APICAL H+-ATPASE ACTIVITY IN THE RABBIT PROXIMAL TUBULE

This study examines Na+-independent acid extrusion mechanisms on the apical membrane of single, isolated, perfused rabbit proximal tubules by monitoring the changes in intracellular pH (pH(i)). The latter was determined using a high resolution video imaging microscopy apparatus to alternately excite the pH-sensitive fluorescent dye 2',7'-bis-2-carboxyethyl-5(and -6)-carboxyfluorescein at 440 and 490 nm, while measuring the fluorescence emission at 530 nm. All experiments were conducted in the nominal absence of HCO3-. S1 segments from both superficial and juxtamedullary nephrons were examined individually. We found that removing Na+ from both the lumen and bath (i.e., basolateral solution) caused pH(i) to fall from 7.19 to 6.82 in the superficial S1 (JS1) segment, and from 7.18 to 6.80 in the juxtamedullary S1 (JS1). The pH remained depressed in both segments until Na+ was returned to the perfusate. In a separate series of experiments, tubules were further acid-loaded by adding NH4Cl to the perfusate following bilateral Na+ removal, pH(i) fell sharply from 6.75 to 6.0 (SS1) and from 6.76 to 6.05 (JS1), respectively. After this additional acid-loading the cells' pH(i) would spontaneously recover in the continued absence of Na+. 150 muM N-ethylmaleimide, an inhibitor of H+-ATPase activity, blocked this Na+-independent pH(i) recovery in both superficial and juxtamedullary segments. We conclude that there is a Na+-independent apical acid extrusion that functions in the nominal absence of HCO3- in the rabbit proximal tubule and that this mechanism is stimulated by extreme acid loads.