Taurocholate transport by basolateral plasma membrane vesicles isolated from human liver

Authors

  • Donald A. Novak,

    1. Division of Pediatric Gastroenterology and Pediatric Surgery, Children's Hospital Research Foundation, Cincinnati, Ohio 45229
    2. Pediatric Gastroenterology/Hepatology Section, Yale University School of Medicine, New Haven, Connecticut 06510
    Current affiliation:
    1. University of South Florida School of Medicine, Department of Pediatrics, MDC Box 15, 12901 Bruce B. Downs Blvd., Tampa, Florida 33612-4799
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  • Frederick C. Ryckman,

    1. Division of Pediatric Gastroenterology and Pediatric Surgery, Children's Hospital Research Foundation, Cincinnati, Ohio 45229
    2. Pediatric Gastroenterology/Hepatology Section, Yale University School of Medicine, New Haven, Connecticut 06510
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  • Frederick J. Suchy M.D.

    Corresponding author
    1. Division of Pediatric Gastroenterology and Pediatric Surgery, Children's Hospital Research Foundation, Cincinnati, Ohio 45229
    2. Pediatric Gastroenterology/Hepatology Section, Yale University School of Medicine, New Haven, Connecticut 06510
    • Pediatric Gastroenterology/Hepatology Section, Yale University School of Medicine, 333 Cedar St., New Haven, Connecticut 06510
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Abstract

Transport of taurocholate into the hepatocyte against unfavorable chemical and electrical gradients occurs via a sodium-dependent, carrier-mediated transport system. Although this cotransporter has been characterized in the rodent, it has not been demonstrated in man. Therefore, we utilized human liver, obtained via multiorgan donation but not used for transplantation, to prepare basolateral (sinusoidal) liver plasma membrane vesicles by a Percoll gradient method. Na+,K+ -ATPase, a marker enzyme for the basolateral domain, was enriched 28.9-fold in the final membrane fraction compared with homogenate, whereas the bile canalicular membrane enzymes Mg++ -ATPase and alkaline phosphatase were enriched only 3.4- and 6.4-fold, respectively. Marker enzyme activities for endoplasmic reticulum, lysosomes and mitochondria were not enriched compared with homogenate. Integrity of the membrane vesicles was confirmed by the demonstration of Na+ -dependent concentrative uptake of the amino acid L-alanine (estimated intravesicular volume of 0.59 μl per mg protein). An inwardly directed 100 mM Na+ gradient stimulated the initial rate of 2.5 μM taurocholate uptake and energized a transient 2-fold accumulation of the bile acid above equilibrium (“overshoot”). In contrast, uptake was slower and no overshoot occurred with a K+ gradient. A negative intravesicular potential, created by altering accompanying anions or by valinomycin-induced K+ diffusion potentials, did not enhance taurocholate uptake, suggesting an electroneutral cotransport mechanism. Chloride as the accompanying anion stimulated the initial rate of uptake compared with anions of lesser or greater lipid permeability. Na+ -dependent taurocholate (4 μM) uptake was significantly inhibited by 250 μM cholate, taurocholate, glycocholate, taurochenodeoxycholate and bromsulfophthalein. Conversely, in the presence of a K+ gradient, only taurochenodeoxycholate and bromsulfophthalein significantly inhibited taurocholate uptake. The initial rate (5 sec) of Na+ -dependent taurocholate uptake, measured as a function of extravesicular taurocholate concentration (1 to 200 μM), demonstrated Michaelis-Menten kinetics with a Km of 33.8 ± 4.4 μM and a Vmax of 1.25 ± 0.99 nmoles per mg protein per min. We conclude from these studies that taurocholate uptake by basolateral plasma membrane vesicles from human liver is sodium dependent, electroneutral, inhibitable by other bile acids and saturable, with an apparent Km near the concentration of taurocholate found in human portal blood.

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