The aim of this proposal is to examine the role of serotonin (5-HT), released from intestinal mucosa enterochromaffin (EC) cells in the regulation of gastrointestinal (GI) transport. The hypothesis is that 5-H released from EC cells induces secretion either directly, by acting on enterocyte-based receptors, or indirectly, by releasing neurotransmitters from submucosal enteric neurons or secretagogues from adjacent paracrine cells. Furthermore, 5-HT released by separate mechanism from intrinsic neurons also mediate transport. For this purpose we plan 1) to identify the mechanisms controlling release of 5-HT from EC cells, 2) to characterize intestinal sites of binding for 5-HT, specifically receptors on the putative target cells the enterocytes, and 3) to determine the mode of action of 5-HT on its mucosal and neural target cells in regulating intestinal transport. Various models will be employed in these studies. 1) 5- HT release from mucosal cells will be examined in two complementary preparations: a. an isolated, vascularly perfused intestinal segment that enables correlation of 5-HT release with changes in intestinal transport, and b. a chambered intestinal mucosal sheet model, employing mucosal sheets stripped of the outer, longitudinal muscle layer so that they are devoid of the attached myenteric plexus with its serotoninergic neurons. 2) 5-HT receptors will be characterized by radioligand binding using plasma membranes isolated from enterocyte and enriched in the basolateral component. 3) The mode of action of 5-HT in regulating intestinal transport will be examined in a modified Ussing chamber using voltage clamp technique and monitoring short circuit current (Isc) and isotopic bidirectional flux (22Na and 36C1). The type of 5-HT receptor(s) involved in regulation of secretion will be evaluated with preferential agonists (5-HT1,2,3) in the presence and absence of tetrodotoxin. Stimulus- secretion 2nd messenger systems will be examined for 5 HT induced enterocyte secretion in epithelial cell lines and enriched enterocytes. The components (afferent, cholinergic interneuron and secretomotor limbs) of a putative neural reflux mediating the secretory response to mucosal 5-HT release in response to luminal stimuli will be dissected in chambered, stripped mucosal sheets using a selective neurotoxin (capsaicin), nicotinic receptor blockade, and receptor antagonists for the presumed effector neurotransmitters, acetylcholine and VIP/PHI. Secretion and neurotransmitter release will be monitored and correlated with release of endogenous 5-HT. The use of these models will provide new and interactive data which will permit concrete conclusions about the mechanisms by which serotonin is released and affects intestinal transport.
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