It is likely that many of the pharmacological actions of ethanol are related to ethanol's ability to disrupt hydrophobic domains of proteins and lipids. This proposal will study the effects of ethanol on a major neurotransmitter and hormonal signal transduction cascade which involves membrane proteins and membranes at each level of the signal cascade. The hydrolysis of a unique class of lipids, the phosphoinositides, appears to be due to the interaction of plasma membrane receptors with intramembrane guanine-nucleotide binding proteins which activate a phosphoinositide phosphodiesterase (e.g. phospholipase C). Calcium flux may also activate a phospholipase. Phosphoinositide hydrolysis results in the formation of at least two second messengers, diacylglycerol and inositol triphosphate. These second messengers may activate a phospholipid dependent protein kinase and release calcium from membrane bound intracellular calcium stores. We have found that ethanol in vitro can inhibit phosphoinositide hydrolysis in both the liver and brain. Radiolabels will be used to follow phosphoinositide hydrolysis stimulated by cholinergic, adrenergic and ionic stimulation in several brain regions. The role of calcium flux and the effects of ethanol on calcium flux will be examined. Parallel studies will be done in the liver which is sensitive to ethanol, but is not responsive to depolarization. The effects of in vitro, acute and chronic ethanol treatment on phosphoinositide hydrolysis will be examined using tissue slices, isolated membrane preparations and receptor binding determinations to establish the sites of action of ethanol on phosphoinositide hydrolysis. Additional studies will follow second messenger release of intracellular calcium and protein kinase changes. The experiments proposed will determine the similarities and differences in the effects of ethanol on the phosphoinositide cascade in liver and brain.
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