This proposal concerns research on the regulation of Na/H exchange and intracellular Ca activity in mitogen-stimulated, cultured human fibroblasts. The long term goals of this project are to elucidate the detailed mechanism by which growth factors activate the amiloride-sensitive Na transport system and induce the mobilization of intracellular Ca, and to assess the role of these early events in regulating mitogenesis in both normal and transformed cells. We propose specifically to: 1) investigate the involvement of calmodulin and/or protein kinase C in the mitogen induced activation of Na/H exchange; 2) investigate the mechanism for regulating Ca uptake into and release from intracellular storage sites; 3) investigate the role of the rise in intracellular pH and Ca activity in subsequent transcriptional events; 4) investigate the role of G proteins in the mitogen-stimulated activation of Na/H exchange and in the stimulation of inositol phosphate release; 5) compare the regulation of Na/H exchange and intracellular Ca activity in normal and transformed cells; 6) investigate the desensitization of the mitogen induced stimulation of Na/H exchange and 7) utilize our recently designed fluorescence macroscope to select Na/H exchange-negative mutants and mutants defective in the regulation of Na/H exchange. Experiments designed to these specific aims involve measurement of ion fluxes across cellular membranes and in membrane vesicles, measurement of intracellular pH and Ca activity using fluorescence indicators, measurement of Ca uptake into digitonin permeabilized cells, measurement of binding of growth factors to membrane receptors, measurement of transcription of mRNA by RNA dot blots, measurement of inositol trisphosphate release, and measurement of protein phosphorylation in cultured human fibroblasts.
Showing the most recent 10 out of 28 publications
