Hepatocytes respond to circulating hormones, growth factors and other stimuli via specific receptors in the plasma membrane. Many of the messages generated by these receptors ultimately activate protein kinases that control the activity of intracellular enzymes. The long term goal of this project is to understand the regulation hepatic metabolism and function by hormones. The work is focused on two areas, the role of protein phosphorylation and dephosphorylation in the control of hepatic function and the nature of the signaling mechanisms used by angiotensin II to provide stimulatory and inhibitory inputs to the hepatocyte. The current proposal will focus on three aspects of the problem. First, the role of specific protein phosphatases, phosphatases-1 and 2A, in controlling the phosphorylation state of hepatic proteins will be examined in the intact cell using a novel, selective, protein phosphatase inhibitor, okadaic acid. Pilot experiments using two-dimensional gels to resolve 32PO4 labeled hepatic proteins have identified 5-7 proteins whose phosphorylation state is increased phosphorylation of these proteins has the potential to uncover new signaling mechanism or protein kinases. Second, the role of protein phosphorylation in regulating inositol lipid metabolism will be examined. Treatment of hepatocytes with okadaic acid markedly reduces the Ca2+ transient and blocks the production of IP3 in response to stimuli that activate phosphatidylinositol breakdown. It also activates one important enzyme of inositol triphosphate metabolism, the inositol (1,4,5) triphosphate 3-kinase. The role of protein phosphorylation in regulating the activity of the inositol (1,4,5) triphosphate 3-kinase will be examined by purifying the protein from liver and examining the effect of certain protein kinases on its activity in vitro. A cDNA for the protein will be cloned from a hepatocyte cDNA library and the primary structure of the protein determined. Third, the signaling systems used by angiotensin II will be explored in detail. Angiotensin II stimulates phospholipase C to generate IP3 in hepatocytes and inhibits hormone stimulated adenylate cyclase. The possibility that different subtypes of angiotensin II receptors couple to these responses will be examined using novel non-peptide antagonists of the angiotensin II receptor. The nature of the G-proteins coupling angiotensin II receptors to inhibition of adenylate cyclase and to phospholipase C will be examined by expressing the G-proteins of interest in the baculovirus system and reconstituting the G-proteins into hepatic membranes modified by prior treatment with pertussis toxin or GTP-gamma-S. Success of the reconstitution will be judged by the ability of the G-protein to recouple angiotensin II inhibition of cyclase in toxin treated membranes or to restore high affinity binding in GTP-gamma-S treated membranes.
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