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 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.
D000818:Animals; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D004848:Epithelium / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000378:metabolism; D007700:Kinetics; D017923:Sodium-Hydrogen Exchangers; D013552:Swine; D014634:Valinomycin / Q000494:pharmacology
D002352:Carrier Proteins; D017923:Sodium-Hydrogen Exchangers; D014634:Valinomycin; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone