ACTIVATION OF PROTON-PUMPING IN HUMAN NEUTROPHILS OCCURS BY EXOCYTOSIS OF VESICLES BEARING VACUOLAR-TYPE H-ATPASES()

Proton pump activity is not measurable in the plasma membrane of unsti mulated neutrophils but becomes readily detectable upon activation by soluble agonists. The mechanism of pump activation was investigated in this report. V-type H+ pump activity, estimated as a bafilomycin A(1) -sensitive elevation of the cytosolic pH, was stimulated in suspended neutrophils by chemotactic peptides and by phorbol esters. Stimulation of pump activity induced by the agonists was greatly enhanced by cyto chalasin B, an agent known to potentiate granular secretion in neutrop hils. We therefore compared the rate and extent of pump activation wit h the pattern of exocytosis of the four types of secretory organelles present in neutrophils, using flow cytometry and enzyme-linked immunos orbent assay, The kinetics of exocytosis of secretory vesicles and sec ondary and tertiary granules but not primary granules paralleled the a ppearance of pump activity, The subcellular localization of the pump w as defined by cellular fractionation and immunoblotting using an antib ody to the C subunit of the V-type ATPase. The pump was abundant in te rtiary granules, with significant amounts present also in primary gran ules and secretory vesicles. The pump was scarce in secondary granules and not detectable in the cytosol. Finally, the agonists failed to st imulate pump activity in neutrophil cytoplasts, which are intact cell fragments devoid of acidic granules, Together, our results suggest tha t the V-type H+-ATPase is not constitutively present in the plasma mem brane of neutrophils but is delivered to the surface membrane by exocy tosis during cellular activation, Tertiary granules and secretory vesi cles are the most likely source of V-ATPases, Following insertion in t he plasma membrane, the pump is poised to effectively extrude the exce ss metabolic acid that is generated during chemotaxis and bacterial ki lling.