ACTIVATION OF MULTIPLE PH-REGULATORY PATHWAYS IN GRANULOCYTES BY A PHOSPHOTYROSINE PHOSPHATASE ANTAGONIST

Activated phagocytes undergo a massive burst of metabolic acid generat ion, yet must be able to maintain their cytosolic pH (pH(i)) within ph ysiological limits. Peroxides of vanadate (V4+-OOH), potent inhibitors of phosphotyrosine phosphatases, have recently been shown to produce activation of the respiratory burst in HL60 granulocytes. We therefore investigated the effects of V4+-OOH on pH(i) homoeostasis in HL60 gra nulocytes, using a pH-sensitive fluorescent dye. V4+-OOH stimulation i nduced a biphasic pH change: a transient cytosolic acidification follo wed by a significant alkalinization. The initial acidification was pre vented by inhibition of the NADPH oxidase and was absent in undifferen tiated cells lacking oxidase activity. Analysis of the alkalinization phase demonstrated the involvement of the Na+/H+ antiporter, and also provided evidence for activation of two alternative H+-extrusion pathw ays: a bafilomycin-sensitive component, likely reflecting vacuolar-typ e H+-ATPase activity, and a Zn2+-sensitive H+-conductive pathway. Our results indicate that V4+-OOH stimulation not only activated the NADPH oxidase but concomitantly stimulated H+-extrusion pathways, enabling the cells to compensate for the massive production of intracellular H associated with the respiratory burst.