Numerous diseases affecting the GI tract, ranging from secretory diarrhea to cystic fibrosis, are characterized by dysregulation of epithelial C1- secretion. This project originally identified that ammonium ion (NH4+, normally present at high concentrations in the colonic lumen) may be a novel endogenous regulator of C1- secretion via effects via effects on K+ channels and begins to define the interaction of NH4+ with the basolateral membrane K+ transporters also required for Cl-secretion. Based on work already accomplished, the current application considers how altered K+ channel regulation may influence various intestinal disease states. Preliminary data indicate that the ammonia-derived oxidant monochloramine (NH2Cl) may contribute to the diarrhea of colitis by potentiating Ca2+- dependent K+ channels. Experiments also suggest that docosahexaenoic acid (DHA, a component of fish oil) can augment Ca+2- dependent K+ channels, finding of particular interest as DHA begins clinical evaluation as therapy in CF. Preliminary findings suggest that the actin cytoskeleton an functionally alter Ca2+-dependent K+ channels, and conversely, that these K+ channels can modulate cell functions such as epithelial restitution that involve actin remodeling. Three sets of studies are proposed. First, the impact of ammonia on colonic epithelial transport will be further characterized in cultured epithelial ells and in human colonic mucosal preparations, with attention to the interaction of NH4+ with the basolateral Na+-k+-2Cl- co- transporter, Na+_K+ ATPase, and K+ channels. Second, potentiation of basolateral Ca+2-dependent K+ channels by cAMP and NH2Cl will be explored using cultured epithelial cells as model systems with the goal of defining a common mechanism for K+ channel potentiation by these seemingly diverse stimuli. The potential for therapeutic modulation of basolateral K + channels will be explored, specifically examining wheth4r docosahexaenoic acid (DHA) can augment Ca2+-dependent Cl- secretion in T84 cells and human colon, and, if so, to determine its mechanism of action. Finally, the studies will define the effect of chemical manipulation of F-actin on Ca2+-dependent K+ channel regulation and extend preliminary findings suggesting that K+ channel regulation affects the actin-regulated process of epithelial restitution. These studies highlight the importance of basolateral K+ channels in the regulation of secretion and other epithelial functions and reinforce their potential as targets for new drug design.
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