Hepatic Na(+)-K(+)-ATPase enzyme activity correlates with polarized beta-subunit expression.
We have examined underlying causes for observations made in hepatocytes in which catalytic subunits of Na(+)-K(+)-ATPase are found both in bile canalicular (apical) and sinusoidal (basolateral) membrane domains, whereas functional activity is associated preferentially with sinusoidal membrane sites. In a series of parallel studies, we determined by both light and electron microscopy that Na(+)-K(+)-ATPase alpha-subunits were localized to both membrane domains of hepatocytes. With the use of purified liver plasma membrane subfractions, ouabain inhibition curves demonstrated similar inhibition constants (inhibition constant 10(-5) M), 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 the alpha 1-subunit, 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. 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 results suggest that functionally competent alpha/beta-complexes form at the sinusoidal domain, whereas only alpha 1-subunits are present at the apical pole.
Simon
F R
FR
Department of Medicine, Denver Veterans Affairs Hospital, Colorado, USA.
Leffert
H L
HL
Ellisman
M
M
Iwahashi
M
M
Deerinck
T
T
Fortune
J
J
Morales
D
D
Dahl
R
R
Sutherland
E
E
D000818:Animals; D005455:Fluorescent Antibody Technique; D007150:Immunohistochemistry; D008099:Liver / Q000166:cytology / Q000201:enzymology*; D008297:Male; D016253:Microscopy, Immunoelectron; D010042:Ouabain / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014018:Tissue Distribution
D012333:RNA, Messenger; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase