Characterization of Serotonin Receptors in Pregnant Human Myometrium Yolande Cordeaux, Dharmintra Pasupathy, Joanne Bacon, D. Stephen Charnock-Jones, and Gordon C. S. Smith Department of Obstetrics and Gynaecology, University of Cambridge, Cambridge, United Kingdom Received July 3, 2008; accepted December 12, 2008 ABSTRACT The monoamine, 5-hydroxytryptamine (5-HT), stimulates con- traction of human uterine smooth muscle (myometrium), but the receptor subtypes involved have not been characterized. We studied the effects of a range of 5-HT receptor subtype-selec- tive agonists and antagonists in isolated strips of myometrium obtained at the time of caesarean section. The 5-HT 1A receptor agonist, 8-hydroxy-2-dipropylaminotetralin, produced an in- crease in contractions that was highly variable, of low potency, and was not significantly inhibited by the 5-HT 1A antagonist WAY100635 [[O-methyl-3H]-N-(2-(4-(2-methoxyphenyl)-1-pipera- zinyl)ethyl)-N-(2-pyridinyl)cyclohexanecarboxamide]. The 5-HT 2 receptor agonist, -methyl-5-hydroxytryptamine (-Me-5-HT), produced a strong, consistent, and concentration-dependent stimulation of contractions (pEC 50 = 7.60  0.10, n = 5). The 5-HT 2A receptor antagonist, ketanserin [3-[2-[4-(4-fluoro benzoyl)- piperidin-1-yl]ethyl]-1H-quinazoline-2,4-dione], caused a parallel shift in the response to -Me-5-HT, with a pK B value consistent with its known affinity for the 5-HT 2A receptor (pK B = 8.47  0.16, n = 5), but it had no effect on the response to oxytocin. The 5-HT 2B and 5-HT 2C receptor agonists, BW723C86 [(-methyl-5- (2-thienylmethoxy)-1H-indole-3-ethanamine)] and Ro-60-01-75 [(S)-2-(6-chloro-5-fluoro-indol-1-yl)-1-methyl-ethylamine fumar- ate], produced inconsistent responses at potencies that were lower than expected for activation of their cognate receptors. The response to -Me-5-HT was unaffected by the 5-HT 2B and 5-HT 2C receptor antagonists, SB204741 [( N-(1-methyl-1H-indolyl-5- yl)-N-(3-methyl-5-isothiazolyl)urea)] and RS102221 [8-[5-(2,4- dimethoxy-5-(4-trifluoromethyl phenylsulphonamido)phenyl-5- oxopentyl]-1,3,8-triazaspiro[4.5]decane-2,4-dione]. The 5-HT 1B/1D receptor agonist, sumatriptan [1-[3-(2-dimethylaminoethyl)-1H- indol-5-yl]-N-methyl-methanesulfonamide], the 5-HT 4 agonist, cisapride [4-amino-5-chloro-N-[1-[3-(4-fluorophenoxy)pro- pyl]-3-methoxy-4-piperidyl]-2-methoxy-benzamide], and the 5-HT 7 agonist, AS19 [(2S)-(+)-5-(1,3,5-trimethylpyrazol-4- yl)-2-(dimethylamino)tetralin], all had no effect on myometrial contractility. 5-HT 2A receptor mRNA and immunoreactivity were identified using reverse transcriptase-polymerase chain reaction, Western blotting, and immunohistochemistry. Spe- cific binding of [ 3 H]ketanserin was demonstrated. This study provides strong evidence for the expression of contractile 5-HT 2A receptors in pregnant human myometrium, and this receptor is a potential target for novel uterotonic therapies. Serotonin (5-hydroxytryptamine) mediates a wide range of physiological effects by activating a large family of receptors that are distributed throughout the body (Hoyer et al., 2002). These cell surface receptors are classified into seven distinct subtypes (5-HT 1 to 5-HT 7 ), and, with the exception of the ligand-gated ion channel 5-HT 3 , they are all members of the G-protein-coupled receptor superfamily (Alexander et al., 2008). In the central nervous system, these receptors are implicated in numerous conditions, including: depression, anxiety, schizophrenia, and migraine (Barnes and Sharp, 1999). Serotonin also plays a role in platelet activation (Kau- This work was supported by the Evelyn Trust and by the National Institute for Health Research Cambridge Comprehensive Biomedical Research Centre. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.108.143040. ABBREVIATIONS: 5-HT, 5-hydroxytryptamine; RT, reverse transcriptase; PCR, polymerase chain reaction; AUC, area(s) under the curve; ANOVA, analysis of variance; RIPA, radioimmunoprecipitation assay; TBST, Tris-buffered saline/Tween 20; HRP, horseradish peroxidase; ketanserin, 3-[2-[4-(4-fluoro benzoyl) piperidin-1-yl]ethyl]-1H-quinazoline-2,4-dione; WAY100635, [O-methyl-3H]-N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)- ethyl)-N-(2-pyridinyl)cyclohexanecarboxamide; 8-OH-DPAT, 8-hydroxy-2-dipropylaminotetralin; AS19, (2S)-(+)-5-(1,3,5-trimethylpyrazol-4-yl)-2- (dimethylamino)tetralin; BW723C86, (-methyl-5-(2-thienylmethoxy)-1H-indole-3-ethanamine); cisapride, 4-amino-5-chloro-N-[1-[3-(4-fluorophenoxy)- propyl]-3-methoxy-4-piperidyl]-2-methoxy-benzamide; Ro 60-0175, (S)-2-(6-chloro-5-fluoro-indol-1-yl)-1-methyl-ethylamine fumarate; RS102221, 8-[5-(2,4-dimethoxy-5-(4-trifluoromethyl phenylsulphonamido)phenyl-5-oxopentyl]-1,3,8-triazaspiro[4.5]decane-2,4-dione; SB269970, (R)-3-(2-(2-(4- methyl-piperidin-1-yl)ethyl)-pyrrolidine-1-sulfonyl)phenol hydrochloride; SB204741, (N-(1-methyl-1H-indolyl-5-yl)-N-(3-methyl-5-isothiazolyl)u- rea); sumatriptan, 1-[3-(2-dimethylaminoethyl)-1H-indol-5-yl]-N-methyl-methanesulfonamide; DMSO, dimethyl sulfoxide; -Me-5-HT, -methyl- 5-hydroxytryptamine; Ro-60-01-75, (S)-2-(6-chloro-5-fluoro-indol-1-yl)-1-methyl-ethylamine fumarate. 0022-3565/09/3283-682–691$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 328, No. 3 Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics 143040/3441367 JPET 328:682–691, 2009 Printed in U.S.A. 682 at ASPET Journals on March 29, 2017 jpet.aspetjournals.org Downloaded from