Na(+)-independent Active H(+) Extrusion and HCO(3)(-)/C1(-) Exchange Contribute to Cytoplasmic pH Regulation in the...

Homeostatic regulation of cytoplasmic pH (pH(eyt)) against acid and alkaline challenges was studied in the human platelet using the intracellular indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Activation of a Na(+)/H(+) exchanger in the plasma membrane is a known mechanism by which the platelet resists cytoplasmic acidification. The present study demonstrates an additional Na(+)-independent H extrusion mechanism which persists at low external Na(+) concentration in the presence of 100 µM ouabain. This mechanism is inhibited by rotenone, oligomycin and an inhibitor of glycolysis, and is tentatively identified as a plasmalemmal H(+)-ATPase. The Na(+)-independent H extrusion mechanism partially restores cytoplasmic pH (pH(eyt)) after the cytoplasm is acidified by addition of 1 µM nigericin. The restoration process is energy-dependent and has a t(1/2) of 7-21 min. The Na(+)-independent H(+) extrusion mechanism is also shown capable of maintaining pH(eyt) ≊ 6.0 against an acidic pH, of 5.3 in an energy-dependent manner. The present study also revealed a Na(+)-independent, DIDS-inhibitable Cl(-)/HCO(3)(-) exchange activity. It removes alkaline equivalents from the cytoplasm with a half-time of 2.0 ± 0.4 min after alkaline loading with 25 mM NH(4)Cl. Its activity was also revealed in chloride to gluconate and gluconate to chloride perturbations of the external medium which raised or lowered pH(eyt) by 0.17 ± 0.05 or 0.14 ± 0.04 units, respectively. The activity of the anion exchanger allows the platelet to maintain pH(ext) = 7.00 ± 0.11 at the alkaline pH(ext) of 8.25. The combined activities of the Na(+)-independent H(+) extrusion and Cl(-)/HCO(3)(-) exchange mechanisms make the platelet cytoplasm very resistant to changes in external pH. For variation of pH(ext) between 5.0 and 8.5, pH(eyt) varies between 6.0 and 7.0, or roughly one-third as much.