The role of vacuolar H+-ATPase in the control of intragranular pH and exocytosis in eosinophils

The presence of vacuolar H + -ATPase (V-ATPase) on exocytotic granules in eosinophils and the role of this enzyme in exocytosis were explored in this study. Antibody against 116-kd subunit of V-ATPase positively stained eosinophil granules in immunofluorescence analysis. When eosinophil lysate was extracted immunomagnetically with the same antibody, the extracted fraction contained a considerable amount of eosinophil peroxidase, a marker of eosinophil-specific granules, which indicates that V-ATPase was present on the membranes of eosinophil exsosomal granules. The pH of the eosinophil granules, measured fluorometrically with acridine orange as a ΔpH-sensitive dye, was estimated to be 5.1. The acidity of the eosinophil granules was perturbed by bafilomycin A 1 , a potent selective inhibitor of V-ATPase, which indicates that the low pH of these granules is maintained by V-ATPase activity. Bafilomycin A 1 and NH 4 Cl, both of which raise the intragranular pH to neutral, inhibited the eosinophil peroxidase exocytosis induced by platelet-activating factor. These agents did not, however, affect the changes in cytosolic free calcium concentration [Ca 2+ ] i induced by platelet-activating factor. These observations suggest that bafilomycin A 1 inhibited a ΔpH-requiring step in eosinophil exocytosis that was preceded by the [Ca 2+ ] i transient in the signal transduction pathway, and, hence, the findings suggest the pivotal role of V-ATPase in maintaining intragranular pH and its function of eosinophil exosomal granules.