The aim of this grant is to extend our understanding of the intracellular regulation of ileal neutral NaC1 absorption and brush border Na+/H+ exchange as a way of providing the necessary background to eventually increase understanding of the pathobiology of diarrheal diseases and develop treatment for these diseases. Mechanistic studies are proposed to test the hypothesis that brush border tyr kinases are involved in regulation of the brush border Na+/H+ exchanger and in the Ca2+-initiated brush border specific signal transduction in the ileal villus Na+ absorbing cell. This proposal is based on our demonstration during the previous grant period that (a) intestinal brush border tyr kinase(s) appear to be involved in short-term regulation of ileal neutral NaCl absorption and brush border Na+/H+ exchange, including in the regulation of basal NaC1 absorption, stimulation of NaC1 absorption by EGF, and the effect of elevated Ca2+ to inhibit NaC1 absorption; and (b) there is a localized brush border component of Ca2+-related signal transduction in the ileal villus Na+ absorbing cell, which is initiated by neurohumoral substances such as carbachol binding to the basolateral membrane but is downstream from the elevation of Ca2+ and represents the level at which final control of Na+ absorption occurs.
AIMS i nclude: I. Role of brush border tyr phosphorylation in regulation of ileal basal Nac1 absorption and its component brush border Na+/H+ exchange, in EGF stimulated absorption, and in Ca2+ inhibited absorption will be studied by a combination of intact tissue measurements of NaC1 absorption, brush border vesicle transport studies emphasizing Na+/H+ exchange, and biochemical determinations in the brush border, including effects on phospholipases (PL), DAG content, and protein kinase C activity. Effects on transport and the biochemical measurements will be determined with several classes of tyr kinase inhibitors including potential tyr kinase inhibitory peptides. Tyr phosphorylation of specific brush border proteins likely involved in regulation of brush border Na+/H+ exchange will be identified. The substrates to be studied are the brush border PI specific PLC, brush border Na+/H+ exchanger isoform and the brush border non-receptor tyr kinase SRC. Physical and functional association of the brush border tyr phosphorylated proteins identified will be carried out as a way to understand the functional role of brush border tyr phosphorylation using co-immunoprecipitation and sequential coimmunoprecipitation with an intermediate kinase assay. II. To direct the biochemical studies in I, the identity of the ileal brush border PL(s) activated by carbachol and/or A23187 and the mechanism of their activation will probed.
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