The long term goals of our research are to answer three fundamental questions: 1) How are substrates transported across the hepatocyte, 2) How many carriers are there and what determines their substrate specificity, an 3) How do estrogens, as in pregnancy and oral contraceptives, inhibit these processes. The type of substrates we are concerned with are the non-bile acid organic anions such as bilirubin and the glucuronide, sulfate and glutathione conjugates of drugs and hormones. Although biliary excretion is a major route of elimination of these potentially toxic waste products, little is known about the mechanisms controlling their transport or how estrogens inhibit these processes. 3H-estradiol-17beta(beta-D-glucuronide) (E217G) will serve as a model organic anion. The major approach to be used is characterization of the transport of E217G in basolateral and canalicula rat liver plasma membrane vesicles (bLPM and cLPM respectively) with respec to the driving forces, kinetic parameters and substrate specificities. The effects of pregnancy on transport of E217G will also be determined. Transport in isolated hepatocytes and the isolated perfused liver will be used to validate results in bLPM and cLPM. We will determine if an anion exchange system, with Cl or OH serving as the counterion, provides the driving force for E217G uptake in bLPM and if an intravesicular negative membrane potential drives efflux of E217G in cLPM. By means of cis- inhibition and trans-stimulation experiments, we will determine what other organic anions, (eg bilirubin and its glucuronide conjugates, the sulfate, glucuronide and glutathione conjugates of estrogens and/or naphthalene), utilize the same carrier as does E217G. Thus we will determine if the substrate specificity resides with the aglycone or the conjugating moiety. Efflux studies in bLPM will test the hypothesis that organic anions are transported from liver to blood. The effect of the cholestatic E217G vs th noncholestatic estradiol-3-glucuronide on efflux of taurocholate and on Cl/HCO3 exchange in cLPM will determine if E217G inhibits bile acid dependent and bile acid independent flow by inhibition of bile acid and HCO transport respectively. In view of the millions of users of oral contraceptives and the potential for toxicity in the maternal and fetal organism in pregnancy, it is in the public health interest to understand th mechanisms by which the liver eliminates these metabolites and the mechanisms by which the estrogens impair these processes.
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