Comparative study on the stimulation of superoxide production in guinea-pig eosinophils by the calcium ionophore A23187

The effect of calcium and/or magnesium on O2- production by guinea-pig eosinophils stimulated by the calcium ionophore A23187 was studied in comparison to neutrophils. In the absence of calcium, A23187 did not stimulate O2- production in resting eosinophils and neutrophils, regardless of the presence of extracellular magnesium. The A23187-induced O2- production by both cells increased linearly with extracellular Ca2+ concentrations. However, the concentration of Ca2+ required for maximum O2- production in eosinophils was about 10-times lower than that required of neutrophils. The addition of Mg2+ strongly inhibited O2- production, especially in eosinophils at low Ca2+ concentrations. The intracellular Ca2+ concentration was lower in eosinophils than in neutrophils in the resting state, and the enhancement of the intracellular Ca2+ concentration in response to A23187 was much lower in eosinophils than in neutrophils. The activation of the NADPH-dependent O2(-)-forming enzyme (NADPH oxidase) in eosinophils depended on extracellular calcium, as observed in O2- production. However, the NADPH oxidase activity in the particulate fraction from A23187-stimulated eosinophils was only slightly affected by the addition of divalent cations or EDTA. The compound W-7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride), a calmodulin antagonist, significantly inhibited O2- production by both cells. On the other hand, the compound H-7 (1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride), a protein kinase C antagonist, was less effective on O2- production than was W-7. H-7 had little effect on O2- production of eosinophils. These findings suggest that NADPH oxidase might be activated by a smaller Ca2+ concentration through the calmodulin-dependent reaction. This was not observed with protein kinase C, at least in eosinophils.     

Divergence of mechanisms regulating respiratory burst in blood and sputum eosinophils and neutrophils from atopic subjects

Eosinophil respiratory burst is an important event in asthma and related inflammatory disorders. However, little is known concerning activation of the respiratory burst NADPH oxidase in human eosinophils. Conversely, neutrophils are known to assemble NADPH oxidase in intracellular and plasma membranes. We hypothesized that eosinophils and neutrophils translocate NADPH oxidase to distinct intracellular locations, consistent with their respective functions in O(2)(-)-mediated cytotoxicity. PMA-induced O(2)(-) release assayed by cytochrome c was 3.4-fold higher in atopic human eosinophils than in neutrophils, although membrane-permeable dihydrorhodamine-123 showed similar amounts of release. Eosinophil O(2)(-) release was dependent on Rac, in that it was 54% inhibited by Clostridium difficile toxin B (400-800 ng/ml). In eosinophils stimulated with PMA, a pronounced shift of cytosolic Rac to p22(phox)-positive plasma membrane was observed by confocal microscopy, whereas neutrophils directed Rac2 mainly to intracellular sites coexpressing p22(phox). Similarly, ex vivo sputum eosinophils from asthmatic subjects exhibited predominantly plasma membrane-associated immunoreactivity for Rac, whereas sputum neutrophils exhibited cytoplasmic Rac2 staining. Thus, activated sputum eosinophils, rather than neutrophils, may contribute significantly to the pathogenesis of asthma by extracellular release of tissue-damaging O(2)(-). Our findings suggest that the differential modes of NADPH oxidase assembly in these cells may have important implications for oxidant-mediated tissue injury.     

Response of superoxide anion production by guinea pig eosinophils to various soluble stimuli: comparison to neutrophils

The effect of various soluble stimuli on the superoxide production by guinea pig eosinophils was studied in comparison to neutrophils. Phorbol myristate acetate, A23187, digitonin, NaF, concanavalin A (Con A), and cytochalasin E stimulated eosinophils and neutrophils to release O2-. The O2- production by these active agents, excluding Con A and cytochalasin E, was much greater in eosinophils than in neutrophils. Formyl-Met-Leu-Phe stimulated the O2- production in neutrophils but not in eosinophils. Neither histamine nor Val/Ala-Gly-Ser-Glu stimulated the O2- production in both types of leukocytes. A23187- or Con A-stimulated O2- production was greatly enhanced by cytochalasin B pretreatment in neutrophils but not in eosinophils. Lineweaver-Burk analysis of NADPH oxidase in particulate fractions showed that eosinophils possessed the same Km values as neutrophils and greater Vmax values than neutrophils, suggesting that eosinophils have a similar, but more active, O2- -generating enzyme system than neutrophils.     

Respiratory burst of rabbit peritoneal neutrophils. Transition from an NADPH diaphorase activity to an .O2(-)-generating oxidase activity

Superoxide (.O2-) production by the NADPH oxidase of a membrane fraction derived from rabbit peritoneal neutrophils activated by 4 beta-phorbol 12-myristate 13-acetate (PMA) was studied at 25 degrees C under different conditions, and measured by the superoxide dismutase inhibitable reduction of cytochrome c. Whereas PMA-activated rabbit neutrophils incubated in a glucose-supplemented medium exhibited a substantial rate of production of .O2-, the membranes prepared by sonication of the activated neutrophils were virtually unable to generate .O2- in the presence of NADPH. Instead, they exhibited an NADPH-dependent diaphorase activity, measured by the superoxide-dismutase-insensitive reduction of cytochrome c. Upon addition of arachidonic acid, which is known to elicit oxidase activation, the NADPH diaphorase activity of the rabbit neutrophil membranes vanished and was stoichiometrically replaced by an NADPH oxidase activity. The emerging oxidase activity was fully sensitive to iodonium biphenyl, a potent inhibitor of the respiratory burst, whereas the diaphorase activity was not affected. Addition of 0.1% Triton X-100 or an excess of arachidonic acid, acting as detergent, resulted in the reappearance of the diaphorase activity at the expense of the oxidase activity. These results indicate that the diaphorase-oxidase transition is reversible. When the rabbit neutrophil membranes were supplemented with rabbit neutrophil cytosol, guanosine 5'-[gamma-thio]triphosphate and Mg2+, in addition to arachidonic acid, not only the NADPH diaphorase activity disappeared, but the emerging NADPH oxidase activity was markedly enhanced (about 10 times compared to that of membranes treated with arachidonic acid alone). The diaphorase-oxidase transition was accompanied by a 10-fold increase in the Km for NADPH, suggesting a change of conformation propagated to the NADPH-binding site during the transition. The treatment of PMA-activated rabbit neutrophils with cross-linking reagents, like glutaraldehyde or 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide, prevented the loss of the PMA-elicited oxidase activity upon disruption of the cells by sonication, suggesting that the interactions between the components of the oxidase complex are stabilized by cross-linking.     

Activation of NADPH-dependent superoxide production in plasma membrane extracts of pig neutrophils by phosphatidic acid

Phosphatidic acid (PA), a molecule that is rapidly produced by the stimulated turnover of phospholipids in a variety of cells including blood neutrophils, elicited NADPH-dependent superoxide anion (O2-) production in detergent extracts from membranes of resting pig neutrophils. The stimulatory effect of PA was independent of cytosolic factors, differing from arachidonic acid and sodium dodecyl sulfate which, on the contrary, absolutely required the presence of cytosol to elicit the same result. The O2(-)-forming activity of the detergent extract activable by PA, as that by sodium dodecyl sulfate and arachidonic acid plus cytosol, was found in the chromatographic fractions containing cytochrome b558 and presented a chromatographic profile identical to that of the activated NADPH oxidase, which was obtained from neutrophils prestimulated with phorbol 12-myristate 13-acetate. The PA-induced NADPH-dependent O2(-)-forming activity showed kinetic properties and sensitivity to the inhibitors similar to the classical ones of the activated neutrophil NADPH oxidase. The data suggest that, in this cell-free system, PA may stimulate O2- formation by direct interaction with latent NADPH oxidase of neutrophils or with some of its regulatory components.     

NADPH oxidase of human neutrophils. Subcellular localization and characterization of an arachidonate-activatable superoxide-generating system

The superoxide-forming NADPH oxidase of human neutrophils was studied in subcellular fractions of unstimulated cells. Purified neutrophils were disrupted by nitrogen cavitation and separated on Percoll density gradients into four fractions: alpha, azurophil granules; beta, mostly specific granules; gamma, plasma membrane, and cytosol. NADPH-dependent O2-. formation by these fractions was quantitated as the rate of superoxide dismutase-inhibitable reduction of ferricytochrome c. In the presence of cytosol, NADPH, and either arachidonic acid (optimum 90 microM) or sodium dodecyl sulfate (optimum 160 microM), 70-75% of the oxidase was in the beta fraction and about 25% was in the gamma fraction. A similar distribution was found for cytochrome b559 and FAD, two putative components of the oxidase. The reaction rates observed with arachidonic acid activation were sufficient to account for 25-75% of the O2-. generated by intact neutrophils. The properties of the beta and gamma enzymes were similar and closely resembled those of the oxidase in intact neutrophils or disrupted prestimulated cells. These included resistance to azide and cyanide, a pH optimum of 7.4, and a preference for NADPH (Km approximately 40-45 microM) rather than NADH (Km approximately 2.5 mM) as the electron donor. The combination of beta and gamma fractions displayed additive activity. The activatable oxidase required Mg2+ but not Ca2+. ATP was required for maximum reaction rates. When beta and gamma membranes were preincubated with cytosol and arachidonic acid in the presence of millimolar Mg2+ and then ultracentrifuged membrane-bound O2-. -forming activity was recovered in the pellet and the enzyme required only NADPH (i.e. no cytosol, arachidonic acid, or Mg2+) for expression of activity. These data suggest that cytosol contains a Mg2+-dependent oxidase-activating factor. Molecular sieve chromatography of cytosol indicated a single peak of activity (i.e. ability to activate O2-. generation by beta and/or gamma fraction) eluting with molecules of about 10,000 daltons.     

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.     

Arachidonate activation of the neutrophil NADPH-oxidase. Synergistic effects of protein phosphatase inhibitors compared with protein kinase activators.

Arachidonate activation of the NADPH-oxidase in intact neutrophils and in a cell-free O2- generation system was compared to synergistic activation in response to arachidonate and agents that effect protein phosphorylation. In intact neutrophils, suboptimal doses of retinal which increase protein phosphorylation, or 4B-phorbol 12-myristate 13-acetate (PMA) an activator of protein kinase C, induced minimal O2- release, but primed neutrophils to release enhanced amounts of O2- in response to 2.5 microM arachidonate. In contrast to retinal or PMA, okadaic acid, a specific inhibitor of serine/threonine protein phosphatases, did not induce any release of O2-, but significantly increased the maximal rate and duration of O2- release in response to arachidonate. In the cell-free system, only arachidonate induced O2- generation. Consistent with previous findings, activation of the cell-free system was dependent of the presence of light membranes, cytosol, NADPH, Mg2+, and 82 microM arachidonate. Pretreatment of neutrophils with suboptimal doses of PMA or retinal had little effect on the arachidonate-stimulated release of O2- in cell-free preparations of these cells. However, cytosol (but not light membranes) from PMA or retinal-primed neutrophils was more effective in completing resting membrane NADPH-oxidase activity when compared to cytosol from resting cells. The addition of protein kinase C inhibitors staurosporine and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine decreased the effectiveness of PMA-primed cytosol to complete the cell-free system, but had little effect on cytosol obtained from cells primed with retinal. The addition of protein phosphatase inhibitors, p-nitrophenyl phosphate or okadaic acid to neutrophil cavitates increased 3-fold the release of O2- in cell-free preparations of these cells. Okadaic acid and p-nitrophenyl phosphate also increased the effectiveness of both cytosol and light membranes to complete the cell-free system when combined with cytosol or light membranes from resting neutrophils, respectively, indicating that both fractions are affected by the inhibition of protein phosphatase activity. These data indicate that increases in protein phosphorylation alone do not lead to the activation of the NADPH-oxidase, but in addition to the requirement of an anionic amphiphile, the release of O2- from intact neutrophils or in the cell-free system is increased by stimulus activation of protein kinase C or more impressively by inhibition of protein phosphatase activity.     

Characterization of the interaction of human eosinophils and neutrophils with opsonized particles

The interaction of human eosinophils with opsonized particles was compared with that of human neutrophils. When eosinophils are stimulated with serum-opsonized zymosan particles, the lag time in H2O2 production is twice as long as found with neutrophils. Moreover, the concentration of these IgG + C3-coated particles required for optimal stimulation is about four times as high for eosinophils as for neutrophils. Under these conditions, the two cell types generate similar amounts of H2O2. However, eosinophils produce twice as much H2O2 as do neutrophils when stimulated with the soluble agent phorbol myristate acetate. Thus, although the oxidase capacity of eosinophils is larger than that of neutrophils, opsonized zymosan is a weak trigger for this activity in eosinophils. This phenomenon may be due to differences between the two cell types in the plasma membrane receptors or in the receptor oxidase transducing signal. The following are indications for the first possibility. i) IgG interacts poorly with the Fc gamma receptors on the eosinophil surface compared with those on neutrophils. This was shown by the inability of IgG-coated zymosan or IgG-coated latex to trigger any substantial H2O2 production by eosinophils unless brought into close contact with these cells by centrifugation. In contrast, neutrophils are stimulated by these particles both in suspension and in a pellet. The dissimilarity of the Fc gamma receptors on eosinophils and neutrophils was also shown with respect to antigenicity, determined by the monoclonal antibodies 3G8 and CLB-FcR-1. ii) Eosinophils contain about half as many receptors for C3b and C3bi on their surface as do neutrophils, also detected with monoclonal antibodies. The interaction of IgG subclasses with functional Fc gamma receptors on eosinophils and neutrophils showed that eosinophils release twice as much H2O2 as do neutrophils upon interaction with IgG1-, IgG2-, or IgG3-coated Sepharose beads, but this difference becomes fivefold with IgG4-coated Sepharose. This might be of relevance to the situation of chronic antigenic stimulation, e.g., in chronic schistosomiasis, in which eosinophil numbers and IgG4 antibody levels are elevated.     

Activation of O2(-)-generating oxidase in an heterologous cell-free system derived from Epstein-Barr-virus-transformed human B lymphocytes and bovine neutrophils. Application to the study of defects in cytosolic factors in chronic granulomatous disease.

Epstein-Barr-virus-transformed human B lymphocytes (EBV B lymphocytes) stimulated by 4 beta-phorbol 12-myristate 13-acetate exhibit an NADPH-dependent oxidase activity capable of generating the superoxide anion O2-, similar to, but less efficient than that of activated neutrophils. A cell-free system of oxidase activation consisting of a membrane fraction and cytosol from EBV B lymphocyte homogenate supplemented with guanosine 5'-[gamma-thio]triphosphate (GTP[S]), arachidonic acid and Mg2+ was found to be competent in the production of O2-, assessed by the superoxide-dismutase-sensitive reduction of cytochrome c in the presence of NADPH. However, cytochrome c reduction was slow and largely insensitive both to superoxide dismutase, and to iodonium biphenyl, a powerful inhibitor of the oxidase activity in neutrophils. A markedly faster reduction of cytochrome c in the presence of NADPH was obtained with a heterologous system consisting of cytosol from EBV B lymphocytes and bovine neutrophil membranes, GTP[S], arachidonic acid and Mg2+; in this system, reduction of cytochrome c was totally inhibited by superoxide dismutase and iodonium biphenyl. These results show that EBV B lymphocytes contain a substantial amount of cytosolic factors of oxidase activation, and that the limiting factors for O2- production in B lymphocytes are the membrane components of the oxidase complex. The heterologous system of EBV B lymphocyte cytosol and bovine neutrophil membranes provided a rapid and convenient method to diagnose cytosolic defects in autosomal forms of chronic granulomatous disease. In addition, it might be a useful tool to explore the mechanism of action of the cytosolic factors in oxidase activation.     

Comparative study on effect of N-ethylmaleimide on the superoxide-generating system of guinea-pig eosinophils stimulated by soluble stimuli

1. The effect of N-ethylmaleimide (MalNEt) modification on O2- production by guinea-pig eosinophils mediated by different soluble stimuli was studied. 2. MalNEt pretreatment inhibited the O2- production stimulated by concanavalin A (Con A), cytochalasin E or digitonin, but not A23187 or sodium fluoride. 3. Particulate fractions from MalNEt-pretreated eosinophils before exposure to the stimulus showed the inhibition of the enhancement of NADPH-dependent O2- production induced by Con A, cytochalasin E or digitonin, but not A23187. 4. Treatment of eosinophils with MalNEt after stimulation had no effect on the NADPH oxidase activity. 5. These findings suggest that at least two pathways exist for the activation of the O2(-)-generating enzyme system, probably the NADPH oxidase system, in guinea-pig eosinophils.     

Involvement of protein kinase Cdelta in the activation of NADPH oxidase and the phagocytosis of neutrophils

This experiment was performed to clarify the role of protein kinase C (PKC) delta in NADPH oxidase-dependent O(2-) production and actin polymerization followed by phagocytosis in neutrophils. Bovine neutrophils and human neutrophil-like differentiated HL-60 (dHL-60) cells were stimulated with serum-opsonized zymosan (OZ) and fMet-Leu-Phe (fMLP), respectively. Rottlerin, a specific inhibitor of PKCdelta, attenuated the production of O(2-) from NADPH oxidase in both neutrophils and dHL-60 cells. However, it did not inhibit the translocation of p47(phox) from the cytosol to the membrane in either type of cell or the phosphorylation of p47(phox) in dHL-60 cells. GF109203X (GFX), an inhibitor of cPKC, attenuated not only the production of O(2-) but also the translocation of p47(phox) in both cells. Furthermore, rottlerin significantly attenuated the ingestion of opsonized particles and the formation of F-actin in OZ-stimulated neutrophils, whereas, GFX did not affect those phagocytic processes. These results suggest that both PKCdelta and cPKC regulate NADPH oxidase through different pathways, but only PKCdelta regulates the phagocytic function in neutrophils.     

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.     

Enhanced activation of the respiratory burst oxidase in neutrophils from hypertensive patients

In neutrophils of patients with essential hypertension the NADPH-dependent O2- production elicited by stimulation with f-Met-Leu-Phe is three to four fold higher in comparison with neutrophils of normotensive control subjects. Neutrophils from hypertensive patients are less responsive to priming, by non-stimulating doses of the agonist, as compared to control cells, which following this pretreatment augment superoxide anion production up to levels close to those expressed by neutrophils from hypertensive patients. No difference in NADPH oxidase activity, between neutrophils from the two groups of subjects, was observed when the rate of O2- production was evaluated in a reconstructed cell-free system containing the membrane fraction and the cytosolic cofactors. These results are consistent with the hypothesis that differences in the functional organization of the oxidase at the membrane level in neutrophils of hypertensive are responsible for the enhanced O2- production following agonist stimulation.     

Fluoride-mediated activation of the respiratory burst in electropermeabilized neutrophils

Electropermeabilization creates small pores in the plasma membrane allowing the introduction of low-molecular-weight modulatory components, such as ions and nucleotides, into the cytosol. The present study investigates fluoride-mediated stimulation of the signal transduction pathway that activates the respiratory burst in electropermeabilized neutrophils. In marked contrast to intact (i.e., non-electropermeabilized) neutrophils, cells permeabilized by this technique demonstrated an immediate and potent stimulation of the superoxide (O2-)-generating NADPH oxidase in response to the addition of fluoride. Furthermore, permeabilization of neutrophils in the presence of exogenously added ATP enhanced the rate of F(-)-mediated O2- production. Fluoride-stimulated O2- production in electropermeabilized neutrophils was antagonized by GDP beta S and dependent upon the presence of Mg2+ in the medium, but was insensitive to pertussis toxin treatment, consistent with the hypothesis that fluoride activates a G protein, probably Gp, by interacting with the nucleotide-binding site on the G alpha subunit. In addition, electropermeabilized neutrophil O2- release triggered by F- was blocked by staurosporine and H-7, indicating that this pathway proceeds largely through protein kinase C activation. However, nucleotide-enhanced O2- production was only partially blocked by these inhibitors, suggesting that under such conditions ATP either competes with the inhibitor-protein kinase interaction or affects the signaling pathway(s) in such a way that protein kinase C may no longer be necessary for the activation of NADPH oxidase.     

Strong glucose dependence of electron current in human monocytes

Reactive oxygen species (ROS) production by human monocytes differs profoundly from that by neutrophils and eosinophils in its dependence on external media glucose. Activated granulocytes produce vast amounts of ROS, even in the absence of glucose. Human peripheral blood monocytes (PBM), in contrast, are suspected not to be able to produce any ROS if glucose is absent from the media. Here we compare ROS production by monocytes and neutrophils, measured electrophysiologically on a single-cell level. Perforated-patch-clamp measurements revealed that electron current appeared after stimulation of PBM with phorbol myristate acetate. Electron current reflects the translocation of electrons through the NADPH oxidase, the main source of ROS production. The electron current was nearly abolished by omitting glucose from the media. Furthermore, in preactivated glucose-deprived cells, electron current appeared immediately with the addition of glucose to the bath. To characterize glucose dependence of PBM further, NADPH oxidase activity was assessed as hydrogen peroxide (H(2)O(2)) production and was recorded fluorometrically. H(2)O(2) production exhibited similar glucose dependence as did electron current. We show fundamental differences in the glucose dependence of ROS in human monocytes compared with human neutrophils.     

The activity of one soluble component of the cell-free NADPH:O2 oxidoreductase of human neutrophils depends on guanosine 5'-O-(3-thio)triphosphate

Neutrophil NADPH:O2 oxidoreductase activity, essential in the killing of bacteria by neutrophils, can be elicited in a cell-free system that requires plasma membranes, cytosol and sodium dodecyl sulfate. In addition, GTP or its nonhydrolyzable analog guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) enhances NADPH oxidase activity. We investigated the mechanism of this effect of GTP gamma S in the cell-free system. Cytosol from human neutrophils was separated in three different soluble oxidase components (SOC I, SOC II, and SOC III). Previously we (Bolscher, B. G. J. M., Van Zwieten, R., Kramer, I. J. M., Weening, R. S., Verhoeven, A. J., and Roos, D. (1989) J. Clin. Invest. 83, 757-763) reported that the cytosol contains two components which act synergistically. We now report that one component (previously labeled SOC II) contains two different components that can be separated by ion exchange chromatography. Immunoblotting with antiserum B-1 (Volpp, B. D., Nauseef, W. M., and Clark, R. A. (1988) Science 242, 1295-1297), directed against a cytosolic complex capable of activating latent membranes in the cell-free system, showed a 47-kDa protein in SOC II and a 67-kDa protein in SOC III. SOC II also contains the 47-kDa phosphoprotein, which indicates that this phosphoprotein and the protein recognized by the antiserum are identical. Low rates of NADPH-dependent O2 consumption can be elicited by SOC II and SOC III in the absence of SOC I. This activity is independent of GTP gamma S. Addition of SOC I increases this activity 3-4-fold, only when GTP gamma S is present. Plasma membranes, incubated with SOC I plus GTP gamma S and re-isolated, showed a similar 3-4-fold enhanced O2 consumption with SOC II and SOC III. The GTP gamma S effect is exerted primarily at the level of the plasma membrane. The concentration of GTP gamma S that causes a half-maximal stimulation was 0.4 mu M. It is concluded that SOC I is a functional component of the NADPH oxidase.     

Diphenylene iodonium as an inhibitor of the NADPH oxidase complex of bovine neutrophils. Factors controlling the inhibitory potency of diphenylene iodonium in a cell-free system of oxidase activation.

Diphenylene iodonium (Ph2I), a lipophilic reagent, is an efficient inhibitor of the production of O2- by the activated NADPH oxidase of bovine neutrophils. In a cell-free system of NADPH oxidase activation consisting of neutrophil membranes and cytosol from resting cells, supplemented with guanosine 5'-[gamma-thio]triphosphate, MgCl2 and arachidonic acid, or in membranes isolated from neutrophils activated by 4 beta-phorbol 12-myristate 13-acetate, addition of a reducing agent, e.g. NADPH or sodium dithionite, markedly enhanced inhibition of the NADPH oxidase by Ph2I. The membrane fraction was found to contain the Ph2I-sensitive component(s). In the presence of a concentration of Ph2I sufficient to fully inhibit O2- production (around 10 nmol/mg membrane protein), addition of catalytic amounts of the redox mediator dichloroindophenol (Cl2Ind) resulted in a by-pass of the electron flow to cytochrome c, the rate of which was about half of that determined in non-inhibited oxidase. A marked increase in the efficiency of this by-pass was achieved by addition of sodium deoxycholate. The Cl2-Ind-mediated cytochrome c reduction was negligible in membranes isolated from resting neutrophils. At a higher concentration of Ph2I (100 nmol/mg membrane protein), the Cl2Ind-mediated cytochrome c reductase activity was only half inhibited, which indicated that, in the NADPH oxidase complex, there are at least two Ph2I sensitive components, differing by their sensitivity to the inhibitor. At low concentrations of Ph2I (less than 10 nmol/mg protein), the spectrum of reduced cytochrome b558 in isolated neutrophil membranes was modified, suggesting that the component sensitive to low concentrations of Ph2I is the heme binding component of cytochrome b558. Higher concentrations of Ph2I were found to inhibit the isolated NADPH dehydrogenase component of the oxidase complex. A number of membrane and cytosolic proteins were labeled by [125I]Ph2I. However, the radiolabeling of a membrane-bound 24-kDa protein, which might be the small subunit of cytochrome b558, responded more specifically to the conditions of activation and reduction which are required for inhibition of O2- production by Ph2I. The O2(-)-generating form of xanthine oxidase was also inhibited by Ph2I. Inhibition of xanthine oxidase, a non-heme iron flavoprotein, by Ph2I had a number of features in common with that of the neutrophil NADPH oxidase, namely the requirement of reducing conditions for inhibition of O2- production by Ph2I and the induction of a by-pass of electron flow to cytochrome c by Cl2Ind in the inhibited enzyme, suggesting some similarity in the molecular organization of the two enzymes.     

Properties of the NADPH dehydrogenase component of the oxidase complex from rabbit peritoneal neutrophils: reconstitution of an oxidase activity with the dehydrogenase component and a membrane extract

A flavin-linked NADPH cytochrome c oxido-reductase of molecular mass 77-kDa was extracted from membranes of rabbit peritoneal neutrophils and purified in the presence of Triton X-100. The redox properties of this enzyme were examined. By some criteria including its high sensitivity to mersalyl, and its relatively high specificity for NADPH compared to NADH, the rabbit neutrophil NADPH cytochrome c reductase resembled NADPH-cytochrome P-450 reductase. Limited proteolysis generated water soluble fragments, with molecular masses of 67-kDa and 57-kDa, which were still endowed with a substantial reductase activity. When added to a lysate of neutrophil membranes in octylglucoside, in the presence of an oxidase activation medium consisting of rabbit neutrophil cytosol, GTP-gamma-S, arachidonic acid and Mg2+, the purified reductase enhanced the production of O2-., suggesting that it forms part of the O2-. generating oxidase.     

Activation of NADPH oxidase and extracellular superoxide production in seizure-induced hippocampal damage

We sought to determine whether the extracellular compartment contributed to seizure-induced superoxide (O2*-) production and to determine the role of the NADPH oxidase complex as a source of this O2*- production. The translocation of NADPH oxidase subunits (p47phox, p67phox and rac1) was assessed by immunoblot analysis and NADPH-driven O2*- production was measured using 2-(4-hydroxybenzyl)-6-(4-hydroxyphenyl)-8-benzyl-3,7-dihydroimidazo [1,2-alpha] pyrazin-3-one-enhanced chemiluminescence. Kainate-induced status epilepticus resulted in a time-dependent translocation of NADPH oxidase subunits (p47phox, p67phox and rac-1) from hippocampal cytosol to membrane fractions. Hippocampal membrane fractions from kainate-injected rats showed increased NADPH-driven and diphenylene iodonium-sensitive O2*- production in comparison to vehicle-treated rats. The time-course of kainate-induced NADPH oxidase activation coincided with microglial activation in the rat hippocampus. Finally, kainate-induced neuronal damage and membrane oxygen consumption were inhibited in mice overexpressing extracellular superoxide dismutase. These results suggest that seizure activity activates the membrane NADPH oxidase complex resulting in increased formation of O2*-.