Phorbol ester-induced changes of cytoplasmic pH in neutrophils: role of exocytosis in Na+-H+ exchange

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Abstract
Cytoplasmic pH homeostasis was studied in intact and granule-free porcine neutrophils following activation with 12-O-tetradecanoyl-phorbol-13-acetate (TPA). In intact cells, TPA activated at least two separate processes: a Na+-independent and amiloride-insensitive acidification, and a compensatory acid extrusion. The latter is Na+ dependent and blocked by amiloride and is likely to represent Na+-H+ exchange. Enucleated and degranulated neutrophils (cytoplasts) were prepared by sedimentation of cytochalasin B-treated neutrophils through a discontinuous density gradient. Cytoplasts responded to an artificially imposed acid load with activation of an amiloride-sensitive Na+-H+ exchange. TPA also activated both acid production and Na+-dependent acid extrusion in cytoplasts. The magnitude of the responses was comparable in intact neutrophils and in cytoplasts. These data suggest that 1) the nucleus and the secretory granules are not involved in the acidifying response to TPA, and 2) exocytosis of secretory vesicles is not required for activation of Na+-H+ exchange during acid loading or following treatment with the phorbol ester TPA.