXNIS LLKHERKNIS LLKHERIUiIS LLKHEFJNIS
IFKREQKAAT SFKREQKAAT IFKREZQKAAT IFKREQKAAT IFKREQKAAT IFKREQKAAT
TLGIIVGAFT TLGIIVGFbFT TLGIIVGAFT TLGIIVGAFT TLGIIVGAFT TLGIIVGAFT
VI VCWLPFFLLS VCWLPFFLLS VCWLPFFPLS VCWLPFFLLS VCWLPFFLLS VCWLPFFLLS
Rat-short Hum-long Hum-short
ALKLAERPBR ALKLARRPER ALKLANRP RR AL-PER
PECVLQNSDY SEFVLQNCDH SEFVLQNSDH SEWL*....
CRKKGHDS* eGKKGHDT* CGKKGHDT' .. . . .. . ..
PSFVLQNADY PEFVL'....
CRKKGHDS* . .._.....
SVSECANLSR EVEECATLSR SVSECANLSR EVEECANLSR EVEE'ZANLSR EVEECANLSR 401
Guin pig Mouse
Rat-long Rat-short Hum-long Hum-short
LTLITLLTIA LTLITLLTIA LTLITLLTIA LTLITLLTIA LTLITLLTIA LTLITLLTIA
FIAMDVMCCT FIAMDVMCCT FIAMDVMYCT FIAMDVMCCT FIAMDVMCCT FIAMDVMCCT
ASIMTLCVIS ASIMTLCVIS ASIMTLCVIS ASIMTLCVIS ASIMTLCVIS ASIMTLCVIS
IDRYLGITRP IDRYLGITRP IDRYLGITRP IDRYLGITRP IDRYLGITRP IDRYLGITRP
LTYPVRQNGK LTYFVRQNGK LTYPVSQNEK LTYPVRQNGK LTYPVRQNGK LTYPVRQNGK
FYIPMSVMLF FYIPMSVMLF
MYYRIYKAAR MYYQIYKAAR MYYQIYKAAR MYYQIYKAAR MYYQIYKAAR MYYQIYKAAR
FPRVQPESII KSAAKHKFSG FPRVQPESVI KSAAKHKFPG FPRVQPESVI KSAAKHKF PG FPRVQPESVI KS AAKHKFPG FPPS'EPDSVI
SLNGVVKLQK SLNGWKLQK ALNGIVKLQK
KSAAKHKFFG
FPRVSPDSVI
ALNGIVKLQK
TARPFICGTA TARPFICGTS TARPFICGTS TARPFICGTS TARPFICGTS TARPFICGTS
CSCIPLWVER CSCIPLWVER CSCIPLWVER CSCIPLWVER CSCIPLWVER CSCIPLWVER
TCLWLGYANS TCLWLGYANS TCLWLGYANS TCLWLGYANS TFLWLGYANS TFLWLGYANS
LINPFIYAFF LINPFIYSFF LINPFIYAFF LINPFIYAFF LINPFIYAFF LINPFIYAFF
NRDLRTTYRS NRDLRTTYRS NRDLRTTYRS NRDLRTTYRS NRDLRTTYRS NRDLRTTYRS
200
III
WIFGHFFCNV
WIFGHFFCNV WIFGHFFCNV
300
FYIPMSVMLF
KSAAKHKFPG
SUZMVKLQK SLNGVVKLQK
400
VII
449
LLQCQYRNIN RKLSAAGMHE LLQCQYPXIN RKLSAAGMHE LLQCQYRNINRKLSAAGMHB LLQCQYRNIN RKLSAAGMHE LLQCQYPXIN LLQCQYFNIN
RAEKWIGSI RVEKWIGSI RVEKVVIGSI RVEKVVIGSI RVEKVVIGSI RVEKVVIGSI
V
301 Guin pig Mouse Rat-long
RXLSAAGMHE ALKLAERPER RKLSAAGMHE ALKLAERPER
Fig. 1. Sequence alignment of predicted amino acid residues for 5-HT,receptors cloned to date. Guin pig, guinea-pig; hum, human.
and femoral veinz7, the Cynomolgus monkey jugular vein% and the beagle dog coronary artery7J9.Responses in other isolated tissues, although less well characterized, may also be mediated by 5-I-I& receptor activation. These tissues include the marmoset aorta3, the sheep pulmonary veinai, the porcine pial vein32 and the guinea-pig trachea33. Moreover, in pig vena cavaz6, homogenates of guinea-pig hippocampus and smooth muscle cells from human uterine artery34, 5-HT receptor activation stimulates the accumulation of CAMPconsistent with coupling to Gs. As in recombinant systems, there is a high degree of correspondence of ligand affinities at these endogenous 5-HT, receptors, implying that the ligand recognition sites are highly conserved between species (Table 2). Notably, however, the affinity values for agonists, such as 5-CT or 5-HT, derived from binding studies
1OQ
SGCGEQINYG SGCGEQINYG SGCGEQINYG SGCGKQINYG SGCGEQINYG SGCGEQINYG
are greater than the potencies seen in functional studies. The reason for this difference is unclear but suggests caution in the use of radiolabelled agonists to define the site. Interestingly, available evidence indicates that the 5-HT, receptor contributes significantly to the systemic haemodynamic effects of 5-I-P; specifically, it appears to mediate the tertiary, prolonged hypotensive response observed in anaesthetized animals after intravenous injection of the monoamine35,~. This vasodilator response was one of the prominent vascular effects of 5-HT described in the earliest studies of Irvine Page and otherss7-39, hence evidence for its existence longpreceded the discovery of the receptor gene. In this sense, the 5-HT, receptor may lay claim to being one of the longest serving orphans of the present scheme for 5-HT receptor nomenclature and classification.
Distribution and function ChJS In terms of distribution, northern blot and in situ hybridization studies show a discrete expression of 5-HT,,,, receptor mRNA in the brain, particularly in CA2 and CA3 pyramidal layers of the hypothalamusi4. In rat40 and guinea-pigQ,Q brain, in situ hybridization studies disclose predominant expression in anteroventral and paraventricular thalamic nuclei. Interestingly, 5-HT,,,, mRNA is highest in the hippocampal CA3 region@, yet high densities of specific [3H]5-CT binding are seen predominantly in the CAI/CA2 regions41,42.These data suggest that the receptor is located presynaptitally in this region, although this has not been confirmed immunohistochemically. The expression of mRNA for the 5-I-U, receptor in midline, thalamic and limbic structures indicates a role in affective
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Table 1. Affinities (p&I of 5HT receptor ligands for recombinant
5HT, receptors from different species
Receptor Host cell Radioligand
h 5-HT,,,,a cos-1 PH]S-HT
h 5-HT,(,,” HE1<293 PH]5-CT
r 5-HT,,,,c COSJ [‘qlsll
r 5-HT,,,, CHO-Kl PH]S-T
r 5-HT,,,,. Cos-M6 [‘ul]LSD
gp 5-HT,uf CHO-Kl PH]ICT .
Agonists 5-HT 5-CT 5MeOT Sumatriptan 8-OH-OPAT
8.1 9.0 8.3 6.0 6.3
9.4 9.7 9.3 -
8.7 9.0 8.8 6.2 7.5
9.2 9.9 9.0 6.3 7.3
8.1 9.1 -
9.6 9.7 9.3 _
7.0
7.4
8.4 8.2 7.7 -
8.9 8.5 7.9
_
6.9 7.3 7.1 7.0 7.3 5.9 -
-
9.0 8.7 8.1 7.9 7.3 7.7 7.9 -
8.9 7.5 7.8 -
8.4 8.2 7.8 7.3 6.9 7.3 7.7 7.3 7.0 6.2 7.8 8.5
6.9
Antagonists Methiothepin Metergoline Mesulergine Clozapine Cyproheptadine Ritanserin Methysergide Spiperone Mianserin Ketanserin LSD Lisuride
7.6 7.7 -
7.2 7.2 7.1 7.7 7.2 c5.1 8.0 8.2
7.0 6.7 -
6.4 -
7.2 7.5 _
9.3
_ _
%?ef. 8, bRef. 15, Ref. 12. dRef. 11, *Ref. 9, ‘Ref. 41. Data are the means from radioligand displacement experiments using recombinant receptors expressed in mammalian cells. h 5-HT,, r 5-HT, and gp 5-HT, refer to the human, rat and guinea-pig primary amino acid sequences, respectively. ‘Long’ and ‘short’ receptor variants are denoted (a) and Lb), respectively. XT, 5carboxamidotryptamine; LSD, o-lysergic acid diethylamide; 5-MeOT, 5-methoxytryptamine; 8-OH-DPAT, (f)B-hydroxy-2dipropylaminotetralin.
behaviour, while the presence of the receptor in the medial geniculate nucleus, the superior and inferior colliculi, the central grey and the spinal trigeminal nuclei suggests a possible involvement in sensory processing. A role of the receptor in regulating circadian phase shifts has been hypothesizedlo, since 5-CT and B-OH-DPAT, both agonists at the 5-HT, receptor, advance neuronal
r
L
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firing in hypothalamic cultures43. Furthermore, electrophysiological recordings from rat suprachiasmatic nuclei show that 5CT or 5-HT inhibit GABA, receptor currents via activation of adenylate cyclasea. This may occur by depression of an inward rectifying potassium current, via activation of protein kinase A, as recently demonstrated in whole cell recordings4. In the hamster, tryptophan-loading
Table 2. Affinity estimates (pKs) for antagonists isolated intact tissues from different species
at 5-HT,
receptors
Antagonist
Monkey jugular vein0
Rabbit femoral veinb
Guinea-pig ileumc
Dog coronary artew
Methiothepin Metergoline Clozapine Mianserin Spiperone Ritanserin Methysergide Ketanserin 8-OH-DPAT LSD
9.7 8.0 7.8 7.7 7.3 7.1 7.0 5.7 <6.0 _
9.5 _ -
_
9.7 _
7.8 _
7.3 7.6 7.6 -
7.8 <6.0 -
7.6 <6.0 6.3 8.1
7.1 7.1 7.1 _ _ _ -
in
aRef. 28, bRef. 7. CRef. 23, dRef. 29. Cynomolgus monkey jugular vein, guinea-pig ileum, 5CT (5carboxamidotryptamine) used as agonist; rabbit femoral vein, 5-HT used as agonist; beagle coronary artery, 5-HT or 5-CT used as agonist. LSD, o-lysergic acid diethylamide; E-OH-DPAT, (i)Bhydroxy-2-dipropylaminotetralin.
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enhances 5-HT release from the suprachiasmatic nucleus and modulates light-induced expression of c--k immunoreactivity. This effect is abolished by ritanserin and metergoline, potentially implicating the 543, receptor in circadian rhythm entrainment45. However, given the nonselective nature of the antagonists used in these studies, participation of other 54-E receptor subtypes cannot be excluded. Little is known about the regulation of the membrane receptor number, although one report has suggested that the receptor is downregulated by chronic treatment with the serotonin reuptake inhibitor fluoxetin&. This has, however, been disputed on the grounds that the concentrations of ligands used to selectively label the receptor were inappropriat&7. In rat frontocortical astrocytes, chronic exposure to antidepressants enhances 5-HT, receptor activation of adenylate cyclase; a finding that may underline the therapeutic action of amitriptyline48. Petiphey Northern blot studies demonstrate high levels of h 5-HT,,,, mRNA
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expression in the human stomach, not to be causally related to the descending colon, ileum and coro- development of bipolar affective disnary arterys. RT-PCR studies also order and schizophreniasz, whilst suggest expression of the receptor in linkage analysis also suggests that the rat renal artery, vena cava, portal, the receptor is not associated with femoral and jugular vein, porcine Tourette syndromei7. The next phase of 5-HT, receptor pulmonary and cerebral arteriesh9, as well as the rabbit retinasO.In con- research will, therefore, undoubtedly trast, mRNA transcripts have not concentrate on the identification of selective agonists and antagonists been detected in the rat urinary bladder, testis, liver, adrenal gland, with which to tease apart physiological and pathophysiological roles. kidney, lung, heart or pituitary cellW. A physiological role in Although this process will be aided cardiovascular function might be by the availability of each human inferred given the extensive distri- 5-I-U, receptor variant, those studies bution in vascular, notably venous, required to define the regulation of smooth muscle. Indeed, it has been the 5-I-IT, receptor gene and, potensuggested that 5-I-IT, receptors in tially, four gene products clearly vascular smooth muscle may serve remain a major challenge. to modulate the vasoconstrictor effects of 5+IT,,,,, or 5-I-U, recep- Selected references tor activation49. In this regard, 1 Rapport, M. M., Green, A. A. and Page, I. H. (1948)Science108,329-330 expression of 5-I-Q receptor mRNA 2 Launav. I. M.. Callebert. I.. Bondoux. D.. ,. _ in the human coronary artery8 and Loric, S. and. Marotea& ‘L. (1994) Cell: its functional presence in the beagle Mol. Biol.40,327-336 3 Kenakin, T. P., Bond, R.A. and Bonner, dog coronary artery729 is intriguing, T. I. (1992)Pharmacol.Rev. 44,351-362 although a specific role in modulat4 Hoyer, D. et al. (1994) Phannacol. Rev. 46, ing myocardial blood flow has not 143-156 5 Bradley, P. B. et al. (1986) Neuropharmabeen established. A functional role in colony 25,563-576 human alimentary tract tissue might 6 Bra&bek, T. (1993)Cum Biol.3,315-317 be anticipated, since the receptor is 7 Martin, G. R. (1994) Pharmacol. Ther. 62, 283-324 ~ ’ expressed in human colon smooth 8 Bard, J. A. et al. (1993) J. Biol. Chem. 31, musck? and it also mediates relax23422-23426 ation in the guinea-pig isolated 9 Lovenberg, T. W. et al. (1993) Neuron 11, 449-458 i1eun-F~.
Concluding remarks It is clear from the studies to date that the 5-HT, receptor is well represented in a diversity of neuronal and non-neuronal tissues where, in at least some tissues, its activation has been shown to elevate intracellular CAMP, as expected for a G,-coupled receptor. However, it is unclear whether these functional studies reflect activation of a single receptor protein, or represent effects mediated by multiple isoforms of the receptor. The possible existence of pathologically important mutations also needs to be explored. In this regard, a preliminary report has described a point mutation in the C-terminal end of the receptor in some alcoholi&. In contrast, two rare, naturally occurring, mutations (Pro279 to Leu; Thr92 to Lys) appear
10 Meyerhof, W., Obermuller, F., Felu, S. and Richter. D. (1993) DNA Cell Biol. 12. 401409 \ 11 Ruat, M. et al. (1993)Proc. N&l. Acad. Sci. U. S. A. 90,8547-8551 12 Shen, Y. ef al. (1993) J. Biol. Chem. 268, 18200-18204 13 Plassat, J-L., Amlaiky, N. and Hen, R. (1993)Mol. Pharmacol.44,229-236 14 Tsou; A-P. et al. (1994)J. Neurochenz.63, 456-464 15 Jasper, J. R., To, A. P., Kosaka, A., Eglen, R. M. and Chang, D. J. Br. J. Phurrnucol.(in press) 16 Peroutka, S. J. and Howell, T. A. (1994) Neurophurmacology33,319-324 17 Gelemter, J. et al. (1995) Genomics 26, 207-209 18 Vanboutte, P. M., Humphrey, P. P. A. and Spedding, M. (1996) Pharmacol. Rev. 48,1-2 19 Heidman, D., Metcalf, M., Kohen, R. and Hamblin, M. (1996) Sot. Neurosci. 22 Abstract 701.8 20 Roth, 8. L. et al. (1994)J, Pharmacol. Exp. Ther. 268,1&x-1410 21 Feniuk, W., Humphrey, P. P. A. and Watts, A. D. (1983) Eur. J. Pharmacol. 96, 71-78 22 Kalkrnan, H. O., Engel, G. and Hoyer, D. (1986)Eur. J. Phurmacol.129,139-145
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The 5=HT,receptor: orphan found
Rilchard M. Eglen, Jeffrey R. Jasper, David J. Chang and Graeme R. Martin
Il. M. Eglen. Vice President and Director, J. R. Jasper, Research Scientist II, D. J. Chang. Research Manager, and 6. Il. Martin. Department Head, Center for Biological Research, Neurobiology Unit, Roche Bioscience. Palo Alto, CA 94304. USA.
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In the 40 or more years since serotonin was first isolated and identified as 5-hydroxytryptamine (5-HT)l, perceptive operational studies have shown that the monoamine elicits a complex array of pharmacological and physiological responses by acting at a diversity of 5-I-IT receptors. Many of these receptors are now widely recognized as important targets for therapeutics2. Whilst the operational characteristics of these 5-m receptors have served for many years as the primary basis for their differentiation, gene cloning techniques now enable the primary structure of the receptors to be determined; this information, together with the operational data, enables unambiguous classification3P. However, a number of functional receptors for 5-m have been described for which a corresponding gene product has yet to be identified. In the absence of complementary structural data these remain ‘orphans’ of the present IUPHAR (International Union of Pharmacology) scheme for classifying and naming 5-HT receptorsd. Conversely, gene products that encode 5-HT receptor proteins have been identified for which an endogenous physiological correlate has yet to be defined. These are identified by a lower case appellation, as in the case of 5-htlE,5-htlp 5-h&*,5-h&and, until recently, 5-ht, gene products. When evidence for endogenous expression and function of the proteins is obtained, they are allocated an upper case notation denoting that they are fully defined transmitter receptors. The most recent orphan 5-HT receptor to achieve ‘upper case’ status is the 5-I-IT, receptor. This adoption was based on the close identity between the operational profile of the 5-HT, gene product expressed in mammalian cells and a long recognized 5-HT receptor, previously designated ‘5-I-IT&e’, which is positively coupled to adenylate cyclase in a variety of neuronal and
TiPS - April 1997 (Vol. 18)
non-neuronal tissuess7. This article briefly reviews the characteristics of endogenous and recombinant 5-HT, receptor systems and highlights emerging new data on the molecular biology of this unique receptor class.
Molecular biology The predicted amino acid sequence of the 5-HT, receptor protein is unique, with low (<40%) overall homology with other 5-HT receptors&15.Like the 5-HT, and putative 5-h& receptors, it preferentially couples to adenylate cyclase via G,. The 5-HT, receptor represents a mammalian homologue of the 5-HT, Drosophila (5-HTdr,,l)receptor (overall homology = 57%), with divergence from a primordial 5-HT receptor predating the evolution of adrenoceptors, muscarinic and dopamine receptorsl6. The receptor gene, located on human chromosome lOq23.3q24.4 (Ref. 17), has two introns in the coding region, one located after the third transmembrane domain and the other close to the C-terminus. In the rat, a five base insertion (GTAAG) at the carboxy region intron introduces an in-frame stop codon, resulting in a 13 amino acid truncated splice variant9 (Fig. 1). Recent studies have identified a clone from a human placental cDNA library that contains the identical five base insertion at the carboxy intron site which results in a similar truncated gene productl5. Consistent with NC-IUPHAR nomenclature guidelines’s the human receptor variants can be named h 5-HT,,,, (long form) and h 5-HT,,, (short form), denoting the order of their discovery. Radioligand binding studies show that in terms of the available ligands, the pharmacology of the h 5-HT,, receptor stably expressed in HEK293 cells, resembles that of the h 5-HT,,,, receptor expressed in Cos7 cell@. Likewise, the pharmacology of the rat short and long variants of the receptor is
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similar (see Table 1). Interestingly, preliminary reverse transcription polymerase chain reaction (RT-PCR) studies in the rat have identified two further novel isoforms of the 5-HT, receptor arising from the insertion of an additional exon which is different in each case (Ref. 19). These novel isoforms also couple to Gs to stimulate adenylate cyclase and reveal no differences in their operational response to 5-HT (Ref. 19). Assuming that they have a physiological role, these isoforms would be named r 5-HT7,,, and r 5-HT,(,, receptor variants.
Pharmacology The recombinant 5-HT, receptor can be radiolabelled with [3H]5-HT, [3H]5_carboxamidotryptamine ([3H]5-CT) or [luI]D-lysergic acid diethylamide ([lEI]LSD). Competition binding studies demonstrate a unique pharmacological profile with good agreement of ligand affinities across several species (Table 1). Operationally, the recombinant receptor is defined by high affinities for 5-CT, LSD, methiothepin, mesulergine and methysergide, as well the antipsychotics clozapine and loxapi&J. Furthermore, (k)B-hydroxy-2dipropylaminotetralin @-OH-DPAT), generally considered a selective agonist for the 5-I-IT,, receptor, can act in some systems as a partial agonist at 5-HT, receptors8-15.In all recombinant systems studied to date, activation of the 5-I-IT,receptor augments intracellular adenylate cyclase activity, with no evidence for coupling of the receptor to other intracellular signalling mechanisms. Operational characterization of the 5-HT7 receptor in recombinant systems has provided an unambiguous profile of ligand affinities. A similar profile is obtained at the orphan 5-HT receptor, previously classified as ‘5-HT,-like’, that mediates smooth muscle relaxation in a variety of tissue@ (Table 2). First characterized in the guinea-pig isolated ileum and cat saphenous vei$*-23, this receptor has subsequently been found in the neonatal pig vena cava24,E,the rabbit jugular26
SO165-6147(97)01043-2
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AWARENESS
I
1 Guin pig MOUSe Rat-long Rat-short Hum-long Hum-short
Guin pig Mouse
Pat-long Rat-short Hum-long Hum-short
Guin pig Mouse Rat-long Rat-short Hum-long Hum-short
MMGVNSSGRP MMDVNSSGRP bQ4DVNSSGRP MMDVNSSGRP MMDVNSSGRP MMDVNSSGRP
DLYGHLHSIL DLYGHLRSLI DLYGHLRSLI DLYGHLRSLI DLYGHLRSFL DLYGHLRSFL
LP..GRGLPD LPSVGRRLQD LPEVGRGLQD LPEVGRGLQD LPEVGRGLPD LPEVGRGLPD
WSPDGGADFG LSPDOOAHSV LSPDGGAHPV LSPXGAHPV LSPDGGADPV LSPDGGADPV
VSTWTPRLLS VSSWMPHLLS VSSWMPHLLS VSSmPHLLS AGSWAPHLLS AGSWAPHLLS
GVPBVAASPS GFPEVTASPA GFLEVTASPA GFLEVTASPA . ..EXl'ASPA . ..EVTASPA
PSWEGTNDNV PTWDAPPDNV PTWDAPPDNV PlWDAPPDNV PTWDAPPDNA PTWDAPPDNA
Ul GNCLWISVC GNCLWISVC GNCLWISVC GNCLWISVC GNCLWISVC GNCLWISVC
FVKKLRQPSN FVKNVRQPSN FVKKLRQPSN FVKKLRQPSN FVKKLRQPSN FVKKLRQPSN
YLIVSLALAD YLIVSLALAD YLIVSLALAD YLIVSLALAD YLIVSLALAD YLIVSLALAD
II LSVAVAVMPF LSVAVAVMPP LSVAVAVMPF LSVAVAVMPF LSVAVAVMPF LSVAVAVMPF
VSVTDLIGGK VSVTDLIGGK VSVTDLIGDK VSVTDLIGGK VSVTDLIGGK VSVTDLIGGK
WIFGHFPCNV WIFGHFFCNV WIFGHFFCNV
201 CMPKMILSVW CMAKMILSVW C!MaKMILSVW CMAKMILSVW CMAKMILSVW CNFdZMILSVW
IV LLSASITLPP PLSASITLPP LLSASITLPP LLSASITLPP LLSASITLPP LLSASITLPP
LFGWAQNVND LFGWAQNVND LFGWAQNVND LFGWAQNVND LFGWAQNVND LFGWAQNVND
DKVCLISQDF DKVCLISQDF DKVCLINQDF DKVCLISQDF DKVCLISQDF DKVCLISQDF
GYTIYSTAVA GYTIYSTAVA GYTIYSTAVA GYTIYSTAVA GYTIYSTAVA GYTIYSTAVA
FYIPNSVMLF FYIPMSVMLF FYIPMSVMLF
LLKHERKNIS LLKHERKNIS LLKH?XRXNIS LLKHERKNIS LLKHERIUiIS LLKHEFJNIS
IFKREQKAAT SFKREQKAAT IFKREZQKAAT IFKREQKAAT IFKREQKAAT IFKREQKAAT
TLGIIVGAFT TLGIIVGFbFT TLGIIVGAFT TLGIIVGAFT TLGIIVGAFT TLGIIVGAFT
VI VCWLPFFLLS VCWLPFFLLS VCWLPFFPLS VCWLPFFLLS VCWLPFFLLS VCWLPFFLLS
Rat-short Hum-long Hum-short
ALKLAERPBR ALKLARRPER ALKLANRP RR AL-PER
PECVLQNSDY SEFVLQNCDH SEFVLQNSDH SEWL*....
CRKKGHDS* eGKKGHDT* CGKKGHDT' .. . . .. . ..
PSFVLQNADY PEFVL'....
CRKKGHDS* . .._.....
SVSECANLSR EVEECATLSR SVSECANLSR EVEECANLSR EVEE'ZANLSR EVEECANLSR 401
Guin pig Mouse
Rat-long Rat-short Hum-long Hum-short
LTLITLLTIA LTLITLLTIA LTLITLLTIA LTLITLLTIA LTLITLLTIA LTLITLLTIA
FIAMDVMCCT FIAMDVMCCT FIAMDVMYCT FIAMDVMCCT FIAMDVMCCT FIAMDVMCCT
ASIMTLCVIS ASIMTLCVIS ASIMTLCVIS ASIMTLCVIS ASIMTLCVIS ASIMTLCVIS
IDRYLGITRP IDRYLGITRP IDRYLGITRP IDRYLGITRP IDRYLGITRP IDRYLGITRP
LTYPVRQNGK LTYFVRQNGK LTYPVSQNEK LTYPVRQNGK LTYPVRQNGK LTYPVRQNGK
FYIPMSVMLF FYIPMSVMLF
MYYRIYKAAR MYYQIYKAAR MYYQIYKAAR MYYQIYKAAR MYYQIYKAAR MYYQIYKAAR
FPRVQPESII KSAAKHKFSG FPRVQPESVI KSAAKHKFPG FPRVQPESVI KSAAKHKF PG FPRVQPESVI KS AAKHKFPG FPPS'EPDSVI
SLNGVVKLQK SLNGWKLQK ALNGIVKLQK
KSAAKHKFFG
FPRVSPDSVI
ALNGIVKLQK
TARPFICGTA TARPFICGTS TARPFICGTS TARPFICGTS TARPFICGTS TARPFICGTS
CSCIPLWVER CSCIPLWVER CSCIPLWVER CSCIPLWVER CSCIPLWVER CSCIPLWVER
TCLWLGYANS TCLWLGYANS TCLWLGYANS TCLWLGYANS TFLWLGYANS TFLWLGYANS
LINPFIYAFF LINPFIYSFF LINPFIYAFF LINPFIYAFF LINPFIYAFF LINPFIYAFF
NRDLRTTYRS NRDLRTTYRS NRDLRTTYRS NRDLRTTYRS NRDLRTTYRS NRDLRTTYRS
200
III
WIFGHFFCNV
WIFGHFFCNV WIFGHFFCNV
300
FYIPMSVMLF
KSAAKHKFPG
SUZMVKLQK SLNGVVKLQK
400
VII
449
LLQCQYRNIN RKLSAAGMHE LLQCQYPXIN RKLSAAGMHE LLQCQYRNINRKLSAAGMHB LLQCQYRNIN RKLSAAGMHE LLQCQYPXIN LLQCQYFNIN
RAEKWIGSI RVEKWIGSI RVEKVVIGSI RVEKVVIGSI RVEKVVIGSI RVEKVVIGSI
V
301 Guin pig Mouse Rat-long
RXLSAAGMHE ALKLAERPER RKLSAAGMHE ALKLAERPER
Fig. 1. Sequence alignment of predicted amino acid residues for 5-HT,receptors cloned to date. Guin pig, guinea-pig; hum, human.
and femoral veinz7, the Cynomolgus monkey jugular vein% and the beagle dog coronary artery7J9.Responses in other isolated tissues, although less well characterized, may also be mediated by 5-I-I& receptor activation. These tissues include the marmoset aorta3, the sheep pulmonary veinai, the porcine pial vein32 and the guinea-pig trachea33. Moreover, in pig vena cavaz6, homogenates of guinea-pig hippocampus and smooth muscle cells from human uterine artery34, 5-HT receptor activation stimulates the accumulation of CAMPconsistent with coupling to Gs. As in recombinant systems, there is a high degree of correspondence of ligand affinities at these endogenous 5-HT, receptors, implying that the ligand recognition sites are highly conserved between species (Table 2). Notably, however, the affinity values for agonists, such as 5-CT or 5-HT, derived from binding studies
1OQ
SGCGEQINYG SGCGEQINYG SGCGEQINYG SGCGKQINYG SGCGEQINYG SGCGEQINYG
are greater than the potencies seen in functional studies. The reason for this difference is unclear but suggests caution in the use of radiolabelled agonists to define the site. Interestingly, available evidence indicates that the 5-HT, receptor contributes significantly to the systemic haemodynamic effects of 5-I-P; specifically, it appears to mediate the tertiary, prolonged hypotensive response observed in anaesthetized animals after intravenous injection of the monoamine35,~. This vasodilator response was one of the prominent vascular effects of 5-HT described in the earliest studies of Irvine Page and otherss7-39, hence evidence for its existence longpreceded the discovery of the receptor gene. In this sense, the 5-HT, receptor may lay claim to being one of the longest serving orphans of the present scheme for 5-HT receptor nomenclature and classification.
Distribution and function ChJS In terms of distribution, northern blot and in situ hybridization studies show a discrete expression of 5-HT,,,, receptor mRNA in the brain, particularly in CA2 and CA3 pyramidal layers of the hypothalamusi4. In rat40 and guinea-pigQ,Q brain, in situ hybridization studies disclose predominant expression in anteroventral and paraventricular thalamic nuclei. Interestingly, 5-HT,,,, mRNA is highest in the hippocampal CA3 region@, yet high densities of specific [3H]5-CT binding are seen predominantly in the CAI/CA2 regions41,42.These data suggest that the receptor is located presynaptitally in this region, although this has not been confirmed immunohistochemically. The expression of mRNA for the 5-I-U, receptor in midline, thalamic and limbic structures indicates a role in affective
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Table 1. Affinities (p&I of 5HT receptor ligands for recombinant
5HT, receptors from different species
Receptor Host cell Radioligand
h 5-HT,,,,a cos-1 PH]S-HT
h 5-HT,(,,” HE1<293 PH]5-CT
r 5-HT,,,,c COSJ [‘qlsll
r 5-HT,,,, CHO-Kl PH]S-T
r 5-HT,,,,. Cos-M6 [‘ul]LSD
gp 5-HT,uf CHO-Kl PH]ICT .
Agonists 5-HT 5-CT 5MeOT Sumatriptan 8-OH-OPAT
8.1 9.0 8.3 6.0 6.3
9.4 9.7 9.3 -
8.7 9.0 8.8 6.2 7.5
9.2 9.9 9.0 6.3 7.3
8.1 9.1 -
9.6 9.7 9.3 _
7.0
7.4
8.4 8.2 7.7 -
8.9 8.5 7.9
_
6.9 7.3 7.1 7.0 7.3 5.9 -
-
9.0 8.7 8.1 7.9 7.3 7.7 7.9 -
8.9 7.5 7.8 -
8.4 8.2 7.8 7.3 6.9 7.3 7.7 7.3 7.0 6.2 7.8 8.5
6.9
Antagonists Methiothepin Metergoline Mesulergine Clozapine Cyproheptadine Ritanserin Methysergide Spiperone Mianserin Ketanserin LSD Lisuride
7.6 7.7 -
7.2 7.2 7.1 7.7 7.2 c5.1 8.0 8.2
7.0 6.7 -
6.4 -
7.2 7.5 _
9.3
_ _
%?ef. 8, bRef. 15, Ref. 12. dRef. 11, *Ref. 9, ‘Ref. 41. Data are the means from radioligand displacement experiments using recombinant receptors expressed in mammalian cells. h 5-HT,, r 5-HT, and gp 5-HT, refer to the human, rat and guinea-pig primary amino acid sequences, respectively. ‘Long’ and ‘short’ receptor variants are denoted (a) and Lb), respectively. XT, 5carboxamidotryptamine; LSD, o-lysergic acid diethylamide; 5-MeOT, 5-methoxytryptamine; 8-OH-DPAT, (f)B-hydroxy-2dipropylaminotetralin.
behaviour, while the presence of the receptor in the medial geniculate nucleus, the superior and inferior colliculi, the central grey and the spinal trigeminal nuclei suggests a possible involvement in sensory processing. A role of the receptor in regulating circadian phase shifts has been hypothesizedlo, since 5-CT and B-OH-DPAT, both agonists at the 5-HT, receptor, advance neuronal
r
L
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firing in hypothalamic cultures43. Furthermore, electrophysiological recordings from rat suprachiasmatic nuclei show that 5CT or 5-HT inhibit GABA, receptor currents via activation of adenylate cyclasea. This may occur by depression of an inward rectifying potassium current, via activation of protein kinase A, as recently demonstrated in whole cell recordings4. In the hamster, tryptophan-loading
Table 2. Affinity estimates (pKs) for antagonists isolated intact tissues from different species
at 5-HT,
receptors
Antagonist
Monkey jugular vein0
Rabbit femoral veinb
Guinea-pig ileumc
Dog coronary artew
Methiothepin Metergoline Clozapine Mianserin Spiperone Ritanserin Methysergide Ketanserin 8-OH-DPAT LSD
9.7 8.0 7.8 7.7 7.3 7.1 7.0 5.7 <6.0 _
9.5 _ -
_
9.7 _
7.8 _
7.3 7.6 7.6 -
7.8 <6.0 -
7.6 <6.0 6.3 8.1
7.1 7.1 7.1 _ _ _ -
in
aRef. 28, bRef. 7. CRef. 23, dRef. 29. Cynomolgus monkey jugular vein, guinea-pig ileum, 5CT (5carboxamidotryptamine) used as agonist; rabbit femoral vein, 5-HT used as agonist; beagle coronary artery, 5-HT or 5-CT used as agonist. LSD, o-lysergic acid diethylamide; E-OH-DPAT, (i)Bhydroxy-2-dipropylaminotetralin.
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enhances 5-HT release from the suprachiasmatic nucleus and modulates light-induced expression of c--k immunoreactivity. This effect is abolished by ritanserin and metergoline, potentially implicating the 543, receptor in circadian rhythm entrainment45. However, given the nonselective nature of the antagonists used in these studies, participation of other 54-E receptor subtypes cannot be excluded. Little is known about the regulation of the membrane receptor number, although one report has suggested that the receptor is downregulated by chronic treatment with the serotonin reuptake inhibitor fluoxetin&. This has, however, been disputed on the grounds that the concentrations of ligands used to selectively label the receptor were inappropriat&7. In rat frontocortical astrocytes, chronic exposure to antidepressants enhances 5-HT, receptor activation of adenylate cyclase; a finding that may underline the therapeutic action of amitriptyline48. Petiphey Northern blot studies demonstrate high levels of h 5-HT,,,, mRNA
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expression in the human stomach, not to be causally related to the descending colon, ileum and coro- development of bipolar affective disnary arterys. RT-PCR studies also order and schizophreniasz, whilst suggest expression of the receptor in linkage analysis also suggests that the rat renal artery, vena cava, portal, the receptor is not associated with femoral and jugular vein, porcine Tourette syndromei7. The next phase of 5-HT, receptor pulmonary and cerebral arteriesh9, as well as the rabbit retinasO.In con- research will, therefore, undoubtedly trast, mRNA transcripts have not concentrate on the identification of selective agonists and antagonists been detected in the rat urinary bladder, testis, liver, adrenal gland, with which to tease apart physiological and pathophysiological roles. kidney, lung, heart or pituitary cellW. A physiological role in Although this process will be aided cardiovascular function might be by the availability of each human inferred given the extensive distri- 5-I-U, receptor variant, those studies bution in vascular, notably venous, required to define the regulation of smooth muscle. Indeed, it has been the 5-I-IT, receptor gene and, potensuggested that 5-I-IT, receptors in tially, four gene products clearly vascular smooth muscle may serve remain a major challenge. to modulate the vasoconstrictor effects of 5+IT,,,,, or 5-I-U, recep- Selected references tor activation49. In this regard, 1 Rapport, M. M., Green, A. A. and Page, I. H. (1948)Science108,329-330 expression of 5-I-Q receptor mRNA 2 Launav. I. M.. Callebert. I.. Bondoux. D.. ,. _ in the human coronary artery8 and Loric, S. and. Marotea& ‘L. (1994) Cell: its functional presence in the beagle Mol. Biol.40,327-336 3 Kenakin, T. P., Bond, R.A. and Bonner, dog coronary artery729 is intriguing, T. I. (1992)Pharmacol.Rev. 44,351-362 although a specific role in modulat4 Hoyer, D. et al. (1994) Phannacol. Rev. 46, ing myocardial blood flow has not 143-156 5 Bradley, P. B. et al. (1986) Neuropharmabeen established. A functional role in colony 25,563-576 human alimentary tract tissue might 6 Bra&bek, T. (1993)Cum Biol.3,315-317 be anticipated, since the receptor is 7 Martin, G. R. (1994) Pharmacol. Ther. 62, 283-324 ~ ’ expressed in human colon smooth 8 Bard, J. A. et al. (1993) J. Biol. Chem. 31, musck? and it also mediates relax23422-23426 ation in the guinea-pig isolated 9 Lovenberg, T. W. et al. (1993) Neuron 11, 449-458 i1eun-F~.
Concluding remarks It is clear from the studies to date that the 5-HT, receptor is well represented in a diversity of neuronal and non-neuronal tissues where, in at least some tissues, its activation has been shown to elevate intracellular CAMP, as expected for a G,-coupled receptor. However, it is unclear whether these functional studies reflect activation of a single receptor protein, or represent effects mediated by multiple isoforms of the receptor. The possible existence of pathologically important mutations also needs to be explored. In this regard, a preliminary report has described a point mutation in the C-terminal end of the receptor in some alcoholi&. In contrast, two rare, naturally occurring, mutations (Pro279 to Leu; Thr92 to Lys) appear
10 Meyerhof, W., Obermuller, F., Felu, S. and Richter. D. (1993) DNA Cell Biol. 12. 401409 \ 11 Ruat, M. et al. (1993)Proc. N&l. Acad. Sci. U. S. A. 90,8547-8551 12 Shen, Y. ef al. (1993) J. Biol. Chem. 268, 18200-18204 13 Plassat, J-L., Amlaiky, N. and Hen, R. (1993)Mol. Pharmacol.44,229-236 14 Tsou; A-P. et al. (1994)J. Neurochenz.63, 456-464 15 Jasper, J. R., To, A. P., Kosaka, A., Eglen, R. M. and Chang, D. J. Br. J. Phurrnucol.(in press) 16 Peroutka, S. J. and Howell, T. A. (1994) Neurophurmacology33,319-324 17 Gelemter, J. et al. (1995) Genomics 26, 207-209 18 Vanboutte, P. M., Humphrey, P. P. A. and Spedding, M. (1996) Pharmacol. Rev. 48,1-2 19 Heidman, D., Metcalf, M., Kohen, R. and Hamblin, M. (1996) Sot. Neurosci. 22 Abstract 701.8 20 Roth, 8. L. et al. (1994)J, Pharmacol. Exp. Ther. 268,1&x-1410 21 Feniuk, W., Humphrey, P. P. A. and Watts, A. D. (1983) Eur. J. Pharmacol. 96, 71-78 22 Kalkrnan, H. O., Engel, G. and Hoyer, D. (1986)Eur. J. Phurmacol.129,139-145
23 Carter, D., Champney, M., Hwang, 8. and Eglen, R. M. (1995)Eur J. Phmmcol. 280, 243-m
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