The rate of metabolic acid generation by neutrophils increases greatly when they are activated. Intracellular acidification is prevented in part by Na+/H' exchange, but a sizable component of H+ extrusion persists in the nominal absence of Na' and HCO3 . In this report we determined the contribution to H+ extrusion of a putative HI conductive pathway and its mode ofactivation. In unstimulated cells, H+ conductance was found to be low and unaffected by depolarization. An experimental system was designed to minimize the metabolic acid generation and membrane potential changes associated with neutrophil activation. By using this system, (3-phorbol esters were shown to increase the H+ (equivalent) permeability of the plasma membrane. The direction of the phorbol ester-induced fluxes was dictated by the electrochemical H+ gradient. Moreover, the parallel migration of a counterion through a rheogenic pathway was necessary for the displacement of measurable amounts of H+ equivalents across the membrane. These frndings suggest that the HI flux is conductive. The effect of ,B-phorbol esters was mimicked by diacylglycerol and mezerein and was blocked by staurosporine, whereas at-phorbol esters were ineffective. Together, these frndings indicate that stimulation of protein kinase C induces the activation of an H+ conductance in the plasma membrane of human neutrophils. Preliminary evidence for activation of a separate, bafilomycin Al-sensitive H+ extrusion mechanism, likely a vacuolar type H+-ATPase, is also presented.Polymorphonuclear leukocytes (neutrophils) are attracted to sites of infection where they destroy invading organisms by a variety of microbicidal mechanisms, including phagocytosis, degranulation, and the production oftoxic oxygen metabolites (1). Activation of neutrophils is associated with a large burst of intracellular H+ generation, due primarily to stimulation of the NADPH oxidase and the associated acceleration of the hexose monophosphate shunt pathway. Despite this massive increase in net H+ production, however, the cytosolic pH (pHi) of activated cells remains at or above the resting level, even in the nominal absence of HCO (2, 3).Maintenance of pH, in the physiological range is due, in part, to a concurrent activation of the Na+/H+ antiport. Indeed, if the antiport is inhibited by amiloride or by suspending the cells in Na+-free medium, a pronounced cytosolic acidification is observed upon activation (2). However, even under these conditions, pHi fails to reach the levels predicted by the amount of acid equivalents generated during the metabolic burst. Approximately 50 nmol of H+ are generated in 5 min by 106 fully activated neutrophils (calculated from the rate of nonmitochondrial 02 consumption of phorbol esterstimulated cells, assuming one H+ released per 02 consumed). By considering the buffering power of the cytosol (-28 mM per pH unit; ref. 4) and the cell volume (-330 ,um3), it can be calculated that pH1 would be expected to drop by over 5 units (to pH 1.6) if the metabolically genera...
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.
H+ conductive pathways have been detected in the plasma membranes of a variety of cell types. The large exquisitely H(+)-selective permeability of the conductive pathway can support sizable net H+ fluxes. Although subtle differences exist among tissues and species, certain common features suggest that related transport systems are involved in all cases. The H+ conductance is gated by depolarizing voltages and is promoted by intracellular acidification. Conversely, extracellular acidification inhibits the conductance. These features facilitate net H+ efflux, while precluding potentially deleterious H+ uptake. In some cell types, activation of the conductance is additionally controlled by physiological ligands and by second messengers. The conductance most likely functions in the regulation of intracellular pH, contributing to the extrusion of H+ during repetitive depolarization of the plasma membrane, as occurs in neurons and muscle cells. This pathway may be particularly relevant in the case of phagocytes. When stimulated, these cells undergo a sustained depolarization, while generating large amounts of metabolic acid. In addition, conductive H+ fluxes may also provide counterions to neutralize the activity of electrogenic enzymes, as suggested for the phagocyte NADPH oxidase.
In phagocytes, superoxide generation by the NADPH oxidase is accompanied by metabolic acid production. Cytoplasmic acidification during this metabolic burst is prevented by a combination of H+ extrusion mechanisms, including a unique H+ conductance. NADPH oxidase is deficient in chronic granulomatous disease (CGD) patients. The burst of acid production is absent in CGD patients lacking the 47-kD (p47-phox) or the 91-kD (gp9l-phox) subunits of the oxidase. Activation of the H+ conductance is also defective in these patients suggesting that (a) the oxidase itself undertakes H' translocation or (b) oxidase assembly is required to stimulate a separate H+ conducting entity. To discern between these possibilities, three rare forms of CGD were studied. In neutrophils expressing nonfunctional cytochrome b, the conductance was activated to near-normal levels, implying that functional oxidase is not required to activate H' extrusion. CGD cells expressing diminished amounts of cytochrome displayed H' conductance approaching normal levels, suggesting that the oxidase itself does not translocate H+ . Finally, the conductance was only partially inhibited in patients lacking the 67-kD subunit, indicating that this component is not essential for stimulation of H+ transport. We propose that normal assembly of the oxidase subunits is required for optimal activation of a closely associated but distinct H+ conducting entity. (J. Clin. Invest. 1994. 93:1770-1775
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