Proc. Nati. Acad. Sci. USAVol. 91, pp. 3680-3684, April 1994Cell Biology

Nitric oxide reacts with intracellular glutathione and activates thehexose monophosphate shunt in human neutrophils: Evidencefor S-nitrosoglutathione as a bioactive intermediaryROBERT M. CLANCY, DAVID LEVARTOVSKY, JOANNA LESZCZYNSKA-PIZIAK, JULIA YEGUDIN,AND STEVEN B. ABRAMSON*Department of Medicine, Division of Rheumatology, New York University Medical Center, and Department of Rheumatology, Hospital for Joint Diseases,New York, NY 10003

Communicated by H. Sherwood Lawrence, December 22, 1993 (receivedfor review October 20, 1993)

ABSTRACT We performed experiments to determinewhether nitric oxide promoted the formation of intracellularS-nitrosothiol adducts in human neutrophils. At concentrationssufficient to inhibit chemoatant-induced superoxide anionproduction, nitric oxide caused a depletion of measurableintracellular glutathione as determined by both the monobro-mobimane HPLC method and the glutathione reductase recy-cling assay. The depletion of glutathione could be shown to bedue to the formation of intracellular S-nitrosoglutathlone asindicated by the ability of sodium borohydride treatment ofcytosol to result in the complete recovery of measurableglutathione. The formation of intracellular S-nitrosylated com-pounds was confirmed by the capacity of cytosol derived fromnitric oxide-treated cells to ADP-ribosylate glyceraldehyde-3-phosphate dehydrogenase. Depletion of intracellular gluta-thione was accompanied by a rapid and concomitant activationof the hexose monophosphate shunt (HMPS) following expo-sure to nitric oxide. Kinetic studies demonstrated that nitricoxide-dependent activation of the IMPS was reversible andparalleled nitric oxide-induced glutathione depletion. Syntheticpreparations of S-nitrosoglutathione shared with nitric oxidethe capacity to inhibit superoxide anion production and acti-vate the HMPS. These data suggest that nitric oxide mayregulate cellular functions via the formation of intracellularS-nitrosothiol adducts and the activation of the HMPS.

Nitric oxide (NO) has been implicated as a cellular mediatorwhich regulates stimulated responses of human neutrophils.Reported effects ofNO on neutrophils include the inhibitionof superoxide anion production and adhesion to endothelialcells (1-3). The biochemical mechanisms by which extracel-lular NO affects intracellular signaling in neutrophils arepoorly understood. NO is a highly reactive (til2 < 15 sec) freeradical which can regulate the activity of proteins through avariety of posttranslational modifications, including the ni-trosylation of transition-metal complexes and thiols (4). Po-tential sites ofNO iron-complex targeting in cells include theheme groups or nonheme iron of enzymes such as aconitaseor other enzymes of the mitochondrial respiratory pathway(4). The attack upon such enzymes may account for NO-dependent cytotoxicity (5). Nitrosylation reactions have alsobeen implicated in signaling: the activation of guanylyl cy-clase by the binding of NO to its heme iron is believed tomediate smooth muscle dilation and inhibition of plateletaggregation. Increases in cyclic GMP, however, do notappear to mediate NO-dependent inhibition of neutrophilsuperoxide anion production (1).A separate reaction of potential importance involves the

S-nitrosylation of free thiol groups (6). In human plasma, the

predominant redox forms ofNO are S-nitrosothiols, the mostabundant of which is S-nitrosoalbumin (6). Such S-nitro-sothiol compounds, which also include S-nitrosocysteine andS-nitrosoglutathione, assume bioactivity through their capac-ity to donate NO and may therefore serve as stable interme-diaries. It has been speculated that this bioactive extracel-lular pool of S-nitroso proteins serves as a source of NO,buffering its free concentration (6). These observations havesuggested that NO also exerts effects within cells by reactingwith intracellular thiols. We therefore examined the effects ofNO on intracellular glutathione, glucose metabolism, andoxidant production in human neutrophils. Our data indicatethat extracellular NO reacts rapidly with intracellular glu-tathione to form a nitrosylated adduct which may regulatecellular functions.

METHODSPreparation of Neutrophils. Neutrophils were isolated from

whole blood (1). In selected experiments, neutrophils werepermeabilized by using a Bio-Rad Pulser cuvette (7).

Neutrophil Function Studies. Superoxide release by acti-vated neutrophils or the cell-free NADPH oxidase was as-sayed as described (1). Activation of the hexose monophos-phate shunt (HMPS) was assessed with [1-14C]glucose at 1pCi/ml (4 mM), obtained from DuPont/NEN (1 juCi = 37kBq). HMPS activity was calculated from the production of14CO2 from [1-14C]glucose (8).

Preparation of NO, S-Nitrocsteine, and S-Nitrogu-tathione. NO solutions were prepared after bubbling NO gasthrough isotonic Hepes buffer (9). NO solutions were quan-titated by using 100 ,uM thionitrobenzoic acid in 0.1 M Tris(pH 8.1) at 370C for 10 min (9). S-Nitrosocysteine and S-ni-trosoglutathione were prepared by reaction of reduced cys-teine or glutathione with red agarose as described below andquantitated by measuring the mercuric chloride release ofnitrite in the Greiss reaction (9).

Glutathione Measurements. Neutrophils were incubatedwith various concentrations of NO at 370C for 5 min. Cellswere lysed by freeze-thaw after addition of an equal volumeof 20 mM Tris (pH 7.4) containing lysophosphatidylcholine(50 pg/ml) and diisopropyl fluorophosphate. After microcen-trifugation at 10,000 x g for 1 min, lysate was assayed forcellular glutathione by the glutathione reductase recyclingassay (10) and by the monobromobimane derivatization ofglutathione and separation by C18 HPLC (11).NaBH4 Treatment. To break an S-nitroso bond (12), one-

half volume of cytosol was incubated with 0.1 M NaBH4 at

Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydroge-nase; HMPS, hexose monophosphate shunt; PMA, phorbol 12-myristate 13-acetate.*To whom reprint requests should be addressed at: Hospital for JointDiseases, 301 East 17th Street, New York, NY 10003.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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370C for 5 min. The solution was acidified to remove unre-acted NaBH4, and NaOH was added to obtain neutral pH.The sample was analyzed for glutathione as described above.Treatment of Protein with Red Agarose. Synthetic S-nitro-

sothiol derivatives were measured utilizing Bio-Gel A-S-nitrosothiol (red agarose) as described (12).

Subceflular Fractionation. Polymorphonuclear leukocyteswere disrupted by N2 cavitation at 350 psi (1 psi = 6.89 kPa)for 20 min at 40C in relaxation buffer (100 mM KCI/3 mMNaCl/3.5mM MgCl2/1 mM ATP/10mM Hepes, pH 7.3) plusprotease inhibitors (phenylmethanesulfonyl fluoride, leupep-tin, pepstatin A, chymostatin, and aprotinin) as described (1).

ADP-Ribosylation. Subcellular fractions (20 ug) were in-cubated for 30 min at 30'C in 40 .l of 5 AM [32P]NAD (20-40Ci/mmol)/50 mM Tris, pH 8.0), in the presence of NO orsamples. Reactions were terminated by the addition ofLaem-mli buffer and boiling for 5 min. ADP-ribosylated proteinswere visualized by SDS/PAGE and autoradiography (13).

RESULTSEffect ofNO and S-Nitrosothiols on Production of Superoxide

Anion. Table 1 illustrates the effects of NO and S-nitrosoglu-tathione on superoxide anion production by human neutro-phils. NO (10-100 ,uM) caused the dose-dependent inhibitionof superoxide generation in response to the chemoattractantfldet-Leu-Phe (0.1 pM). Preincubation of neutrophils withS-nitrosoglutathione had no effect on superoxide production.However, S-nitrosoglutathione effectively inhibited the bro-ken-cell NADPH oxidase superoxide-generating system: theaddition of S-nitrosoglutathione (80 pmol/,ig of protein) 10min before arachidonate activation (16 min before NADPHinitiation), reduced superoxide release from 507 ± 75 to 272 +41 nmol/min per mg of protein (P = 0.0056). The potency ofS-nitrosoglutathione in the cell-free system was equivalent tothat of authentic NO. We hypothesized that the differencebetween whole-cell and broken-cell reconstitution measure-ments was due to the inability ofintact neutrophils to transportextracellular glutathione (14). Therefore, neutrophils werepermeabilized prior to incubation with NO and its derivative.Electropermeabilized neutrophils produced significantamounts of superoxide anion in response to fMet-Leu-Phe (16± 3 nmol per 106 electropermeabilized cell). Exposure ofelectropermeabilized neutrophils to S-nitrosoglutathione be-fore addition offMet-Leu-Phe significantly reduced stimulatedsuperoxide production (Table 1). Electropermeabilization didnot enhance the capacity of NO to inhibit superoxide. Re-

Table 1. Effect of NO and S-nitrosoglutathione on

fMet-Leu-Phe-stimulated superoxide release inelectropermeabilized (EP) and intact neutrophils

Superoxide release, nmol/5 min

Conc., per 106 cellsAgent ,M Intact EP

NO 10 19.2 0.6* 11.2 1.0**30 13.0 1.0* 8.8 0.8**100 9.6 0.4* 6.1 1.8**

SNO-GSH 10 24.0 1.2 14.4 + 0.330 25.0 + 1.7 11.5 0.4**100 22.0 1.9 7.7 + 0.6**

Intact or electropermeabilized neutrophils (1.25 x 106 per ml) wereincubated in the absence (control) or presence of NO, or S-nitroso-glutathione (SNO-GSH) (concentration varied, 5 min, 37°C) beforeexposure to 0.1 pM fMet-Leu-Phe. fMet-Leu-Phe-stimulated super-oxide release (control, data not shown) was 24.0 ± 5 nmol and 16 ±3 nmol of cytochrome c reduced in 5 min for 106 intact and 106electropermeabilized neutrophils, respectively. *, P < 0.003 vs.control; **, P < 0.01 vs. control.

duced glutathione did not inhibit superoxide production in anysystem tested.

Extraceflular NO Depletes Intracellular Glutathione. Wenext performed a series of studies to examine whether NOinteracted with glutathione to form an S-nitroso intermediate.Fig. 1 illustrates the dose-response of NO-induced depletionof cellular glutathione, as measured by the glutathione re-ductase recycling assay (10). As shown, measurable glu-tathione fell to 25% of control values in the presence of 100,uM NO; half-maximal depletion was observed at 30 ,M NO.This observation was confirmed with the monobromobimaneHPLC method: the addition of 100 ,uM NO for 2 mindecreased glutathione from 1.8 ± 0.5 to 0.4 ± 0.2 nmol per106 cells (P < 0.01).To test the hypothesis that a decrease in measurable

cellular glutathione was due to the formation of intracellularS-nitrosoglutathione, we treated cytosol prepared from intactneutrophils previously exposed to NO with NaBH4, whichbreaks S-nitrosothiol bonds (12). As shown by glutathionereductase recycling assay (Fig. 1) and confirmed with themonobromobimane HPLC method (data not shown), NaBH4treatment of such cytosol resulted in a complete recovery ofmeasurable glutathione.These data indicate that the apparent decrease of total

measurable glutathione in NO-treated neutrophils can beaccounted for by the conversion of glutathione to a nitrosy-lated species not reported by either the monobromobimaneHPLC or glutathione reductase recycling assays.

Effect ofNO and S-Nitrosothiols on the HMPS Pathway. Wenext performed studies to determine whether the decline ofcellular glutathione reported by our assays was also"sensed" by the cell, as would be reflected by activation ofthe HMPS (14, 15). The addition of NO provoked a rapidactivation of the HMPS in resting neutrophils (Fig. 2). Thiseffect was reversed in the presence of extracellular 200 ,uMhemoglobin, which scavenges NO. Sodium nitroprusside andS-nitrosocysteine, but not S-nitrosoglutathione, also acti-

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FIG. 1. Effect of NO on cellular glutathione. Neutrophils (5 x106) were incubated with various concentrations ofNO (time varied,37°C). Cells were lysed by freeze-thaw after addition of an equalvolume of 20 mM Tris (pH 7.4) containing lysophosphatidylcholine(50 pg/ml) and phenylmethanesulfonyl fluoride (1 pg/ml). Aftermicrocentrifugation at 10,000 x g for 1 min, lysate was processed forglutathione measurement. One-half volume of cytosol was incubatedwith 0.1M NaBH4 at 37°C for 5 min, to break S-nitroso (SNO) bonds.The solution was acidified to remove unreacted NaBH4, and NaOHwas added to obtain neutral pH. Untreated cytosol and NaBH4-treated cytosol were assayed for total glutathione by the glutathionereductase recycling assay, which measures the total glutathione(reduced and oxidized, GSH and GSSG) (10). Control neutrophilcytosol (in absence of NO exposure) contained 1.4 0.7 nmol ofglutathione per 106 cells (equals 100%6 total glutathione).

(o) DIRECT MEASUREMENT (GSH + GSSG)(0) PROCESSED TO BREAK SNO BOND

T~~~~IT.*

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CONTROL

NO 50 pMNO 100 gM

SNO-CYS 100 gMSNO-GSH 100pM

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FIG. 2. NO, S-nitrosocysteine, and sodium nitroprusside acti-vate the HMPS in intact human neutrophils. Neutrophils (4 x 107 perml) were incubated in the absence (control) or presence of NOS-nitrosocysteine (SNO-CYS), S-nitrosoglutathione (SNO-GSH),phorbol 12-myristate 13-acetate (PMA), sodium nitroprusside (SNP),or the prostaglandin E1 analog misoprostol (PGE1) for 60 min at 37TC.HMPS activity was tested by determining the amount of 14CO2evolved from [1-14C]glucose (8). S-Nitrosoglutathione did activatethe HMPS in electropermeabilized neutrophils (see text). Datarepresent the mean and SEM of at least three separate determina-tions in cells from different donors.

vated the HMPS. Interestingly, however, exposure of elec-tropermeabilized neutrophils to 0.1 mM S-nitrosoglutathionefor 5 min at 370C did activate the HMPS (1270 cpm to 7070cpm). Activation of the HMPS by NO and its derivatives wasofa magnitude comparable to that provoked byPMA (Fig. 2).The effects ofNO were compared with those of misoprostol,a synthetic prostaglandin El analog. At concentrations suf-ficient to inhibit superoxide generation (70 ± 9%o control, P< 0.01), 1 1LM misoprostol did not activate the HMPS.Misoprostol also did not lower intracellular levels of glu-tathione (103 ± 7% control, n = 3).The kinetics ofNO-dependent activation of the HMPS and

depletion of cellular glutathione were analyzed. Exposure ofneutrophils to 100 ,uM NO resulted in the rapid release of14CO2, detectable at 30 sec and complete by 5 min (Fig. 3A).Following exposure to NO, total cellular glutathione reachedits nadir between 2 and 5 min and returned to baseline by 15min. PMA exposure resulted in a gradual decrease in totalcellular glutathione that was first measurable at 5 min andthat, unlike the response to NO, continued during the 60-minobservation period (Fig. 3B).

S-Nitrosylation of Glutathione and Thiols in Cytosol Pro-duces S-Nitroso Adducts with NO-Like Activity. S-nitrosyla-tion of proteins in purified cytosol preparations. To deter-mine whether thiol-containing proteins present in neutrophilcytosol could serve as targets for S-nitrosylation, we utilizedthe red-agarose technique to produce S-nitrosothiol species(12). We analyzed the biological activity of the red-agarosereaction product derived from a variety of substrates, includ-ing preparations of purified neutrophil cytosol. We used as ameasure of NO activity the capacity to ADP-ribosylate a37-kDa cytosolic protein which has been identified in othercell types as glyceraldehyde-3-phosphate dehydrogenase(GAPDH) (16). NO stimulated the ADP-ribosylation ofGAPDH, as expected (Fig. 4). The red-agarose reactionproducts of the thiol-containing substrates S-nitrosoglu-tathione, S-nitrosocysteine, S-nitrosoalbumin, including un-identified S-nitroso adducts formed in cytosol fractions, alsoADP-ribosylated GAPDH. Neither oxidized glutathione norred agarose-treated oxidized glutathione induced the ADP-ribosylation ofGAPDH (data not shown). These results show

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FIG. 3. Kinetics of NO-dependent glutathione depletion andHMPS activation. Neutrophils (4 x 106 per ml) were incubated in thepresence of NO (0.1 mM) (A) or PMA (50 ng/ml) (B) (time varied,37'C). HMPS activity was tested as described (8). Total cellularglutathione was measured as described in Fig. 1. Data are represen-tative of four experiments performed in duplicate. PMN, polymor-phonuclear leukocytes (neutrophils).

that S-nitrosoglutathione can serve as an intermediary ofNOactivity and that stable S-nitroso adducts can be formed inneutrophil cytosol.

S-nitrosylation ofproteins in intact neutrophils exposed toextracellular NO. We next utilized the ADP-ribosylation ofGAPDH as an assay to determine whether extracellular NOinduced the formation of intracellular S-nitroso intermedi-ates. Utilizing the information provided by the kinetics ofdepletion of cellular glutathione, we examined the lysatesof neutrophils in the GAPDH assay after 0, 2, or 15 min of

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FIG. 4. S-Nitroso adducts formed in cytosol ADP-ribosylateGAPDH. Cysteine (1 ,umol), glutathione (1 .nol), albumin (1 mg),and cytosol (1 mg) were incubated for 10 min at 37°C with red agarose(1 ml) as described in Methods. A separate preparation of cytosol (10pg) was incubated for 30 min at 30'C in ADP-ribosylation buffer [5AM [32P]NAD (20-40 ACilpmol)/50 mM Tris, pH 8.0] with com-pounds to be tested for the capacity to ADP-ribosylate the 37-kDaGAPDH. ADP-ribosylated proteins were visualized by SDS/PAGEand autoradiography. The concentration of each compound was asfollows: pertussis toxin (5 pg), sodium nitrite (NaNO2, 30 PM), NO(30 PM), glutathione (GSH, 100 ,uM), S-nitrosoglutathione (SNO-GSH, 100 jsM), cysteine (100 ,uM), S-nitrosocysteine (SNO-cysteine,100 pM), albumin (3 pg), S-nitrosoalbumin (SNO-albumin, 3 Pg),cytosol (3 ag), or S-nitrosylated cytosol (SNO cytosol, 3 pg).

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FIG. 5. Intracellular S-nitroso adducts formed in neutrophilsexposed to extracellular NO ADP-ribosylate GAPDH. Neutrophils(107) were incubated in the absence (lane 1) or presence ofNO (37rC,time varied). After NO exposure, cells were lysed as described inFig. 1. Lysate was examined for the capacity to ADP-ribosylateGAPDH. Cytosol (5 pg) plus lysate (1/50th of total volume) werecombined with the ADP-ribosylation buffer and analyzed as de-scribed in Fig. 4. Intervals of exposure to NO were 0 min (lane 2),2 min (lane 3), and 15 min(lane 4).

exposure to 30 gM NO. Lysates prepared from neutrophilsexposed to NO for 2 min induced ADP-ribosylation ofGAPDH (Fig. 5). The extent of ADP-ribosylation was of amagnitude comparable to that observed when purified cyto-sol was exposed directly to a NO donor (Fig. 4). In contrast,lysates prepared from neutrophils exposed to NO for 0 minor 15 min did not promote the ADP-ribosylation of GAPDHand were similar in activity to the control lysates. These dataindicate that the S-nitrosylation of cytosolic protein is re-versible at 15 min, consistent with the kinetics of the replen-ishment of glutathione (Fig. 5).

DISCUSSIONIn the extracellular compartment there is evidence that thereaction between NO and extracellular thiol-containing pro-teins results in the formation of stable S-nitroso adductswhich have the properties of endothelium-derived relaxingfactor (6). It has been suggested that extracellular S-nitrosoproteins, present in human plasma at micromolar concentra-tions, prolong the half-life ofNO in the blood and tissues (4,6). The studies reported here provide evidence that NOreacts with thiols in human neutrophils, including glu-tathione, to form stable, bioactive intermediaries which exerteffects on oxidant production and glucose metabolism. NOinhibited superoxide generation by intact neutrophils ex-posed to the chemoattractant fMet-Leu-Phe. S-Nitrosoglu-tathione, which had no effect on the intact cell, did inhibitsuperoxide release in the reconstituted broken-cell NADPHoxidase system. Since it is known that glutathione is notreadily taken up by cells (14), the absence of an S-nitroso-glutathione effect on intact neutrophils was most likely due toan inability to gain access to the intracellular compartment.Indeed, we demonstrated that the introduction of S-nitroso-glutathione into the cytosol by means of electroporationresulted in an effective inhibition of the respiratory burst. Thecapacity of S-nitrosoglutathione to serve as a NO interme-diary in the intracellular compartment was further supportedby its ability to activate the HMPS in electropermeabilizedneutrophils, to ADP-ribosylate GAPDH in purified cytosolpreparations, and, as previously reported, to activate theguanylyl cyclase of human lymphocytes (17) and inhibitplatelet aggregation (12).

In separate studies we demonstrated that thiols present incytosol assumed bioactivity following S-nitrosylation. Weused as an assay ofNO activity the capacity of S-nitrosylatedproteins to ADP-ribosylate GAPDH (16). There is recentcontroversy regarding whether the modification of GAPDHrepresents the covalent binding of NAD rather than ADP-ribose (18). However, in either case, the modification ofGAPDH is NO-dependent. By means of the red-agarosemethod of S-nitrosothiol preparation, we generated com-pounds in purified cytosol preparations which induced theADP-ribosylation ofGAPDH. Ofpotentially greater interest,we demonstrated S-nitrosylation of proteins in intact neutro-phils which had been exposed to extracellular NO by thecapacity of such cytosol to ADP-ribosylate GAPDH. Inter-estingly, the kinetics of S-nitrosylation of neutrophil thiolsparalleled the kinetics of glutathione depletion and replen-ishment. Only lysates derived from neutrophils exposed toNO for 2 min, corresponding to the nadir of measurableglutathione, were capable of promoting ADP-ribosylation ofGAPDH. Since the S-nitrosothiols detected in lysates 2 miafter NO exposure were stable (tin > 2 hr), their absence 15min after NO indicates an active process which reversesS-nitrosylation in vivo. The identity(ies) of the biologicallyactive, nitrosylated species generated in cytosol followingNO exposure requires investigation. However, our datasuggest that intracellular S-nitrosoglutathione is among theproducts of the reaction between extracellularly derived NOand intracellular glutathione.The above studies demonstrated that (i) synthetic S-ni-

trosoglutathione could function as NO donor and (ii) thatthiols present in neutrophil cytosol could serve as targets ofS-nitrosylation and function as NO intermediaries. Separatestudies indicated that neutrophils exposed to extracellularNO converted intracellular glutathione to an S-nitroso ad-duct. Using two independent methods, the monobromobi-mane HPLC method and the glutathione reductase recyclingassay, we showed that the exposure of intact neutrophils toNO caused a dose-dependent depletion of measurable intra-cellular glutathione. The dose dependence for NO depletionof glutathione was similar to that observed for NO-dependentinhibition of superoxide production (EC50 30 pM). In bothglutathione assay systems, NaBH4 treatment of cytosol de-rived from neutrophils exposed to NO completely restoredmeasurable glutathione to control levels. Since NaBH4 isknown to break S-nitrosothiol bonds (12), these data indicatethat the depletion of cytosolic glutathione is due to theformation of S-nitrosoglutathione not reported by the mono-bromobimane HPLC method (which requires reduced sulf-hydryl) or the glutathione reductase recycling assay (whichmeasures both reduced and oxidized glutathione).The biochemical and HPLC evidence that NO decreased

intracellular glutathione was supported by physiological evi-dence: the rapid and concomitant activation of the HMPS inresting neutrophils following exposure to nitric oxide. Acti-vation of the HMPS in intact cells was shared by the NOdonors sodium nitroprusside and S-nitrosocysteine, but not byS-nitrosoglutathione or a prostaglandin analog. However,consistent with its effects on superoxide production, S-ni-trosoglutathione did activate the HMPS when introduced intothe cytosol after electropermeabilization. The very rapid (sec-onds) decline of glutathione and activation of the HMPSfollowing exposure to NO contrasted with the more gradual(minutes) changes observed following exposure to PMA. Thekinetics of the PMA effect are consistent with its capacity toprovoke oxidant production by neutrophils, an effect whichalso requires a lag phase of 2-3 min. The activation of therespiratory burst in response to PMA would be expected tooxidize glutathione, decrease intracellular NALPIH, andthereby trigger the activity of the HMPS pathway. In contrastto PMA, NO does not activate the respiratory burst in resting

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neutrophils (1), and therefore the mechanism by which itdepletes reduced glutathione and activates the HMPS requiresan alternative explanation. Concomitant with the conversionof S-nitrosoglutathione to oxidized glutathione is the utiliza-tion of NADPH and activation of the HMPS which rapidlyrestores reduced glutathione levels to baseline levels.Our observations are consistent with those of Albina and

Mastrofrancesco (19), who demonstrated that treatment ofelicited rat macrophages with N"'-monomethyl-L-arginineinhibited basal activity of the HMPS. Mauel and Corradin(20) have demonstrated in cytokine activated macrophagesan increase in the production of nitrite that is accompanied byactivation of the HMPS. We suggest that the observationsreported by those authors can be explained by the capacityof NO, in either an autocrine or paracrine fashion, to reactwith and deplete intracellular glutathione.Our data have implications for both signaling and for

susceptibility to NO-dependent cytotoxicity. With regard tosignaling, S-nitrosoglutathione may serve as a stable (til2 > 2hr) source of NO, able to exert effects on cellular function(e.Ig., superoxide production, activation of guanylyl cyclase,and ADP-ribosylation). The formation of S-nitrosothiolscould also protect the cell against injury. NO in this form isless reactive with oxygen and superoxide anion, reducing thelikelihood of toxic peroxynitrite formation (4). In addition,the reaction of NO with glutathione, the latter present inmillimolar concentrations within the cell, could compete withthe nitrosation ofiron-containing proteins, the inactivation ofwhich is implicated in cytotoxicity (4, 21).The kinetics ofglutathione depletion and HMPS activation

may'also have implications regarding cell injury. In neutro-phils, rapid activation of the HMPS would be expected toprotect the cell from the susceptibility to oxidant injury whichwould otherwise result from glutathione depletion. There-fore, the capacity of different cell types to replenish reducedglutathione stores via activation of the HMPS could be animportant determinant of susceptibility to the cytotoxic ef-fects ofNO. This hypothesis is supported by the observationthat macrophages and neutrophils, which utilize NO formicrobial killing, have high HMPS activity and are resistantto attack by NO (22, 23).

In summary, our data indicate that NO reacts with intra-cellular glutathione and activates the HMPS. As has beensuggested for the extracellular compartment, S-nitrosothiolcompounds such as S-nitrosoglutathione may function asstable intracellular intermediates of NO activity and, per-haps, protect against NO-dependent cytotoxicity.

We thank Dr. Gerald Weissmann for helpful insights and Mrs.Maddy Rios for support in preparation of the manuscript.

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