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

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.