The objective of the proposed research is to clarify the cause-effect relationships of cyclic AMP and arachidonic acid (AA) metabolites along with serotonin as mediators of the physiological effects of cholera toxin (CT) on the small intestine. After stimulation with CT, the time of appearance and types of eicosanoids present in the lamina propria versus the epithelium of rabbit intestinal segments will be compared with that of cAMP and serotonin. These biochemical alterations will be correlated with the effects of CT on intestinal motility in addition to absorption and secretion of water and electrolytes. The action of CT, eicosanoids, and serotonin will be evaluated further in an in vitro cell culture model, in which transepithelial currents can be measured. Selected inhibitors will be used to block cholera toxin-induced adenylate cyclase, phospholipases, cyclo-oxygenase, serotonin binding, and protein synthesis, while measuring their capacity to block physiological effects of the toxin and synthesis of the various mediators. We will probe the biochemical mechanism of CT- induced phospholipase stimulation that leads to prostaglandin synthesis by 1) focusing on a newly described CT-induced protein, the synthesis of which is required for CT-induced activation of phospholipase activity, 2) analyzing the phospholipid composition of CT-treated versus control enterocytes, and 3) characterizing the arachidonic acid metabolites formed in CT-treated enterocytes. Finally, our studies will determine if related diarrheal diseases might involved the synthesis of intestinal eicosanoids and the above mentioned mediators and whether drug intervention might alter the clinical course of these infections. These studies will improve our understanding of the biochemical basis of enterotoxin-mediated diarrheas and may lead to improvements in treatment of prophylaxis.
