In the intestine, electrogenic Cl-secretion forms the basis for a variety of pathologic states characterized by secretory diarrhea, including antibiotic-associated colitis, bacterial toxin-mediated diarrhea, ostomy diarrhea, certain neuroendocrine syndromes, and numerous inflammatory diseases of the intestine. Current models of electrogenic Cl secretion emphasize regulation of electrogenic Cl secretion at the level of apical membrane Cl channels because of the recently recognized relationship of this pathway to the pathogenesis of cystic fibrosis. In contrast, little is known about the regulation of basolateral membrane transporter function. In order to maintain cellular composition during high rates of secretion, basolateral Cl entry and apical Cl exit must be precisely matched, yet the mechanism by which coordination of apical and basolateral transport events (""""""""crosstalk"""""""") is accomplished is incompletely understood. The general goal of this proposal is to examine at a fundamental level the regulation of the Na+/K+/2Cl cotransporter, the basolateral Cl uptake mechanism of Cl secreting epithelial cells. The Na+/K+/2Cl cotransporter is a highly conserved membrane transporter which, in addition to its integral role in salt and water transport in both secretory and absorptive epithelia, may be involved in cell volume regulation in both epithelial and non-epithelial cells. Accumulating evidence suggests that the regulation of the Na+/K+/2Cl cotransporter is a complex process involving protein phosphorylation and alteration in numbers of transporters in the plasmalemma. Preliminary data for this project suggest the novel possibility that the F-actin cytoskeleton may dynamically influence these events. The studies proposed will be carried out using established cultured human intestinal cell lines as model systems. The specific goals of this project are 1) to characterize functionally the regulation of the basolateral membrane Na+/K/2Cl cotransporter in response to cAMP-mediated agonist, non-cAMP secretagogue, and osmotic perturbation, 2) to correlate regulated changes in cotransporter function with changes in the number of active cotransporter sites on the basolateral membrane, 3) to define the possible role of the F-actin microfilament system in regulation of cotransporter number and activity, and, 4) to identify some of the intracellular signalling pathways involved in modulation of cotransporter function. The propose studies should improve our understanding of electrogenic Cl secretion by elucidating the mechanism for regulation of Na+/K+/2Cl cotransport in intestinal epithelial cells. By determining the interaction between an ion-transporting integral membrane protein (the cotransporter) and the F-actin microfilament system, these experiments may prove to have broader implications for other important ion transport processes and should yield insight into the function of the cytoskeleton in epithelial cells. It is hoped that an understanding of fundamental mechanisms underlying electrogenic Cl secretion will establish a rational basis for designing new therapeutic approaches to the problem of secretory diarrhea.
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