ATP-dependent pHi recovery in lung macrophages: evidence for a plasma membrane H(+)-ATPase.
We have previously shown that cytoplasmic pH (pHi) recovery in pulmonary macrophages, under nominally HCO3(-)-free conditions, after acute intracellular acidification is Na+ and amiloride insensitive and is blocked by nonspecific proton adenosinetriphosphatase (ATPase) inhibitors N-ethyl-maleimide and N,N'-dicyclohexylcarbodiimide [Am. J. Physiol. 257 (Cell. Physiol. 26): C65-C76, 1989]. To further delineate the mechanism of H+ extrusion across plasma membranes of pulmonary macrophages, we investigated the effects of metabolic inhibitors of oxidative phosphorylation and glycolysis on cellular ATP content and pHi recovery from an intracellular acid load under nominally HCO3(-)-free conditions. Dose-dependent reductions in ATP levels and in the rate of pHi recovery were obtained in the presence of KCN (50% inhibition, 10(-4) M). Parallel reductions in ATP content and the rate of pHi recovery were noted in the presence of antimycin A, rotenone, oligomycin, and iodoacetate. However, inhibition by iodoacetate was reduced in the presence of pyruvate. The more specific vacuolar H(+)-ATPase inhibitors, bafilomycin A1 and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, resulted in no decrement in cellular ATP levels but significantly inhibited pHi recovery. These studies demonstrate that recovery from an acid load is ATP dependent and provide support for a plasmalemmal proton ATPase, perhaps of the vacuolar type, that participates in regulation of pHi in pulmonary macrophages.
Bidani
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Department of Internal Medicine, University of Texas Medical Branch, Galveston 77550.
Brown
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D009327:4-Chloro-7-nitrobenzofurazan / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008168:Lung; D018942:Macrolides; D008264:Macrophages / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D008297:Male; D011190:Potassium Cyanide / Q000494:pharmacology; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011817:Rabbits; D013050:Spectrometry, Fluorescence
D000900:Anti-Bacterial Agents; D018942:Macrolides; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D009327:4-Chloro-7-nitrobenzofurazan; D011190:Potassium Cyanide