Asymmetrical distribution of hepatic nak-atpase subunits and enzyme
activity correlates with polarized [beta] subunit expression.
Simon, Francis R., Hyam L. Leffert, Mark Ellisman, Mieko Iwahashi, Tom
Deerinck, John Fortune, Denise Morales, Rolf Dahl, and Eileen
Sutherland.
Department of Medicine, Denver Veterans Affairs Hospital and
Hepatobiliary Research Center, University of Colorado School of
Medicine, Denver, CO 80262 and Departments of Pharmacology and Center
for Molecular Genetics and Neurobiology, University of California at
San Diego, La Jolla, CA 92093
APStracts 2:0113C, 1995.
We have examined underlying causes for observations made in
hepatocytes in which catalytic subunits of sodium-potassium adenosine
triphosphatase (NaK-ATPase) are found both in bile canalicular
(apical) and sinusoidal (basolateral) membrane domains, whereas
functional activity is associated preferentially at sinusoidal
membrane sites. In a series of parallel studies, using fixed tissues
from adult rats examined by both light and electron microscopy, and
both fluorescent and gold-labelled mono-specific anti-catalytic
monoclonal and polyclonal antibodies, NaK-ATPase [alpha]-subunits
were localized to both membrane domains of hepatocytes. This bipolar
distribution was not due to different hepatic isoforms of [alpha]
subunit. Using purified liver plasma membrane subfractions, ouabain
inhibition curves demonstrated similar inhibition constants (Ki= 10
-5M), and immunoblots using [alpha]1, [alpha]2 and [alpha]3 polyclonal
and monoclonal antibodies demonstrated antigenic sites predominantly
for [alpha]1 in both membrane fractions. Also, Northern blot
hybridization analysis revealed only the [alpha]1 isoform in
hepatocytes. In contrast to the bipolar distribution of [alpha]1, the
[beta] subunit was identified only at the sinusoidal surface using
fluorescence labeling with a monoclonal antibody. The [beta]1 isoform
was demonstrated by Northern blot analysis and was present
predominantly at the sinusoidal domain by immunoblotting with
polyclonal antibodies. The [beta]2 isoform was not detected by either
immunoblotting after deglycosylation or by Northern blots. In
addition to the bipolar distribution of [alpha]1, immunoblotting of
liver plasma membrane subfractions demonstrated a symmetrical
distribution of fodrin, ankyrin, actin and E-cadherin at both
domains. These and previous results (Sutherland, et al. Proc. Natl.
Acad. Sci. USA 85:8673-8677, 1988) suggest functionally competent
[alpha]/[beta] complexes form at the sinusoidal domain, while only
[alpha]1 subunits are present at the apical pole.
Received 31 March 1994; accepted in final form 19 December 1994.
APS Manuscript Number C182-4.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 7 March 1995.