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 i n 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 eosin ophil lysate was extracted immunomagnetically with the same antibody, the extracted fraction contained a considerable amount of eosinophil p eroxidase, a marker of eosinophil-specific granules, which indicates t hat V-ATPase was present on the membranes of eosinophil exsosomal gran ules. The pH of the eosinophil granules, measured fluorometrically wit h acridine orange as a Delta pH-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 t he low pH of these granules is maintained by V-ATPase activity. Bafilo mycin A(1) and NH4Cl, both of which raise the intragranular pH to neut ral, inhibited the eosinophil peroxidase exocytosis induced by platele t-activating factor. These agents did not, however, affect the changes in cytosolic free calcium concentration [Ca2+](i) induced by platelet -activating factor. These observations suggest that bafilomycin A(1) i nhibited a Delta pH-requiring step in eosinophil exocytosis that was p receded by the [Ca2+](i) transient in the signal transduction pathway, and, hence, the findings suggest the pivotal role of V-ATPase in main taining intragranular pH and its function of eosinophil exosomal granu les.