This application for a new PPG represents an ongoing comprehensive multidisciplinary research program in epithelial cell and molecular biology which is occurring among the GI and Renal Divisions and the Department of Physiology of the Johns Hopkins University School of Medicine. The theme is description of epithelial transport proteins, regulation of epithelial transport, differentiation of epithelial cells, including studies to understand the contribution of the cystic fibrosis gene product to epithelial cell transport responses. Correlation of observations made in individual projects will feed on each other since the same cells and transport processes are being studied by more than one investigator. Five Projects supported by 3 Cores are proposed. The projects will: 1) probe structure/function aspects of the three isoform Na/H exchangers identified (epithelial and housekeeper) using the molecular biological approach of chimeras and point mutations in stably transfected fibroblasts and epithelial cells; 2) probe the genetic control of Na-betaine/Na-GABA cotransporter, which regulates renal cell accumulation of the non- perturbing osmolyte betaine; 3) examine the role of the cystic fibrosis gene product (CFTR) in intestinal Cl- secretion regulated by second messengers, using cells with different amounts of CFTR; 4) determine whether CFTR contributes to HT29 cell Cl- secretion, and study in detail the stimulatory (Ca2+ and cAMP) transduction pathways which modulate its activity; 5) study the transcriptional regulation of differentially expressed genes in the human colon cancer cell line HT29, as a way of studying intestinal epithelial cell differentiation. The three Cores are an Administrative Core; a Cell Culture Core, and a Fluorescence Core, with fluorescence microscopy/imaging and a computerized fluorometer which are used by multiple investigators to understand the intracellular signals in epithelial cells involved in regulation of transport and differentiation.
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