It is proposed to investigate the hypothesis that the function of the family of related signaling proteins that translocate from the cytosol to the liver plasma membrane is modified as a result of chronic ethanol ingestion. These proteins, which include protein kinase C (PKC), phospholipase A2 (PLA2), diacylglycerol kinase, rafkinase, phospholipase Cy etc., require phosphati-dylserine and calcium for the translocation, have homology in Ca2+ -binding and/or zinc finger domain regions and are implicated in chronic ethanol induced effects on signal transduction. Preliminary and published data indicates that both PKC and PLA2 activities are modified as a result of chronic ethanol-induced lipid alteration in a manner consistent with the hypothesis. It has been established from our previous studies that: (a) the effect on PLA2 is due to a modification primarily in phosphatidylserine (PS) and (b) that the modification consists of an increase in fatty acyl unsaturation (increased docosahexaenoyl (n-3 22:6) and decreased in arachidonyl (n-6 20:4) containing PS-molecular species). Preliminary studies indicate that PKC, which requires PS for activity, is highly sensitive to PS-unsaturation, determined using highly purified-recombinant PKC and defined lipid bilayer vesicle systems. Further studies are proposed to more closely define (a) which of the Ca2+ -dependent and -independent PKC molecular species that occur in the liver cell are affected. The type and amount of each PKC isoform that associates with liver plasma membranes from livers of rats fed ethanol chronically will be determined along with and the PS-unsaturation. A lipid test system will be developed and used as a model for further studies to determine if the effects of chronic ethanol induced modifications interfere with PKC-membrane association and PKC activator-interaction using recently published methodology. Finally, we will explore the possibility that chronic ethanol-feeding affects the range of signaling proteins that translocate to the membrane in a PS dependent manner thus providing a mechanism that may underlie many of the effects of chronic ethanol ingestion on liver cell signal transduction.
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