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 is mediated primarily by the 5-HT4 receptor. Since this receptor is known to be positively coupled to adenylate cyclase, we postulated that 5-HT should induce generation of cAMP, which should be inhibited by 5-HT, antagonists. Method. Mucosal cells from rat distal colon were taken by a sequential calcium chelation technique for enrichment of crypt cells. Cytokeratin stains demonstrated that >99% of cells were colonocytes. [H-3]Thymidine uptake studies demonstrate a fivefold increased incorporation in this cell preparation compared to earlier fractions, 3-Isobutyl-l-methylxanthine (IBMX, 100 mu M) was added to all cell suspensions in order to prevent cAMP metabolism. Cell suspensions were incubated 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 measured 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 concentrations greater than or equal to 0.3 mu M when compared to cells incubated with IBMX alone. In the second series of experiment, 5-HT-induced generation 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 colonocytes. (C) 1998 Academic Press.