Diarrheal diseases are the largest cause of mortality and one of the greatest causes of morbidity on a global scale, particularly among young children. Recognized mechanisms leading to intestinal secretion fall into primarily two categories, cyclic nucleotide-mediated and calcium-mediated. Protein kinase C (PKC) activation has been hypothesized to result in intestinal secretion, but until recently no direct association between intestinal secretion and PKC stimulation had been reported. In our laboratory, we have measured PKC enzymatic activity in isolated intestinal epithelial cells. Using the phorbol esters (PE) which directly activate PKC, we have demonstrated that PKC activation causes significant intestinal water and ion secretion in vivo. The overall objective of this proposal is to characterize the effect of PKC activation in the intestinal epithelial cell. Subsequent studies will investigate the relationship of PKC to intestinal secretion mediated by microbial toxins and calcium-dependent agonists important in human diarrheal diseases.
The specific aims are: I) to characterize the effect of PKC activation in the intestinal epithelial cell; and II) to determine if certain microbial toxins and calcium-dependent secretagogues may interact with PKC. The direct effects of PKC activation in isolated rat jejunal enterocytes will be established by measuring two outcomes consistent with PKC activation in other cell systems: 1) translocation of PKC from cytosol to membrane; and 2) phosphorylation of substrates by PKC in the cell. This series of experiments will determine the distribution of PKC in unstimulated enterocytes and in entercytes stimulated with PE, and will identify in vivo and in vitro substrates of PKC using one and two dimensional gel electrophoresis. After characterizing the effects of PKC activation in isolated enterocytes, we will study how cholera toxin, Escherichia coli STa and serotonin affect this assay system by: 1) determining if these agonists alter the intracellular PKC distribution suggesting involvement of PKC in their mechanism of action; and 2) identifying cellular substrates of these secretory effectors. Comparison of the substrates phosphorylated in vivo and in vitro in response to PKC, cyclic nucleotides or calcium represents a new approach to analyzing the interrelationships of the secretory pathways in the small intestine and may identify common target proteins for these agonists. These studies should have future application to the development of effective antisecretory agents for adjunctive therapy of human diarrheal diseases.
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