Bile salts are ionic sterol detergents which can disrupt membranes and damage cells. Hepatocellular injury by bile salts may be import in pathogenesis of cholestatic liver disease. Ursodeoxycholate (UDC), a hydrophilic bile salt which is a relatively poor detergent, improves liver function in patients with chronic cholestasis. We have shown that conjugates of UDC prevent cholestasis caused by taurochenodeoxycholate or taurodeoxycholate in vivo in bile fistula rats and block lysis of isolated rat hepatocytes, human erythrocytes, or cholesterol/phospholipid vesicles by these bile salts in vitro. We now propose to elucidate the mechanism of this potentially important protective interaction. Questions to be addressed include: i) how is the susceptibility of membranes to disruption by bile salts influenced by bile salt structure, composition of the aqueous phase, and composition of the membrane? ii) does UDC prevent membrane disruption by preventing partition of more toxic bile salts into membranes? iii) do UDC and other bile salts at non-disruptive concentrations alter physical properties of membranes (fluorescence anisotropy gradient)? iv) does UDC block the effects of more hydrophobic bile salts on exchange of cholesterol and phospholipid between membranes/ and v) how specific is the hepatoprotective action of UDC? Studies will employ a variety of in vivo (rat) and in vitro experimental systems. Membrane disruption will be assessed by leakage of permeability markers from vesicles or cytosolic enzymes from cells. Bile salt/membrane partition will be quantified using four complementary methods (rapid ultrafiltration, dialysis, density gradient ultracentrifugation, gel chromatography). Fluidity gradients of viable cells will be determined using a new flow cytometry method recently developed at this institution. We will quantify effects of UDC on bile salt-mediated exchange of cholesterol and phospholipid between membrane vesicles. Finally, we will investigate in primate hepatocyte culture the protective effects (if any) of UDC against a wide variety of cytotoxins. The ultimate purposes of this work ar to expand our basic understanding of bile salt:membrane interactions, to elucidate the mechanism of bile salt toxicity, and to establish a scientific rationale for use of UDC in treatment of human liver diseases.
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