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Julie Y. Crider, Gary W. Williams, Colene D. Drace, Parvaneh Katoli, Michelle Senchyna, Najam A. Sharif; Pharmacological Characterization of a Serotonin Receptor (5-HT7) Stimulating cAMP Production in Human Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2003;44(11):4837-4844. doi: 10.1167/iovs.02-1292.
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purpose. To study the mRNA and pharmacology of a serotonin (5-HT) receptor positively coupled to adenylyl cyclase in normal, primary (P-CEPI), and immortalized human corneal epithelial cells (CEPI-17-CL4), by using numerous 5-HT agonists and antagonists. To determine and compare cloned human 5-HT7 receptor binding affinities of compounds with their functional potency data.
methods. RT-PCR was used to detect the presence of an mRNA for the human 5-HT7 receptor in CEPI-17-CL4 cells. Receptor-mediated production of cAMP in cultured cells was measured using an enzyme immunoassay. Compound binding affinities were determined using [3H]-lysergic acid diethylamide ([3H]-LSD) binding to cell membranes of human embryonic kidney (HEK-293) cells expressing the cloned human 5-HT7 receptor.
results. RT-PCR revealed the presence of a 5-HT7 receptor mRNA in CEPI-17-CL4 cells. Normal P-CEPI cells generated cAMP in response to 5-HT (−log EC50; pEC50 = 7.6), 5-carboxamidotryptamine (5-CT; pEC50 = 7.8), 5-methoxy-tryptamine (pEC50 = 7.0) and 5-methoxy-dimethyl-tryptamine (pEC50 = 5.7). In CEPI-17-CL4 cells, serotonergic agonists also stimulated cAMP production with different potencies (pEC50): 5-CT (7.4) > 5-HT (6.5) ≥ 5-methoxy-tryptamine (6.1) > 5-methoxy-dimethyl-tryptamine (5.4) ≥ 8-OH-DPAT (<5.0) = α-methyl-5-HT (<5.0). Various 5-HT receptor antagonists inhibited cAMP production induced by 5-CT in CEPI-17-CL4 cells with different potencies (pKi): methiothepin (8.5) > mesulergine (8.1) = metergoline (8.0) > spiperone (7.4) ≥ clozapine (7.2) = SB-258719 (7.2) > mianserin (6.9) > ketanserin (6.3). Antagonist pKi values in P-CEPI cells were methiothepin (8.7), spiperone (7.4) and SB-258719 (6.6). The rank order of affinity for displacement of [3H]-LSD from the cloned human 5-HT7 receptor was: methiothepin > ritanserin > mesulergine = clozapine ≥ metergoline = 5-HT > SB-258719 ≥ spiperone > mianserin ≥ ketanserin. The functional agonist and antagonist potency data obtained from CEPI-17-CL4 cells correlated well with cloned human 5-HT7 receptor binding affinity data (r = 0.69), with P-CEPI cell functional data (r = 0.85), and with functional potency data in the literature for the cloned human 5-HT7 receptor (r = 0.88).
conclusions. These collective data support the presence of a pharmacologically defined, adenylyl cyclase-coupled 5-HT7 receptor in the CEPI-17-CL4 cells that may have relevance to physiological and/or pathologic functions of 5-HT7 receptors in the human cornea.
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