Intestinal epithelial cell Na-H exchanger (NHE) isoforms have very specialized functions, regulatory systems, cellular distribution, and regional patterns of expression which are inherently important in adapting to acute and chronic metabolic demands and changes in liminal Na load. Until now, the regulation of NHE isoforms has largely been studied in mutant, NHE-deficient, non-epithelial cells where some findings, particularly with the epithelial-specific NHE-2 and NHE-3 isoforms, substantially differ from NHE function and regulation in intact enterocytes. These isoforms are believed to be the major route for non-nutrient dependent Na absorption by the gut. Therefore, the goal of this proposal is to test the hypothesis that the role and regulation of intestinal epithelial NHE isoforms are unique and best studied in intestinal epithelial cells in vivo and in vitro. The first Specific Aim is to characterize the role of intestinal epithelial NHEs in mediating the adaptation of intestinal Na absorption following massive small bowel resection and chronic metabolic acidosis in the rat. Preliminary studies show striking isoform-, region-, and cell-specific changes in NHE function and expression in response to these conditions. Additional studies will be performed to define the cellular and molecular bases for these adaptive responses. Potential determinants of the adaptive changes in the NHE expression, i.e. alterations in Na luminal load or metabolic/hormonal perturbations, will be experimentally assessed. In Specific Aim #2, Caco-2/C2BBE (C2) intestinal epithelial cell monolayers will be used to examine specific mechanisms of NHE function, regulation, and expression in response to extracellular metabolic perturbations and stimuli. C2 cells spontaneously differentiate in culture, have endogenous NHE-1, but little apical NHE expression, and, when transfected with NHE-2 or NHE-3, demonstrate correct sorting and functional and regulatory characteristics of these proteins that correlate with observations made in native enterocytes. NHE isoform activities expressed in C2 cells can also be separately measured. The roles of protein phosphorylation, membrane recycling, post-translational modifications, and potential regulatory roles of NHE binding proteins in mediating NHE responses to hormonal stimuli, luminal Na load, metabolic acidosis, and states of cellular differentiation will be examined. These studies will provide important insights into the gut-specific cellular and molecular mechanisms for regulation of intestinal epithelial NHEs and into physiologically-relevant mechanisms mediating the Na absorptive response of the gut to acute and chronic metabolic perturbations.