The long-term goal of this research is to elucidate the molecular mechanisms by which neurotransmitters regulate neuronal metabolism. This research will focus on the regulation of tyrosine hydroxylase (TH) activity by muscarinic agonists in PC12 pheochromocytoma cells. Because TH is the rate limiting enzyme in the synthesis of catecholamines and because catecholamines released from sympathetic neurons regulate cardiac contractility, heart rate and peripheral vasoconstriction, TH plays an important role in the regulation of the cardiovascular system. PC12 cells are widely used as a model system to study the function of sympathetic neurons. Muscarine increases TH activity and also increases the hydrolysis of inositol-containing phospholipids in PC12 cells. Phospholipid hydrolysis is thought to increase the accumulation of inositol-1,4,5-trisphosphate (IP3) and diacylglycerol in cells. IP3 is believed to cause the release of Ca2+ from intracellular stores and so could activate Ca2+/calmodulin-dependent protein kinase and diacylglycerol is thought to activate a phospholipid-dependent protein kinase, protein kinase C. This research will test the hypothesis that the activation of TH by muscarinic agonists is mediated by the hydrolysis of inositol-containing phospholipids, the accumulation of IP3 and/or of diacylglycerol, and the subsequent phosphorylation of TH by Ca2+/calmodulin-dependent protein kinase and/or by protein kinase C. This proposal will focus on the following questions: (1) Does muscarine stimulate the formation of inositol-1,4,5-trisphosphate and diacylglycerol? Cells will be prelabeled with either (3H)inositol or (3H)arachidonic acid. (3H)inositol phosphates and (3H)diacylglycerol will be analyzed. (2) Does muscarine cause the release of intracellular Ca2+? Cells will be preloaded with either 45Ca2+ or the fluorescent Ca2+ chelator, quin2, and the release of radioactivity or the increase in fluorescence will be monitored. (3) Does muscarine activate TH by increasing the activity of Ca2+/Calmodulin-dependent protein kinase and/or protein kinase C? TH activity will be monitored by measuring 3,4-dihydroxyphenylalanine production. Protein kinase activity will be assayed by measuring the incorporation of 32Pi into proteins separated by gel electrophoresis.
