MECHANISMS OF BILE PIGMENT EXCRETION

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Using cell biological, biochemical, biophysical and immunomorphological methods, we seek to continue studies of the mechanisms involved in the transfer of bilirubin and other organic anions from blood to bile. (1) The role of hepatic endothelial cell fenestrae in controlling the uptake of bilirubin and other substances will be studied based upon characterization of fenestral dynamics in culture and effects on uptake mechanisms in isolated perfused rat liver. (2) Unique peptides from different isoforms of UDP glucuronyl transferase from normal and mutant (Gunn) rats will be sequenced and used for synthesis of oligonucleotide probes to identify isoform-specific cDNAs; the long range goal is to correct defective bilirubin UDPGI activity in Gunn rats and patients with the Crigler-Najjar syndrome, Type I. (3) Study of cellular mechanisms involved in cholestasis will be performed using isolated hepatocytes and vesicles derived from the sinusoidal and canalicular domains of the plasma membrane of hepatocytes; specifically, the role of membrane lipids, fluidity, polarity and the relation between transport events in the sinusoidal domain and observed defects in canalicular structure and function. A novel hypothesis that a group of canalicular plasma membrane ecto-"ATPases" protect hepatocytes from extracellular ATP concentrations will also be tested. (4) Functional and structural studies will be performed using sinusoidal and canalicular plasma membrane vesicles from animals with autosomal recessively inherited defects in membrane transport affecting bilirubin and other organic anions. The group includes heterozygotes and homozygotes for an "uptake defect" in Southdown sheep, the Dubin-Johnson syndrome in Corriedale sheep, and newly described conjugated hyperbilirubinemia in Wistar rats and cholestasis in homozygous athymic:heterozygous Gunn rat hybrids after administration of ethinyl estradiol. The goal is to define basic transport defects in these mutants.