Adenosinetriphosphatase (ATPase) activity stimulated by K+ and inhibited by Sch-28080 (SCH), omeprazole (OME), and vanadate has been measured in microsomes from mammalian renal medulla and attributed to a kidney isoform of the H(+)-K(+)-ATPase. To determine whether the H(+)-K(+)-ATPase inhibitors could also inhibit the vacuolar (V)-type H(+)-adenosinetriphosphatase (H(+)-ATPase, i.e., H+ pump) in mammalian intracellular vesicles, we examined their effects on bafilomycin-sensitive acidification in renal cortical vesicles (CEV) and medullary endocytic vesicles (MEV). Rats were injected with fluorescein isothiocyanate-labeled dextran, and labeled endosomes were enriched from kidney tissue homogenates by differential and Percoll density gradient centrifugation. In the CEV, the V-type H+ pump was inhibited 25% by SCH and 30% by OME (100 microM each). Whereas the inhibition by OME was concentration and time dependent, the inhibition by SCH was only concentration dependent. Inhibition by these compounds was similar in the presence of 50 mM K+ (in = out) and in the complete absence of K+, thus ruling out a significant involvement of H(+)-K(+)-ATPase-mediated acidification. Inhibition, however, was not observed with 10 microM SCH and OME. The sensitivity of the V-type H+ pump to 100 microM SCH and OME in CEV was confirmed by the comparable inhibitions of intravesicular acidification observed in acridine orange fluorescence quench studies and by inhibition of Pi liberation in an ATPase assay. We also found that the V-type H+ pump in isolated rat liver endosomes is sensitive to 100 microM SCH and OME to a similar degree. In the MEV, acidification was only weakly affected by 100 microM SCH and OME, thus suggesting that H(+)-ATPases in endosomes from cortical and medullary tubules are different, possibly due to a previously described selective expression of subunit isoforms. Our finding indicates the importance of using low concentrations (< 10 microM) of OME and SCH in studies of H(+)-K(+)-ATPase in nongastric tissues to avoid misinterpretation of the data due to nonspecific inhibition of V-type H(+)-ATPases.

The Gs alpha and Gi alpha 1-3 subunits of GTP-binding proteins were localized in sections of rat kidney using antibodies against unique synthetic decapeptides from the different G alpha subunits. All of the G alpha subunits were found to have a polarized distribution on renal tubule epithelial cells, and staining was typically found on either basolateral or apical membranes in a given cell type. Gi alpha 1 was localized to the apical pole of both thick ascending limb cells and cells forming the papillary epithelium, Gi alpha 2 labeled the basolateral plasma membrane and the cytoplasm of collecting duct principal cells, and Gi alpha 3 was most abundant in the apical region of proximal tubule cells of the S1 segment, where it was concentrated in sub-brush-border invaginations. It was also found in the perinuclear Golgi complex in these cells. Gs alpha was heavily concentrated on the basolateral plasma membranes of thick ascending limb cells and both principal and intercalated cells of the collecting duct. Less intense subapical staining of G alpha s was also found in proximal tubule cells. The cells of the macula densa had a unique G protein distribution that was distinct from the surrounding cells of the thick ascending limb of Henle. Antibodies specific for the Gi alpha 1 and Gi alpha 3 subunits both stained intracellular vesicles clustered at the basal pole of the cell. A heterogeneous distribution of G alpha subunits was also found by Western blotting on isolated cortical membrane fractions.

Brush-border (BBMV) and basolateral membrane vesicles (BLMV) from rat renal cortex exhibit an ecto-ATPase activity that is distinct from other ATPases. We have examined the cellular and regional distribution of this enzyme in rat kidney using antibodies against rat liver ecto-ATPase. In isolated vesicles, the distribution shown by biochemical assays of ATPase activity was confirmed by immunocytochemistry and Western blotting. Indirect immunofluorescence and immunogold labeling showed that brush borders of the S1 and S3 segments of the proximal tubule (PT) were stained, but the S2 segment was negative. Staining was most intense in the S3 segment. The luminal membrane of the initial part of the thin descending limb of Henle also showed a marked staining. Surprisingly, basolateral plasma membranes of PT had no detectable staining. However, the plasma membrane of endothelial cells was heavily stained, both in larger vessels and in peritubular capillaries. Using an antibody against rat thrombomodulin, a marker for endothelial cell plasma membranes, we showed that preparations of BBMV, BLMV, and endocytic vesicles are all contaminated with these membranes. This may explain, at least partially, the biochemically measured ecto-ATPase activity in renal cortical membrane vesicles. Finally, no specific staining in the kidney was found using polyclonal antipeptide antibodies against the "long form" of liver ecto-ATPase, either by immunocytochemistry or by Western blotting. This indicates either that there is no long isoform of the ecto-ATPase in the kidney or that the intracellular domains of the long form are different in the two tissues.

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