5-HT INDUCES CAMP PRODUCTION IN CRYPT COLONOCYTES AT A 5-HT4 RECEPTOR

Previous studies demonstrate that both 5-hydroxytryptamine (5-HT) and cyclic AMP (cAMP) induce chloride efflux from crypt colonocytes in the rat distal colon; antagonist studies suggest that the 5-HT response i s mediated primarily by the 5-HT4 receptor. Since this receptor is kno wn to be positively coupled to adenylate cyclase, we postulated that 5 -HT should induce generation of cAMP, which should be inhibited by 5-H T, antagonists. Method. Mucosal cells from rat distal colon were taken by a sequential calcium chelation technique for enrichment of crypt c ells. Cytokeratin stains demonstrated that >99% of cells were colonocy tes. [H-3]Thymidine uptake studies demonstrate a fivefold increased in corporation in this cell preparation compared to earlier fractions, 3- Isobutyl-l-methylxanthine (IBMX, 100 mu M) was added to all cell suspe nsions in order to prevent cAMP metabolism. Cell suspensions were incu bated for 2 min at 37 degrees C with different concentrations of 5-HT (n = 7). cAMP was measured by enzyme immunoassay. In another series of experiments, 5-HT (0.3 mu M) stimulation of cAMP was similarly measur ed in the presence and absence of 5-HT receptor antagonists: 10 mu M 5 -HTP-DP (5-HT1P; n = 4), 0.1 mu M ketanserin (5-HT2A; n = 4), 0.3 mu M ondansetron (5-HT3; n = 4), 3 mu M tropisetron (5-HT3 and 5-HT4; n = 4), and 10 nM GR-113808 (5-HT4; n = 5). Results. 5-HT produced a dose- dependent increase in cAMP. The increase was significant at concentrat ions greater than or equal to 0.3 mu M when compared to cells incubate d with IBMX alone. In the second series of experiment, 5-HT-induced ge neration of cAMP at a dose of 0.3 mu M was significantly inhibited in the presence of GR-113808 and tropisetron, Conclusion. 5-HT acts at a 5-HT4 receptor to induce production of cAMP in rat distal crypt colono cytes. (C) 1998 Academic Press.