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-HT4 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. [3H]Thymidine uptake studies demonstrate a fivefold increased incorporation in this cell preparation compared to earlier fractions. 3-Isobutyl-l-methylxanthine (IBMX, 100 microM) was added to all cell suspensions in order to prevent cAMP metabolism. Cell suspensions were incubated for 2 min at 37 degreesC with different concentrations of 5-HT (n = 7). cAMP was measured by enzyme immunoassay. In another series of experiments, 5-HT (0.3 microM) stimulation of cAMP was similarly measured in the presence and absence of 5-HT receptor antagonists: 10 microM 5-HTP-DP (5-HT1P; n = 4), 0.1 microM ketanserin (5-HT2A; n = 4), 0.3 microM ondansetron (5-HT3; n = 4), 3 microM 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 >/=0.3 microM 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 microM 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.
Albuquerque
F C
FC
Department of Surgery, Medical College of Virginia/VCU, Richmond, Virginia, 23298-0161, USA.
Smith
E H
EH
Kellum
J M
JM
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D002712:Chlorides / Q000378:metabolism; D003106:Colon / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D000242:Cyclic AMP / Q000096:biosynthesis*; D004305:Dose-Response Relationship, Drug; D007211:Indoles / Q000494:pharmacology; D007413:Intestinal Mucosa / Q000737:chemistry / Q000201:enzymology; D007633:Keratins / Q000032:analysis; D007650:Ketanserin / Q000494:pharmacology; D008297:Male; D017294:Ondansetron / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011985:Receptors, Serotonin / Q000378:metabolism*; D044422:Receptors, Serotonin, 5-HT4; D012701:Serotonin / Q000494:pharmacology*; D012702:Serotonin Antagonists / Q000494:pharmacology; D013194:Staining and Labeling; D013449:Sulfonamides / Q000494:pharmacology; D013936:Thymidine / Q000494:pharmacology; D014316:Tritium
D002712:Chlorides; D007211:Indoles; D011985:Receptors, Serotonin; D012702:Serotonin Antagonists; D013449:Sulfonamides; D014316:Tritium; D044422:Receptors, Serotonin, 5-HT4; D012701:Serotonin; D017294:Ondansetron; D007633:Keratins; D007650:Ketanserin; D000242:Cyclic AMP; D000262:Adenylyl Cyclases; D013936:Thymidine; C082871:GR 113808