Many neurotransmitters and growth factors produce intracellular second messengers through the polyphosphoinositide cycle. Through diacylglycerol activation of protein kinase C, neuronal cell division and differentiation is controlled. The diacylglycerol stimulus is removed either by phosphorylation using diacylglycerol kinase or by the hydrolytic production of additional second messengers using diacylglycerol lipase and monoacylglycerol lipase. The second messenger profile is dependent on the regulation of diacylglycerol lipase, a glycoprotein present in neuronal plasma and nuclear membranes with a localization resembling protein kinase C. The long term goal is to understand the regulation of these lipases, the cause of their very high activities in Alzheimer membranes, and their role in neuronal degeneration. Bradykinin causes receptor-mediated 4-fold increased activities of these lipases in primary neuronal-enriched cell cultures. The generality and mechanism of this stimulation will be determined. Bradykinin and other neurotransmitters linked with protein kinase C activation will be tested for acylhydrolase (diacylglycerol lipase, monoacylglycerol lipase, and phospholipase A2) stimulation in primary neuronal and glial cultures. The most likely mechanism for stimulation is phosphorylation of the lipases or of an endogenous protein inhibitor by protein kinase. This will be tested with intact cells and with purified enzymes. The exact location of the phosphate on diacylglycerol lipase will be determined using our sequence information and antibodies. Synthesis of new enzyme will be evaluated with diacylglycerol lipase antibodies. Known inhibitors of diacylglycerol lipase, C-MT peptide, RHC-80267, and R-59022, will be used with intact and permeabilized cells to determine if basal and stimulated activities of diacylglycerol lipase can be inhibited. Activities of mono- and diacylglycerol lipase and phospholipase A2 will be determined in skin fibroblasts from Alzheimer's disease (AD) patients. If increased activities are observed, then experiments will be devised to explain the activation on the basis of receptor stimulation.
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