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

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Nitric Oxide Reacts with Intracellular Glutathione and Activates the Hexose Monophosphate Shunt in Human Neutrophils 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 acti- vate 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 dehydroge- nase; 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,
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