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Abstract

The presence of membrane vesicles containing an ATP-driven H⁺ pump was demonstrated in rat kidney cortex homogenate using the ΔpH-sensitive dye acridine orange (AO). These vesicles were purified by differential and Percoll density gradient centrifugation. ATP-driven H⁺ uptake was about 20-fold enriched compared with the homogenate. Determination of marker enzyme activities indicated that these vesicles do not originate from brush border and basolateral membranes, lysosomes, endoplasmic reticulum, mitochondria, Golgi membranes, or red blood cells. The identity with brush border membranes was further excluded by the absence of Na⁺-H⁺ exchange. Renal cortical endocytotic vesicles that had taken up horseradish peroxidase or fluorescein isothiocyanate-labeled dextran (FITC-dextran) after injection of these substances into rats in vivo comigrated with the H⁺ pump activity on the Percoll gradient. Similar characteristics of the H⁺ pump demonstrated by the AO method and by fluorescence changes of in vivo trapped FITC-dextran proved the identity of H⁺ pump-containing vesicles with endocytotic vesicles. ATP-driven H⁺ uptake into endocytotic vesicles was stimulated by Cl^- and weakly inhibited by oligomycin. N-ethylmaleimide, dicyclohexylcarbodiimide, and Dio-9 were stronger inhibitors. Histochemical studies revealed that horseradish peroxidase-filled endocytotic vesicles are localized in the apical region of proximal tubule cells. An H⁺ pump with similar characteristics, but much lower activity, was found in brush border membranes, basolateral membranes, and mitochondria isolated by standard techniques, suggesting a possible contamination of these preparations with endocytotic vesicles.