A sodium-hydrogen exchange system in isolated apical membrane from LLC-PK1 epithelia

A. Moran, J. Biber, H. Murer

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Abstract

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 K(i) = 100 ± 10 μM for amiloride and an apparent K(m) for Na+ of ~ 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.

Original languageEnglish
Pages (from-to)F1003-F1008
JournalAmerican Journal of Physiology - Renal Fluid and Electrolyte Physiology
Volume251
Issue number6 (20/6)
DOIs
StatePublished - 1 Jan 1986
Externally publishedYes

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