Previous work from this laboratory has shown that neurotransmitter can activate phospholipase D and, thereby, cause the rapid generation of products that can act as second messengers. Phospholipase D has a unique property called transphosphatidylation. This means that phosphatidyl groups can be transferred to other nucleophilic acceptors besides water. In the presence of ethanol, transphosphatidylation leads to the production of phosphatidylethanol. Neurotransmitter cause a rapid increase in phosphatidylethanol in both neuronal tissue culture and brain. This unique phospholipid C that specifically hydrolyzes inositol- containing phospholipids, phospholipase D specifically hydrolyzes phosphatidylcholine. One route for the synthesis of the phospholipase D substrate, phosphatidylcholine, is via methylation of phosphatidylethanolamine. Using an in vitro assay, indicates that the methylated intermediates are also good substrates for phospholipase D. Preliminary data presented in this proposal shows that increased levels of these methylated intermediates lead to increased neurotransmitter- stimulated phosphatidylethanol production even in the intermediates lead to increased neurotransmitter-stimulated phosphatidylethanol production even in the intact cell. Furthermore, Beta-adrenergic receptor stimulation which is known to activate the methylation pathway also enhances phosphatidylethanol production. It is, therefore, possible that catecholamine can increase phospholipase D activity by acting at both alpha- and Beta-receptors. Studies are planned for both neuronal tissue culture and brain cortical slices. The level of phosphatidyl-N-methylethanolamine in neuronal tissue culture will be increased by incubation with methylethanolamine and Beta-adrenergic receptor agonists. The cells will then be incubated with agents that are known to activate phospholipase D. Phosphatidylethanol production will be measured. The brain cortical slice model will be used to investigate the effect of norepinephrine on phosphatidylethanol production. The cortical slice model is appropriate to study the potential interaction of alpha and Beta-adrenergic receptors activation because norepinephrine is known to stimulate both these receptor in the cortex. This slice model will also be used to investigate the effect of chronic ethanol consumption on phospholipid methylation and phosphatidylethanol production. The goal of this proposal is to understand the role of these methylated intermediates as substrates for phospholipase D and to understand the potential role of Beta-receptor mediated phospholipid methylation in the production of phosphatidylethanol.
| Levine, G H; Maglio, J J; Horwitz, J (2000) Differential effects of ethanol on signal transduction. Alcohol Clin Exp Res 24:93-101 |
| Davis, L L; Maglio, J J; Horwitz, J (1998) Phospholipase D hydrolyzes short-chain analogs of phosphatidylcholine in the absence of detergent. Lipids 33:223-7 |
| Jacobs, T Q; Passarello, B; Horwitz, J (1998) Increased levels of methylated intermediates of phosphatidylcholine lead to enhanced phospholipase D activity. Neurochem Res 23:1099-105 |
| Ischiropoulos, H; Duran, D; Horwitz, J (1995) Peroxynitrite-mediated inhibition of DOPA synthesis in PC12 cells. J Neurochem 65:2366-72 |
| Horwitz, J; Passarello, B; Corso, M (1995) Bradykinin stimulates phospholipase D in PC12 cells by a mechanism which is independent of increases in intracellular Ca2+. Neurochem Res 20:1041-8 |
