Coexistence of contractile and relaxant 5-hydroxytryptamine receptors coupled to distinct signaling pathways in intestinal muscle cells: convergence of the pathways on Ca2+ mobilization.
Muscle cells were dispersed separately from circular and longitudinal muscle layers of guinea pig intestine, and 5-hydroxytryptamine (5-HT) receptors were characterized in naive cells and in cells in which one receptor type was preserved by selective receptor protection. In naive cells from both regions, 5-HT caused contraction and stimulated increases in cytosolic free calcium concentration ([Ca2+]i) (3-fold; p < 0.01) and cAMP levels (40-60%; p < 0.01) that were inhibited, respectively, by the 5-HT2 antagonist ketanserin and the 5-HT1p antagonist N-acetyl-5-hydroxytryptophyl 5-hydroxytryptophan amide (5-HTP-DP). In circular muscle cells, where agonist-induced increase in [Ca2+]i is mediated by Ca2+ release from inositol (1,4,5)trisphosphate-sensitive stores, 5-HT caused an increase in inositol (1,4,5)trisphosphate levels that was inhibited by ketanserin. In cells maximally contracted with a non-5-HT agonist (cholecystokinin octapeptide), 5-HT caused relaxation when the contractile effect mediated by 5-HT2 receptors was blocked with ketanserin; relaxation and the concomitant increase in cAMP were inhibited by 5-HTP-DP. The singular contributions of the Ca2+ and cAMP signaling pathways were identified in cells where only one receptor type was preserved. In cells with only 5-HT2 receptors, 5-HT caused contraction and an increase in [Ca2+]i but not in cAMP levels; contraction and the increase in [Ca2+]i were inhibited by ketanserin. Conversely, in cells with only 5-HT1p receptors, 5-HT caused relaxation and an increase in cAMP levels but not in [Ca2+]i; relaxation and the increase in cAMP levels were inhibited by 5-HTP-DP. The two signaling pathways were functionally linked, converging to regulate the level of [Ca2+]i. Thus, the increase in [Ca2+]i was augmented 1) when cAMP production was inhibited by 5-HTP-DP in naive cells or 2) when cAMP production was suppressed in cells where 5-HT1p receptors were inactivated and only 5-HT2 receptors were preserved. The results imply that the increase in cAMP levels mediated by 5-HT1p receptors acted to attenuate the increase in [Ca2+]i mediated by 5-HT2 receptors. We conclude that the response to 5-HT in muscle cells is a compound effect involving activation of two receptor types coupled to distinct signaling pathways that converge on [Ca2+]i as the determinant of mechanical activity.
Kuemmerle
J F
JF
Department of Physiology, Medical College of Virginia, Richmond 23298-0711.
Martin
D C
DC
Murthy
K S
KS
Kellum
J M
JM
Grider
J R
JR
Makhlouf
G M
GM
D006916:5-Hydroxytryptophan / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D006168:Guinea Pigs; D007413:Intestinal Mucosa / Q000378:metabolism*; D007650:Ketanserin / Q000494:pharmacology; D009119:Muscle Contraction / Q000187:drug effects; D009126:Muscle Relaxation / Q000187:drug effects; D009130:Muscle, Smooth / Q000378:metabolism*; D011985:Receptors, Serotonin / Q000378:metabolism*; D015398:Signal Transduction
D011985:Receptors, Serotonin; C022197:N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophanamide; D007650:Ketanserin; D006916:5-Hydroxytryptophan; D000242:Cyclic AMP; D002118:Calcium