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Pharmacological Characterization of a Serotonin Receptor (5-HT₇) Stimulating cAMP Production in Human Corneal Epithelial Cells

¹From the Molecular Pharmacology Unit, Alcon Research, Ltd., Fort Worth, Texas; and the ²School of Optometry, University of Waterloo, Waterloo, Ontario, Canada.

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) = -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 (pK_i): 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 pK_i 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.