Effect of molecular charge on para- and transcellular access of horseradish peroxidase into rat bile

Authors

  • William G. M. Hardison M.D.,

    Corresponding author
    1. Department of Medicine, Section of Gastroenterology, Veterans Administration Medical Center and University of California, San Diego, California 92161
    • Veterans Administration Medical Center, Department of Medicine (111-D), San Diego, California 92161
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  • Philip J. Lowe,

    1. Department of Medicine, Section of Gastroenterology, Veterans Administration Medical Center and University of California, San Diego, California 92161
    Current affiliation:
    1. Ciba-Geigy Pharmaceuticals, Wimblehurst Road, Horsham, West Sussex, RH12-4AB England
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  • Miya Shanahan

    1. Department of Medicine, Section of Gastroenterology, Veterans Administration Medical Center and University of California, San Diego, California 92161
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Abstract

The permeability pathway into the biliary tree for small inert molecules exhibits a charge selectivity. Using a method which distinguishes trans- from paracellular access, we have examined the charge selectivity of biliary access pathways for the 40-kD protein horseradish peroxidase (pI 7.5), which was derivatized to strongly anionic (pI < 3.5) and strongly cationic (pI > 9.5) isoenzymes. Each isoenzyme was injected as a bolus into the perfusate of an isolated rat liver perfused in situ with a nonrecirculating Krebs-Ringer buffer. Bile was collected at intervals and horseradish peroxidase activity was measured. Its appearance allowed differentiation of paracellular from transcellular access, and the amount entering via each pathway was quantified. The species of enzyme entering bile was the same as that injected as determined by cation-exchange high-performance liquid chromatography of biliary horseradish peroxidase. Paracellular biliary access of anionic horseradish peroxidase was less than 50% that of neutral and cationic horseradish peroxidase both in the control state and when paracellular entry was augmented with 10−10M vasopressin. Transcellular access of anionic horseradish peroxidase was similarly restricted. To determine whether this restriction of anionic transcellular access was brought about by diminished hepatocellular uptake or augmented catabolism, we studied these parameters in 4-hr primary hepatocyte cultures. The uptake rates of all species were similar. Little or no degradation or efflux of any horseradish peroxidase species occurred over 30 min in the cultured cells. We conclude that access is charge selective for macromolecules and that this selectivity holds for trans- as well as for paracellular pathways. The selectivity of transcellular access may occur at the stage of intracellular sorting for biliary excretion.

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