Interference of Carboxy-PTIO with Nitric Oxide- and Peroxynitrite-Mediated Reactions

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Free Radical Biology & Medicine, Vol. 22, No. 5, pp. 787–794, 1997Copyright q 1997 Elsevier Science Inc.Printed in the USA. All rights reserved

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Original Contribution

INTERFERENCE OF CARBOXY-PTIO WITH NITRIC OXIDE-AND PEROXYNITRITE-MEDIATED REACTIONS

Silvia Pfeiffer,* Eva Leopold,* Benjamin Hemmens,* Kurt Schmidt,*Ernst R. Werner,† and Bernd Mayer*

*Institut fur Pharmakologie und Toxikologie, Karl-Franzens-Universitat Graz, Universitatsplatz 2, A-8010 Graz, Austria; and†Institut fur Medizinische Chemie und Biochemie, Universitat Innsbruck, Fritz-Pregl-Straße 3, A-6020 Innsbruck, Austria

(Received 4 June 1996; Revised 6 August 1996; Accepted 6 August 1996)

Abstract—Carboxy-PTIO reacts rapidly with NO to yield NO2 and has been used as a scavenger to test the impor-tance of nitric oxide (NO) in various physiological conditions. This study investigated the effects of carboxy-PTIOon several NO- and peroxynitrite-mediated reactions. The scavenger potently inhibited NO-induced accumulationof cGMP in endothelial cells but potentiated the effect of the putative peroxynitrite donor SIN-1. Carboxy-PTIOcompletely inhibited peroxynitrite-induced formation of 3-nitrotyrosine from free tyrosine (EC50 Å 36 { 5 ) asmMwell as nitration of bovine serum albumin. Peroxynitrite-mediated nitrosation of GSH was stimulated by the drugwith an EC50 of 0.12 { 0.03 mM, whereas S-nitrosation induced by the NO donor DEA/NO (0.1 mM) was inhibitedby the scavenger with an IC50 of 0.11 { 0.03 mM. Oxidation of NO with carboxy-PTIO resulted in formation ofnitrite without concomitant production of nitrate. Our results demonstrate that the effects of carboxy-PTIO are diverseand question its claimed specificity as NO scavenger. Copyright q 1997 by Elsevier Science Inc.

Keywords—Carboxy-PTIO, Nitric oxide, 3-Nitrotyrosine, Peroxynitrite, SIN-1, S-Nitrosogluthatione

INTRODUCTION

NO has several important biological functions, includ-ing the regulation of vascular tone, inhibition of plateletaggregation, modulation of synaptic transmission in thebrain, and neurotransmission in the peripheral nervoussystem.1 NO can undergo a variety of reactions withincells, and to understand NO-mediated regulatory ef-fects it is necessary to study the rates of these reactionsand the activity of their products.2 The reactions withmolecular oxygen, superoxide, and iron proteins arealready known to be physiologically significant.3 Thereaction with oxygen is second order with respect toNO,4–6 and yields intermediates with potential cyto-toxic and mutagenic properties.7,8 It is not yet clearwhich of the possible nitrogen oxides are intermediatesin this reaction.4,9

One of the fastest reactions of NO is the nearly dif-fusion-controlled combination with superoxide to form

Address correspondence to: Dr. Bernd Mayer, Institut fur Phar-makologie und Toxikologie, Karl-Franzens-Universitat Graz, Univ-ersitatsplatz 2, A-8010 Graz, Austria.

peroxynitrite,10,11 a potent oxidant of many biologicalmolecules.12 In a metal-catalyzed reaction, peroxyni-trite nitrates several phenolic compounds including freeand peptide tyrosine.13–15 Peroxynitrite is protonatedwith a pKa of 6.8, and the corresponding peroxynitrousacid rapidly decomposes to form a potent oxidant withhydroxyl radical-like properties.10 The precise mecha-nism of peroxynitrite decomposition is not clear yet,but it appears that free radical intermediates are notinvolved.12

Several reports indicate that peroxynitrite has simi-lar physiological effects to NO. It induces NO-like re-laxation of vascular smooth muscle,16,17 inhibits plateletaggregation,17 and stimulates purified soluble guanylylcyclase.18 These effects may be due to formation ofsmall amounts of NO during peroxynitrite decompo-sition16 or result from S-nitrosation of thiols followedby release of free NO from the corresponding thioni-trites.18,19 Thus, selective NO scavengers are requiredto discriminate between free NO and other species ex-hibiting NO-like biological activity. Reduced hemoglo-bin was described as a potent and specific scavenger of

788 S. PFEIFFER et al.


NO,20 but authentic peroxynitrite reacts rapidly withhemoglobin in a manner indistinguishable from NO-induced formation of methemoglobin.21 Another classof NO scavengers are nitronyl nitroxides.22 The pro-totypes of such stable free radicals are PTIO and itswater-soluble derivative carboxy-PTIO, which reactrapidly with NO to yield the corresponding imidazoli-neoxyl and free NO2 radical.23 Based on susceptibilityto carboxy-PTIO, it was reported that EDRF was iden-tical with NO, whereas the nitrergic neurotransmitterproducing relaxation of certain smooth muscles wasnot.23,24 To find out whether carboxy-PTIO can be re-garded as a specific NO scavenger, we have studied theeffect of this drug on several NO- and peroxynitrite-mediated reactions.

MATERIAL AND METHODS

Materials

Solutions of peroxynitrite (80–100 mM) were pre-pared as described21 and diluted to 10 mM with H2Oprior to experiments. GSNO and carboxy-PTIO werepurchased from ALEXIS Corp., Switzerland. DEA/NOwas from NCI Chemical Carcinogen Repository, Kan-sas City, MO; 10-fold concentrated stock solutions ofthe NO donor were prepared daily in 10 mM NaOH.SIN-1, a generous gift from Cassella-Riedel, Frankfurt,Germany, was dissolved at pH 5.0 prior to use. Themonoclonal nitrotyrosine antibody was a generous giftfrom Dr. J. S. Beckman. The ECL Western blottingdetection system was from Amersham. All other chem-icals were from Sigma.

Culture of endothelial cells and determination ofintracellular cGMP

Porcine aortic endothelial cells were cultured as pre-viously described.25 Endothelial cells were isolated byenzymatic treatment (0.1% collagenase) and culturedup to three passages in Dulbecco’s MEM (SEBAKGmbH, Suben, Austria) containing 10% fetal calf se-rum and antibiotics. Prior to experiments, endothelialcells were subcultured in 24-well plastic plates andgrown to confluence (Ç21 105 cells/well). The culturemedium was removed, and the cells were washed onceand equilibrated in incubation buffer (isotonic 50 mMHEPES buffer, pH 7.4, containing 2.5 mM CaCl2, 1mM MgCl2, 1 mM 3-isobutyl-1-methylxanthine, and 1

indomethacin) in the absence or presence of 0.1mMmM carboxy-PTIO. After 15 min, Ca2/ ionophore A23187 or DEA/NO were added to give initial final con-centrations of 0.3 and 1 , respectively. ReactionsmMwere stopped 4 min later by removal of the incubation

buffer and treatment for 1 h with 1 ml of 0.01 N HCl.Intracellular cGMP was measured in the supernatantsof the lysed cells by radioimmunoassay.

Nitrosation of GSH and HPLC analysis of GSNO

GSH (1 mM) was dissolved in 100 mM phosphatebuffer (pH 7.4) in the presence of 0.1 mM neocuproine,a selective Cu(I) chelator preventing GSNO decomposi-tion,19 and carboxy-PTIO at concentrations ranging from1 to 1 mM. The samples were preincubated for 5 minmMat 377C, followed by addition of 0.1 mM DEA/NO or 1mM peroxynitrite, rapid vortexing and incubation for 1 hat 377C. Changes of buffer pH were õ 0.1 units. Deter-mination of GSNO was performed by HPLC and UVdetection as described previously.18 Samples (30- ) weremlinjected onto a 250 1 4 mm C18 reversed phase columnequipped with a 4 1 4 mm C18 guard column for HPLCanalysis (LiChrospher 100 RP-18, 5 particle size,mmMerck, Vienna, Austria). Elution was with 20 mM so-dium phosphate buffer, pH 7.4, containing 2% (v/v)methanol and 0.1 mM EDTA at a flow rate of 0.75 ml/min. GSNO was detected by its absorbance at 338 nm.Calibration of the method with authentic GSNO, freshlydissolved in 20 mM phosphate buffer (pH 7.4), yieldedlinear responses of peak area to concentration in the rangeof 1 to 100 .mM

Tyrosine nitration and HPLC analysis of 3-nitrotyrosine

Solutions of 1 mM D,L-tyrosine in 50 mM phosphatebuffer (pH 7.4) were preincubated for 5 min at 377C inthe absence or presence of carboxy-PTIO (1 to 1mMmM). Alkaline stock solution of peroxynitrite (1 mM finalconcentration) was added dropwise with vigorous vortex-ing. Changes of buffer pH were õ 0.1 units. Reactionmixtures were incubated for 1 h at 377C and then placedon ice until HPLC analysis. Samples (30- ) were in-mljected onto a 250 1 4 mm C18 reversed phase columnequipped with a 4 1 4 mm C18 guard column for HPLCanalysis (LiChrospher 100 RP-18, 5 particle size,mmMerck, Vienna, Austria). Elution was with 50 mMKH2PO4/H3PO4 buffer (pH 3) containing 6% (v/v) meth-anol at a flow rate of 1.0 ml/min. 3-Nitrotyrosine wasdetected by its absorbance at 274 nm. Identification andquantification of 3-nitrotyrosine was based on calibrationcurves established with the authentic compound (1–100

) under the same conditions.mM

Determination of nitrite and nitrate

Because carboxy-PTIO interferred strongly with theGriess reaction, nitrite and nitrate were determined by

789Effects of carboxy-PTIO on NO- and peroxynitrite-mediated reactions


Fig. 1. Effect of carboxy-PTIO on NO- and SIN-1-induced accu-mulation of cGMP in endothelial cells. Endothelial cells were prein-cubated in the absence or presence of 0.1 mM carboxy-PTIO. After15 min DEA/NO (1 ), A 23187 (0.3 ), or SIN-1 (1mM) weremM mMadded and cells were incubated for 4 min at 377C. Cyclic GMP wasmeasured by radioimmunoassay. Data represent mean values { SEof three experiments performed in duplicate.

HPLC.26,27 Samples (50- ) were injected onto a 2501ml4 mm C18 reversed phase column (LiChrospher 100RP-18, 5 particle size, Merck, Vienna, Austria) andmmeluted with 5% (w/v) NH4Cl, pH 7.0 at a flow rate of0.7 ml/min. Nitrite was detected by postcolumn deri-vatization with the stable Griess-Ilosvay reagent(Merck) (0.7 ml/min), heating to 607C, and measure-ment of the absorbance at 546 nm. For determinationof nitrite / nitrate, samples were reduced with a cad-mium reactor (cadmium, 0.3–0.8 mm, 20–50 meshASTM, Merck, washed with 0.1 N HCl, and packed ina Pharmacia HR 5/5 glass column) prior to postcolumnderivatization.

Nitration of bovine serum albumin and detection byimmunoblotting

Bovine serum albumin (10 mg/ml) was incubatedin 50 mM phosphate buffer pH 7.4 in a total volumeof 1 ml for 1 h at 377C in the presence of 1 mMperoxynitrite and carboxy-PTIO at concentrations asindicated. Samples were diluted with a 0.125 M Tris/HCl buffer (pH 6.8) containing 4% (w/v) SDS, 20%(w/v) glycerol, 0.02% bromphenol blue and 10% (v/v) 2-mercaptoethanol, and kept at 0207C until use.Samples containing 6 of protein were subjectedmgto SDS-PAGE on 10% slab gels followed by transferonto nitrocellulose membranes in 25 mM Tris/HCl(pH 8.3), containing 192 mM glycine, 0.02% (w/v)SDS, and 20% (v/v) methanol at 250 mA for 1 h.Blots were blocked by overnight incubation of themembranes at 47C with 3% (w/v) ovalbumin (Sigma#A 5253) in 20 mM Tris/HCl, pH 7.7, containing 137mM NaCl and 0.1% (w/v) Tween 20 (buffer A). Themembranes were washed twice for 5 min with bufferA, followed by incubation for 5 h with the mono-clonal nitrotyrosine antibody28 (kindly provided byJ. S. Beckman) diluted 1:300 in buffer A containing1.5% (w/v) ovalbumin. After washing three times for20 min with buffer A, the membranes were incubatedfor 1 h with horseradish peroxidase-labeled antirab-bit-IgG antibody, which had been diluted 1:8,000 inbuffer A containing 1.5% (w/v) ovalbumin. The blotswere washed three times for 20 min with buffer Aand peroxidase was detected with the ECL systemaccording to manufacturer’s instructions. Relativestaining intensities were determined by densitometricanalysis of the blots with the vds 800 video systemand H1D-software of Hirschmann (AnalysentechnikHirschmann, Taufkirchen, Germany).

Kinetic simulations

Kinetic simulations of N2O3 formation were per-formed using a second order Runge-Kutta algorithm

from the software package Mathematica (Version2.2.2., Wolfram Research Inc., Champaign, IL). Themodel was based on oxidation of NO released from 0.1mM DEA/NO at pH 7.4 and 377C (k Å 5.2 1 1003 s01)in the presence of 1.85 1 1004 M O2 and 1 mM GSH.Based on the rate constant of overall NO autoxidationdetermined at 377C with an NO sensitive Clark-typeelectrode (4 1 kaq Å 1.36 1 107 M02 s01; Schmidt, K.and Mayer, B.; unpublished), a third-order rate constantof 6.8 1 106 M02 s01 (2 1 kaq)29 was used to accountfor the reaction 2NO / O2 ! 2NO2. The followingsecond-order rate constants (M01 s01) were used to ac-count for the reactions of NO2 with NO, NO2, and GSH,respectively: 1.1 1 109,30 4.5 1 108,30 and 1.0 1 108.31

The same simulation was performed for NO oxidationin the presence of 0.1 mM carboxy-PTIO assuming arate constant for the reaction with NO of 1.01 104 M01

s01.23 The model does not account for reactions down-stream of N2O3 formation and yields theoretical con-centrations, only.

RESULTS

The ability of carboxy-PTIO to scavenge NO wasassessed by determination of NO-induced accumu-lation of cGMP in cultured porcine aortic endothelialcells in the absence and presence of the drug. BasalcGMP levels of unstimulated cells were 2.2 { 0.7pmol/106 cells. As shown in Fig. 1, 1 DEA/NOmMinduced a 20-fold increase in cGMP (44{ 12.5 pmol/106 cells) and this effect was inhibited by 0.1 mMcarboxy-PTIO to 16.3 { 4.8% of the control. In the



Fig. 2. Effect of carboxy-PTIO on peroxynitrite-induced nitration oftyrosine. Tyrosine (1 mM) was incubated at 377C for 1 h in 50 mMphosphate buffer (pH 7.4) in the presence of 1 mM peroxynitriteand carboxy-PTIO at the indicated concentrations. Data representmean values { SE of three experiments performed in duplicate.

Fig. 3. Effect of carboxy-PTIO on peroxynitrite-induced nitration ofBSA. Bovine serum albumin (0.15 mM) was incubated at 377C for1 h in 50 mM phosphate buffer (pH 7.4) with 1 mM peroxynitritein the absence or presence of carboxy-PTIO followed by SDS-PAGEand immunoblotting (6 of protein per lane) as described in Ma-mgterials and Methods. Lane A: control; lane B: 0.1 mM carboxy-PTIO;lane C: 0.05 mM carboxy-PTIO. The blot shown is representativeof three.

Fig. 4. Effect of carboxy-PTIO on peroxynitrite-induced S-nitrosa-tion of GSH. GSH (1 mM) was incubated at 377C for 1 h in 0.1 Mphosphate buffer (pH 7.4), containing 0.1 mM neocuproine, in theabsence or presence of carboxy-PTIO at the indicated concentrations.Data represent mean values { SE of three experiments performedin duplicate.

presence of the Ca2/ ionophore A 23187 (0.3 ),mMwhich induces Ca2/-mediated formation and releaseof EDRF,32 cGMP levels were increased to 19.3 {3.7 pmol/106 cells. Again, the effect was inhibited bythe NO scavenger (0.1 mM) to 43 { 3.6% of control.The effect of DEA/NO and A 23187 was completelyinhibited by 1 mM carboxy-PTIO (data not shown).The sydnonimine SIN-1 (1 mM), a putative peroxy-nitrite donor, also induced accumulation of endothe-lial cGMP albeit to a smaller degree (7.3 { 0.2 pmol/106 cells). In contrast to the effects of DEA/NO andthe Ca2/ ionophore, the effect of SIN-1 was not in-hibited but significantly potentiated by carboxy-PTIO (to 35.4 { 2.9 pmol cGMP/106 cells at 0.1mM). Carboxy-PTIO did not potentiate SIN-1– in-duced stimulation of purified soluble guanylyl cy-clase (data not shown).

Potentiation of cGMP accumulation by SIN-1 indi-cated that carboxy-PTIO may interfere with reactionsof peroxynitrite. To address this issue more directly,we studied the effect of carboxy-PTIO on tyrosine ni-tration by authentic peroxynitrite. Figure 2 shows thatperoxynitrite (1 mM final initial concentration) led toformation of 60.6 { 1.9 3-nitrotyrosine and thatmMcarboxy-PTIO produced a concentration-dependent in-hibition of tyrosine nitration with an IC50 of 36 { 5

. Inhibition was complete at 0.6 mM of the drug.mMComparable data were obtained when tyrosine nitrationof BSA was monitored by immunoblotting with a mon-oclonal antibody that specifically reacts with peroxy-nitrite-modified albumin.28 As shown in Fig. 3, onestrong immunoreactive band (67 kDa) was observed

upon treatment of 0.15 mM BSA with 1 mM peroxy-nitrite (lane A). Immunostaining was inhibited to 13.6and 5.8% of control by 0.05 (lane C) and 0.1 mM (laneB) carboxy-PTIO, respectively.

Reaction of peroxynitrite with thiols results mainlyin formation of the corresponding disulfides and sulf-oxides,33 but small amounts of the corresponding thio-nitrites are also formed.18 We studied the effect of car-



boxy-PTIO on the peroxynitrite-induced S-nitrosationof GSH. Addition of peroxynitrite to 1 mM GSH (pH7.4) gave rise to formation of 5.8 { 0.2 GSNOmM(Fig. 4). Carboxy-PTIO increased the yield of thioni-trite up to twofold with an EC50 of 119 { 31 .mM

A potent nitrosating intermediate (NOx) is gen-erated in the course of NO autoxidation.34,35 To seewhether carboxy-PTIO would also affect NOx-me-diated S-nitrosation, we measured formation ofGSNO from GSH and the NO donor DEA/NO in thepresence of carboxy-PTIO. Incubation of 1 mM GSHwith 0.1 mM DEA/NO (1 h; pH 7.4; 377C) led toformation of 17.2 { 2.7 GSNO (Fig. 5). Car-mMboxy-PTIO inhibited this nitrosation reaction with anIC50 of 110 { 31 . Inhibition was virtually com-mMplete at 0.2 mM carboxy-PTIO.

Determination of nitrite and nitrate formed fromDEA/NO (0.1 mM) confirmed that nitrite is the solemetabolite of NO autoxidation. Presence of up to 1 mMcarboxy-PTIO did not induce detectable formation ofnitrate.

DISCUSSION

Our results on inhibition of NO-induced cGMP ac-cumulation in cultured cells and inhibition of NO-stim-ulated soluble guanylyl cylcase confirm that carboxy-PTIO is a potent scavenger of NO, but the action profileof this compound may be more complex than hithertoassumed. Unexpectedly, carboxy-PTIO did not inhibitbut potentiated the effect of SIN-1 on cGMP accumu-lation in endothelial cells. Decomposition of SIN-1yields NO and superoxide,36 suggesting that the syd-nonimine may be a donor of peroxynitrite. Because car-boxy-PTIO did not potentiate stimulation of purifiedsoluble guanylyl cyclase by SIN-1, the drug appears tointerfere with a reaction downstream of peroxynitriteformation. Based on our observation that carboxy-PTIO enhanced peroxynitrite-induced S-nitrosation,we suppose that a similar reaction may account for theeffect of SIN-1 on accumulation of cGMP in cells, butthis remains to be investigated.

A major finding of this study was the identificationof carboxy-PTIO as inhibitor of peroxynitrite-in-duced tyrosine nitration. The effect of the drug wasobserved using two different assay systems (i.e.,HPLC determination of 3-nitrotyrosine and immu-noblotting of peroxynitrite-modified BSA). Becauseinhibition occurred at substoichiometric concentra-tions of carboxy-PTIO, the drug probably does notreact with peroxynitrite itself but with an interme-diate produced during decomposition. Clearly, thisreactive species must be a key intermediate in per-oxynitrite-induced tyrosine nitration, but the precise

mechanism of peroxynitrite decomposition is a mat-ter of ongoing controversy,12 and it is premature tospeculate which of the proposed intermediates couldbe trapped by carboxy-PTIO.

We observed that carboxy-PTIO inhibits DEA/NO-induced nitrosation of GSH. NO does not nitrosate thi-ols under anaerobic conditions,37 but in the presence ofoxygen, a nitrosating intermediate (NOx), is generated,which reacts 10,000-fold faster with GSH than withH2O.34 The intermediate has not been identified so far,but because nitrite is formed as the sole stable end-product of NO autoxidation, it was suggested that thereaction may proceed according to Eqs. (1)–(3).9,38,39

2NO / O ! 2NO (1)2 2

2NO / 2 NO ! 2N O (2)2 2 3

0 /2N O / 2H O ! 4NO /4H (3)2 3 2 2

N2O3 is regarded as a possible key intermediate of NO/O2-mediated nitrosation reactions,40,41 although kineticanalyses have provided evidence against formation ofthis species during NO autoxidation.4

There is general agreement that carboxy-PTIO con-verts NO to NO2.23,42 For Eq. (4), a second order rateconstant of 1 1 104 M01 s01 was calculated.23

CH‹CH‹CH‹CH‹

©

Oı

O2

©COO2 1 NON1

N

CH‹CH‹CH‹CH‹

©

Oı

©COO2 1 NO¤ (4)N

N

NO2 can react either with NO (k Å 1.1 1 109 M01 s01)or dimerize (k Å 4.5 1 108 M01 s01)23 (Eq. 2 and 5respectively).

NO / NO ! N O (5)2 2 2 4

N2O3 reacts rapidly with water to yield NO20, whereas

N2O4 spontaneously dismutates to yield equimolaramounts of NO2

0 and NO30. From the rate constants of

reactions (2) and (5), it is expected that the ratio of NO20/

NO30 produced from NO in the presence of carboxy-

PTIO should increase with increasing concentrations of



Fig. 6. Kinetic simulation of N2O3 formation in the course of NOoxidation with or without 0.1 mM carboxy-PTIO. Simulations wereperformed using a second order Runge-Kutta algorithm from thesoftware package Mathematica (Version 2.2.2., Wolfram Research,Inc., Champaign, IL).

Fig. 5. Effect of carboxy-PTIO on DEA/NO-induced S-nitrosationof GSH. GSH (1 mM) was incubated with 0.1 mM DEA/NO at 377Cfor 1 h in 50 mM phosphate buffer (pH 7.4), containing 0.1 mMneocuproine, in the absence or presence of carboxy-PTIO at theindicated concentrations. Data represent mean values { SE of sixexperiments performed in duplicate.

free NO. Akaike et al. reported that equimolar amountsof nitrite and nitrate were formed and concluded thatthe stoichiometry of the reaction is 1:1.23 However, ki-netic simulations performed by Hogg et al.42 suggestthat this is only true for rates of NO generationõ 10013

M s01, whereas, at higher rates, 2 equivalents of NOare consumed by 1 equivalent of the scavenger. Fur-thermore, Hogg et al. calculated that nitrate productionshould become significant only at very low rates of NOgeneration (õ 10017 M s01). Their kinetic model is sup-ported by our findings showing that decomposition of0.1 mM DEA/NO in the presence of 0.2–1.0 mM car-boxy-PTIO led to formation of nitrite without detect-able production of nitrate. Based on the rate constantof DEA/NO decomposition at pH 7.4 and 377C (5.2 11003 s01),43 the initial rate of NO release from 0.1 mMDEA/NO was Ç0.5 s01 and, hence, several ordersmMof magnitude higher than the threshold calculated byHogg et al. (10017 M s01).

Presence of GSH is expected to render NO oxidationmore complex because the generated NO2 can oxidizeGSH according to Eq. (6) with a rate constant ofÇ108

M01 s01.31

0 /NO / 2GSH ! NO /2GSSG/2H (6)2 2

In spite of this rapid oxidation of GSH by NO2,N2O3 may be formed via reaction (2) in amounts suf-ficient to support S-nitrosation by the NO/O2 system.In the presence of carboxy-PTIO, however, oxidationof NO to NO2 may be fast enough to render Eq. (6)

the predominant fate of NO2 and, hence, prevent for-mation of N2O3. This assumption was confirmed bya kinetic simulation of N2O3 formation using theknown rate constants for NO release from DEA/NO,the reactions of NO with O2 and carboxy-PTIO, thereactions of NO2 with NO and GSH as well as NO2

dimerization. Of note, because reactions downstreamof N2O3 formation were not considered, the simula-tion yields theoretical N2O3 concentrations, only. Asshown in Fig. 6, decomposition of 0.1 mM DEA/NOis predicted to result in a theoretical N2O3 concentra-tion of Ç10 . In the presence of 0.1 mM carboxy-mMPTIO, this value was reduced to less than 0.3 mMN2O3. Accordingly, inhibition of S-nitrosation bycarboxy-PTIO may be explained by a pronounced re-duction of N2O3 formation, but our data do not ruleout that another potent nitrosating species is gener-ated during NO autoxidation.

Acknowledgements — We thank an anonymous referee for hiscomments on oxidation of GSH by NO2 and Margit Rehn for ex-cellent technical assistance. This work was supported by Grants10655, 10859, 11478 (to B.M.), F712 (K.S.), and 11301 (toE.R.W.) of the Fonds zur Forderung der Wissenschaftlichen For-schung in Osterreich.

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ABBREVIATIONS

BSA—bovine serum albuminCarboxy-PTIO — 2-(4-carboxyphenyl)-4,4,5,5-tetra-

methylimidazoline-1-oxyl-3-oxide

cGMP—guanosine-3*,5*-cyclic monophosphateDEA/NO — 2,2-diethyl-1-nitroso-oxyhydrazine so-

dium saltEDRF—endothelium-derived relaxing factorGSNO—S-nitrosoglutathioneHPLC—high-performance liquid chromatographyNO—nitric oxidePAGE—polyacrylamide gel electrophoresisPTIO — 2-phenyl-4,4,5,5-tetramethylimidazoline-1-

oxyl-3-oxideSIN-1—3-(4-morpholinyl)-sydnonimine-

hydrochloride

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Interference of Carboxy-PTIO with Nitric Oxide- and Peroxynitrite-Mediated Reactions