Abstract
Endocytic vesicles are acidified by an electrogenic proton pump and a parallel chloride conductance; however, acidification might be decreased if electrogenic transporters, such as Na,K-ATPase, that increase vesicle interior-positive membrane potential were also present. We examined this issue in early rat liver endosomes using ion substitution and inhibitors to alter Na,K-ATPase activity. These early endosomes, labeled for 2 min with the fluorescent fluid-phase marker fluorescein isothiocyanate-dextran, consistently acidified faster than endosomes similarly labeled for a 10-min period. In chloride-free media initial rates of acidification of early endosomes were faster in K+ media than in Na+ medium, although addition of K+ to Na+ or Na+ to K+ media to allow Na,K-ATPase to function did not decrease the rate of acidification. In chloride-containing media, rates were the same regardless of cation composition. The Na,K-ATPase inhibitor vanadate was prepared from orthovanadate by several methods, all of which inhibited liver ATPase activity. Two hundred mumol/L vanadate, prepared Cl(-)-free, tended to decrease rates of acidification in all media tested and these effects achieved statistical significance in Cl(-)-free media containing 150 mmol/L K+ or mixtures of Na+ and K+ and in 145 mmol/L KCl/5 mmol/L NaCl medium. Vanadate stocks pH-adjusted with hydrogen chloride increased rates of acidification in sodium gluconate buffers, probably as a result of the effects of the included Cl-. Five mmol/L ouabain (loaded into vesicles by endocytosis) and the membrane-permeable analog strophanthidin (2 mmol/L) both markedly inhibited endosome acidification, regardless of buffer ion composition. Collectively, these results suggest that Na,K-ATPase does not regulate acidification of rat liver early endocytic vesicles, that vanadate may modestly inhibit endosome acidification and that ouabain at high concentrations may inhibit acidification from the vesicle interior face.
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