Diarrheal diseases are the largest cause of mortality and morbidity on a global scale. Mechanisms leading to intestinal secretion fall into primarily two categories, cyclic nucleotide -mediated and calcium-mediated. Our long range goal is to understand how protein kinase C (PKC) activation alters the response of the intestinal epithelium to microbial enterotoxins which increase cyclic nucleotide production. PKC is reversibly activated by calcium, phospholipid and diacylglycerol. We hypothesize that secretion stimulated by mucosal enteric pathogens occurs in the environment of the enteric nervous and immune systems resulting, most likely, in the simultaneous activation of multiple intracellular signaling pathways. The outcome of this second messenger """"""""cross talk"""""""" in the intestinal epithelial cell may contribute to clinical disease. Using T84 cells, a human colonic carcinoma cell line, as a model system, our studies demonstrate that PKC activation potentiates the physiologic and biochemical responses to the guanylate cyclase-activating heat stable enterotoxin of Escherichia coli (STa).
The specific aims of this proposal are: I. To examine the effect of PKC activation on the physiologic response to STa. The effect of carbachol, an acetylcholine analogue, and histamine, a mast cell mediator, on STa-stimulated chloride secretion will be examined. Both carbachol and histamine are likely physiologic regulators of PKC. Our preliminary data indicate that carbachol acts synergistically with STa to stimulate chloride secretion and that there is a unique directionality to this synergistic response. The roles of calcium and PKC activation in the synergy with STa will be examined by using calcium ionophores, phorbol ester, a diacylglycerol analogue, and inhibitors of PKC and intracellular calcium; and II. To examine the biochemical mechanisms by which PKC activators augment STa-stimulated cyclic GMP production and chloride secretion in T84 cells. Our studies indicate that PKC activators potentiate the cyclic GMP response to STa. We will examine four potential mechanisms for this response: 1) an alteration in cell receptors for STa; 2) an effect on guanylate cyclase; 3) involvement of a guanine nucleotide binding protein (G protein); and in stimulation of gene expression and new protein synthesis. Our preliminary results indicate that PKC activation increases the number of STa receptors in T84 cell membranes, augments particulate guanylate cyclase activity and indices new gene expression in T84 cells. STa also induces new gene expression. These results suggest multiple biochemical mechanism for synergy between PKC activators and STa.
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