The long-term objective of this proposal is to define the role of protein phosphorylation in the regulation of intestinal ion transport and in the pathogenesis of secretory diarrhea. A differentiated human colonic epithelial cell line, T84, will be used as an experimental model. T84 cell monolayers grown on mounted filters will be used to directly correlate biochemical and physiologic responses in cells stimulated under identical conditions.
The specific aims are as follows. 1/ Demonstrate cholinergic regulation of protein phosphorylation in intact T84 cells and correlate this with Ca++-dependent events in cell fractions. 2/ Determine which carbachol-stimulated phosphorylation events are mediated by protein kinase C (PKC). 3/ Determine whether PKC mediates (ie, is necessary and sufficient for) cholinergic effects on ion transport in T84 cells. 4/ Determine the distribution of pp83 in stimulated and unstimulated cells as a clue to its function. pp83 is a protein shown in preliminary studies to be a PKC substrate which exhibits increased phosphorylation in cholinergically stimulated T84 cells and for which specific antisera has been obtained. 5/ Determine the effects of bile acids on protein phosphorylation in T84 cells. 6/ Develop a brush border membrane preparation procedure for T84 cells using carcinoembryonic antigen as a marker. This procedure will be used to identify brush border proteins which are subject to secretagogue-dependent phosphorylation and to examine the possible regulation of brush border ion transport by such phosphorylation events. Protein phosphorylation changes will be examined using two-dimensional gel electrophoresis and pp83 phosphorylation will be estimated by immunoprecipitation. Ion transport effects will be measured in Ussing chambers. The distribution of pp83 will be determined by immunoblotting and by immunohistochemical methods. This study should identify protein kinases and phosphorylated cell proteins which are directly involved in regulating intestinal ion transport. This information should lead to new approaches to treating diarrhea and constipation based on the pharmacologic manipulation of these protein kinases and phosphorylated proteins.
