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.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD013250-15
Application #
3312160
Study Section
Pharmacology A Study Section (PHRA)
Project Start
1979-09-30
Project End
1995-04-30
Budget Start
1993-05-01
Budget End
1994-04-30
Support Year
15
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of Kentucky
Department
Type
Schools of Medicine
DUNS #
832127323
City
Lexington
State
KY
Country
United States
Zip Code
40506
Vore, M; Liu, Y; Huang, L (1997) Cholestatic properties and hepatic transport of steroid glucuronides. Drug Metab Rev 29:183-203
Vore, M; Hoffman, T; Gosland, M (1996) ATP-dependent transport of beta-estradiol 17-(beta-D-glucuronide) in rat canalicular membrane vesicles. Am J Physiol 271:G791-8
Liu, Y; Huang, L; Hoffman, T et al. (1996) MDR1 substrates/modulators protect against beta-estradiol-17beta-D-glucuronide cholestasis in rat liver. Cancer Res 56:4992-7
Liu, Y; Ganguly, T; Hyde, J F et al. (1995) Prolactin increases mRNA encoding Na(+)-TC cotransport polypeptide and hepatic Na(+)-TC cotransport. Am J Physiol 268:G11-7
Ganguly, T C; Liu, Y; Hyde, J F et al. (1994) Prolactin increases hepatic Na+/taurocholate co-transport activity and messenger RNA post partum. Biochem J 303 ( Pt 1):33-6
Vore, M; Hoffman, T (1994) Carrier-mediated electrogenic transport of estradiol-17 beta-glucuronide in rat liver BMV. Am J Physiol 267:G546-51
Gosland, M; Tsuboi, C; Hoffman, T et al. (1993) 17 beta-estradiol glucuronide: an inducer of cholestasis and a physiological substrate for the multidrug resistance transporter. Cancer Res 53:5382-5
Ganguly, T; Hyde, J F; Vore, M (1993) Prolactin increases Na+/taurocholate cotransport in isolated hepatocytes from postpartum rats and ovariectomized rats. J Pharmacol Exp Ther 267:82-7
Zimniak, P; Ziller 3rd, S A; Panfil, I et al. (1992) Identification of an anion-transport ATPase that catalyzes glutathione conjugate-dependent ATP hydrolysis in canalicular plasma membranes from normal rats and rats with conjugated hyperbilirubinemia (GY mutant). Arch Biochem Biophys 292:534-8
Liu, Y; Hyde, J F; Vore, M (1992) Prolactin regulates maternal bile secretory function post partum. J Pharmacol Exp Ther 261:560-6

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