Regulation of NADPH oxidase activity by Rac GTPase activating protein(s).

Activation of the NADPH oxidase of phagocytic cells requires the action of Rac2 or Rac1, members of the Ras superfamily of GTP-binding proteins. Rac proteins are active when in the GTP-bound form and can be regulated by a variety of proteins that modulate the exchange of GDP for GTP and/or GTP hydrolysis. The p190 Rac GTPase Activating Protein (GAP) inhibits human neutrophil NADPH oxidase activity in a cell-free assay system with a K1 of approximately 100 nM. Inhibition by p190 was prevented by GTP gamma S, a nonhydrolyzable analogue of GTP. Similar inhibition was seen with a second protein exhibiting Rac GAP activity, CDC42Hs GAP. The effect of p190 on superoxide (O2-) formation was reversed by the addition of a constitutively GTP-bound Rac2 mutant or Rac1-GTP gamma S but not by RhoA-GTP gamma S. Addition of p190 to an activated oxidase produced no inhibitory effect, suggesting either that p190 no longer has access to Rac in the assembled oxidase or that Rac-GTP is not required for activity once O2- generation has been initiated. These data confirm the role of Rac in NADPH oxidase regulation and support the view that it is the GTP form of Rac that is necessary for oxidase activation. Finally, they raise the possibility that NADPH oxidase may be regulated by the action of GAPs for Rac proteins.     

Human monocytes use Rac1, not Rac2, in the NADPH oxidase complex

Phagocyte NADPH oxidase is critical for defense against pathogens and contributes to inflammatory tissue injury. One component of the NADPH oxidase complex is the small GTP-binding protein Rac. There are two isoforms of Rac, and Rac2 is the predominant isoform in neutrophils and has been shown to be essential for NADPH oxidase activity. In primary human monocytes we report that in contrast to neutrophils, Rac1 is the predominantly expressed isoform. Upon monocyte activation by a variety of agents, we found that Rac1 dissociates from Rho GDP dissociation inhibitor (RhoGDI) and translocates to the membrane. We also found that Rac1 interacts with two other NADPH oxidase components, p67phox and p47phox, upon monocyte activation. These data indicate that Rac1, and not Rac2, is a component of the activated NADPH oxidase in monocytes. This finding suggests that it may be possible to selectively interfere with either monocyte or neutrophil NADPH oxidase activity, thereby selectively targeting chronic versus acute inflammatory processes.     

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.     

Requirement for posttranslational processing of Rac GTP-binding proteins for activation of human neutrophil NADPH oxidase.

Rac1 and Rac2 are closely related, low molecular weight GTP-binding proteins that have both been implicated in regulation of phagocyte NADPH oxidase. This enzyme system is composed of multiple membrane-bound and cytosolic subunits and when activated catalyzes the one-electron reduction of oxygen to superoxide. Superoxide and its highly reactive derivatives are essential for killing microorganisms. Rac proteins undergo posttranslational processing, primarily the addition of an isoprenyl group to a carboxyl-terminal cysteine residue. We directly compared recombinant Rac1 and Rac2 in a human neutrophil cell-free NADPH oxidase system in which cytosol was replaced by purified recombinant cytosolic components (p47-phox and p67-phox). Processed Rac1 and Rac2 were both highly active in this system and supported comparable rates of superoxide production. Under different cell-free conditions, however, in which suboptimal amounts of cytosol were present in the assay mixture, processed Rac2 worked much better than Rac1 at all but the lowest concentrations. This suggests that a factor in the cytosol may suppress the activity of Rac1 but not of Rac2. Unprocessed Rac proteins were only weakly able to support superoxide generation in either system, but preloading of Rac1 or Rac2 with guanosine 5'-O-(3-thio-triphosphate) (GTP gamma S) restored activity. These results indicate that processing is required for nucleotide exchange but not for interaction with oxidase components.     

A neutrophil GTP-binding protein that regulates cell free NADPH oxidase activation is located in the cytosolic fraction

The dormant O2(-)-generating oxidase in plasma membranes from unstimulated neutrophils becomes activated in the presence of arachidonate and a multicomponent cytosolic fraction. This process is stimulated by nonhydrolyzable GTP analogues and may involve a pertussis toxin insensitive GTP-binding protein. Our studies were designed to characterize the putative GTP-binding protein, localizing it to either membrane or cytosolic fraction in this system. Exposure of the isolated membrane fraction to guanosine-5'-(3-O-thio)triphosphate (GTP gamma S), with or without arachidonate, had no effect on subsequent NADPH oxidase activation by the cytosolic fraction. Preexposure of the cytosolic fraction to GTP gamma S alone did not enhance activation of the membrane oxidase. However, preexposure of the cytosol to GTP gamma S then arachidonate caused a four-fold enhancement of its ability to activate the membrane oxidase. This enhancement was evident after removal of unbound GTP gamma S and arachidonate, and was not augmented by additional GTP gamma S during membrane activation. A reconstitution assay was developed for cytosolic component(s) responsible for the GTP gamma S effect. Cytosol preincubated with GTP gamma 35S then arachidonate was fractionated by anion exchange chromatography. A single peak of protein-bound GTP gamma 35S was recovered that had reconstitutive activity. Cytosol preincubated with GTP gamma 35S alone was similarly fractionated and the same peak of protein-bound GTP gamma 35S was observed. However, this peak had no reconstitutive activity. We conclude that the GTP-binding protein regulating this cellfree system is located in the cytosolic fraction. The GTP gamma S-liganded form of this protein may be activated or stabilized by arachidonate.     

Evaluation of the process for superoxide production by NADPH oxidase in human neutrophils: evidence for cytoplasmic origin of superoxide

We present an up-to-date insight into the function of NADPH oxidase in human neutrophils, the signalling pathways involved in activation of this enzyme and the process of association of its components with the cytoskeleton. We also discuss the functional implications of morphological studies revealing localization of the sites of NADPH oxidase activity. An original model of the process of superoxide (O2*-) production in human neutrophils is shown. Organization of NADPH oxidase is associated with several components. Upon stimulation, tri-phox cytosolic components of NADPH oxidase (p40-phox, p47-phox and p67-phox) bind to actin filaments. This process involves other actin-binding proteins, such as cofilin and coronin. Activated protein kinase C, translocated from the plasma membrane, phosphorylates cytosolic components at a scaffold of cytoskeleton. Subsequently, p40-phox, responsible for maintaining the resting state of NADPH oxidase, is separated from other two cytosolic phox proteins following an attachment of the active form of small GTP-binding protein Rac to p67-phox. Cytosolic duo-phox proteins (p47-phox and p67-phox) conjugate with membrane components (gp91-phox, p22-phox and Rapla) of NADPH oxidase residing within membranes of intracellular compartments. This chain of events triggers production of O2*-. Then, oxidant-producing intracellular compartments associate with the plasma membrane. Eventually, intracellularly produced O2*- is released to the extracellular environment through the orifice formed by fusion of oxidant-producing compartments with the plasma membrane. Intracellular movement of the oxidant-producing compartments may be regulated by myosin light chain kinase. The review emphasizes that functional assembly of NADPH oxidase and, therefore, generation of O2*- is accomplished essentially within the intracellular compartments. Upon neutrophil stimulation, intracellularly generated O2*- is transported to the plasma membrane to be released and to ensure host defense against infection.     

Evaluation of the process for superoxide production by NADPH oxidase in human neutrophils: evidence for cytoplasmic origin of superoxide

Abstract

We present an up-to-date insight into the function of NADPH oxidase in human neutrophils, the signalling pathways involved in activation of this enzyme and the process of association of its components with the cytoskeleton. We also discuss the functional implications of morphological studies revealing localization of the sites of NADPH oxidase activity. An original model of the process of superoxide (O2) production in human neutrophils is shown. Organization of NADPH oxidase is associated with several components. Upon stimulation, tri-phox cytosolic components of NADPH oxidase (p40-phox, p47-phox and p67-phox) bind to actin filaments. This process involves other actin-binding proteins, such as cofilin and coronin. Activated protein kinase C, translocated from the plasma membrane, phosphorylates cytosolic components at a scaffold of cytoskeleton. Subsequently, p40-phox, responsible for maintaining the resting state of NADPH oxidase, is separated from other two cytosolic phox proteins following an attachment of the active form of small GTP-binding protein Rac to p67-phox. Cytosolic duo-phox proteins (p47-phox and p67-phox) conjugate with membrane components (gp91-phox, p22-phox and Rap1a) of NADPH oxidase residing within membranes of intracellular compartments. This chain of events triggers production of O2. Then, oxidant-producing intracellular compartments associate with the plasma membrane. Eventually, intracellularly produced O2 is released to the extracellular environment through the orifice formed by fusion of oxidant-producing compartments with the plasma membrane. Intracellular movement of the oxidant-producing compartments may be regulated by myosin light chain kinase. The review emphasizes that functional assembly of NADPH oxidase and, therefore, generation of O2 is accomplished essentially within the intracellular compartments. Upon neutrophil stimulation, intracellularly generated O2 is transported to the plasma membrane to be released and to ensure host defense against infection.     

Role of Mg2+ in activation of NADPH oxidase of human neutrophils: evidence that Mg2+ acts through G-protein.

The membrane fraction and three cytosolic proteins of neutrophils, p47-phox, p67-phox and a G-protein, are involved in the cell-free activation of the O2(-)-generating NADPH oxidase in the presence of SDS, though it has been controversial whether the G-protein is required or just enhancing the activity. We have used the three cytosolic factors, the solubilized membrane fraction, GTP gamma S and SDS, and found that both G-protein and GTP gamma S are essential for the activation of the NADPH oxidase. The effect of GTP gamma S is modified by Mg2+: the cations enhance the O2- generation at low concentrations of GTP gamma S, whereas they attenuate the activity at higher concentrations of GTP gamma S. In presence of 10 microM GTP gamma S, the maximal activity is observed at 0.1 microM Mg2+, which is several-fold higher than that at 1 mM Mg2+. The omission of Mg2+ followed by the chelation with EDTA results in loss of the activation, which is completely restored by the addition of Mg2+. Thus, Mg2+ seems to modulate the activation of the NADPH oxidase at the level of the G-protein.     

Regulation of neutrophil NADPH oxidase activation in a cell-free system by guanine nucleotides and fluoride. Evidence for participation of a pertussis and cholera toxin-insensitive G protein

Guanine nucleotide-binding regulatory proteins (G proteins) transduce a remarkably diverse group of extracellular signals to a relatively limited number of intracellular target enzymes. In the neutrophil, transduction of the signal following fMet-Leu-Phe receptor-ligand interaction is mediated by a pertussis toxin substrate (Gi) that activates inositol-specific phospholipase C. We have utilized a plasma membrane-containing fraction from unstimulated human neutrophils as the target enzyme to explore the role of G proteins in arachidonate and cytosolic cofactor-dependent activation of the NADPH-dependent O-2-generating oxidase. When certain guanine nucleotides or their nonhydrolyzable analogues were present during arachidonate and cytosolic cofactor-dependent activation, they exerted substantial dose-dependent effects. The GTP analogue, GTP gamma S, caused a 2-fold increase in NADPH oxidase activation (half-maximal stimulation, 1.1 microM). Either GDP or its nonhydrolyzable analogue, GDP beta S, inhibited up to 80% of the basal NADPH oxidase activation (Ki GDP = 0.12 mM, GDP beta S = 0.23 mM). GTP caused only slight and variable stimulation, whereas F-, an agent known to promote the active conformation of G proteins, caused a 1.6-fold stimulation of NADPH oxidase activation. NADPH oxidase activation in the cell-free system was absolutely and specifically dependent on Mg2+. Although O2- production in response to fMet-Leu-Phe was inhibited greater than 90% in neutrophils pretreated with pertussis toxin, cytosolic cofactor and target oxidase membranes from neutrophils treated with pertussis toxin showed no change in basal- or GTP gamma S-stimulated NADPH oxidase activation. Cholera toxin treatment of neutrophils also had no effect on the cell-free activation system. Our results suggest a role for a G protein that is distinct from Gs or Gi in the arachidonate and cytosolic cofactor-dependent NADPH oxidase cell-free activation system.     

Reversible activation of NADPH oxidase in membranes of HL-60 human leukemic cells

NADPH oxidase in membranes of undifferentiated and dimethylsulphoxide-differentiated HL-60 cells was activated by arachidonic acid (AA) in the presence of Mg2+ and a cytosolic cofactor (CF) found in differentiated HL-60 cells. Basal superoxide (O2-) formation was enhanced several-fold by addition of the stable GTP-analogue, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), prior to AA and was completely prevented by that of GDP. Basal and GTP gamma S-stimulated O2- formation was terminated by GDP. In the presence of Mg2+ or EDTA, basal O2- formation ceased after 25 or 10 min, respectively, and was reinitiated by GTP gamma S or GTP gamma S plus Mg2+. Albumin terminated O2- formation, which was reactivated by AA in the presence of GTP gamma S. Our results show that (1) activation of NADPH oxidase in HL-60 membranes is dependent on endogenous GTP, Mg2+, AA and CF, which is induced during myeloid differentiation, and that (2) NADPH oxidase activation is a reversible process modulated by exogenous guanine nucleotides at various stages of activity of NADPH oxidase. We suggest crucial roles of guanine nucleotide-binding proteins in the activation, deactivation and reactivation of the enzyme.     

Activation of neutrophil NADPH oxidase in a cell-free system. Partial purification of components and characterization of the activation process

The superoxide-generating enzyme of human neutrophils, NADPH oxidase, is converted from an inactive to an active form upon stimulation of the neutrophil. This activation process was examined using a recently developed cell-free system in which dormant oxidase is activated by arachidonic acid in the presence of a soluble factor from the neutrophil (Curnutte, J. T. (1985) J. Clin. Invest. 75, 1740-1743). NADPH oxidase from unstimulated human neutrophils was detected only in the membrane fraction. The soluble activation factor was localized entirely to the cytosolic fraction and exhibited two peaks of activity when partially purified under nondenaturing conditions: a major peak with a molecular mass of approximately 250 kDa and a variable minor peak with a mass of approximately 40 kDa. Both forms activated NADPH oxidase in a similar manner and did not exhibit synergy when combined. The cytosolic factor is not protein kinase C (or another kinase) as both peaks of factor activity could be resolved from the protein kinase C peak and neither required calcium or ATP to activate the oxidase. Activation of NADPH oxidase did require the simultaneous presence of the membrane fraction, the cytosolic factor, arachidonic acid, and magnesium. Following activation, however, only the membrane fraction was then required for O2- production. Cytosolic factor levels were normal in five patients with either X-linked or autosomal recessive cytochrome b-negative chronic granulomatous disease. In contrast, the membrane fractions from each failed to generate O2-, indicating that the defects in these two genetic forms of chronic granulomatous disease reside either in the oxidase itself or in a membrane component required for activation.     

The S100A8/A9 protein as a partner for the cytosolic factors of NADPH oxidase activation in neutrophils.

In a previous study, the S100A8/A9 protein, a Ca2+- and arachidonic acid-binding protein, abundant in neutrophil cytosol, was found to potentiate the activation of the redox component of the O2- generating oxidase in neutrophils, namely the membrane-bound flavocytochrome b, by the cytosolic phox proteins p67phox, p47phox and Rac (Doussière J., Bouzidi F. and Vignais P.V. (2001) Biochem. Biophys. Res. Commun.285, 1317-1320). This led us to check by immunoprecipitation and protein fractionation whether the cytosolic phox proteins could bind to S100A8/A9. Following incubation of a cytosolic extract from nonactivated bovine neutrophil with protein A-Sepharose bound to anti-p67phox antibodies, the recovered immunoprecipitate contained the S100 protein, p47phox and p67phox. Cytosolic protein fractionation comprised two successive chromatographic steps on hydroxyapatite and DEAE cellulose, followed by isoelectric focusing. The S100A8/A9 heterodimeric protein comigrated with the cytosolic phox proteins, and more particularly with p67phox and Rac2, whereas the isolated S100A8 protein displayed a tendancy to bind to p47phox. Using a semirecombinant cell-free system of oxidase activation consisting of recombinant p67phox, p47phox and Rac2, neutrophil membranes and arachidonic acid, we found that the S100A8/A9-dependent increase in the elicited oxidase activity corresponded to an increase in the turnover of the membrane-bound flavocytochrome b, but not to a change of affinity for NADPH or O2. In the absence of S100A8/A9, oxidase activation departed from michaelian kinetics above a critical threshold concentration of cytosolic phox proteins. Addition of S100A8/A9 to the cell-free system rendered the kinetics fully michaelian. The propensity of S100A8/A9 to bind the cytosolic phox proteins, and the effects of S100A8/A9 on the kinetics of oxidase activation, suggest that S100A8/A9 might be a scaffold protein for the cytosolic phox proteins or might help to deliver arachidonic acid to the oxidase, thus favoring the productive interaction of the cytosolic phox proteins with the membrane-bound flavocytochrome b.     

Enhancement by Ca2+ or Mg2+ of catalytic activity of the superoxide-producing NADPH oxidase in membrane fractions of human neutrophils and monocytes

To examine the role of divalent cations in the generation of superoxide anion (O2-) by the NADPH oxidase system of phagocytic cells, membrane-rich fractions were prepared from human neutrophils and monocytes. O2- generation by the fractions in sucrose was enhanced by addition of Ca2+ or Mg2+. EDTA inhibited most of the O2- generation; Ca2+ or Mg2+ reversed the inhibition. Zn2+, Mn2+, or Cu2+ completely inhibited O2- production. Neutrophil membrane fraction solubilized with Triton X-100, then passed through a chelating column, lost 80% of its oxidase activity; the loss could be reversed by addition of Ca2+ or Mg2+. Addition of 0.3 mM Ca2+ or Mg2+ protected against thermal instability of the enzyme. Kinetic analysis of the neutrophil oxidase activity as a function of NADPH and Ca2+ or Mg2+ concentrations showed that cation did not interact with NADPH in solution or affect the binding of NADPH to the oxidase; rather, cation bound directly to the oxidase, or to some associated regulatory component, to activate the enzyme. For the neutrophil oxidase, the Km for NADPH was 51 +/- 6 (S.D.) microM. Hyperbolic saturation was observed with Ca2+ and Mg2+, and the Kd values were 1.9 +/- 0.3 and 2.9 +/- 0.3 microM, respectively, suggesting that the oxidase, or some associated component, has a relatively high-affinity binding site for Ca2+ and Mg2+.     

Interaction between p21-activated protein kinase and Rac during differentiation of HL-60 human promyelocytic leukemia cell induced by all-trans-retinoic acid.

Undifferentiated human promyelocytic leukemia HL-60 cells show little or no superoxide production, but generate a very low O(2)(-) concentration upon incubation with all-trans-retinoic acid (ATRA). Its production reaches a maximum within 20 h, and thereafter is maintained at an almost constant level. The differentiated cells show phorbol 12-myristate 13-acetate (PMA)-stimulated NADPH oxidase activity consistent with the amount of gp91phox (phagocytic oxidase) expressed in the plasma membrane. Three isoforms of p21-activated serine/threonine kinases, PAK68, PAK65 and PAK62, were found in both cytosolic and membrane fractions, and their contents were significantly increased during induced differentiation. The amount of Rac identified in the two fractions was also markedly enhanced by ATRA- induced differentiation. In contrast, neither PAK nor Rac was seen in the plasma membrane of undifferentiated HL-60 or human neutrophil, but they were abundant in the cytoplasmic fraction. Binding of Rac with PAK isoforms was shown in the membrane upon induced differentiation of HL-60 cells. Direct binding of purified Rac1 to PAK68 was quantified using a fluorescent analog of GTP (methylanthraniloyl guanosine-5'-[beta,gamma-imido]triphosphate) bound to Rac as a reporter group. Rac1 bound to PAK68 with a 1 : 1 stoichiometry and with a K(d) value of 6.7 nm.     

Resolution of a low molecular weight G protein in neutrophil cytosol required for NADPH oxidase activation and reconstitution by recombinant Krev-1 protein

Activation of the membrane-associated NADPH oxidase in intact human neutrophils requires a receptor-associated heterotrimeric GTP-binding protein that is sensitive to pertussis toxin. Activation of this NADPH oxidase by arachidonate in a cell-free system requires an additional downstream pertussis toxin-insensitive G protein (Gabig, T. G., English, D., Akard, L. P., and Schell, M. J. (1987) (J. Biol. Chem. 262, 1685-1690) that is located in the cytosolic fraction of unstimulated cells (Gabig, T. G., Eklund, E. A., Potter, G. B., and Dykes, J. R. (1990) J. Immunol. 145, 945-951). In the present study, immunodepletion of G proteins from the cytosolic fraction of unstimulated neutrophils resulted in a loss of the ability to activate NADPH oxidase in the membrane fraction. The activity in immunodepleted cytosol was fully reconstituted by a partially purified fraction from neutrophil cytosol that contained a 21-kDa GTP-binding protein. Purified human recombinant Krev-1 p21 also completely reconstituted immunodepleted cytosol whereas recombinant human H-ras p21 or yeast RAS GTP-binding proteins had no reconstitutive activity. Rabbit antisera raised against a synthetic peptide corresponding to the effector region of Krev-1 (amino acids 31-43) completely inhibited cell-free NADPH oxidase activation, and this inhibition was blocked by the synthetic 31-43 peptide. An inhibitory monoclonal antibody specific for ras p21 amino acids 60-77 (Y13-259) had no effect on cell-free NADPH oxidase activation. Activation of the NADPH oxidase in intact neutrophils by stimulation with phorbol myristate acetate caused a marked increase in the amount of membrane-associated antigen recognized by 151 antiserum on Western blot. Thus a G protein in the cytosol of unstimulated neutrophils antigenically and functionally related to Krev-1 may be the downstream effector G protein for NADPH oxidase activation. This system represents a unique model to study molecular interactions of a ras-like G protein.     

S-glutathionylation regulates GTP-binding of Rac2

Phagocyte NADPH oxidase catalyzes the reduction of molecular oxygen to superoxide and is essential for defense against microbes. Rac2 is a low molecular weight GTP-binding protein that has been implicated in the regulation of phagocyte NADPH oxidase. Here we report that Cys(157) of Rac2 is a target of S-glutathionylation and that this modification is reversed by dithiothreitol as well as enzymatically by thioltransferase in the presence of GSH. S-glutathionylated Rac2 enhanced the binding of GTP, presumably due to structural alterations. These results elucidate the redox regulation of cysteine in Rac2 and a possible mechanism for regulating NADPH oxidase activation.     

NADPH oxidase activation increases the sensitivity of intracellular Ca2+ stores to inositol 1,4,5-trisphosphate in human endothelial cells

Many stimuli that activate the vascular NADPH oxidase generate reactive oxygen species and increase intracellular Ca(2+), but whether NADPH oxidase activation directly affects Ca(2+) signaling is unknown. NADPH stimulated the production of superoxide anion and H(2)O(2) in human aortic endothelial cells that was inhibited by the NADPH oxidase inhibitor diphenyleneiodonium and was significantly attenuated in cells transiently expressing a dominant negative allele of the small GTP-binding protein Rac1, which is required for oxidase activity. In permeabilized Mag-indo 1-loaded cells, NADPH and H(2)O(2) each decreased the threshold concentration of inositol 1,4,5-trisphosphate (InsP(3)) required to release intracellularly stored Ca(2+) and shifted the InsP(3)-Ca(2+) release dose-response curve to the left. Concentrations of H(2)O(2) as low as 3 microm increased the sensitivity of intracellular Ca(2+) stores to InsP(3) and decreased the InsP(3) EC(50) from 423.2 +/- 54.9 to 276.9 +/- 14. 4 nm. The effect of NADPH on InsP(3)-stimulated Ca(2+) release was blocked by catalase and by diphenyleneiodonium and was not observed in cells lacking functional Rac1 protein. Thus, NADPH oxidase-derived H(2)O(2) increases the sensitivity of intracellular Ca(2+) stores to InsP(3) in human endothelial cells. Since Ca(2+)-dependent signaling pathways are critical to normal endothelial function, this effect may be of great importance in endothelial signal transduction.     

The translocation of respiratory burst oxidase components from cytosol to plasma membrane is regulated by guanine nucleotides and diacylglycerol.

The respiratory burst oxidase is a multimeric enzyme responsible for O2- production by stimulated neutrophils and a few other cell types. In the resting neutrophil, the oxidase is dormant, and its subunits are distributed between the cytosol, in which they appear to exist in the form of a multisubunit complex, and the plasma membrane; but, when the neutrophil is activated, the cytosolic complex translocates to the membrane to assemble the active enzyme. Using a cell-free system in which oxidase activity was elicited with SDS, we examined the effects of GTP gamma S and dioctanoylglycerol (DiC8) on both the activation of O2- production and the transfer of the cytosolic oxidase components p47phox and p67phox to the plasma membrane. GTP (added as undialyzed cytosol) and GTP gamma S augmented the transfer of the oxidase components to the plasma membrane and was essential for the acquisition of O2- producing activity by the oxidase. DiC8 also supported the SDS-mediated transfer of oxidase components to the membrane, but O2- production did not take place unless GTP or GTP gamma S was present. In the presence of these nucleotides, however, DiC8 augmented both translocation and O2- production. We interpreted these results in terms of a mechanism in which 2 membrane-binding sites are created during the activation of the cytosolic complex, one for diacylglycerol and the other for a second site on the membrane. Development of the second membrane-binding site depends upon the action of a G protein and is essential for the expression of oxidase activity. The results further suggested that the priming of the respiratory burst oxidase in intact neutrophils might be due to an increase in membrane diacylglycerol concentration that occurs in response to the priming stimulus. Because of the increased diacylglycerol content, a larger than usual amount of active respiratory burst oxidase could be assembled on the primed plasma membrane when the neutrophil is fully activated.     

The guanine nucleotide exchange factor trio activates the phagocyte NADPH oxidase in the absence of GDP to GTP exchange on Rac. "The emperor's nw clothes"

The superoxide-generating NADPH oxidase complex of phagocytes consists of a membrane-associated flavocytochrome b(559) and four cytosolic components as follows: p47(phox), p67(phox), p40(phox), and the small GTPase Rac (1 or 2). Activation of the oxidase is the result of assembly of the cytosolic components with cytochrome b(559) and can be mimicked in vitro by mixtures of membrane and cytosolic components exposed to an anionic amphiphile, serving as activator. We reported that prenylation of Rac1 endows it with the ability to support oxidase activation in conjunction with p67(phox) but in the absence of amphiphile and p47(phox). We now show the following 6 points. 1) The Rac guanine nucleotide exchange factor Trio markedly potentiates oxidase activation by prenylated Rac1-GDP. 2) This occurs in the absence of exogenous GTP or any other source of GTP generation, demonstrating that the effect of Trio does not involve GDP to GTP exchange on Rac1. 3) Trio does not potentiate oxidase activation by prenylated Rac1-GTP, by nonprenylated Rac1-GDP in the presence or absence of amphiphile, and by a prenylated [p67(phox)-Rac1] chimera in GDP-bound form. 4) Rac1 mutants defective in the ability to bind Trio or to respond to Trio by nucleotide exchange fail to respond to Trio by enhanced oxidase activation. 5) A Trio mutant with conserved Rac1-binding ability but lacking nucleotide exchange activity fails to enhance oxidase activation. 6) The effect of Trio is mimicked by displacement of Mg(2+) from Rac1-GDP. These results reveal the existence of a novel mechanism of Rac activation by a guanine nucleotide exchange factor and suggest that the induction by Trio of a conformational change in Rac1, in the absence of nucleotide exchange, is sufficient for enhancing its effector function.     

Fatty-acid-induced activation of NADPH oxidase in plasma membranes of human neutrophils depends on neutrophil cytosol and is potentiated by stable guanine nucleotides

Both cis and trans unsaturated fatty acids and sodium dodecyl sulfate activated NADPH oxidase in plasma membranes of human neutrophils in the presence of neutrophil cytosol. In contrast, 5,8,11,14-icosatetraynoic acid, saturated fatty acids, esters, peroxides and 4 beta-phorbol 12-myristate 13-acetate, a potent activator of protein kinase C, were inactive. 5,8,11,14-icosatetraynoic acid inhibited superoxide formation elicited by fatty acids. Guanosine 5'[gamma-thio]triphosphate (GTP[gamma S]), a potent activator of guanine-nucleotide-binding proteins (N-proteins) enhanced superoxide formation elicited by fatty acids up to fourfold, supporting our previous suggestion that NADPH oxidase is regulated by an N-protein [Seifert, R. et al. (1986) FEBS Lett. 205, 161-165]. Cytosols from various tissues, soybean lipoxygenase and protein kinase C, purified from chicken stomach, did not substitute neutrophil cytosol. The activity of neutrophil cytosol was destroyed by heating at 95 degrees C. Superoxide formation was not affected by the inhibitor of protein kinase C 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7). Removal of cytosolic ATP by preincubation with hexokinase and glucose, dialysis of neutrophil cytosol or chelation of calcium with EGTA did not abolish the stimulatory effect of arachidonic acid and GTP[gamma S]. Thus, the cytosolic cofactor appears to be a neutrophil-specific and heat-labile protein, which is neither a lipoxygenase nor protein kinase C.