Taurocholate transport by basolateral plasma membrane vesicles isolated from human liver.
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 microliter per mg protein). An inwardly directed 100 mM Na+ gradient stimulated the initial rate of 2.5 microM 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 microM) uptake was significantly inhibited by 250 microM cholate, taurocholate, glycocholate, taurochenodeoxycholate and bromsulfophthalein.(ABSTRACT TRUNCATED AT 250 WORDS)
Division of Pediatric Gastroenterology and Pediatric Surgery, Children's Hospital Research Foundation, Cincinnati, Ohio 45229.
D A
DA
Novak
F C
FC
Ryckman
F J
FJ
Suchy
D000328:Adult; D000409:Alanine / Q000378:metabolism; D000838:Anions / Q000494:pharmacology; D001693:Biological Transport, Active; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism / Q000648:ultrastructure; D002648:Child; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008099:Liver / Q000201:enzymology / Q000378:metabolism* / Q000648:ultrastructure; D008564:Membrane Potentials; D013656:Taurocholic Acid / Q000378:metabolism*
D000838:Anions; D013656:Taurocholic Acid; D000409:Alanine