BILE SALTS, MEMBRANES, AND CYTOTOXICITY

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Bile salts adsorb to membranes, at high concentrations causing membrane disruption. Adsorption of bile salts to intracellular membranes may determine many of their physiological effects, and bile salt induced membrane injury may be important in pathogenesis of cholestatic liver disease and gallstones. We have studied the adsorption of bile salts to lecithin-cholesterol vesicles and have developed and validated a quantitative model which predicts the distribution of bile salt taurine conjugates in mixed bile salt solutions between lecithin-cholesterol bilayers and the aqueous phase. In the studies proposed, this model will be generalized to a broad array of bile acids and other organic anions, membrane lipids, and solution conditions. Using large unilamellar vesicles of varying lipid composition, we will examine the relationship between membrane binding of bile salts, mixed micellar dissolution of membrane lipids (observed with quasielastic light scattering) and altered membrane permeability (release of trapped soluble markers assessed by ultrafiltration) to determine if the mixed micellar threshold concentration and the permeation threshold at which membrane leakage begins are predictable consequences of the membrane-bound ionized bile salt/lecithin ratio. Pure protein kinase C isoenzymes (alpha, betaII, delta, epsilon) prepared in a baculovirus system will be employed to test the hypothesis that bile salts activate protein kinase C isoenzymes by binding to membranes and serving as a "bridge" between the enzymes and membrane lipids. The model of bile salt-lecithin interactions will be extended beyond the limits of the two phase (monomer-membrane) region into micellar regions of the phase diagram by combining techniques of gel filtration and ultrafiltration, in order to permit modelling of detergent effects of mixed bile salt solutions. Using synthetic vesicles, isolated canalicular plasma membranes, and living cells (erythrocytes, cultured neoplastic gallbladder epithelia) we will test the hypothesis that lecithin in bile normally protects high cholesterol plasma membranes from bile salt injury by depressing the non-lecithin- associated bile salt concentration to non-toxic levels, and that this protective effect declines predictably as the cholesterol content of biliary vesicles increases. Finally the hepatoprotective role of biliary lipids and biliary bile salt-lipid interactions will be studied in two in vivo models of bile salt-induced liver injury: acute infusion of bile salts in the choline deficient bile fistula rat and chronic feeding of bile salts in hamsters fed lithogenic diets. The ultimate goal of these studies is to provide a conceptual framework for understanding the toxic and protective properties of bile salts and the role of bile salt toxicity in human disease.