©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Complete and Reversible Inhibition of NADPH Oxidase in Human Neutrophils by Phenylarsine Oxide at a Step Distal to Membrane Translocation of the Enzyme Subunits (*)

(Received for publication, July 22, 1994; and in revised form, November 14, 1994)

Véronique Le Cabec (§) Isabelle Maridonneau-Parini (¶)

From the Centre National de la Recherche Scientifique, UPR 8221, 205 route de Narbonne, 31077 Toulouse, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The effects of the trivalent arsenical phenylarsine oxide (PAO) on the activity of NADPH oxidase in human neutrophils were studied. PAO caused a rapid dosedependent inhibition of superoxide generation which was maximal at a concentration of 1 µM, irrespective of the stimulating agent. This inhibitory effect was not due to impaired transduction of activation signals since neither degranulation nor phagocytosis were modified. When cytosolic and membrane fractions from resting neutrophils were combined to reconstitute the NADPH oxidase, O(2)generation was inhibited by PAO while translocation of the NADPH oxidase components to the plasma membrane fraction was not affected. The inhibition was completely and specifically reversed by 2,3-dimercaptopropanol, not by dithiothreitol or beta-mercaptoethanol, indicating that PAO binds covalently to spatially vicinal thiol groups. PAO inhibited the plasma membrane's capacity to initiate O(2)generation while it apparently did not affect the cytosol. When PAO was added subsequently to NADPH oxidase activation, no inhibition was observed, indicating that PAO cannot reach its target once the oxidase is functionally assembled. In conclusion, PAO is the first complete and reversible inhibitor of NADPH oxidase which could provide the basis for new therapeutical approaches in inflammatory diseases.


INTRODUCTION

Reactive oxygen species such as superoxide, hydrogen peroxide, hypohalides, and hydroxyl radicals, formed by neutrophils play a primary role in host defense against disease. They are also implicated in the pathogenesis of many diseases, e.g. respiratory distress syndromes, rheumatoid arthritis, and ischemia-reperfusion injury(1) . Therefore, there is great interest in the development of methods for control of their production.

The enzyme responsible for neutrophil-derived oxygen radical production is NADPH oxidase. This multicomponent electron-transfer complex, found exclusively in phagocytes and B-lymphocytes, catalyses the reduction of oxygen to superoxide anions (O(2)) using NADPH as the electron donor (see (2) for review). The oxidase is dormant in resting neutrophils but acquires catalytic activity and releases O(2)outside the cells or inside phagosomes when the cells are exposed to appropriate stimuli (see (3) and (4) for review). The known components necessary for the respiratory burst include a unique low potential b-type cytochrome, cytochrome b, present in the membrane of both specific granules (5) and secretory vesicles(6) , and at least three cytosolic proteins p47, p67, and p21(7, 8, 9) . Recently, an additional cytosolic protein, p40, has been proposed to modulate the activity of NADPH oxidase(10) . Except for p21, for which the recruitment to the plasma membrane is still debated(11, 12, 13, 14, 15) , the other oxidase components undoubtedly associate together at the plasma membrane upon cell activation.

The molecular details of the pathways regulating the activation, the assembly, and the deactivation to NADPH oxidase are still unknown. Distinct pathways for transduction of activation signals to the NADPH oxidase have been identified (see (16) for review). Inhibitors of several transducing proteins (17, 18, 19) only partially inhibit O (2) generation. Protease inhibitors such as antichymotrypsin(20, 21, 22) , arylating agents such as iodonium halides (23, 24) , or the chrysotherapeutic agent auranofin (25) have been proposed to inhibit O (2) generation. However, these molecules often provided incomplete and irreversible inhibition and they did not directly and specifically affect the NADPH oxidase complex (26) .

Recent data suggest that phosphorylation of proteins on tyrosine residues may play an important role in the activation of neutrophils (27, 28) . We sought to manipulate the tyrosine phosphorylation pathway by inhibiting tyrosine phosphatase activity to increase the level of tyrosine-phosphorylated proteins in human neutrophils and study whether this can modify the NADPH oxidase activity. A recent study of Oetken and co-workers (29) on the effects of phenylarsine oxide (PAO) (^1)on phosphorylations showed that PAO augments tyrosine phosphorylations in hematopoietic cells. PAO, a membrane-permeable trivalent arsenical that specifically complexes vicinal sulfhydryl groups of proteins to form stable ring structures, has been widely used as an inhibitor of phosphotyrosine phosphatases (30, 31) . Although PAO cannot be considered as a specific inhibitor of tyrosine phosphatases since it also affects glucose transport(32, 33) , inhibits receptor endocytosis(34, 35) , and blocks insulin-mediated activation of p21(36) , we studied its effect on the O (2) generation in human neutrophils. Surprisingly, we observed that, when PAO is used at concentrations well below those which produce the effects described above, it inhibits fully and reversibly the NADPH oxidase without any other detectable effect.


EXPERIMENTAL PROCEDURES

Reagents and Antibodies

PAO was obtained from Aldrich-Chemie, Steinheim, Germany. Bovine erythrocyte superoxide dismutase, NADPH, ferricytochrome c, ATP, GTP, GTPS, phorbol 12-myristate 13-acetate (PMA), N-formyl-Met-Leu-Phe (fMLP), the calcium ionophore A23187, and arachidonic acid were obtained from Sigma. Anti-p47, anti-p67 and anti-p22, anti-p91 antibodies were kindly provided by A. Segal and A. Verhoeven, respectively. Antiphosphotyrosine antibodies were obtained from UBI, Lake Placid, NY.

Isolation of Neutrophils

Neutrophils were isolated from blood collected from healthy donors after dextran T-500 sedimentation and centrifugation through Ficoll separating solution as described previously (37) and resuspended in Hepes-buffered minimal essential medium. Cells were stimulated with PMA (stock solution 1 mg/ml Me(2)SO stored at -80 °C), A23187 (stock solution 10M in Me(2)SO), fMLP (stock solution 10M in PBS), or zymosan opsonized in human serum as described previously(37) . Neutrophils were treated before or after cell stimulation with different doses of PAO (stock solution 25 mM in Me(2)SO).

Whole Cell Superoxide Production

The generation of superoxide by intact neutrophils was determined by the superoxide dismutase-inhibitable cytochrome c reduction assay as described previously for continuous (19) or discontinuous (37) measurement using a double-beam spectrophotometer Uvikon 930 (Kontron, France). The increase in absorbance at 550 nm was converted using the extinction coefficient of 21.1 mM cm.

Degranulation and Phagocytosis

Enzyme-linked immunosorbent assay detection of lactoferrin was performed in the extracellular and intracellular compartments as described previously(11, 38) . The rate of phagocytosis was evaluated by counting the particles of opsonized zymosan ingested by untreated and PAO-treated neutrophils fixed in cold methanol(39) .

Measurement of Intracellular ATP Levels

Intracellular ATP was measured in cytosolic fractions from control or PAO-treated neutrophils by the luciferin/luciferase chemiluminescence method. Samples or ATP (for standard curves) at different concentrations were incubated with phosphate-buffered saline (PBS) containing 10 mM MgCl(2), 15% glycerol, 1 mM EDTA, 1 mM dithiothreitol, 0.3 mM AMP, and 5 µg/ml luciferase. Next, luciferin (84 µg/ml) was added and the photon emission was measured in a Lumat LB9501 Berthold Luminometer. The concentration of ATP in the samples was calculated using the standard curves.

Measurement of the Phosphatase Activity

Cytosolic, membrane, and granule fractions (5 times 10^7 cell equivalents) were mixed (1/2, v/v) with the phosphatase buffer (50 mM Tris, 0.25 M sucrose, 2 mM MgCl(2), 1 mM ZnCl(2), 1000 IU/ml aprotinin, 1 µg/ml pepstatin, 3 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 0.5 mM EGTA, pH 7.0) and incubated with 10 mM of the phosphatase substrate para-nitrophenyl phosphate for 1 h at 37 °C. The released para-nitrophenol was measured at 410 nm.

Subcellular Fractionation

Subcellular fractionation of neutrophils on discontinuous Percoll gradient was performed as described previously(5, 39) . Briefly, neutrophils resuspended in relaxation buffer (10^8 cells/ml) were disrupted at 4 °C by cavitation in a nitrogen bomb at 375 p.s.i., collected in a tube containing EGTA (6 mM, final concentration) and centrifuged in order to remove debris, nuclei, and intact cells. The supernatant was subjected to centrifugation at 48,000 times g for 20 min at 4 °C on a discontinuous isotonic Percoll gradient; four fractions corresponding to the cytosol (S3), the plasma membrane-enriched fraction (), the specific (beta) and the azurophil granules (alpha) were collected and centrifuged at 245,000 times g for 90 min at 4 °C. The granule and membrane fractions free of Percoll were resuspended in 0.34 M sucrose, 10 mM Pipes, pH 7.2 (5 times 10^8 cell equivalents/ml). For studies analyzing the translocation of the NADPH oxidase subunits to the plasma membrane by immunoblot, subcellular fractionation was performed by differential centrifugation at 15,600 times g to spin down the granules, and subsequently at 200,000 times g to separate the cytosol from cell membranes as described(11) .

Markers (Class I HLA molecules for the plasma membrane, lactoferrin for the specific granules, and beta-glucuronidase for the azurophil granules) were assayed as described previously (11) to verify that the membrane fraction was essentially free (<10%) of contaminating granules. Protein concentration was measured by the Bio-Rad protein assay as described by the supplier.

Preparation of Active NADPH Oxidase from PMA-stimulated Neutrophils

NADPH oxidase activity was assessed in a membrane preparation as described(40) . Briefly, neutrophils were suspended at a concentration of 2 times 10^7 cells/ml in modified Eagle's medium supplemented with 20 mM Hepes and stimulated with PMA (0.5 µg/ml) at 37 °C to activate the oxidase. Five minutes after the addition of PMA, the cells were sedimented, resuspended to a concentration of 2 times 10^7 cells/ml in PBS supplemented with 1 mM EDTA, 1000 IU/ml aprotinin, 1 µg/ml pepstatin, 3 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride, and disrupted by sonication using 10 pulses at 20% power of a Branson sonifier 450. The post-nuclear supernatant was centrifuged at 27,000 times g for 20 min at 4 °C. The pellet containing the membrane fraction was then resuspended at concentration of 1 times 10^8 cell eq/ml. Activated membranes were assayed by adding 150 µM NADPH to initiate the generation of O(2).

Cell Free Assay of NADPH Oxidase

The activation of NADPH oxidase was studied in a cell-free system using the membrane and cytosolic fractions isolated from unstimulated neutrophils after Percoll gradient, as described previously(41) . The plasma membrane-enriched fraction (25 µg) and the cytosolic fraction (50 µg) both suspended in PBS supplemented with 1 mM ATP, 1 mM MgCl(2), and 0.6 mM CaCl(2) in a final volume of 500 µl were preincubated with arachidonic acid (final concentration 25 µg/ml) for 2 min. Superoxide dismutase (75 IU) was added to the reference cuvette. Cytochrome c (final concentration 0.75 mg/ml) and NADPH (final concentration 150 µM) were then added to the sample and reference cuvettes and the production of O(2)was measured. Where indicated, the membrane and the cytosolic fractions were both incubated with PAO in the cuvettes 3 min prior to the addition of arachidonic acid.

In some experiments, cytosol and membrane fractions were treated separately for 10 min at 4 °C with or without 1 µM PAO. Unbound PAO was then eliminated by two consecutive centrifugations of the membrane fraction and by running the cytosol on a Sephadex G-25 column as described by the manufacturer. Briefly, 2.5 ml of PBS containing cytosolic proteins (140 µg/ml) were loaded onto Pharmacia PD-10 columns. Proteins were eluted with 3.5 ml of PBS. The final concentration of cytosolic proteins was 100 µg/ml (dilution factor 1.4) indicating that proteins were not retained on the column. We verified that the amount of p47 and p67 recovered in the eluted cytosol did not change compared to control. The reconstitution assay was made by combining 0.5 ml of the eluted cytosolic proteins with 25 µg of membrane proteins in the presence of ATP, MgCl(2), CaCl(2), NADPH, and arachidonic acid as described above. Addition of 0.5 mM GTP and/or 10 µM GTPS did not improve the assay.

SDS-Polyacrylamide Gel Electrophoresis and Immunoblot Analyses

Proteins solubilized by boiling in SDS sample buffer for 10 min were separated on SDS-polyacrylamide gel electrophoresis (12% polyacrylamide gel) using the Mini-Protean II cell (Bio-Rad) and transferred to nitrocellulose. The nitrocellulose sheets were subsequently treated as described previously (42) with either antiphosphotyrosine antibodies or anti-p47, -p67, -p22, or -p91 antibodies.


RESULTS

PAO Inhibits Superoxide Generation Induced by a Variety of Activators

Activation of neutrophils by a variety of stimuli elicits the assembly of an active NADPH oxidase which generates O(2). We examined the dose-dependent effect of PAO on O(2)generation. As shown in Fig. 1A, concentrations as low as 1 µM PAO completely inhibited O(2)production. We first verified that this inhibition was not due to a toxic effect of PAO. None of the PAO concentrations used in Fig. 1A caused cell death even when the incubation period was extended to 30 min. Then, we verified that PAO did not interfere with the reduction of cytochrome c. The inhibition persisted even when neutrophils were preincubated with PAO, washed free of PAO, and then stimulated with PMA (Fig. 1A, inset).


Figure 1: PAO inhibits dose dependently the O(2)generation initiated by different stimuli. A, neutrophils were incubated with different concentrations of PAO for 10 min at 37 °C and subsequently stimulated. NADPH oxidase activity was measured in resting cells and in cells stimulated with PMA (100 ng/ml). The results of four experiments are expressed as mean ± S.D. Inset, resting and PMA-stimulated neutrophils were pretreated or not (none) with 1 µM PAO and O(2)production was measured. In the last line (PAO/wash), cells were treated with 1 µM PAO, washed free of PAO by dilution of the incubation medium (1/5, v/v), centrifugation, and resuspension of the cells in PAO-free medium. B, neutrophils were incubated with different concentrations of PAO for 10 min at 37 °C, and subsequently stimulated with PMA (100 ng/ml), A23187 (2.5 µM), opsonized zymosan (OZ) (3 mg/ml), fMLP (2.5 µM), or a combination of PMA and A23187 (50 ng/ml and 2.5 µM, respectively). One experiment representative of three is shown.



To further characterize the inhibitory effect of PAO on O(2)generation, cells were stimulated by a variety of stimuli described to activate the NADPH oxidase through distinct pathways of signal transduction(37, 43, 44) . We observed that O(2)generation initiated by either surface receptor-mediated agonists (fMLP and opsonized zymosan) or stimuli which act at sites distal to plasma membrane receptors (PMA and A23187) did not show different sensitivity to PAO (Fig. 1B). These data suggested that either a step common to these different transduction pathways or the NADPH oxidase itself was sensitive to PAO.

Lack of PAO Effect on Degranulation and Phagocytosis

We also examined if the inhibitory effect of PAO on O(2)generation could reflect a general mechanism inhibiting the transduction of activation signals in neutrophils. Therefore, the effects of 1 µM PAO were tested on the processes of degranulation and phagocytosis. As shown on Table 1, O(2)production was completely inhibited, while in parallel, degranulation was not affected. Furthermore, we observed that PAO did not modify the number of ingested serum-opsonized zymosan particles (data not shown).



PAO Does Not Modify Phosphatase Activities, Tyrosine Phosphorylation, and Intracellular ATP Concentration in Neutrophils

PAO was shown to exert its effect on tyrosine phosphorylation by inhibiting tyrosine phosphatases located in several subcellular compartments (30, 45, 46) or by activating a tyrosine kinase activity(47) . All these effects were observed at concentrations of PAO higher than those used to inhibit O(2)generation in the present study. Furthermore, concentrations up to 10 µM PAO were reported to be unable to modify the amount of phosphotyrosine in the leukemic cell line HL60(29) . Nevertheless, we tested whether 1 µM PAO can affect the phosphatase activity and the profile of tyrosine-phosphorylated proteins in neutrophils. Under experimental conditions shown to completely inhibit the O(2)production, PAO did not appear to modify the phosphatase activities in cytosolic, membrane, and granule fractions (Fig. 2A). Additionally, by Western blot analyses with antiphosphotyrosine antibodies, we did not observe any differences in the patterns of tyrosine-phosphorylated proteins in cells pretreated or not with 1 µM PAO and stimulated with PMA (Fig. 2B) or fMLP (data not shown).


Figure 2: PAO does not modify the phosphatase activity, the profile of tyrosine-phosphorylated proteins, or the intracellular concentration of ATP. A, neutrophils were treated with or without (control) 1 µM PAO for 10 min at 37 °C before PMA stimulation and submitted to subcellular fractionation as described under ``Experimental Procedures.'' The cytosolic (1), membrane (2), and granular fractions (3) (5 times 10^7 cell equivalent) were then assayed for their phosphatase activity. B, neutrophils pretreated or not with 1 µM PAO and stimulated with PMA were incubated with 100 µM vanadate and solubilized in SDS sample buffer containing 1 mM vanadate. Proteins were separated by gel electrophoresis and transferred to nitrocellulose membrane. The tyrosine-phosphorylated proteins were revealed with antiphosphotyrosine antibodies. Molecular weight standards are shown on the left. C, cytosols were isolated from resting and PMA-activated neutrophils pretreated or not with PAO (1 µM) and intracellular ATP levels were measured by the luciferin/luciferase assay. The results of three experiments are expressed as mean ± S.D.



PAO was reported to decrease the intracellular ATP concentration in several cell systems. Although this was generally observed when PAO was used at concentrations well above 1 µM(33, 48) , e.g. at 18 µM it had a 10-15% inhibitory effect on adipocyte ATP content(49) , we examined whether inhibition of O (2)production could result from the decrease in cellular ATP. As shown on Fig. 2C, 1 µM PAO did not cause any modification in neutrophil ATP levels in resting or PMA-activated cells.

Reversibility of PAO Inhibition on NADPH Oxidase Activity by 2,3-Dimercaptopropanol (DMP)

The dithiol compound DMP, a molecule containing vicinal sulfhydryls, was described to be able of competing for PAO binding(50, 51) . To determine whether the inhibitory effect of PAO on O (2) generation was reversible, neutrophils were incubated for 10 min with 1 µM PAO. Unbound PAO was removed by sedimenting the cells which were resuspended in fresh medium containing 10 µM DMP. Under these conditions, the level of O (2) generation upon PMA stimulation was restored indicating that the effect of PAO was fully reversed by DMP. Neither 1,4-dithiothreitol, which does not contain contiguous sulfhydryl groups, nor the monothiol reagent beta-mercaptoethanol used at the same thiol-equivalents (Table 2) were able to reverse the inhibitory effect of PAO, indicating a selective reversion of PAO effects by DMP. In addition, reversibility of the inhibitory effect of PAO by DMP confirms that PAO did not nonspecifically affect the plasma membrane or irreversibly damaged the NADPH oxidase.



PAO Is Unable to Inhibit Preactivated NADPH Oxidase in Isolated Membranes or Intact Cells

From the above results, it appears that 1 µM PAO did not modify other neutrophil metabolic pathways than O (2) generation. Therefore, we postulated that PAO could directly inhibit the NADPH oxidase complex. Neutrophils were stimulated with PMA to obtain an active NADPH oxidase complex in the membrane. Cells were then disrupted and the effect of PAO was tested on the 27,000 times g membrane fraction containing the O (2) generation capacity. We observed that, when added to the isolated membranes, PAO did not cause inhibition of the preassembled oxidase complex, even when the time of incubation (Table 3A) or the concentration of PAO (Table 3B) were increased. This suggests that once the oxidase is assembled, PAO cannot exert its inhibitory action anymore. In addition, these data confirmed that PAO has no free radical scavenger effect since the reduction of cytochrome c persists normally in its presence.



In intact cells, the oxidase activity is maintained by the continuous recruitment of new functional enzyme units(52, 53, 54) . We examined the effect of PAO when added after the activating agent on intact cells (Fig. 3). We observed that when PAO was added to cell suspensions 30 s after PMA (during the lag phase, i.e. before the generation of O (2)), O (2) production was strongly inhibited. When added 2 or 5 min after PMA, when the oxidase was active, the cytochrome c reduction flattened but did not plateau immediately.


Figure 3: Addition of PAO on PMA-activated cells inhibits the formation of new functional oxidase units. The effect of PAO on the O (2) production was measured in PMA-stimulated neutrophils. PAO was added at different times after the stimulus (30 s, 2 min, and 5 min).



Translocation of p47, p67, p22, and p91 to the Plasma Membrane Is Not Modified by PAO

A possible role of PAO could be that translocation of the oxidase subunits to the plasma membrane is impaired. To test this hypothesis, immunoblots with anti-p47, -p67, -p22, and -p91 antibodies were performed on the cytosolic and plasma membrane-enriched fractions prepared from resting or PMAactivated cells pretreated or not with PAO. Upon activation with PMA, the oxidase subunits were detected in similar amounts in the membrane of control or PAO-treated cells (Fig. 4A). Since secretory vesicles co-fractionate with the plasma membrane, the small enrichment in cytochrome b (p91 and p22) in this fraction isolated from activated cells resulted from mobilization of the specific granule pool. We can, however, conclude from our results that cytochrome b translocated to the plasma membrane to a similar extent in control or PAO-treated cells, since translocation of p47 which absolutely requires the presence of cytochrome b, and translocation of p67 which binds to p47(55, 56) , were not affected by the presence of PAO. Similarly, in the cell-free assay, 1 µM PAO did not modify the association of p47 and p67 to the membrane fraction (Fig. 4B).


Figure 4: PAO does not inhibit translocation of p22, p91, p47, and p67 to the plasma membrane in intact cells or in the cell-free system. A, neutrophils pretreated or not with 1 µM PAO and stimulated or not with PMA were fractionated by differential centrifugation. Proteins from the cytosol and the plasma membrane-enriched fraction were immunoblotted with affinity-purified anti-p47 antibodies and anti-p67 antiserum (left panel) or with anti-p22 and anti-p91 (right panel) antibodies. An additional unidentified band at approximately 42 kDa (right panel) was observed. In parallel, the inhibitory effect of PAO on the generation of O(2)by intact cells was measured (93 ± 9%, n = 3). B, cytosol and membrane fractions from resting neutrophils were obtained by centrifugation on discontinuous Percoll gradient. These fractions were coincubated in the presence or absence of 1 µM PAO followed or not by the addition of arachidonic acid for 2 min. Cytochrome c and NADPH were added and the generation of O(2)was measured (see Table 4, lines 1 and 2). After activation, membrane and cytosolic fractions were separated by centrifugation and proteins were immunoblotted as in A.





The small G protein p21 is required for NADPH oxidase activation(7, 8) , however, we did not test it in the above assays since we recently demonstrated that it is not recruited to the plasma membrane when intact neutrophils are activated(11) . However, we explored the possibility that PAO could inhibit the O(2)production by interacting with the GTP binding site of p21 since vicinal thiols, critical for the enzyme activity, have been identified in the GTP-binding site of phosphoenolpyruvate carboxykinase(57) . Reconstitution experiments of the oxidase activity were performed using cytosolic fractions preincubated with 10 µM GTPS to hypothetically protect the GTP-binding site from the subsequent addition of PAO. Under these conditions the inhibitory effect of 1 µM PAO was unchanged suggesting that the GTP-binding site of p21 is not the target of PAO (data not shown).

PAO Inhibits a Membrane Component Necessary for the Oxidase Activity

Next, we investigated whether PAO acts on the membrane-associated or on the cytosolic components of the oxidase. Cytosol and membrane fractions were treated separately for 10 min at 4 °C with 1 µM PAO and unbound PAO was then eliminated by centrifugation of the membrane fractions and by running the cytosols through Sephadex G-25 columns. PAO-treated membranes were combined with control cytosol and control membranes with PAO-treated cytosol. Under these experimental conditions, PAO was able to inhibit O(2) generation when added to the membrane fraction (Table 4, line 4), in a similar extent to that when added to the mixture of membrane and cytosol (Table 4, line 2) or to intact cells (Fig. 1). Despite the partial lost of the O(2)-generating capacity of G25-eluted cytosols (Table 4, line 5) one can see that the rate of O (2) production was similar in untreated and PAO-treated cytosols (lines 5 and 6) suggesting that only membrane-associated components of the oxidase were PAO-sensitive.


DISCUSSION

In several cell systems, when PAO is used at concentrations over 10 µM, it has been described to affect multiple sites(30, 31, 32, 33, 34, 35, 36) , such as modification of the phosphatase activities, the pattern of tyrosine-phosphorylated proteins, or the ATP concentration which are potentially involved in the regulation of NADPH oxidase(58) . Here in neutrophils, PAO was used at 1 µM, a concentration which provided a complete inhibition of the O (2) generation without detectable effect on the above mentioned PAO targets. In addition, it did not affect other neutrophil functions which are initiated upon cell activation, i.e. phagocytosis and degranulation. Therefore, using PAO at 1 µM in neutrophils appeared to have a limited effect on O (2) generation. Furthermore, PAO also prevented the O (2) generation in human monocytes (data not shown). We observed that PAO inhibited indiscriminately the generation of O (2) initiated by several stimuli reported to activate the oxidase through different signaling pathways(37, 43, 44) . It also inhibited the O (2) production by the reconstituted oxidase in the cell-free system in which transduction signals such as phosphorylation events are bypassed(59, 60, 61, 62) . From these results, it emerged that PAO could directly affect the NADPH oxidase complexes.

Whether PAO was affecting the membrane or the cytosolic components of the oxidase was determined. We report that, although chromatography through Sephadex G-25 columns induced a decrease in the O (2) generating capacity of the cytosol, no inhibitory effect of PAO was detectable in this fraction. This eliminates the possibility that PAO could interact with cytosolic proteins including the recently identified p40, a cytosolic protein not required for activity of the oxidase in the cell-free system. In contrast, the capacity of the membrane fraction to induce O (2) generation in the cell-free assay was affected by PAO. This indicated that one or more of the membrane components of the oxidase were targeted by PAO such as cytochrome b, a membrane-associated heterodimer which constitutes the electron transport apparatus of the NADPH oxidase(63) . Other proteins which may regulate the oxidase activity such as rap1 or membrane-associated cytoskeletal proteins(14, 64, 65) , have also to be considered as potential PAO targets. The identity of the PAO-sensitive protein(s) remains to be established.

In our attempt to characterize the inhibitory mechanism of PAO, the following results were obtained: 1) PAO had no effect on the generation of O (2) when added to isolated membranes containing the activated oxidase complex; 2) when PAO was added to the cell suspension after the O (2) generation had initiated, the production of O (2) rapidly decreased but did not cease, indicating that new units of active oxidase are not formed anymore, whereas functional enzymes remained active for few more minutes; 3) the components of the NADPH oxidase appeared to associate in a similar extent with the plasma membrane in the presence or absence of PAO in both intact cells or the cell-free system while the generation of O (2) was fully inhibited; 4) the inhibitory effect of PAO which reacts specifically with vicinal sulfhydryl groups to form stable five-ring structures (50) was reversed by the vicinal dithiol competitor DMP, while monothiols, or dithiols which do not have the correct space (49, 50) between the sulfhydryl groups, did not react efficiently with PAO. Taken together, these observations suggest that PAO may act at a step of the activation process distal to the translocation of the oxidase subunits and proximal to the starting point of O (2) generation by complexing spatially vicinal sulfhydryl groups. This step could correspond to a conformational change needed to initiate of the electron transport chain activity. This hypothesis is supported by the observation that the activated oxidase is PAO-insensitive, suggesting that the PAO-reactive sulfhydryl groups are not vicinal anymore. As an alternative hypothesis, it can be proposed that assembly of the oxidase components in the plasma membrane is sufficient to drive the electron transport but, once assembled, a steric hindrance impairs PAO to reach its target and inhibit the electron transfer.

N-Ethylmaleimide (NEM), a known monothiol alkylating compound, has been described to inhibit irreversibly the generation of O (2) in neutrophils(66) . Its mechanism of action can be distinguished from that of PAO according to the following observations: (i) NEM inhibits fMLP-induced production of O (2) but is a poor inhibitor when the cells are stimulated with serum-opsonized zymosan(67) , (ii) NEM irreversibly inhibits the capacity of the cytosol to drive the O (2) generation in the cell-free assay(66) , and (iii) in the presence of NEM, translocation of p47 and p67 to the plasma membrane during neutrophil activation is inhibited(54) . Therefore, PAO and NEM do not act on the same targets. In addition, NEM was used at 150 µM to induce a complete inhibition of the O (2) generation in intact neutrophils, a concentration producing multiple effects in cells(50) .

In conclusion, we describe the first complete and reversible inhibitor of the human NADPH oxidase. Reversal of the inhibition by DMP indicates that PAO affects the NADPH oxidase through its association with a protein, or a protein complex, containing vicinal thiol groups. This PAO-sensitive site is likely to be membrane-associated. The observation that PAO did not inhibit the O (2) generation once the oxidase is activated suggests that the vicinal thiol groups are no longer accessible, due to a steric hindrance, or adjacent, as the result of a potential conformational change occurring to activate the enzyme.

The respiratory burst of phagocytes has a critical role in tissue damage in inflammatory diseases. The prevention of inappropriate generation of oxygen reactive species is therefore likely to be an important homeostatic mechanism. Arsenical compounds have been applied for human therapy of a variety of protozoan infections(50) . Whether PAO produces anti-inflammatory effects in vivo is currently in progress in the laboratory. In addition, the possibility to use a complete and reversible inhibitor of the oxidase will be an important tool to progress in the understanding of the role of oxygen radicals as mediators of signal transduction in phagocytes.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by the French Ministery for Research and Teaching.

To whom correspondence should be addressed. Tel.: 33-61-17-59-77; Fax: 33-61-17-59-94.

(^1)
The abbreviations used are: PAO, phenylarsine oxide; DMP, 2,3dimercaptopropanol; fMLP, formylmethionylleucylphenylalanine; GTPS, guanosine 5`-3-O-(thio)triphosphate; NEM, N-ethylmaleimide; PBS, phosphate-buffered saline; Pipes, piperazine-N,N`-bis(2-ethanesulfonic acid); PMA, phorbol 12-myristate 13-acetate.


ACKNOWLEDGEMENTS

We gratefully acknowledge A. Segal and A. Verhoeven for providing anti-p47 and anti-p67, and anti-p22 and anti-p91 antibodies, respectively, and Heidi Möhn for critical reading of the manuscript.


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