Luminal and cytosolic pH feedback on proton pump activity and ATP affinity of V-type ATPase from Arabidopsis

Proton pumping of the vacuolar-type H(+)-ATPase into the lumen of the central plant organelle generates a proton gradient of often 1-2 pH units or more. Although structural aspects of the V-type ATPase have been studied in great detail, the question of whether and how the proton pump action is controlled by the proton concentration on both sides of the membrane is not understood. Applying the patch clamp technique to isolated vacuoles from Arabidopsis mesophyll cells in the whole-vacuole mode, we studied the response of the V-ATPase to protons, voltage, and ATP. Current-voltage relationships at different luminal pH values indicated decreasing coupling ratios with acidification. A detailed study of ATP-dependent H(+)-pump currents at a variety of different pH conditions showed a complex regulation of V-ATPase activity by both cytosolic and vacuolar pH. At cytosolic pH 7.5, vacuolar pH changes had relative little effects. Yet, at cytosolic pH 5.5, a 100-fold increase in vacuolar proton concentration resulted in a 70-fold increase of the affinity for ATP binding on the cytosolic side. Changes in pH on either side of the membrane seem to be transferred by the V-ATPase to the other side. A mathematical model was developed that indicates a feedback of proton concentration on peak H(+) current amplitude (v(max)) and ATP consumption (K(m)) of the V-ATPase. It proposes that for efficient V-ATPase function dissociation of transported protons from the pump protein might become higher with increasing pH. This feature results in an optimization of H(+) pumping by the V-ATPase according to existing H(+) concentrations.     

Salmonella pathogenicity island 2-encoded type III secretion system mediates exclusion of NADPH oxidase assembly from the phagosomal membrane

Salmonella typhimurium requires a type III secretion system encoded by pathogenicity island (SPI)-2 to survive and proliferate within macrophages. This survival implies that S. typhimurium avoids or withstands bactericidal events targeted to the microbe-containing vacuole, which include intraphagosomal production of reactive oxygen species (ROS), phagosomal acidification, and delivery of hydrolytic enzymes to the phagosome via fusion with lysosomes. Recent evidence suggests that S. typhimurium alters ROS production by murine macrophages in an SPI-2-dependent manner. To gain insights into the mechanism by which S. typhimurium inhibits intraphagosomal ROS production, we analyzed the subcellular distribution of NADPH oxidase components during infection of human monocyte-derived macrophages by wild-type (WT) or several SPI-2 mutant strains of S. typhimurium. We found that the membrane component of the NADPH oxidase, flavocytochrome b(558), was actively excluded or rapidly removed from the phagosomal membrane of WT-infected monocyte-derived macrophages, thereby preventing assembly of the NADPH oxidase complex and intraphagosomal production of superoxide anion. In contrast, the NADPH oxidase assembled on and generated ROS in phagosomes containing SPI-2 mutant S. typhimurium. Subversion of NADPH oxidase assembly by S. typhimurium was accompanied by increased bacterial replication relative to that of SPI-2 mutant strains, suggesting that the ability of WT S. typhimurium to prevent NADPH oxidase assembly at the phagosomal membrane represents an important virulence factor influencing its intracellular survival.     

Phagosomal acidification is mediated by a vacuolar-type H(+)-ATPase in murine macrophages

The mechanism underlying phagosomal acidification was studied in thioglycolate-elicited murine macrophages. The pH of the phagosomal compartment (pHp) was measured fluorimetrically in macrophage suspensions following ingestion of fluorescein isothiocyanate-labeled Staphylococcus aureus. At 37 degrees C, pHp decreased rapidly, reaching a steady state value of 5.8-6.1, while the cytoplasmic pH remained near neutrality, pH 7.1. The phagosome to cytosol pH gradient could be collapsed by addition of nigericin, monensin, or weak bases. The substrate dependence and inhibitor sensitivity profile of phagosomal acidification were investigated in intact and permeabilized cells. Phagosomal acidification was inhibited when ATP was depleted using metabolic inhibitors or permeabilizing the plasma membrane by electroporation. In permeabilized cells, acidification could be initiated by readdition of both Mg2+ and ATP. Neither adenosine 5'-(beta,gamma-imido)triphosphate nor adenosine 5'-(gamma-thio)triphosphate supported phagosomal acidification. Inhibitors of F1F0-type H(+)-ATPase such as oligomycin and azide, and the E1E2-type H(+)-ATPase inhibitor vanadate had no effect on phagosomal acidification. In contrast, the rate of phagosomal acidification was reduced by micromolar concentrations of N-ethylmaleimide and N,N'-dicyclohexylcarbodiimide. In permeabilized cells, nitrate inhibited the acidification with an apparent Ki of 25 mM. Phagosomal acidification was also effectively blocked by the macrolide antibiotic bafilomycin A1, with an apparent Ki of approximately 3 mM in both intact and electroporated cells. In this concentration range, bafilomycin A1 selectively inhibits vacuolar H(+)-ATPases. The substrate requirement and inhibitor susceptibility profile of phagosomal acidification strongly suggest that proton translocation across the phagosomal membrane is mediated by a vacuolar-type H(+)-ATPase.     

Na+/H+ exchange and pH regulation in the control of neutrophil chemokinesis and chemotaxis

Large proton fluxes accompany cell migration, but their precise role remains unclear. We studied pH regulation during the course of chemokinesis and chemotaxis in human neutrophils stimulated by attractant peptides. Activation of cell motility by chemoattractants was accompanied by a marked increase in metabolic acid generation, attributable to energy consumption by the contractile machinery and to stimulation of the NADPH oxidase and the ancillary hexose monophosphate shunt. Despite the increase in acid production, the cytosol underwent a sizable alkalinization, caused by acceleration of Na(+)/H(+) exchange. The development of the alkalinization mirrored the increase in the rate of cell migration, suggesting a causal relationship. However, elimination of Na(+)/H(+) exchange by omission of external Na(+) or by addition of potent inhibitors was without effect on either chemokinesis or chemotaxis, provided the cytosolic pH remained near neutrality. At more acidic levels, cell motility was progressively inhibited. These observations suggest that Na(+)/H(+) exchange plays a permissive role in cell motility but is not required for the initiation or development of the migratory response. Chemokinesis also was found to be exquisitely sensitive to extracellular acidification. This property may account for the inability of neutrophils to access abscesses and solid tumors that have been reported to have inordinately low pH.     

Charge compensation during the phagocyte respiratory burst

The phagocyte NADPH oxidase produces superoxide anion (O(2)(.-)) by the electrogenic process of moving electrons across the cell membrane. This charge translocation must be compensated to prevent self-inhibition by extreme membrane depolarization. Examination of the mechanisms of charge compensation reveals that these mechanisms perform several other vital functions beyond simply supporting oxidase activity. Voltage-gated proton channels compensate most of the charge translocated by the phagocyte NADPH oxidase in human neutrophils and eosinophils. Quantitative modeling of NADPH oxidase in the plasma membrane supports this conclusion and shows that if any other conductance is present, it must be miniscule. In addition to charge compensation, proton flux from the cytoplasm into the phagosome (a) helps prevent large pH excursions both in the cytoplasm and in the phagosome, (b) minimizes osmotic disturbances, and (c) provides essential substrate protons for the conversion of O(2)(*-) to H(2)O(2) and then to HOCl. A small contribution by K+ or Cl- fluxes may offset the acidity of granule contents to keep the phagosome pH near neutral, facilitating release of bactericidal enzymes. In summary, the mechanisms used by phagocytes for charge compensation during the respiratory burst would still be essential to phagocyte function, even if NADPH oxidase were not electrogenic.     

Intracellular pH regulation during spreading of human neutrophils

The regulation of the intracelluar pH (pHi) during spreading of human neutrophils was studied by a combination of fluorescence imaging and video microscopy. Spreading on adhesive substrates caused a rapid and sustained cytosolic alkalinization. This pHi increase was prevented by the omission of external Na+, suggesting that it results from the activation of Na+/H+ exchange. Spreading-induced alkalinization was also precluded by the compound HOE 694 at concentrations that selectively block the NHE-1 isoform of the Na+H+ antiporter. Inhibition of Na+/H+ exchange by either procedure unmasked a sizable cytosolic acidification upon spreading, indicative of intracellular acid production. The excess acid generation was caused, at least in part, by the activation of the respiratory burst, since the acidification closely correlated with superoxide production, measured in single spreading neutrophils with dihydrorhodamine-123, and little acid production was observed in the presence of diphenylene iodonium, a blocker of the NADPH oxidase. Moreover, neutrophils from chronic granulomatous disease patients, which do not produce superoxide, failed to acidify. Comparable pHi changes were observed when beta 2 integrins were selectively activated during spreading on surfaces coated with anti-CD18 antibodies. When integrin engagement was precluded by pretreatment with soluble anti-CD18 antibody, the pHi changes associated with spreading on fibrinogen were markedly reduced. Inhibition of microfilament assembly with cytochalasin D precluded spreading and concomitantly abolished superoxide production and the associated pHi changes, indicating that cytoskeletal reorganization and/or an increase in the number of adherence receptors engaged are required for the responses. Neutrophils spread normally when the oxidase was blocked or when pHi was clamped near physiological values with nigericin. Spreading, however, was strongly inhibited when pHi was clamped at acidic values. Our results indicate that neutrophils release superoxide upon spreading, generating a burst of intracellular acid production. The concomitant activation of the Na+/H+ antiport not only prevents the deleterious effects of the acid released by the NADPH oxidase, but induces a net cytosolic alkalinization. Since several functions of neutrophils are inhibited at an acidic pHi, the coordinated activation of pHi regulatory mechanisms along with the oxidase is essential for sustained microbicidal activity.     

The antibacterial activity of human neutrophils and eosinophils requires proton channels but not BK channels

Electrophysiological events are of central importance during the phagocyte respiratory burst, because NADPH oxidase is electrogenic and voltage sensitive. We investigated the recent suggestion that large-conductance, calcium-activated K(+) (BK) channels, rather than proton channels, play an essential role in innate immunity (Ahluwalia, J., A. Tinker, L.H. Clapp, M.R. Duchen, A.Y. Abramov, S. Page, M. Nobles, and A.W. Segal. 2004. Nature. 427:853-858). In PMA-stimulated human neutrophils or eosinophils, we did not detect BK currents, and neither of the BK channel inhibitors iberiotoxin or paxilline nor DPI inhibited any component of outward current. BK inhibitors did not inhibit the killing of bacteria, nor did they affect NADPH oxidase-dependent degradation of bacterial phospholipids by extracellular gIIA-PLA(2) or the production of superoxide anion (O(2*)(-)). Moreover, an antibody against the BK channel did not detect immunoreactive protein in human neutrophils. A required role for voltage-gated proton channels is demonstrated by Zn(2+) inhibition of NADPH oxidase activity assessed by H(2)O(2) production, thus validating previous studies showing that Zn(2+) inhibited O(2*)(-) production when assessed by cytochrome c reduction. In conclusion, BK channels were not detected in human neutrophils or eosinophils, and BK inhibitors did not impair antimicrobial activity. In contrast, we present additional evidence that voltage-gated proton channels serve the essential role of charge compensation during the respiratory burst.     

Novel insight into current models of NADPH oxidase regulation, assembly and localization in human polymorphonuclear leukocytes.

We review herein the definition of the NADPH oxidase-activating site in human neutrophils and eosinophils, together with the new biochemical findings of the assembly of NADPH oxidase components and the signal transduction for the activation of NADPH oxidase. The activation of this enzyme is associated with multiple interrelated signaling pathways. Upon cell stimulation, the second messengers act on the assembly of NADPH oxidase components. The cytosolic components are first phosphorylated, and then associated with the membrane components. Small GTP-binding proteins and cytoskeletal components also participate in the activation of the NADPH oxidase. The cytochemical findings demonstrate that the superoxide generated by NADPH oxidase activity is initially localized in distinct types of intracellular granules, and not at the plasma membrane as previously believed. Thus, the assembly of NADPH oxidase components possibly occurs at the limiting membrane of the intracellular compartments. The oxidant-producing compartments mobilize and become associated with the plasma membrane upon cell stimulation with soluble stimulants, or fuse to phagosomes upon stimulation with particulate stimulants. Accordingly, superoxide is released to the extracellular space and into phagosomes in proportion to the oxidant-producing intracellular granule association with the plasma membrane and with the phagosomal membrane, respectively.     

Mechanism of hydrogen peroxide formation catalyzed by NADPH oxidase in thyroid plasma membrane

The thyroid plasma membrane contains a Ca2(+)-regulated NADPH-dependent H2O2 generating system which provides H2O2 for the thyroid peroxidase-catalyzed biosynthesis of thyroid hormones. The plasma membrane fraction contains a Ca2(+)-independent cytochrome c reductase activity which is not inhibited by superoxide dismutase. But it is not known whether H2O2 is produced directly from molecular oxygen (O2) or formed via dismutation of super-oxide anion (O2-). Indirect evidence from electron scavenger studies indicate that the H2O2 generating system does not liberate O2-, but studies using the modified peroxidase, diacetyldeuteroheme horseradish peroxidase, to detect O2- indicate that H2O2 is provided via the dismutation of O2-. The present results provide indirect evidence that the cytochrome c reductase activity is not a component of the NADPH-dependent H2O2 generator, since it was removed by washing the plasma membranes with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid without affecting H2O2 generation. Spectral studies with diacetyldeuteroheme-substituted horseradish peroxidase showed that the thyroid NADPH-dependent H2O2 generator does not catalyze superoxide anion formation. The O2- adduct compound (compound III) was formed but was completely inhibited by catalase, indicating that the initial product was H2O2. The rate of NADPH oxidation also increased in the presence of diacetylheme peroxidase. This increase was blocked by catalase and was greatly enhanced by superoxide dismutase. The O2- adduct compound (compound III) was produced in the presence of NADPH when glucose-glucose oxidase (which does not produce O2-) was used as the H2O2 generator. NADPH oxidation occurred simultaneously and was enhanced by superoxide dismutase. We conclude that O2- formation occurs in the presence of an H2O2 generator, diacetylheme peroxidase and NADPH, but that it is not the primary product of the H2O2 generator. We suggest that O2- formation results from oxidation of NADPH, catalyzed by the diacetylheme peroxidase compound I, producing NADP degree, which in turn reacts with O2 to give O2-.     

Internal pH changes associated with the activity of NADPH oxidase of human neutrophils. Further evidence for the presence of an H+ conducting channel.

The internal pH (pHi) of cytoplasts, derived from human neutrophils, falls 0.05 pH units upon activation of the superoxide-generating NADPH oxidase. The decrease in pHi is absent in diphenyleneiodonium-treated cytoplasts and therefore it is likely to arise directly from the activity of the oxidase. The addition of amiloride, to diminish the Na+/H+ exchanger, enhanced the extent of the internal acidification but not the initial rate. However the electroneutral Na+/H+ exchanger cannot be a contributor to H+ efflux to compensate for charge translocated by the oxidase. In the presence of Cd ions or valinomycin, phorbol-induced acidification of the cytosol was greatly increased, suggesting an inability to translocate the cytosolic H+ generated by an electrogenic oxidase. In the presence of both Cd and valinomycin the cytoplasts retained 0.8 H+ per O2-. generated. The rate of acidification of the external medium by stimulated cytoplasts is greatly reduced in the presence of Zn and valinomycin. Our results support the view that the plasma membrane of neutrophils contains Zn2+- or Cd2+-sensitive proton-conducting channels which maintain a stable membrane potential and pHi during the activity of the electrogenic NADPH oxidase.     

Phagosome neutrality in host defense

Activation of proteases in the phagosomes of neutrophils occurs by neutralization of the phagosomal pH by NADPH oxidase. In this issue, Savina et al. (2006) show that dendritic cell phagosomes also recruit NADPH oxidase but with different results. Here, the neutralization of phagosomal pH reduces protease activity, which preserves antigens for crosspresentation on class I MHC molecules.     

Absence of proton channels in COS-7 cells expressing functional NADPH oxidase components

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is an enzyme of phagocytes that produces bactericidal superoxide anion (O(2)(-)) via an electrogenic process. Proton efflux compensates for the charge movement across the cell membrane. The proton channel responsible for the H(+) efflux was thought to be contained within the gp91(phox) subunit of NADPH oxidase, but recent data do not support this idea (DeCoursey, T.E., V.V. Cherny, D. Morgan, B.Z. Katz, and M.C. Dinauer. 2001. J. Biol. Chem. 276:36063-36066). In this study, we investigated electrophysiological properties and superoxide production of COS-7 cells transfected with all NADPH oxidase components required for enzyme function (COS(phox)). The 7D5 antibody, which detects an extracellular epitope of the gp91(phox) protein, labeled 96-98% of COS(phox) cells. NADPH oxidase was functional because COS(phox) (but not COS(WT)) cells stimulated by phorbol myristate acetate (PMA) or arachidonic acid (AA) produced superoxide anion. No proton currents were detected in either wild-type COS-7 cells (COS(WT)) or COS(phox) cells studied at pH(o) 7.0 and pH(i) 5.5 or 7.0. Anion currents that decayed at voltages positive to 40 mV were the only currents observed. PMA or AA did not elicit detectable H(+) current in COS(WT) or COS(phox) cells. Therefore, gp91(phox) does not function as a proton channel in unstimulated cells or in activated cells with a demonstrably functional oxidase.     

Myeloperoxidase in human neutrophil host defence

Human neutrophils represent the predominant leucocyte in circulation and the first responder to infection. Concurrent with ingestion of microorganisms, neutrophils activate and assemble the NADPH oxidase at the phagosome, thereby generating superoxide anion and hydrogen peroxide. Concomitantly, granules release their contents into the phagosome, where the antimicrobial proteins and enzymes synergize with oxidants to create an environment toxic to the captured microbe. The most rapid and complete antimicrobial action by human neutrophils against many organisms relies on the combined efforts of the azurophilic granule protein myeloperoxidase and hydrogen peroxide from the NADPH oxidase to oxidize chloride, thereby generating hypochlorous acid and a host of downstream reaction products. Although individual components of the neutrophil antimicrobial response exhibit specific activities in isolation, the situation in the environment of the phagosome is far more complicated, a consequence of multiple and complex interactions among oxidants, proteins and their by‐products. In most cases, the cooperative interactions among the phagosomal contents, both from the host and the microbe, culminate in loss of viability of the ingested organism.     

Charge compensation during the phagocyte respiratory burst

The phagocyte NADPH oxidase produces superoxide anion (O₂^·−) by the electrogenic process of moving electrons across the cell membrane. This charge translocation must be compensated to prevent self-inhibition by extreme membrane depolarization. Examination of the mechanisms of charge compensation reveals that these mechanisms perform several other vital functions beyond simply supporting oxidase activity. Voltage-gated proton channels compensate most of the charge translocated by the phagocyte NADPH oxidase in human neutrophils and eosinophils. Quantitative modeling of NADPH oxidase in the plasma membrane supports this conclusion and shows that if any other conductance is present, it must be miniscule. In addition to charge compensation, proton flux from the cytoplasm into the phagosome (a) helps prevent large pH excursions both in the cytoplasm and in the phagosome, (b) minimizes osmotic disturbances, and (c) provides essential substrate protons for the conversion of O₂^·− to H₂O₂ and then to HOCl. A small contribution by K⁺ or Cl⁻ fluxes may offset the acidity of granule contents to keep the phagosome pH near neutral, facilitating release of bactericidal enzymes. In summary, the mechanisms used by phagocytes for charge compensation during the respiratory burst would still be essential to phagocyte function, even if NADPH oxidase were not electrogenic.     

Electron and proton transport by NADPH oxidases

The NADPH oxidase is the main weapon of phagocytic white blood cells that are the first line of defence of our body against invading pathogens, and patients lacking a functional oxidase suffer from severe and recurrent infections. The oxidase is a multisubunit enzyme complex that transports electrons from cytoplasmic NADPH to molecular oxygen in order to generate superoxide free radicals. Electron transport across the plasma membrane is electrogenic and is associated with the flux of protons through voltage-activated proton channels. Both proton and electron currents can be recorded with the patch-clamp technique, but whether the oxidase is a proton channel or a proton channel modulator remains controversial. Recently, we have used the inside-out configuration of the patch-clamp technique to record proton and electron currents in excised patches. This approach allows us to measure the oxidase activity under very controlled conditions, and has provided new information about the enzymatic activity of the oxidase and its coupling to proton channels. In this chapter I will discuss how the unique characteristics of the electron and proton currents associated with the redox activity of the NADPH oxidase have extended our knowledge about the thermodynamics and the physiological regulation of this remarkable enzyme.     

Superoxide anion is the initial product in the hydrogen peroxide formation catalyzed by NADPH oxidase in porcine thyroid plasma membrane

The plasma membrane fraction from porcine thyroid is known to exhibit an NADPH-dependent production of hydrogen peroxide (H2O2), which is utilized for the oxidative biosynthesis of thyroid hormones catalyzed by thyroid peroxidase. The H2O2 formation is cyanide-insensitive, ATP-activatable, and Ca2+-dependent (Nakamura, Y., Ogihara, S., and Ohtaki, S. (1987) J. Biochem. (Tokyo) 102, 1121-1132). It remains unknown, however, whether H2O2 is produced directly from molecular oxygen (O2) or formed via dismutation of superoxide anion (O2-). We therefore attempted to analyze the mechanism of H2O2 formation by utilizing a new method for the simultaneous measurement of O2- and H2O2, in which diacetyldeuteroheme-substituted horseradish peroxidase was employed as the trapping agent for both oxygen metabolites. When NADPH was incubated with the membrane fraction in the presence of the heme-substituted peroxidase, a massive O2 consumption was observed together with the formation of compound III, and O2- adduct of the peroxidase. The amounts of compound III formed and O2 consumed were stoichiometric with each other, while formation of compound II, an indicative of H2O2, was not observed during the reaction. On the other hand, when an excess amount of superoxide dismutase was included in the reaction mixture, compound II was produced with complete suppression of the compound III formation. NADH minimally supported both O2 consumption and formation of compound III or II. These results indicate that the NADPH oxidase in the plasma membrane of thyroid produces O2- as the primary metabolite of O2 and hence that H2O2 required for the thyroid hormone synthesis provided through the dismutation of O2-.     

Implication of cytosolic phospholipase A2 (cPLA2) in the regulation of human synoviocyte NADPH oxidase (Nox2) activity

NADPH oxidase Nox2 is involved in the production of superoxide by rheumatoid synovial cells, constitutively and after pro-inflammatory cytokine treatment. The aims of the study were to evaluate the capacity of these cells to produce the superoxide anion in response to arachidonic acid (AA), and to study the involvement of cytosolic phospholipase A(2) (cPLA(2)) in the cytokine regulation of Nox2. Superoxide production was quantified in synovial cells obtained from six patients with rheumatoid arthritis (RA) and six with osteoarthritis (OA), stimulated with (i) AA, and (ii) PLA(2) inhibitors prior to IL-1beta or TNF-alpha treatment. Total cellular AA concentrations and PLA(2) activity were measured; effects of cytokines and NADPH oxidase inhibitors on the AA-activatable proton channel opening were also studied. Our results demonstrated that AA enhanced superoxide production in RA and OA cells; this production was significantly inhibited by iodonium diphenyl and apocynin. cPLA(2) inhibitors inhibited both IL-1beta and TNF-alpha-induced superoxide production in RA and OA cells. Basal PLA(2) activity was significantly more important in RA cells than in OA cells; PLA(2) activity was increased in IL-1beta and TNF-alpha pre-treated RA cells, and cPLA(2) inhibitors inhibited this activity. Opening of the AA-activatable proton channel was amplified when RA cells were pre-treated with both IL-1beta and TNF-alpha, and iodonium diphenyl and apocynin inhibited these cytokine effects. We concluded that AA is an important cofactor for synovial NADPH oxidase activity. Despite their direct effects on p47-phox phosphorylation, cytokines can also regulate the Nox2 activity though the AA-activatable associated H(+) channel.     

Characterization of nitric oxide consumption pathways by normal,chronic granulomatous disease and myeloperoxidase-deficient human neutrophils

The detailed mechanisms by which acutely activated leukocytes metabolize NO and regulate its bioactivity are unknown. Therefore, healthy, chronic granulomatous disease (CGD) or myeloperoxidase (MPO)-deficient human neutrophils were examined for their ability to consume NO and attenuate its signaling. fMLP or PMA activation of healthy neutrophils caused NO consumption that was fully blocked by NADPH oxidase inhibition, and was absent in CGD neutrophils. Studies using MPO-deficient neutrophils, enzyme inhibitors, and reconstituted NADPH oxidase ruled out additional potential NO-consuming pathways, including Fenton chemistry, PGH synthase, lipoxygenase, or MPO. In particular, the inability of MPO to consume NO resulted from lack of H(2)O(2) substrate since all superoxide (O(2)(-.) reacted to form peroxynitrite. For healthy or MPO-deficient cells, NO consumption rates were 2- to 4-fold greater than O(2)(-.) generation, significantly faster than expected from 1:1 termination of NO with O(2)(-.). Finally, fMLP or PMA-stimulated NO consumption fully blocked NO-dependent neutrophil cGMP synthesis. These data reveal NADPH oxidase as the central regulator of NO signaling in human leukocytes. In addition, they demonstrate an important functional difference between CGD and either normal or MPO-deficient human neutrophils, namely their inability to metabolize NO which will alter their ability to adhere and migrate in vivo.     

The gp91phox component of NADPH oxidase is not the voltage-gated proton channel in phagocytes, but it helps

During the "respiratory burst," the NADPH oxidase complex of phagocytes produces reactive oxygen species that kill bacteria and other invaders (Babior, B. M. (1999) Blood 93, 1464-1476). Electron efflux through NADPH oxidase is electrogenic (Henderson, L. M., Chappell, J. B., and Jones, O. T. G. (1987) Biochem. J. 246, 325-329) and is compensated by H(+) efflux through proton channels that reportedly are contained within the gp91(phox) subunit of NADPH oxidase. To test whether gp91(phox) functions as a proton channel, we studied H(+) currents in granulocytes from X-linked chronic granulomatous disease patients lacking gp91(phox) (X-CGD), the human myelocytic PLB-985 cell line, PLB-985 cells in which gp91(phox) was knocked out by gene targeting (PLB(KO)), and PLB-985 knockout cells re-transfected with gp91(phox) (PLB(91)). H(+) currents in unstimulated PLB(KO) cells had amplitude and gating kinetics similar to PLB(91) cells. Furthermore, stimulation with the phorbol ester phorbol 12-myristate 13-acetate increased H(+) currents to a similar extent in X-CGD, PLB(KO), and PLB(91) cells. Thus, gp91(phox) is not the proton channel in unstimulated phagocytes and does not directly mediate the increase of proton conductance during the respiratory burst. Changes in H(+) channel gating kinetics during NADPH oxidase activity are likely crucial to the activation of H(+) flux during the respiratory burst.