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Abstract

The characteristics of the H⁺ pump in isolated rat renal endocytotic vesicles were studied by the delta pH-sensitive dye acridine orange, the voltage-sensitive dye 3,3'-dipropylthiadicarbocyanine iodide, and by a coupled optical ATPase assay. Intravesicular acidification depended on ATP and Mg²⁺ concentrations with half-maximal activations at 73 and 77 microM, respectively. CTP, GTP, UTP, and ITP partially supported acidification, but ADP and AMP did not. Ouabain, ethoxzolamide, levamisole, and vanadate did not inhibit H⁺ uptake into endocytotic vesicles. Oligomycin inhibited partially. Depending on concentration and preincubation time, Dio-9, filipin, N-ethylmaleimide (NEM), and dicyclohexylcarbodiimide (DCCD) inhibited H⁺ uptake completely. Filipin and, partially, DCCD acted nonspecifically by dissipating pH gradients. A specific cation was not required for the H⁺ pump; Zn²⁺ inhibited. Compared with mannitol, ATP-driven H⁺ uptake was stimulated by SCN- greater than Cl^- greater than Br^- greater than I^- much greater than HPO₄^2- = gluconate = HCO₃^- = F^-, but not by SO₄^2-, NO₃^-, CH₃COO^-, S2O₃^2-, and S4O6^2-. Chloride stimulated H⁺ uptake from the outside of the vesicles with an apparent K_m of 27 mM. In the absence of Cl^-, ATP-driven proton uptake was increased by intravesicular K⁺ and valinomycin, suggesting that the pump is electrogenic. The electrogenicity, however, could not be demonstrated with voltage-sensitive dyes. The vesicle membrane contains no significant K⁺ and Cl^- conductances; only a conductance for H⁺ was found. The vesicles exhibited an ouabain-, oligomycin-, and vanadate-insensitive ATPase activity that was inhibited by DCCD and NEM. Our data indicate the presence of an electrogenic H⁺ pump in endocytotic vesicles from rat renal proximal tubules with similar characteristics as H⁺ pumps present in various intracellular (nonmitochondrial) membranes.