The chronic administration of ethanol results in changes that have been observed at behavorial, metabolic, and cellular levels. In vitro studies have shown that membrane fluidity is increased in the presence of ethanol. Treatment of animals with ethanol in vivo results in changes in the lipid composition of plasma membranes, and the membranes become resistant to the in vitro fluidizing effects of ethanol. These phenomena will be investigated by culturing mammalian cells for varying periods of time in the presence of varying concentrations of ethanol. Experiments will be carried out with L6 myoblasts because they have a high density of Beta-adrenergic receptors, and with S49 lymphoma cells. The advantage of these cells derives from the existence of variants with a defective coupling mechanism between the receptor and the catalytic moiety of adenylate cyclase. In addition to assessing the effects of chronic exposure to carefully controlled concentrations of ethanol on the viability of the cells, changes in the structural and functional properties of cell membranes will be determined. The use of cultured cells will make it possible to expose cells continuously to known concentrations of ethanol. The formation of acetaldehyde will be determined to control for toxic effects fo this metabolite. Fatty acid and phospholipid compositions will be determined after exposure of cells to ethanol. Effects on membrane fluidity will be investigated using the techniques of steady-state fluorescence polarization and nanosecond time-resolved fluorescence anisotropy decay. Functional effects of exposure to ethanol will be assessed by examining effects on the Beta adrenergic receptor/adenylate cyclase system. These studies will involve the use of radioligand binding techniques to determine the density and properties of Beta-adrenergic receptors. The coupling of these receptors to the adenylate cyclase system will also be examined. These experiments will employ both radiolabelled agonists and antagonists, and the interactions of these ligands with receptors will be assessed in membranes and on intact cells. These experiments are designed to reveal functional and biochemical alterations that may occur as a consequence of continuous exposure to ethanol under defined conditions. Membrane fluidity will also be altered by growing cells resistant to the effect of 25-hydroxycholesterol in the presence of cholesterol and by the addition of a series of structurally dissimilar agents known to affect membrane fluidity. The effects of these manipulations on the receptor/adenylate cyclase system will be compared to the effects of ethanol on these systems. The goal of these experiments is to use cultured cells to characterize chronic effects of ethanol. The results may increase our understanding of the molecular basis of tolerance and physical dependence.
| Bode, D C; Molinoff, P B (1988) Effects of chronic exposure to ethanol on the physical and functional properties of the plasma membrane of S49 lymphoma cells. Biochemistry 27:5700-7 |
| Bode, D C; Molinoff, P B (1988) Effects of ethanol in vitro on the beta adrenergic receptor-coupled adenylate cyclase system. J Pharmacol Exp Ther 246:1040-7 |
| Neve, K A; McGonigle, P; Molinoff, P B (1986) Quantitative analysis of the selectivity of radioligands for subtypes of beta adrenergic receptors. J Pharmacol Exp Ther 238:46-53 |
| Romano, C; Molinoff, P B (1986) Adenylate kinase increases adenylate cyclase activity in membranes from rat lung. J Cyclic Nucleotide Protein Phosphor Res 11:63-73 |
| Rabin, R A; Bode, D C; Molinoff, P B (1986) Relationship between ethanol-induced alterations in fluorescence anisotropy and adenylate cyclase activity. Biochem Pharmacol 35:2331-5 |
