The long term objective of this project is to elucidate the molecular mechanisms and physiological significance of hormone activated signaling systems in hepatocytes. A common mechanism by which many hormones and growth factors exert their effects on cell function is by phospholipase C (PLC)-mediated activation of inositol lipid breakdown, changes of Ca2+ flux and covalent modification of proteins. Complex feedback mechanisms regulate receptor-mediated cell signaling processes and an imbalance between these factors results in abnormal metabolism or cell growth. An elucidation of these fundamental biochemical events is important for understanding the etiology of disease states such as diabetes and cancer. This proposal will address the following specific aims: 1) the mechanism of signal transduction of hepatocyte growth factor (HGF) in rat hepatocytes, 2) the mechanisms of Ca2+ influx into hepatocytes mediated by Ca2+ mobilizing hormones (co-mitogens) and growth factors, 3) the mechanisms of synergistic or inhibitory interactions between growth factors and co-mitogenic hormones and 4) receptor coupling to phospholipase C isoenzymes alpha and delta. It is hypothesized that the signal transduction pathway involves tyrosine phosphorylation of PLC-gamma and possibly a G-protein for enhanced production of inositol 1,4,5-P3 and diacylglycerol with associated intracellular Ca2+ mobilization and Ca2+ influx. This hypothesis will be tested using anti-phosphotyrosine and anti-PLC-gamma antibodies for immunoprecipitation and Western blotting, together with analytical procedures and Ca2+ measurements using hepatocytes loaded with fura-2. Ca2+ influx mechanisms mediated by phenylephrine and peptide hormones, which overproduce Ins 1,4,5-P3 will be compared with those mediated by HGF and other growth factors to determine whether separate processes are involved that may account for synergistic effects on cell growth by augmenting cytosolic free Ca2+ during the cell cycle. The techniques to be used for measurement of hormone-mediated Ca2+ influx include whole cell patch clamp, Mn2+ quench of the fura-2 fluorescence and Ca2+ readdition to Ca2+-depleted cells. Inhibitory interactions between transforming growth factor beta (TGFbeta) and mitogenic growth factors will be investigated by measuring effects on Ca2+ flux and the effects of okadaic acid to inhibit protein phosphatases. The hypothesis that receptors for co-mitogenic hormones are coupled to PLC isoenzymes other than PLC-gamma will be investigated by use of immunoprecipitating antibodies to PLC-alpha and PLC-delta with solubilized rat liver membranes to identify complexes between alpha1-adrenergic, vasopressin and angiotensin II receptors, G-proteins and particular PLC isoenzymes.
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