The long term objective of this project is to elucidate the mechanisms and physiological significance of hormonally-activated intracellular signalling systems in hepatocytes. It is now known that the effects of a variety of Ca2+-mobilizing hormones are mediated by activation of phospholipase C, which causes a rapid breakdown of phosphatidylinositol polyphosphates in the plasma membrane with a simultaneous production of the Ca2+ -mobilizing second messenger, inositol 1, 4,5-trisphosphate, and the protein kinase C activator, 1,2-diacylglycerol. The major purpose of the present proposal will be a) to investigate the nature of the coupling mechanism whereby receptor occupancy causes an activation of phospholipase C, and b) to characterize the functional effects and nature of target phosphorylated proteins induced by diacylglycerol and phorbol ester-mediated activation of protein kinase C. One of the hypotheses to be investigated is that the molecular coupling between the plasma membrane receptor and inositol phosphate metabolism is mediated by a GTP-binding protein that is distinct from the stimulatory (Gs) and inhibitory (Gi) GPT-binding proteins known to interact with adenylate cyclase. This putative GTP-binding protein will be isolated, purified and its effects on phospholipase C characterized by reconstitution experiments using purified phospholipase C inserted into phospholipid vesicles. Other possible target sites for interaction of the GTP-binding protein will be investigated together with possible modulation of the effects exerted by the alpha and beta subunits of GTP-binding proteins by cAMP-mediated protein kinase and by protein kinase C. The kinetics of the agonist-stimuated turnover of the inositol lipids will be investigated using isotopic techniques and the significance of possible activation of inositol lipid kinases by protein kinase C evaluated. The mechanism responsible for hormone-activated influx of Ca2+ into the cell will be investigated and the hypothesis evaluated that Ca2+ influx occurs by a Ca2+/Na+ exchange made energetically favorable by a protein kinase C-mediated activation of Na+/H+ exchange. Further studies relate to investigations of the localization and possible heterogeneity of Ins-1,4,5-P3-mediated calcium pools in intact cells using microspectrofluorometry techniques; the mechanism of glucagon-mediated Ca2+ mobilization in hepatocytes; and insulin-mediated effects. The propoed studies are directly relevant to the mechanisms of hormone action and stimulus-secretion coupling within the area of diabetes related research.
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