The long-term goal of the investigations described in this proposal is to define the precise intracellular mechanisms responsible for the control of active transcellular transport of chloride ions by the intestinal epithelium. This transport mechanism is the major driving force for the movement of water into the intestinal lumen and, accordingly, its over- expression is the primary basis for secretory diarrhea. Studies accomplished during the current funding period have identified both positive and negative intracellular signals, or messengers, that regulate the overall level of chloride secretion in response to hormones that increase intracellular levels of cytoplasmic calcium. It is now proposed to examine the exact nature of these signals, how they are generated within the cell, and the mechanisms whereby they exert their positive or negative effects. Two specific hypotheses will be tested. First, that inositol (3,4,5,6) tetrakisphosphate (InsP4) is a negative signal that is mobilized by calcium.mobilizing agonists such as carbachol, via the tyrosine phosphorylation of phospholipase C-gamma, and that this molecule may interact with basolateral potassium channels and/or apical chloride channels to limit secretion. Second, that cyclic AMP (cAMP) can potentiate the secretory effects of carbachol by modifying the production of putative negative signals, such as the influx of calcium from the extracellular environment and/or the synthesis of InsP4. The general strategy that will be employed to address these hypotheses will be to correlate biochemical and physiological parameters in a well-characterized cell culture model of the intestinal epithelium, the T84 cell line. Techniques to be employed include Ussing chamber, isotope flux and patch clamp studies of ion transport, biochemical analyses of inositolphosphates and tyrosine phosphorylation, and fluorescence imaging of intracellular calcium concentrations. The significance of the studies lies in their potential to increase our understanding of the cellular abnormalities underlying secretory diarrhea. Ultimately, improved knowledge of such abnormalities might be expected to suggest better therapeutic approaches to this condition.
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