Brush Border membrane vesicles isolated from organ donor intestinal mucosa will be employed to elucidate the influx processes for peptides, amino acids and electrolytes in the human small intestine at the membrane level. In addition, the afflux processes for these substrates will be examined by utilizing human intestinal basolateral membrane vesicles. Transport mechanisms for dipeptides containing acidic and basic amino acids at the N- terminal end and for selected tripeptides will be investigated to fully understand the absorptive processes for di- and tripeptides in the human small intestine. Control, papain-and inhibitor (peptidase)-treated membrane vesicles will be employed for these studies. The requirements for peptide transport, mode of energization and number of transport systems for di- and tripeptides in the luminal membrane will be determined. Specific transport systems for free amino acids have not been investigated at the membrane level in the human intestine. Using model amino acids as probes, transport systems for amino acids in brush border membrane vesicles will be examined to determine the number of Na+-dependent, Na+independent and specific transport systems for individual amino acids. Transport studies using basolateral membrane vesicles will be carried out to delineate the afflux processes for selected amino acids and peptides. Our studies of the transport of Na+ and Cl- in human intestinal brush border and basolateral membrane vesicles will focus on the Na+/H+ and cl- /HCO3- exchangers and evaluating the mechanism of coupling of Na+ and Cl- transport in the absorption of NaCl in the human intestine. Transport of Na+ and Cl- will be investigated using 22Na, 36Cl, 82Br, acridine orange and Cl-sensitive fluorescent dye studies to determine the presence of conductive processes, Na+/H+ and Cl-/HCO3- exchange processes. Transport characteristics for selected anions such as sulfate, oxalate, formate, propionate and butyrate will be analyzed to evaluate the specificity, interactions with Cl- and number of independent anion exchangers in the human small intestine. Studies to phosphorylate membrane proteins by loading vesicles with ATP, and selected combinations of Ca++, calmodulin, cyclic AMP and protein kinase C will be carried out and such vesicles will be used to measure transport of Na+ and Cl- to determine the direct effects of regulators on Na+/H+ and c/-/HCO3- exchangers and Na+ and Cl- conductive pathways in brush-border and basolateral membranes. Our proposed studies on the mechanisms of intestinal transport of peptides, amino acids and electrolytes using human intestine will be of great importance in our understanding of protein nutrition and electrolyte absorption in man and will be essential for future study of diseased states of the gastrointestinal system.
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