Active, microtubule-dependent secretion of a fluorescent bile salt
derivative in skate hepatocyte clusters.
Miller, David S., Gert Fricker, Ursula Schramm, John H. Henson, David
N. Hager, Surajit Nundy, Nazzareno Ballatori, and James L. Boyer.
Laboratory of Cellular and Molecular Pharmacology, National
Institute of Environmental Health Sciences, National Institutes of
Health, Research Triangle Park, North Carolina 27709, Sandoz Pharma
AG, CH-4002 Basle, Switzerland, Department of Biology, Dickinson
College, Carlisle, PA 17013, Department of Medicine and Liver Center,
Yale University School of Medicine, New Haven, CT 06510, Department
of Environmental Medicine, University of Rochester School of
Medicine, Rochester, NY 14642, Mount Desert Island Biological
Laboratory, Salsbury Cove, Maine 04672
APStracts 2:0236G, 1995.
Fluorescence microscopy and video image analysis were used to study
the transport of a fluorescent bile acid derivative (N-{7-(4
-nitrobenzo-2-oxa-1,3-diazol)}-7[beta]-amino-3[alpha],12[alpha]-
dihydroxy-5[beta]-cholan-24-oyl)-2-aminoethanesulfonate; NBD-TC) in
isolated clusters of hepatocytes from the little skate, Raja
erinacea.. Analysis of images of hepatocyte clusters that were
incubated in medium with 0.5-1 [mu]M NBD-TC showed that the
fluorescent derivative accumulated in the cells and that the clusters
retained a patent canalicular lumen as well as the ability to
actively transport the bile acid derivative from the cells into the
lumen, i.e. lumen/cell fluorescence ratio greatly exceeded unity.
NBD-TC uptake by hepatocytes was inhibited by several organic anions,
of which taurocholate was the most effective. Uptake was also blocked
by metabolic inhibitors and by incubation in the cold. Neither Na
-replacement nor increased medium K, which depolarizes the membrane
electrical potential (PD), affected NBD-TC accumulation by
hepatocytes. Transport of NBD-TC into the canalicular lumen was
inhibited by incubation in the cold and was substantially reduced by
high K medium; these blocks were removed by warming and transfer to
normal K medium, respectively. Treatment of hepatocytes with 20-40
[mu]M nocodazole, a drug that reversibly depolymerizes microtubules,
reduced cellular NBD-TC accumulation and blocked its secretion into
the canalicular lumen; nocodazole effects were reversed by washing
the hepatocyte clusters in drug-free medium. Thus, uptake of NBD-TC
by skate hepatocytes is active and carrier-mediated, but not
dependent on the PD or Na gradient. NBD-TC secretion from cell to
canalicular lumen also appears to be active and carrier-mediated.
Canalicular secretion appears to be driven at least in part by the PD
and is highly dependent on an intact microtubular system in this
marine species.
Received 3 October 1994; accepted in final form 5 November 1995.
APS Manuscript Number G393-4.
Article publication pending Am. J. Physiol. (Gastrointest. Liver
Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 30 November 95