Increases in cytosolic calcium (Cai) and activation of protein kinase C (PKC) appear to have a major role in regulating intestinal Na absorption. Our previous investigations have shown that neurohumoral-stimulated increases in Cai can occur by at least 3 different mechanisms: (1) stimulation of phosphatidylinositol (PI) metabolism, (2) increased plasma membrane Ca permeability, and (3) stimulation of endogenous Ca release by cyclic nucleotides. Furthermore, they demonstrated that pharmacological activation of PKC with phorbol esters causes a significant inhibition of Na absorption by blocking brush-border membrane (BBM) Na/H exchange. We would now like to address many of these issues in greater detail. We wish to define the cellular events following physiological activation of PI metabolism in isolated chicken enterocytes. We will try to elucidate the relative roles PKC and increased Cai in the regulation of Na/H exchange. These studies will involve correlative measurements of Cai and pH in intact cells using fluorescent indicators, Na transport studies in BBM vesicles, PKC translocation determinations and biochemical studies of physiologically-relevant PKC- and Cai-dependent phosphoproteins. To further address these issues, studies will also be performed in isolated enterocytes and in spontaneously-differentiating Caco-2 colon cells where changes in Cai are buffered or where PKC has been downregulated. Next, we will determine why hormonally-stimulated PKC translocation from cytosol to the membrane fraction is transient and whether this may involve the subsequent formation of a soluble, but activatable PKC proteolytic fragment we recently identified. The mechanisms of cyclic nucleotide-stimulated increases in Cai and increased plasma membrane Ca permeability following stimulated PI hydrolysis will be studied more extensively in microsomal preparations and in basolateral membrane vesicles. Finally, we will investigate 2 previously unexplored areas of enterocyte ion transport, i.e., the distribution and relevant roles of PKC isoenzymes along the villus-crypt axis and the identification of the BBM Na/H exchanger. These studies will help define the physiological mechanisms by which intestinal peptides and neurotransmitters regulate intestinal salt and water transport. This information may widen application to understanding the pathophysiological basis of diarrheal diseases and to the formulation of strategies to treat them.
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