NA-INDEPENDENT ACTIVE H+ EXTRUSION AND HCO3-()CL- EXCHANGE CONTRIBUTETO CYTOPLASMIC PH REGULATION IN THE HUMAN PLATELET/

Homeostatic regulation of cytoplasmic pH (pH(cyt)) against acid and al kaline challenges was studied in the human platelet using the intracel lular indicator 2',7'- bis(carboxyethyl)-5(6)-carboxyfluorescein (BCEC F), Activation of a Na+/H+ exchanger in the plasma membrane is a known mechanism by which the platelet resists cytoplasmic acidification, Th e present study demonstrates an additional Na+-independent H+ extrusio n mechanism which persists at low external Na+ concentration in the pr esence of 100 mu M ouabain, This mechanism is inhibited by rotenone, o ligomycin and an inhibitor of glycolysis, and is tentatively identifie d as a plasmalemmal H+-ATPase. The Na+-independent H+ extrusion mechan ism partially restores cytoplasmic pH (pH(cyt) after the cytoplasm is acidified by addition of 1 mu M 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(cyt) appr oximate to 6.0 against an acidic pH(ext) of 5.3 in an energy-dependent manner, The present study also revealed a Na+-independent, DIDS-inhib itable Cl-/HCO3- exchange activity, It removes alkaline equivalents fr om the cytoplasm with a half-time of 2.0 +/- 0.4 min after alkaline lo ading with 25 mM NH4Cl. Its activity was also revealed in chloride to gluconate and gluconate to chloride perturbations of the external medi um which raised or lowered pH(cyt) by 0.17 +/- 0.05 or 0.14 +/- 0.04 u nits, respectively. The activity of the anion exchanger allows the pla telet to maintain pH(cyt) = 7.00 +/- 0.11 at the alkaline pH(ext) of 8 .25, The combined activities of the Na+-independent H+ extrusion and C l-/HCO3-, exchange mechanisms make the platelet cytoplasm very resista nt to changes in external pH, For variation of pH(ext) between 5.0 and 8.5, pH(cyt) varies between 6.0 and 7.0, or roughly one-third as much .