Nitric Oxide Reacts with Intracellular Glutathione and Activates the Hexose Monophosphate Shunt in Human Neutrophils

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Proc. Nati. Acad. Sci. USA Vol. 91, pp. 3680-3684, April 1994 Cell Biology Nitric oxide reacts with intracellular glutathione and activates the hexose monophosphate shunt in human neutrophils: Evidence for S-nitrosoglutathione as a bioactive intermediary ROBERT 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 determine predominant redox forms ofNO are S-nitrosothiols, the most whether nitric oxide promoted the formation of intracellular abundant of which is S-nitrosoalbumin (6). Such S-nitro- S-nitrosothiol adducts in human neutrophils. At concentrations sothiol compounds, which also include S-nitrosocysteine and sufficient to inhibit chemoatant-induced superoxide anion S-nitrosoglutathione, assume bioactivity through their capac- production, nitric oxide caused a depletion of measurable ity to donate NO and may therefore serve as stable interme- intracellular glutathione as determined by both the monobro- diaries. It has been speculated that this bioactive extracel- mobimane HPLC method and the glutathione reductase recy- lular pool of S-nitroso proteins serves as a source of NO, cling assay. The depletion of glutathione could be shown to be buffering its free concentration (6). These observations have due to the formation of intracellular S-nitrosoglutathlone as suggested that NO also exerts effects within cells by reacting indicated by the ability of sodium borohydride treatment of with intracellular thiols. We therefore examined the effects of cytosol to result in the complete recovery of measurable NO on intracellular glutathione, glucose metabolism, and glutathione. The formation ofintracellular S-nitrosylated com- oxidant production in human neutrophils. Our data indicate pounds was confirmed by the capacity of cytosol derived from that extracellular NO reacts rapidly with intracellular glu- nitric oxide-treated cells to ADP-ribosylate glyceraldehyde-3- tathione to form a nitrosylated adduct which may regulate phosphate dehydrogenase. Depletion of intracellular gluta- cellular functions. thione was accompanied by a rapid and concomitant activation of the hexose monophosphate shunt (HMPS) following expo- METHODS sure to nitric oxide. Kinetic studies demonstrated that nitric oxide-dependent activation of the IMPS was reversible and Preparation ofNeutrophils. Neutrophils were isolated from paralleled nitric oxide-induced glutathione depletion. Synthetic whole blood (1). In selected experiments, neutrophils were preparations of S-nitrosoglutathione shared with nitric oxide permeabilized by using a Bio-Rad Pulser cuvette (7). the capacity to inhibit superoxide anion production and acti- Neutrophil Function Studies. Superoxide release by activate the HMPS. These data suggest that nitric oxide may vated neutrophils or the cell-free NADPH oxidase was as- regulate cellular functions via the formation of intracellular sayed as described (1). Activation of the hexose monophos- S-nitrosothiol adducts and the activation of the HMPS. phate shunt (HMPS) was assessed with [1-14C]glucose at 1 pCi/ml (4 mM), obtained from DuPont/NEN (1 juCi = 37 kBq). HMPS activity was calculated from the production of Nitric oxide (NO) has been implicated as a cellular mediator 14CO2 from [1-14C]glucose (8). which regulates stimulated responses of human neutrophils. Preparation of NO, S-Nitrocsteine, and S-Nitrogu- Reported effects of NO on neutrophils include the inhibition tathione. NO solutions were prepared after bubbling NO gas of superoxide anion production and adhesion to endothelial through isotonic Hepes buffer (9). NO solutions were quan- cells (1-3). The biochemical mechanisms by which extracel- titated by using 100 ,uM thionitrobenzoic acid in 0.1 M Tris lular NO affects intracellular signaling in neutrophils are (pH 8.1) at 370C for 10 min (9). S-Nitrosocysteine and S-ni- poorly understood. NO is a highly reactive (til2 < 15 sec) free trosoglutathione were prepared by reaction of reduced cys- radical which can regulate the activity of proteins through a teine or glutathione with red agarose as described below and variety of posttranslational modifications, including the ni- quantitated by measuring the mercuric chloride release of trosylation of transition-metal complexes and thiols (4). Po- nitrite in the Greiss reaction (9). tential sites ofNO iron-complex targeting in cells include the Glutathione Measurements. Neutrophils were incubated heme groups or nonheme iron of enzymes such as aconitase with various concentrations of NO at 370C for 5 min. Cells or other enzymes of the mitochondrial respiratory pathway were lysed by freeze-thaw after addition of an equal volume (4). The attack upon such enzymes may account for NO- of 20 mM Tris (pH 7.4) containing lysophosphatidylcholine dependent cytotoxicity (5). Nitrosylation reactions have also (50 pg/ml) and diisopropyl fluorophosphate. After microcen- been implicated in signaling: the activation of guanylyl cy- trifugation at 10,000 x g for 1 min, lysate was assayed for clase by the binding of NO to its heme iron is believed to cellular glutathione by the glutathione reductase recycling mediate smooth muscle dilation and inhibition of platelet and aggregation. Increases in cyclic GMP, however, do not assay (10) by the monobromobimane derivatization of appear to mediate NO-dependent inhibition of neutrophil glutathione and separation by C18 HPLC (11). superoxide anion production (1). NaBH4 Treatment. To break an S-nitroso bond (12), one- A separate reaction of potential importance involves the half volume of cytosol was incubated with 0.1 M NaBH4 at S-nitrosylation offree thiol groups (6). In human plasma, the Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HMPS, hexose monophosphate shunt; PMA, phorbol 12- The publication costs ofthis article were defrayed in part by page charge myristate 13-acetate. payment. This article must therefore be hereby marked "advertisement" *To whom reprint requests should be addressed at: Hospital forJoint in accordance with 18 U.S.C. §1734 solely to indicate this fact. Diseases, 301 East 17th Street, New York, NY 10003. 3680 Downloaded by guest on September 25, 2021 Cell Biology: Clancy et al. Proc. Natl. Acad. Sci. USA 91 (1994) 3681

370C for 5 min. The solution was acidified to remove unre- duced glutathione did not inhibit superoxide production in any acted NaBH4, and NaOH was added to obtain neutral pH. system tested. The sample was analyzed for glutathione as described above. Extraceflular NO Depletes Intracellular Glutathione. We Treatment of Protein with Red Agarose. Synthetic S-nitro- next performed a series of studies to examine whether NO sothiol derivatives were measured utilizing Bio-Gel A-S- interacted with glutathione to form an S-nitroso intermediate. nitrosothiol (red agarose) as described (12). Fig. 1 illustrates the dose-response of NO-induced depletion Subceflular Fractionation. Polymorphonuclear leukocytes of cellular glutathione, as measured by the glutathione re- were disrupted by N2 cavitation at 350 psi (1 psi = 6.89 kPa) ductase recycling assay (10). As shown, measurable glu- for 20 min at 40C in relaxation buffer (100 mM KCI/3 mM tathione fell to 25% of control values in the presence of 100 NaCl/3.5 mM MgCl2/1 mM ATP/10 mM Hepes, pH 7.3) plus ,uM NO; half-maximal depletion was observed at 30 ,M NO. protease inhibitors (phenylmethanesulfonyl fluoride, leupep- This observation was confirmed with the monobromobimane tin, pepstatin A, chymostatin, and aprotinin) as described (1). HPLC method: the addition of 100 ,uM NO for 2 min ADP-Ribosylation. Subcellular fractions (20 ug) were in- decreased glutathione from 1.8 ± 0.5 to 0.4 ± 0.2 nmol per cubated for 30 min at 30'C in 40 .l of 5 AM [32P]NAD (20-40 106 cells (P < 0.01). Ci/mmol)/50 mM Tris, pH 8.0), in the presence of NO or To test the hypothesis that a decrease in measurable samples. Reactions were terminated by the addition ofLaem- cellular glutathione was due to the formation of intracellular mli buffer and boiling for 5 min. ADP-ribosylated proteins S-nitrosoglutathione, we treated cytosol prepared from intact were visualized by SDS/PAGE and autoradiography (13). neutrophils previously exposed to NO with NaBH4, which breaks S-nitrosothiol bonds (12). As shown by glutathione reductase recycling assay (Fig. 1) and confirmed with the RESULTS monobromobimane HPLC method (data not shown), NaBH4 Effect ofNO and S-Nitrosothiols on Production ofSuperoxide treatment of such cytosol resulted in a complete recovery of Anion. Table 1 illustrates the effects of NO and S-nitrosoglu- measurable glutathione. tathione on superoxide anion production by human neutro- These data indicate that the apparent decrease of total phils. NO (10-100 ,uM) caused the dose-dependent inhibition measurable glutathione in NO-treated neutrophils can be accounted for by the conversion of glutathione to a nitrosy- of superoxide generation in response to the chemoattractant fldet-Leu-Phe (0.1 Preincubation of neutrophils with lated species not reported by either the monobromobimane pM). HPLC or glutathione reductase recycling assays. S-nitrosoglutathione had no effect on superoxide production. Effect ofNO and S-Nitrosothiols on the HMPS Pathway. We However, S-nitrosoglutathione effectively inhibited the bro- next performed studies to determine whether the decline of system: ken-cell NADPH oxidase superoxide-generating the cellular glutathione reported by our assays was also addition of S-nitrosoglutathione (80 pmol/,ig of protein) 10 "sensed" by the cell, as would be reflected by activation of min before arachidonate activation (16 min before NADPH the HMPS (14, 15). The addition of NO provoked a rapid initiation), reduced superoxide release from 507 ± 75 to 272 + activation of the HMPS in resting neutrophils (Fig. 2). This 41 nmol/min per mg of protein (P = 0.0056). The potency of effect was reversed in the presence of extracellular 200 ,uM S-nitrosoglutathione in the cell-free system was equivalent to hemoglobin, which scavenges NO. Sodium nitroprusside and that of authentic NO. We hypothesized that the difference S-nitrosocysteine, but not S-nitrosoglutathione, also acti- between whole-cell and broken-cell reconstitution measurements was due to the inability ofintact neutrophils to transport 120 (o) DIRECT MEASUREMENT (GSH + GSSG) extracellular glutathione (14). Therefore, neutrophils were (0) PROCESSED TO BREAK SNO BOND permeabilized prior to incubation with NO and its derivative. Electropermeabilized neutrophils produced significant 100 U -j amounts of superoxide anion in response to fMet-Leu-Phe (16 zo 0 cc 80 ± 3 nmol per 106 electropermeabilized cell). Exposure of z < 0 IT electropermeabilized neutrophils to S-nitrosoglutathione before addition offMet-Leu-Phe significantly reduced stimulated 60 T~~~~ superoxide production (Table 1). Electropermeabilization did (9 z .* not enhance the capacity of NO to inhibit superoxide. Re- ~-w 40 O a: Table 1. Effect of NO and S-nitrosoglutathione on I_ _- fMet-Leu-Phe-stimulated superoxide release in 20 electropermeabilized (EP) and intact neutrophils Superoxide release, nmol/5 min 0 0 50 100 150 200 250 Conc., per 106 cells *P < 0.022 Agent ,M Intact EP NITRIC OXIDE (pM) NO 10 19.2 0.6* 11.2 1.0** FIG. 1. Effect of NO on cellular glutathione. Neutrophils (5 x 30 13.0 1.0* 8.8 0.8** 106) were incubated with various concentrations of NO (time varied, 100 9.6 0.4* 6.1 1.8** 37°C). Cells were lysed by freeze-thaw after addition of an equal SNO-GSH 10 24.0 1.2 14.4 + 0.3 volume of 20 mM Tris (pH 7.4) containing lysophosphatidylcholine 30 25.0 + 1.7 11.5 0.4** (50 pg/ml) and phenylmethanesulfonyl fluoride (1 pg/ml). After 100 22.0 1.9 7.7 0.6** microcentrifugation at 10,000 x g for 1 min, lysate was processed for + glutathione measurement. One-half volume of cytosol was incubated Intact or electropermeabilized neutrophils (1.25 x 106 per ml) were with 0.1 M NaBH4 at 37°C for 5 min, to break S-nitroso (SNO) bonds. incubated in the absence (control) or presence of NO, or S-nitroso- The solution was acidified to remove unreacted NaBH4, and NaOH glutathione (SNO-GSH) (concentration varied, 5 min, 37°C) before was added to obtain neutral pH. Untreated cytosol and NaBH4- exposure to 0.1 pM fMet-Leu-Phe. fMet-Leu-Phe-stimulated super- treated cytosol were assayed for total glutathione by the glutathione oxide release (control, data not shown) was 24.0 ± 5 nmol and 16 ± reductase recycling assay, which measures the total glutathione 3 nmol of cytochrome c reduced in 5 min for 106 intact and 106 (reduced and oxidized, GSH and GSSG) (10). Control neutrophil electropermeabilized neutrophils, respectively. *, P < 0.003 vs. cytosol (in absence of NO exposure) contained 1.4 0.7 nmol of control; **, P < 0.01 vs. control. glutathione per 106 cells (equals 100%6 total glutathione). Downloaded by guest on September 25, 2021 3682 Cell Biology: Clancy et A Proc. Natl. Acad. Sci. USA 91 (1994)

50000 CONTROL NO 1.6 NO 50 pM 40000 H, NO 100 gM Em1w- 30000 Uz 0 SNO-CYS 100 8 gM 20000 Z < E SNO-GSH 100 pM 10000 D cC I0- 0 PMA 50 ng/ml I I- C -J 0 o 0 SNP 10 mM W* E C PGE1 1 pM 0 15000 30000 45000 50000 1.6 vs. control PMA U *p < 0.03 HMPS ACTIVITY (cpm) 40000 X,0 **p < 0.005 30000 0.8Izs FIG. 2. NO, S-nitrosocysteine, and sodium nitroprusside acti- 20000 vate the HMPS in intact human neutrophils. Neutrophils (4 x 107 per a~~~~~~~~d~~ ~ H Z ml) were incubated in the absence (control) or presence of NO HzL 10000 S-nitrosocysteine (SNO-CYS), S-nitrosoglutathione (SNO-GSH), ,,"d~ . DJ-r 0 II CL phorbol 12-myristate 13-acetate (PMA), sodium nitroprusside (SNP), 0 or the prostaglandin E1 analog misoprostol (PGE1) for 60 min at 37TC. E HMPS activity was tested by determining the amount of 14CO2 evolved from [1-14C]glucose (8). S-Nitrosoglutathione did activate 0 10 20 30 40 50 60 the HMPS in electropermeabilized neutrophils (see text). Data represent the mean and SEM of at least three separate determina- FIG. 3. Kinetics of NO-dependent glutathione depletion and tions in cells from different donors. HMPS activation. Neutrophils (4 x 106 per ml) were incubated in the presence of NO (0.1 mM) (A) or PMA (50 ng/ml) (B) (time varied, vated the HMPS. Interestingly, however, exposure of elec- 37'C). HMPS activity was tested as described (8). Total cellular tropermeabilized neutrophils to 0.1 mM S-nitrosoglutathione glutathione was measured as described in Fig. 1. Data are represen- tative of four experiments performed in duplicate. PMN, polymor- for 5 min at 370C did activate the HMPS (1270 cpm to 7070 phonuclear leukocytes (neutrophils). cpm). Activation ofthe HMPS by NO and its derivatives was ofa magnitude comparable to that provoked by PMA (Fig. 2). that S-nitrosoglutathione can serve as an intermediary ofNO The effects of NO were compared with those of misoprostol, activity and that stable S-nitroso adducts can be formed in a synthetic prostaglandin El analog. At concentrations suf- neutrophil cytosol. ficient to inhibit superoxide generation (70 ± 9%o control, P S-nitrosylation ofproteins in intact neutrophils exposed to < 0.01), 1 1LM misoprostol did not activate the HMPS. Misoprostol also did not lower intracellular levels of glu- extracellular NO. We next utilized the ADP-ribosylation of GAPDH as an assay to determine whether extracellular NO tathione (103 ± 7% control, n = 3). The kinetics ofNO-dependent activation ofthe HMPS and induced the formation of intracellular S-nitroso intermedi- depletion of cellular glutathione were analyzed. Exposure of ates. Utilizing the information provided by the kinetics of neutrophils to 100 ,uM NO resulted in the rapid release of depletion of cellular glutathione, we examined the lysates 14CO2, detectable at 30 sec and complete by 5 min (Fig. 3A). of neutrophils in the GAPDH assay after 0, 2, or 15 min of Following exposure to NO, total cellular glutathione reached its nadir between 2 and 5 min and returned to baseline by 15 z LU j min. PMA exposure resulted in a gradual decrease in total z z cellular glutathione that was first measurable at 5 min and 00(n I Z Z m that, unlike the response to NO, continued during the 60-min 0 Zn 0z_ >. - -J 0 0 < observation period (Fig. 3B). 0z LW = 0 S-Nitrosylation of Glutathione and Thiols in Cytosol Pro- I~z o C'n 6 m o 6 z -j EczO a0 >- z z > z duces S-Nitroso Adducts with NO-Like Activity. S-nitrosyla- n En < 0n L0 tion ofproteins in purified cytosol preparations. To determine whether thiol-containing proteins present in neutrophil 37- 4w 41 4K 4W GAPDH cytosol could serve as targets for S-nitrosylation, we utilized the red-agarose technique to produce S-nitrosothiol species (12). We analyzed the biological activity of the red-agarose 1 2 3 4 5 6 7 8 9 10 11 reaction product derived from a variety of substrates, includ- FIG. 4. S-Nitroso adducts formed in cytosol ADP-ribosylate ing preparations of purified neutrophil cytosol. We used as a GAPDH. Cysteine (1 ,umol), glutathione (1 .nol), albumin (1 mg), measure of NO activity the capacity to ADP-ribosylate a and cytosol (1 mg) were incubated for 10 min at 37°C with red agarose 37-kDa cytosolic protein which has been identified in other (1 ml) as described in Methods. A separate preparation ofcytosol (10 cell types as glyceraldehyde-3-phosphate dehydrogenase pg) was incubated for 30 min at 30'C in ADP-ribosylation buffer [5 (GAPDH) (16). NO stimulated the ADP-ribosylation of AM [32P]NAD (20-40 ACilpmol)/50 mM Tris, pH 8.0] with com- GAPDH, as expected (Fig. 4). The red-agarose reaction pounds to be tested for the capacity to ADP-ribosylate the 37-kDa products of the thiol-containing substrates S-nitrosoglu- GAPDH. ADP-ribosylated proteins were visualized by SDS/PAGE and autoradiography. The concentration of each compound was as tathione, S-nitrosocysteine, S-nitrosoalbumin, including un- follows: pertussis toxin (5 pg), sodium nitrite (NaNO2, 30 PM), NO identified S-nitroso adducts formed in cytosol fractions, also (30 PM), glutathione (GSH, 100 ,uM), S-nitrosoglutathione (SNO- ADP-ribosylated GAPDH. Neither oxidized glutathione nor GSH, 100 jsM), cysteine (100 ,uM), S-nitrosocysteine (SNO-cysteine, red agarose-treated oxidized glutathione induced the ADP- 100 pM), albumin (3 pg), S-nitrosoalbumin (SNO-albumin, 3 Pg), ribosylation ofGAPDH (data not shown). These results show cytosol (3 ag), or S-nitrosylated cytosol (SNO cytosol, 3 pg). Downloaded by guest on September 25, 2021 Cell Biology: Clancy et al. Proc. Natl. Acad. Sci. USA 91 (1994) 3683

4 z I.-4z In separate studies we demonstrated that thiols present in cytosol assumed bioactivity following S-nitrosylation. We c N T- used as an assay ofNO activity the capacity of S-nitrosylated proteins p a; to ADP-ribosylate GAPDH (16). There is recent 0 - controversy regarding whether the modification of GAPDH o 0 0 0 represents the covalent binding of NAD rather than ADP- ribose (18). However, in either case, the modification of 4 GAPDH is NO-dependent. By means of the red-agarose 0O En 4 u 4 z method of S-nitrosothiol preparation, we generated compounds in purified cytosol preparations which induced the ADP-ribosylation ofGAPDH. Ofpotentially greater interest, 37 M-mm .-- we demonstrated S-nitrosylation ofproteins in intact neutrophils which had been exposed to extracellular NO by the capacity of such cytosol to ADP-ribosylate GAPDH. Inter- estingly, the kinetics of S-nitrosylation of neutrophil thiols 1 2 3 4 paralleled the kinetics of glutathione depletion and replen- FIG. 5. Intracellular S-nitroso adducts formed in neutrophils ishment. Only lysates derived from neutrophils exposed to exposed to extracellular NO ADP-ribosylate GAPDH. Neutrophils NO for 2 min, corresponding to the nadir of measurable (107) were incubated in the absence (lane 1) or presence of NO (37rC, glutathione, were capable of promoting ADP-ribosylation of time varied). After NO exposure, cells were lysed as described in GAPDH. Since the S-nitrosothiols detected in lysates 2 mi Fig. 1. Lysate was examined for the capacity to ADP-ribosylate after NO exposure were stable (tin > 2 hr), their absence 15 GAPDH. Cytosol (5 pg) plus lysate (1/50th of total volume) were min after NO indicates an active process which reverses combined with the ADP-ribosylation buffer and analyzed as de- S-nitrosylation in vivo. The identity(ies) of the biologically scribed in Fig. 4. Intervals of exposure to NO were 0 min (lane 2), active, nitrosylated species generated in cytosol following 2 min (lane 3), and 15 min(lane 4). NO exposure requires investigation. However, our data suggest that intracellular S-nitrosoglutathione is among the exposure to 30 gM NO. Lysates prepared from neutrophils exposed to NO for 2 min induced ADP-ribosylation of products of the reaction between extracellularly derived NO and intracellular glutathione. GAPDH (Fig. 5). The extent of ADP-ribosylation was of a The above studies demonstrated that (i) synthetic S-ni- magnitude comparable to that observed when purified cyto- trosoglutathione could function as NO donor and (ii) that sol was exposed directly to a NO donor (Fig. 4). In contrast, thiols present in neutrophil cytosol could serve as targets of lysates prepared from neutrophils exposed to NO for 0 min S-nitrosylation and function as NO intermediaries. Separate or 15 min did not promote the ADP-ribosylation of GAPDH studies indicated that neutrophils exposed to extracellular and were similar in activity to the control lysates. These data NO converted intracellular glutathione to an S-nitroso ad- indicate that the S-nitrosylation of cytosolic protein is re- duct. Using two independent methods, the monobromobi- versible at 15 min, consistent with the kinetics of the replen- mane HPLC method and the glutathione reductase recycling ishment of glutathione (Fig. 5). assay, we showed that the exposure of intact neutrophils to NO caused a dose-dependent depletion of measurable intra- DISCUSSION cellular glutathione. The dose dependence for NO depletion ofglutathione was similar to that observed for NO-dependent In the extracellular compartment there is evidence that the inhibition of superoxide production (EC50 30 pM). In both reaction between NO and extracellular thiol-containing pro- glutathione assay systems, NaBH4 treatment of cytosol de- teins results in the formation of stable S-nitroso adducts rived from neutrophils exposed to NO completely restored which have the properties of endothelium-derived relaxing measurable glutathione to control levels. Since NaBH4 is factor (6). It has been suggested that extracellular S-nitroso known to break S-nitrosothiol bonds (12), these data indicate proteins, present in human plasma at micromolar concentra- that the depletion of cytosolic glutathione is due to the tions, prolong the half-life of NO in the blood and tissues (4, formation of S-nitrosoglutathione not reported by the mono- 6). The studies reported here provide evidence that NO bromobimane HPLC method (which requires reduced sulf- reacts with thiols in human neutrophils, including glu- hydryl) or the glutathione reductase recycling assay (which measures both reduced and oxidized glutathione). tathione, to form stable, bioactive intermediaries which exert The biochemical and HPLC evidence that NO decreased effects on oxidant production and glucose metabolism. NO intracellular glutathione was supported by physiological evi- inhibited superoxide generation by intact neutrophils ex- dence: the rapid and concomitant activation of the HMPS in posed to the chemoattractant fMet-Leu-Phe. S-Nitrosoglu- resting neutrophils following exposure to nitric oxide. Acti- tathione, which had no effect on the intact cell, did inhibit vation of the HMPS in intact cells was shared by the NO superoxide release in the reconstituted broken-cell NADPH donors sodium nitroprusside and S-nitrosocysteine, but not by oxidase system. Since it is known that glutathione is not S-nitrosoglutathione or a prostaglandin analog. However, readily taken up by cells (14), the absence of an S-nitroso- consistent with its effects on superoxide production, S-ni- glutathione effect on intact neutrophils was most likely due to trosoglutathione did activate the HMPS when introduced into an inability to gain access to the intracellular compartment. the cytosol after electropermeabilization. The very rapid (sec- Indeed, we demonstrated that the introduction of S-nitroso- onds) decline of glutathione and activation of the HMPS glutathione into the cytosol by means of electroporation following exposure to NO contrasted with the more gradual resulted in an effective inhibition ofthe respiratory burst. The (minutes) changes observed following exposure to PMA. The capacity of S-nitrosoglutathione to serve as a NO interme- kinetics of the PMA effect are consistent with its capacity to diary in the intracellular compartment was further supported provoke oxidant production by neutrophils, an effect which by its ability to activate the HMPS in electropermeabilized also requires a lag phase of 2-3 min. The activation of the neutrophils, to ADP-ribosylate GAPDH in purified cytosol respiratory burst in response to PMA would be expected to preparations, and, as previously reported, to activate the oxidize glutathione, decrease intracellular NALPIH, and guanylyl cyclase of human lymphocytes (17) and inhibit thereby trigger the activity of the HMPS pathway. In contrast platelet aggregation (12). to PMA, NO does not activate the respiratory burst in resting Downloaded by guest on September 25, 2021 3684 Cell Biology: Clancy et al. Proc. Natl. Acad. Sci. USA 91 (1994) neutrophils (1), and therefore the mechanism by which it We thank Dr. Gerald Weissmann for helpful insights and Mrs. depletes reduced glutathione and activates the HMPS requires Maddy Rios for support in preparation of the manuscript. an alternative explanation. Concomitant with the conversion of S-nitrosoglutathione to oxidized glutathione is the utiliza- 1. Clancy, R. M., Leszczynska-Piziak, J. & Abramson, S. B. tion of NADPH and activation of the HMPS which rapidly (1992) J. Clin. Invest. 90, 1116-1121. restores reduced glutathione levels to baseline levels. 2. Kubes, P. M., Suzuki, M. & Granger, D. N. (1991) Proc. Natl. Our observations are consistent with those of Albina and Acad. Sci. USA 88, 4651-4655. Mastrofrancesco (19), who demonstrated that treatment of 3. Stefanovic-Racic, M., Stadler, J. & Evans, C. H. (1993) Ar- elicited rat macrophages with N"'-monomethyl-L-arginine thritis Rheum. 36, 1036-1044. 4. Stamler, J. S., Singel, D. J. & Loscalzo, J. (1992) Science 258, inhibited basal activity of the HMPS. Mauel and Corradin 1898-1902. (20) have demonstrated in cytokine activated macrophages 5. Nathan, C. & Hibbs, J. B. (1991) Curr. Opin. Immunol. 3, an increase in the production ofnitrite that is accompanied by 65-70. activation of the HMPS. We suggest that the observations 6. Stamler, J. S., Simon, D. I., Jaraki, O., Osborne, J. A., Fran- reported by those authors can be explained by the capacity cis, S., Mullins, M., Singel, D. & Loscalzo, J. (1992) Proc. of NO, in either an autocrine or paracrine fashion, to react Natl. Acad. Sci. USA 89, 8087-8091. with and deplete intracellular glutathione. 7. Smolen, J. E. & Sandborg, R. R. (1990) Biochim. Biophys. Our data have implications for both signaling and for Acta 1052, 133-142. susceptibility to NO-dependent cytotoxicity. With regard to 8. Babior, B. M. & Cohen, H. J. (1981) in Methods in Hemoa- signaling, S-nitrosoglutathione may serve as a stable (til2 > 2 tology: Leukocyte Function, ed. Cline, M. J. (Churchill Liv- hr) source of NO, able to exert effects on cellular function ingstone, New York), pp. 1-39. (e.Ig., superoxide production, activation of guanylyl cyclase, 9. Clancy, R. M., Miyazaki, Y. & Cannon, P. J. (1990) Anal. and ADP-ribosylation). The formation of S-nitrosothiols Biochem. 191, 138-143. could also protect the cell against injury. NO in this form is 10. Tietz, F. (1969) Anal. Biochem. 27, 502-522. less reactive with oxygen and superoxide anion, reducing the 11. Newton, G. L., Dorian, R. & Fahey, R. C. (1981) Anal. Bio- likelihood of toxic peroxynitrite formation (4). In addition, chem. 114, 383-387. the reaction of NO with glutathione, the latter present in 12. Clancy, R. M. & Abramson, S. B. (1992) Anal. Biochem. 204, millimolar concentrations within the cell, could compete with 365-371. 13. Clancy, R. M., Leszczynska-Piziak, J. & Abramson, S. B. the nitrosation ofiron-containing proteins, the inactivation of (1993) Biochem. Biophys. Res. Commun. 191, 847-852. which is implicated in cytotoxicity (4, 21). 14. Meister, A. & Anderson, M. E. (1983)Annu. Rev. Biochem. 52, The kinetics ofglutathione depletion and HMPS activation 711-760. may'also have implications regarding cell injury. In neutro- 15. Mize, C. E. & Langdon, R. G. (1962) J. Biol. Chem. 237, phils, rapid activation of the HMPS would be expected to 1589-1595. protect the cellfrom the susceptibility to oxidant injury which 16. Molina-Vedia, L., McDonald, B., Reep, B., Brune, B., DiSil- would otherwise result from glutathione depletion. There- vio, M., Billiar, T. R. & Lapetina, E. G. (1992)J. Biol. Chem. fore, the capacity of different cell types to replenish reduced 267, 24929-24932. glutathione stores via activation of the HMPS could be an 17. Merryman, P. F., Clancy, R. M., He, X. & Abramson, S. B. important determinant of susceptibility to the cytotoxic ef- (1993) Arthritis Rheum. 36, 1414-1422. fects ofNO. This hypothesis is supported by the observation 18. McDonald, L. J. & Moss, J. (1993) Proc. Natl. Acad. Sci. USA that macrophages and neutrophils, which utilize NO for 90, 6238-6241. 19. Albina, J. E. & Mastrofrancesco, B. (1993)Am. J. Physiol. 363, microbial killing, have high HMPS activity and are resistant c1594-c1599. to attack by NO (22, 23). 20. Mauel, J. & Corradin, S. B. (1991) J. Immunol. 146, 279-285. In summary, our data indicate that NO reacts with intra- 21. Drapier, J. C., Pellat, C. & Henry, Y. (1991)J. Biol. Chem. 266, cellular glutathione and activates the HMPS. As has been 10162-10167. suggested for the extracellular compartment, S-nitrosothiol 22. Stuehr, D. J., Gross, S. S., Sakuma, I., Levin, R. & Nathan, compounds such as S-nitrosoglutathione may function as C. F. (1989) J. Exp. Med. 169, 1011-1020. stable intracellular intermediates of NO activity and, per- 23. Malawista, S. E., Montgomery, R. R. & Van Blarican, G. haps, protect against NO-dependent cytotoxicity. (1992) J. Clin. Invest. 90, 631. Downloaded by guest on September 25, 2021