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

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-HT 7 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-HT 7 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 [ 3 H]-lysergic acid diethylamide ([ 3 H]-LSD) binding to cell membranes of human embryonic kidney (HEK-293) cells expressing the cloned human 5-HT z receptor. RESULTS. RT-PCR revealed the presence of a 5-HT 7 receptor mRNA in CEPI-17-CL4 cells. Normal P-CEPI cells generated cAMP in response to 5-HT (-log EC 50 ; pEC 50 = 7.6), 5-carboxamidotryptamine (5-CT; pEC 50 = 7.8), 5-methoxy-tryptamine (pEC 50 = 7.0) and 5-methoxy-dimethyl-tryptamine (pEC 50 = 5.7). In CEPI-17-CL4 cells, serotonergic agonists also stimulated cAMP production with different potencies (pEC 50 ): 5-CT (7.4) > 5-HT (6.5) ≥ 5-methoxy-tryptamine (6.1) > 5-methoxy-dimethyl-tryptamine (5.4) ≥ 8-OH-DPAT ( 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 [ 3 H]-LSD from the cloned human 5-HT 7 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-HT 7 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-HT 7 receptor (r = 0.88). CONCLUSIONS. These collective data support the presence of a pharmacologically defined, adenylyl cyclase-coupled 5-HT 7 receptor in the CEPI-17-CL4 cells that may have relevance to physiological and/or pathologic functions of 5-HT 7 receptors in the human cornea.