Activation of Proton Pumping in Human Neutrophils Occurs by Exocytosis of Vesicles Bearing Vacuolar-type H+-ATPases

Abstract

Proton pump activity is not measurable in the plasma membrane of unstimulated 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 cytochalasin B, an agent known to potentiate granular secretion in neutrophils. We therefore compared the rate and extent of pump activation with the pattern of exocytosis of the four types of secretory organelles present in neutrophils, using flow cytometry and enzymelinked immunosorbent assay. The kinetics of exocytosis of secretory vesicles and secondary and tertiary granules but not primary granules paralleled the appearance of pump activity. The subcellular localization of the pump was defined by cellular fractionation and immunoblotting using an antibody to the C subunit of the V-type ATPase. The pump was abundant in tertiary granules, with significant amounts present also in primary granules and secretory vesicles. The pump was scarce in secondary granules and not detectable in the cytosol. Finally, the agonists failed to stimulate pump activity in neutrophil cytoplasts, which are intact cell fragments devoid of acidic granules. Together, our results suggest that the V-type H ؉ -ATPase is not constitutively present in the plasma membrane of neutrophils but is delivered to the surface membrane by exocytosis during cellular activation. Tertiary granules and secretory vesicles are the most likely source of V-ATPases. Following insertion in the plasma membrane, the pump is poised to effectively extrude the excess metabolic acid that is generated during chemotaxis and bacterial killing.

High affinity capture and concentration of quinacrine in polymorphonuclear neutrophils via vacuolar ATPase-mediated ion trapping: Comparison with other peripheral blood leukocytes and implications for the distribution of cationic drugs

“…Activating PMNLs via the formylated peptide receptors forces the exocytosis of V-ATPase to the cell surface mainly from tertiary granules and secretory vesicles, rendering PMNLs capable of secreting H + (Nanda et al, 1996). Pre-stimulation of PMNL with fMLF failed, however, to modify quinacrine uptake in PMNLs (Fig.…”

“…The neutrophil, representing the vast majority of PMNLs, is rich in various types of granules, and V-ATPase is present in primary (lysosomal) and tertiary granules as well as secretory vesicules (Nanda et al, 1996). Activating PMNLs via the formylated peptide receptors forces the exocytosis of V-ATPase to the cell surface mainly from tertiary granules and secretory vesicles, rendering PMNLs capable of secreting H + (Nanda et al, 1996).…”

“…While V-ATPase is mainly vacuolar and intracellular in most cell types, the translocation of V-ATPase from specific vesicles to the cells surface has been observed in response to N-formyl-Met-Leu-Phe (fMLF) stimulation in human neutrophils, which acquire the capacity to secrete protons in the extracellular fluid (Nanda et al, 1996). Thus, this form of stimulation may redistribute the proton pump in such a way that the cell upake of quinacrine is reduced if the mobilized vacuoles are responsible for a large fraction of it.…”

Analysis of the Membrane Topology of Transmembrane Segments in the C-terminal Hydrophobic Domain of the Yeast Vacuolar ATPase Subunit a (Vph1p) by Chemical Modification

“…Acidification of intracellular compartments is important for membrane traffic processes, protein degradation and processing, coupled transport of small molecules, and the entry of various pathogens, including envelope viruses like influenza virus and bacterial toxins like anthrax toxin (4). V-ATPases are also present at the plasma membrane of a variety of cell types, including renal-intercalated cells, osteoclasts, macrophages, and neutrophils, epididymal clear cells, insect goblet cells, and certain tumor cells (5)(6)(7)(8)(9)(10). Plasma membrane V-ATPases play a critical role in processes such as urinary acidification, bone resorption, sperm maturation, pH homeostasis, coupled transport, and tumor metastasis.…”

Do Hv1 proton channels regulate the ionic and redox homeostasis of phagosomes?

“…Additionally, phagosomal-localized ROS production was shown to impair the fusion of granules with phagosomes and to affect the insertion of V-ATPases, resulting in lower rates of H + pumping (Jankowski et al, 2002). In neutrophils, secretory vesicles, primary and tertiary granules, but not secondary granules contain V-ATPases (Nanda et al, 1996). Hv1 channels were found in secondary and specific granules, but not in primary (azurophilic) granules of neutrophils, a distribution that mirrors that of NOX2, and accumulated in phagosomes together with NOX2 (Petheo et al, 2010).…”