AJP - Renal Physiology, Vol 251, Issue 6 1003-F1008, Copyright © 1986 by American Physiological Society
A sodium-hydrogen exchange system in isolated apical membrane from LLC-PK1 epithelia
A. Moran, J. Biber and H. Murer
We have monitored transmembrane pH gradients using acridine orange
fluorescence quenching and traced Na+ flux to study the properties of
Na+-H+ exchange in apical membrane vesicles isolated from LLC-PK1
epithelia. The membranes have low conductance for Na+, H+, and K+ ions. An
outwardly directed K+ gradient in the presence of valinomycin and carbonyl
cyanide p-trifluoromethoxyphenyl hydrazone produced intravesicular
acidification. This pH gradient was collapsed by addition of extravesicular
Na+ or Li+ ions but not by tetramethylammonium. Amiloride (10(-4) M)
inhibited the effect of both Na+ and Li+. An outwardly directed Na+
gradient stimulated H+ influx, which was also inhibited by 10(-4) M
amiloride. Membrane short-circuit conditions affected neither Na+ nor H+
flux, consistent with transport mediated by an electroneutral process. The
interaction of amiloride and sodium is consistent with noncompetitive
inhibition with Ki = 100 +/- 10 microM for amiloride and an apparent Km for
Na+ of approximately 20 mM. This finding is in agreement with previous
studies of intact LLC-PK1 epithelia but differs from observations in
brush-border membrane vesicles isolated from kidney proximal tubule in
which competitive and mixed inhibition have been reported. These observed
differences can be reconciled if two types of Na+-H+ exchange systems exist
along the nephron, one with competitive and the other with noncompetitive
inhibition, and if only the latter is expressed in the homogeneous cultured
cells.
