AJP - Cell Physiology, Vol 236, Issue 3 157-C162, Copyright © 1979 by American Physiological Society
Localization of [3H]ouabain-sensitive Na+ pump sites in cultured pig kidney cells
J. W. Mills, A. D. Macknight, J. M. Dayer and D. A. Ausiello
Pig kidney cells, LLC-PK1, grown by standard tissue-culture techniques form
monolayers and maintain morphological features characteristic of epithelia.
Cultures exposed to 2 X 10(-6) M [3H]ouabain for 30 min at 37 degrees C
bound 7.77 +/- 0.37 pmol/mg protein. This could be reduced by 58% by
incubation in the presence of 45 mM K+. Freeze-dry radioautographic
localization of [3H]ouabain-binding sites revealed grains distributed only
along that fraction of the plasmalemma directly facing the culture-dish
surface. Binding and localization of [3H]ouabain were correlated with an
inhibition of the Na+ pump in these cells because analysis of cellular
electrolytes in control cultures versus those exposed to 10(-3) M ouabain
revealed a fall in K+ from 419 +/- 9 to 173 +/- 4 mmol/kg dry wt with a
reciprocal increase in Na+. There was no change in cell H2O. Similarly,
oxygen consumption was reduced by 32% after exposure to ouabain. These
results provide direct evidence that in epithelial cells in culture the
membrane facing the culture dish corresponds to the basolateral membrane of
epithelial cells in vivo.
