The overall objective is to better understand the pathophysiology of diarrheal diseases and to discover drugs potentially useful for treating these diseases. This will be approached by understanding regulation of basal intestinal electrolyte absorption and stimulated secretion and using this information to modify active intestinal transport in such a way as to be useful in therapy of diarrheal diseases; that is, stimulating absorption or inhibiting secretion. Emphasis in probing regulation will be on how intracellular calcium affects intestinal Na and C1 absorption and C1 secretion. Studies will be done in rabbit ileum. The bulk of the studies will define the role of second messengers on ileal Na and C1 absorption using brush border membrane vesicles - emphasis will be on the effect of Ca++/calmodulin, protein kinase C, cAMP and cGMP on Na and C1 influx processes. By performing these studies with and without ATP, it will be determined whether Ca++/calmodulin and protein kinase C act by regulating phosphorylation/dephosphorylation in the brush border. We will define the role of protein kinase C in regulation of rabbit ileal transport using intact tissue studies and correlating measurements of protein kinase C activity in ileal absorptive cells with changes in the linked NaC1 absorptive process localized to the same cells. In order to understand the role of calcium in regulation of transport, levels of free calcium in the cytosol of single intestinal epithelial cells will be measured with a fluorescence microscope-digitizing system-computer using the fluorescent dye Fura-2. Methodologies to be used include: radioisotopic flux measurements of Na and C1 using the Ussing chamber-voltage clamp technique to measure active electrolyte transport; Na and C1 influx measurements into purified brush border membrane vesicles over short periods of time to quantify the initial rates of the entry processes; identification of phosphorylated peptides by SDS-PAGE; and measurements of cytosol free Ca++ by fluorescence microscopy-image processing.
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