quantification of superoxide radicals and peroxynitrite in vascular cells using oxidation of...

NITRIC OXIDE: Biology and ChemistryVol. 1, No. 5, October, pp. 423–431 (1997)Article No. NO970139

Quantification of Superoxide Radicals and Peroxynitrite inVascular Cells Using Oxidation of Sterically HinderedHydroxylamines and Electron Spin Resonance

S. Dikalov,*,1 M. Skatchkov,† B. Fink,† and E. Bassenge†

*Institute of Chemical Kinetics and Combustion, Novosibirsk, Russia; and †Institute of Applied Physiology,University of Freiburg, Germany

Presented at the First International Conference on the Chemistry and Biology of Peroxynitrite, Ascona, Switzerland,May 11–16, 1997

glycerol trinitrate (GTN) as an NO donor. It wasThe reactions of two hydroxylamines, 1-hydroxy- shown that both the acute addition of GTN (0.5 mM)

3-carboxy-pyrrolidine (CP-H) and 1-hydroxy-2,2,6,6- to vascular cells and the incubation of smooth mus-tetramethyl-4-oxo-piperidine (TEMPONE-H), with cle or endothelial cells in culture with 0.1 mM GTNsuperoxide radicals and peroxynitrite were studied. for 24 h enhance significantly the formation of reac-In these reactions corresponding stable nitroxyl tive oxygen species in cells. The rates of of superox-radicals 3-carboxy-proxyl (CP) and 1-hydroxy- ide radical formation were increased at least in two2,2,6,6-tetramethyl-4-oxopiperidinoxyl (TEMPONE) times and peroxynitrite was detected. Hydroxyl-are formed and the amount of them can be quanti- amines TEMPONE-H and CP-H can be used as non-fied by electron spin resonance (ESR). It was found toxic compounds in ESR assay capable of quantify-that CP-H and TEMPONE-H provide almost the ing the formation of superoxide radicals and per-same efficacy in assaying peroxynitrite by ESR in oxynitrite in suspensions of cells and in the wholevitro at pH 7.4. The formation of superoxide radicals blood with high sensitivity. q 1997 Academic Press

in suspensions of cells was discriminated from thatof peroxynitrite using superoxide dismutase or di-methyl sulfoxide as competitive reagents. The sta- The release of nitric oxide in cells in the presencebility of the radicals CP and TEMPONE in the pres- of superoxide radicals causes the formation ofence of ascorbate or thiols was studied in vitro. The peroxynitrite which is a strong oxidant (1–3). Thereduction rate of CP by ascorbate was 66-fold slower extracellular formation of superoxide radicals and ofthan the rate of reduction of TEMPONE. Therefore, peroxynitrite plays an important role in the develop-the quantification of the formation of superoxide ment of diseases such as atherosclerosis, inflamma-radicals and of peroxynitrite is much less affected

tion, or ischemia (2, 3). However, a direct quantifi-by ascorbic acid when CP-H, but not TEMPONE-H,cation of the peroxynitrite formation in biologicalis used. Both TEMPONE-H and CP-H were used tosystems is difficult (4). Recently, it was reporteddetermine the formation rates of superoxide radi-that using hydroxylamines 1-hydroxy-2,2,6,6-tet-cals and peroxynitrite in suspensions of culturedramethyl-4-oxopiperidine (TEMPONE-H)2 and 1-hy-aortic smooth muscle cells and endothelial cells, in

washed ex vivo platelets, and in blood treated with2 Abbreviations used: TEMPONE-H, 1-hydroxy-2,2,6,6-tetra-

methyl-4-oxopiperidine; CP-H, 1-hydroxy-3-carboxypyrrolidine;1 To whom correspondence should be addressed. Fax: 007-3832- TEMPONE, 2,2,6,6-tetramethyl-4-oxopiperidinoxyl; CP, 3-car-

boxy-proxyl; ESR, electron spin resonance; GTN, glycerol trini-352350. E-mail: [email protected].

4231089-8603/97 $25.00Copyright q 1997 by Academic PressAll rights of reproduction in any form reserved.

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424 DIKALOV ET AL.

droxy-3-carboxypyrrolidine (CP-H) (5, 6) one canquantify the formation of superoxide radicals and ofperoxynitrite by detection of stable nitroxyl radicals2,2,6,6-tetramethyl-4-oxopiperidinoxyl (TEMPONE)or 3-carboxy-proxyl (CP).

The aim of our study was to use ESR spectroscopyto determine the rate of formation of superoxide rad-ical and of peroxynitrite in various vascular celltypes which metabolize glycerol trinitrate (GTN) SCHEME 1. Chemical structures of CP-H and TEMPONE-H.with a subsequent release of NO. GTN is a well-known nitrovasodilator which is used for the therapy

cells and following the incubation of cultured smoothof various cardiovascular diseases, including myo-muscle and endothelial cells with GTN was studied.cardial ischemia. However, success in the treatment

of patients with GTN or other organic nitrates isMATERIALS AND METHODSlimited by the development of nitrate tolerance, es-

pecially during nonintermittent nitrate administra- Hydroxylamines TEMPONE-H and CP-H weretion (7). There is increasing evidence that the devel- supplied by Alexis Corp. (U.S.A.). CP, TEMPONE,opment of nitrate tolerance is due to an enhanced dimethyl sulfoxide (DMSO), diethyltetraaminopen-generation of reactive oxygen species during GTN taacetic acid (DTPA), bovine erythrocytes superox-metabolism in vascular cells, particularly of super- ide dismutase SOD (S 2515), and xanthine were ob-oxide radicals in endothelial cells (8). GTN is metab- tained from Sigma (Deisenhofen, Germany). Peroxy-olized in smooth muscle cells (SMC) and at a lower nitrite was from Alexis Corp. Smooth muscle cellsrate in endothelial cells (EC) and in platelets (9, 10) and endothelial cells were washed from aortas ofwith a subsequent release of nitric oxide. It was Wistar rats and prepared as in Ref. (13). Solutionsfound that GTN metabolism enhances the formation of GTN in water containing 0.9% NaCl was fromof superoxide radicals due to the activation of Pohl Boskamp (Hohenlockstedt, Germany). Hydra-NADH-oxidases in EC (8). Thus, superoxide radicals lazine (antihypertensive drug) was from Ciba-Geigyformed by activated NADH-oxidases (that are (Basel, Switzerland). Concentrations of GTN men-known to form superoxide radicals extracellularly) tioned in the text are final concentrations of GTNcan react with NO derived from GTN with a subse- used in experiments.quent formation of peroxynitrite. The formation ofperoxynitrite in GTN-treated ex vivo platelets (11)

Preparation of CP-H and TEMPONE-H Stockand in human blood under GTN therapy was re-Solutionscently reported (12). However, there are no quantita-

tive data on the formation of superoxide radical and CP-H and TEMPONE-H were dissolved in oxygen-peroxynitrite during GTN metabolism in endothelial free (nitrogen-bubbled) 0.1 M sodium phosphatecells or in smooth muscle cells. buffer (PBS), pH 7.4, in the presence of 0.15 M NaCl

In this project TEMPONE-H and CP-H (Scheme and 1 mM DTPA. DTPA was used to decrease the1) were used to determine the formation rates of self-oxidation rate of hydroxylamines catalyzed bysuperoxide radicals and of peroxynitrite in suspen- traces of ions of transition metals. The concentrationsions of cultured smooth muscle and endothelial of CP-H and TEMPONE-H in stock solutions was 10cells, in ex vivo platelets, and in whole blood. The mM. Stock solutions were frozen before experimentsformation of superoxide radicals and of peroxynitrite or kept in a cool place without contact with air.following an acute GTN administration to vascular

ESR Experimentstrate; SMC, smooth muscle cells; EC, endothelial cells; DMSO,

All ESR samples were mesured in 100-ml glassdimethyl sulfoxide; DTPA, diethyltetraaminopentaacetic acid;PBS, sodium phosphate buffer; X/XO, xanthine/xanthine oxidase. capillars (internal diameter 1 mm). In order to in-

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12-03-97 11:32:40 noa AP: NO

425QUANTIFICATION OF SUPEROXIDE RADICALS AND PEROXYNITRITE

hibit reactions catalyzed by transition metals DTPA of nitroxyl radical generation monitoring the ampli-tude of low field component of ESR spectrum. Exper-was added to all samples (final concentration 0.2

mM). The ESR measurements were performed at imental concentrations of CP and TEMPONE invitro were determined from the dependence of ampli-room temperature using an EMX-A ESR spectrome-

ter (Bruker, Karlsruhe, Germany). The ESR settings tude of ESR spectrum on the concentration of CP andTEMPONE obtained from Sigma. All measurementswere the following: field sweep 50 G, microwave fre-

quency 9.72 GHz, microwave power 20 mW, modula- were performed in sodium phosphate buffer (50 mM)in the presence of 0.15 M NaCl and 0.2 mM DTPAtion amplitude 2 G, conversion time 327 ms, detector

time constant 655 ms, receiver gain 105. Calibration at pH 7.4.of the sensitivity of ESR spectrometer was carriedout using calibration probes (Bruker). Processing of Cells

Endothelial and smooth muscle cells were washedSource of Superoxide Radicals and Peroxynitrite from rat aortas and were grown in culture (Gibco

BRL Cat. No. 31095-029, Life Technologies, Eg-Xanthine/xanthine oxidase (X/XO) system gener-genstein, Germany) as in Ref. (13). Washed ex vivoating superoxide radicals contained xanthine oxi-platelets were obtained from the blood of dogs as indase (0.004 units activity), xanthine (100 mM), DTPARef. (15). Endothelial cells (4000 cells/ml), smooth(1 mM) in 0.3 M PBS, pH 7.4. The rate of superoxidemuscle cells (2500 cells/ml), and platelets (100,000/radical formation was checked using cytochrome cml) were incubated with 0.5 mM GTN in buffer forassay.30 min in the presence of 20 mM cysteine. Long-termAs a source of peroxynitrite the stock solution oftreatment (24 h) of endothelial and smooth muscleONOONa (1 mM stabilized in 0.3 M NaOH) wascell cultures by GTN was performed with an initialused. The concentrations of peroxynitrite in 0.3 Mconcentration of GTN of 0.1 mM in culture media.NaOH were determined spectrophotometrically us-Proteins were determined as in Ref. (16).ing extinction coefficient e(302nm) Å 1670 M01 cm01.

Blood SamplesDetermination of Rate Constants by CompetitiveReactions Blood was drawn from the carotid artery of dogs

into a citric acid solution (6:1, v/v) as in Ref. (15).The rate constant for the reaction of CP-H with thesuperoxide radical was determined by competitivekinetics as described in Ref. (14) using SOD as a RESULTS AND DISCUSSIONcompetitive reagent. The rate constant for the reac-

Reactions of Hydroxylamines with Peroxynitritetion of CP-H with peroxynitrite was determined us-ing DMSO as a competitive reagent for peroxynitrite During the reaction of CP-H or TEMPONE-H with(4). Concentrations of DMSO mentioned in the text peroxynitrite the corresponding nitroxyl radicals CPare the final concentrations used. All measurements or TEMPONE are formed (Fig. 1). DMSO was anwere carried out at pH 7.4. effective competitive inhibitor of peroxynitrite-in-

duced oxidation of CP-H and TEMPONE-H (6). Thereactivity of CP-H and TEMPONE with peroxyni-Quantification of Superoxide Radicals and oftrite were compared to the reactivity of peroxynitritePeroxynitrite in Vitrowith DMSO (Fig. 2). The ratios of the rate constants

The rates of formation of superoxide radical and kCP-H/kDMSO and kCP-H/kDMSO were calculated from theof peroxynitrite in vitro were determined from the plot depicting the rate of formation of nitroxyl radi-oxidation of hydroxylamines CP-H (1 mM) and TEM- cals (CP or TEMPONE) on the DMSO concentrationPONE-H (1 mM) to its corresponding nitroxyl radi- (Fig. 2) using the following equation:cals CP and TEMPONE. The rates of formation ofCP and TEMPONE were measured from the kinetics (A0/A)-1 Å kCP-Hr[CP-H]/kDMSOr[DMSO],

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426 DIKALOV ET AL.

FIG. 1. ESR spectra of samples initially containing 2 mM CP-H (a), 2 mM CP-H / 50 mM peroxynitrite (b), 2 mM TEMPONE- FIG. 3. Inhibition of CP-H and TEMPONE-H oxidation in pres-H (c), 2 mM TEMPONE-H / 50 mM peroxynitrite (d). ESR set- ence of SOD as competitive scavenger of superoxide radical. Su-tings are described under Materials and Methods. Hyperfine ESR peroxide radicals were generated in xanthine/xanthine-oxidasesplitting constants of the triplet signals are: aN Å 16.1 G for system as described under Materials and Methods. V0 and V rep-spectrum (b) and aN Å 16.2 G for spectrum (d). resent the rates of formation of nitroxyl radicals CP or TEMPONE

in the absence and presence of SOD, respectively.

where A0 and A represent the amplitudes of the low-field component of the ESR spectra of the nitroxyl In the reaction of 2 mM CP-H with 50 mM of per-radical in the absence and presence of DMSO, re- oxynitrite the concentration of CP determined by ESRspectively. The second order rate constants kCP-H and was 6.2 mM, whereas during the reaction of 2 mMkDMSO are of the reactions of peroxynitrite with CP- TEMPONE-H with 50 mM of peroxynitrite the con-H and DMSO, respectively. centration of TEMPONE was 5.5 mM (Fig. 1). There-

It was found that the ratio of the rate constant fore, the efficacy of the reaction of peroxynitrite withkCP-H/kDMSO is 3.46 and that of kTEMPONE-H/kDMSO is 2 mM CP-H and with 2 mM TEMPONE-H is almost4.62 (Fig.2). identical in phosphate buffer at pH 7.4, 207C.

Reaction of Hydroxylamines with SuperoxideRadicals

In the reactions of CP-H and TEMPONE-H withsuperoxide radicals the corresponding nitroxyl radi-cals CP and TEMPONE are formed. High concentra-tions of SOD (1000 U/ml) completely inhibited theformation of CP and TEMPONE from hydroxyl-amines CP-H or TEMPONE-H in the presence ofsuperoxide radicals formed in the X/XO system (6).SOD was used to determine the rate constant for thereaction of hydroxylamine derivatives with superox-

FIG. 2. Inhibition of CP-H and TEMPONE-H oxidation in pres- ide radicals (14). Using the same method the rateence of DMSO as competitive scavenger of peroxynitrite. Peroxy- constants for the reactions of CP-H and TEMPONE-nitrite (50 mM) was added to the samples containing 2 mM CP-

H with superoxide radicals were calculated from theH or 2 mM TEMPONE-H. A0 and A represent the amplitudes ofdependence of the rate of the formation nitroxyl radi-the low-field components of the ESR spectra of CP or TEMPONE

observed in the absence and presence of DMSO, respectively. cals on SOD concentrations (see Fig. 3):

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TABLE I

Rate of Reduction (mM/min) of Nitroxyl Radicals CP and TEMPONE by Cysteine, Glutathione,Ascorbate, and by Smooth Muscle Cells, pH 7.4, 207C

Smooth muscle cells,Cysteine, 1 mM Glutathione, 1 mM Ascorbate, 1 mM 4000 cells/1 ml

CP, 40 mM 0.012 0.012 0.23 0.06TEMPONE, 40 mM 0.013 0.021 15.7 0.13

Note. The data are means of five experiments.

the reduction rates of CP and TEMPONE in 1 mM(V0/V ) 0 1 Å k*CP-Hr[CP-H]/kSODr[SOD],solution of cysteine were the same while the reduc-

where V0 and V represent the rates of formation of tion rate of CP by 1 mM glutathione and by smoothnitroxyl radicals in the absence and the presence of muscle cells was twofold slower than the reductionSOD, respectively. The second order rate constants rate of TEMPONE (Table I).k*CP-H and kSOD are of the reactions of superoxide radi- The most prominent difference in the reductioncals with CP-H and SOD, respectively. rates of CP and TEMPONE was observed in the

It was found that k*TEMPONE-H Å 1.2 1 104 M01s01presence of 1 mM ascorbate (Table I). The reduction

and k*CP-H Å 3.2 1 103 M01s01 (5, 6). It is known that rates of the radicals CP and TEMPONE were lin-at pH 7.4 the rate constant for the reaction of super- early proportional to both ascorbate and nitroxyloxide radicals with the spin trap DMPO is 35 M01s01

concentrations. The rate constants for the reactions(17). Therefore, both TEMPONE-H and CP-H react of CP and TEMPONE with ascorbate are 0.11 0.01with superoxide radicals much more effectively than and 7.2 0.8 M01s01, respectively (6). Therefore, theDMPO. Thus, we can use lower concentrations of assay based on the oxidation of CP-H is much lessCP-H and TEMPONE-H than DMPO in the ESR affected by the presence of ascorbate than the assayassay for reactive oxygen species. Moreover both CP- based of the oxidation of TEMPONE-H. This is par-H and TEMPONE-H provide a 10-fold higher sensi- ticularly important when the hydroxylamine/ESRtivity in the ESR detection of peroxynitrite and su- assay is used for quantification of ROS in plateletsperoxide radicals than DMPO. This is because the or in leukocytes which contain high intracellularlifetime of CP or TEMPONE radicals is much greater concentrations of ascorbate.than the lifetime of DMPO-OH spin adduct in aer-ated solutions at pH 7.4.

Experiments with Smooth Muscle Cells

Reduction of Nitroxyl Radicals CP or TEMPONE During GTN metabolism in vascular cells superox-by Cysteine, Glutathione, Ascorbate and Smooth ide radicals and peroxynitrite can be formed (11, 12).Muscle Cells TEMPONE-H reacts with all these reactive oxygen

species with the formation of nitroxyl TEMPONE.In biological systems the nitroxyl radicals CP andTEMPONE can be reduced to hydroxylamines CP- TEMPONE-H reacts also with NO0

2 radicals and theTEMPONE radical is formed (5). The formation ofH and TEMPONE-H which are ESR silent. This pro-

cess can lead to an underestimation of the formation superoxide radicals can be discriminated from thatof peroxynitrite using SOD or DMSO as competitiveof superoxide radicals and of peroxynitrite and to a

decrease in the sensitivity of our ESR assay. We reagents. DMSO will inhibit the reaction of TEM-PONE-H with peroxynitrite while SOD will preventcompared the stability of the nitroxyl radicals CP

and TEMPONE in the presence of the reductants the reaction of TEMPONE-H with superoxide radi-cals. The formation of reactive oxygen species wasabundant in biological systems. For this purpose the

rates of the reduction of CP or TEMPONE by cys- measured as TEMPONE generation in culturedsmooth muscle cells (2500 cells per 1 ml) after addi-teine, glutathione, ascorbate, and smooth muscle

cells were determined (Table I). It was found that tion of 0.5 mM GTN; 0.5 mM GTN / 1000 U/ml

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428 DIKALOV ET AL.

Experiments with Endothelial Cells

The formation of reactive oxygen species was mea-sured as the rate of TEMPONE formation in cul-tured endothelial cells (4000 cells per 1 ml) after theaddition of 0.5 mM GTN; 0.5 mM GTN / 1% DMSO;0.5 mM GTN/ 1000 U/ml SOD (Fig. 5). It was foundthat the addition of 0.5 mM GTN increased the for-mation of reactive oxygen species by 254% (from 11.8to 30 nmol/min). The addition of 1000 U/ml SODeffectively reduced TEMPONE formation. The addi-tion of DMSO (1% final concentration) inhibitedTEMPONE formation from 254 to 203%. Therefore,the addition of GTN to endothelial cells increasesthe formation of superoxide radicals which partially

FIG. 4. Determination of reactive oxygen species in smooth reacts with NO yielding peroxynitrite.muscle cells measured as the amount of TEMPONE radicals

It is interesting that the inhibition of the forma-formed in cultured smooth muscle cells (2500 cells per 1 ml) aftertion of the nitroxyl radical TEMPONE in smooththe addition of GTN (0.5 mM), GTN (0.5 mM) / 1000 U/ml SOD,muscle cells is much greater than that in endothelialin cells after 24 h of treatment with GTN (0.1 mM) and resus-

pended in buffer free from GTN. Data are means SE, n Å 6. cells. We suppose that the rate of GTN-induced for-l, SMC from control culture; s, SMC from control culture / 0.5 mation of peroxynitrite in smooth muscle cells is sig-mM GTN; ., SMC from control culture / SOD / 0.5 mM GTN; nificantly higher than in endothelial cells.,, SMC cultured with 0.1 mM GTN for 24 h.

Therefore, the GTN-induced formation of superox-ide radicals and of peroxynitrite in endothelial cells

SOD; following a 24-h incubation with GTN (Fig.4). and in smooth muscle cells in GTN-treated vesselIt was found that an addition of 0.5 mM GTN in- walls can lead to oxidative damage of these cells.creased the formation of reactive oxygen species up Oxidative damage can contribute to the developmentto 184% (from 13.6 to 25 nmol/min). The additionof 1000 U/ml SOD effectively inhibited TEMPONEformation. This means that all trapped reactive oxy-gen species originate from superoxide radicals. Theaddition of DMSO (final concentration 1%) reducedthe formation of TEMPONE from 184 to 131% (datanot shown). Therefore GTN in smooth muscle cellsincreases the formation of superoxide radicals, a sig-nificant part which will react with released NO toform peroxynitrite.

It is interesting that in smooth muscle cells incu-bated for 24 h with 0.1 mM GTN and resuspendedin PBS free from GTN, the formation rate of reactiveoxygen species was significantly greater than afterthe addition of 0.5 mM GTN to control cells. Wesuppose that a long-term treatment with GTN re-sults in a much greater enhanced formation rate

FIG. 5. Reactive oxygen species (ROS) formed in cultured endo-of reactive oxygen species than an acute additionthelial cells quantified as the rate of formation of nitroxyl radicalsof GTN.TEMPONE after the addition of GTN (0.5 mM), GTN (0.5 mM)

Thus, the formation of superoxide radicals and of / DMSO (1%), GTN (0.5 mM) / 1000 U/ml SOD to suspensionsperoxynitrite in GTN-treated smooth muscle cells of cells (4000 cells per 1 ml). Data are means SE, n Å 6. Ω,

control; …, GTN;

, GTN / DMSO; h, GTN / SOD.can lead to pronounced oxidative cell damage.

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It is interesting that an addition of hydralazine asan inhibitor of NADH-oxidases (8) to platelets beforeGTN treatment reduced the formation of CP from365 to 250%. We suppose that this effect of hydral-azine observed in our experiments supports the hy-pothesis of a GTN-induced enhancement of superox-ide radical formation via activation of NADH-oxi-dases (8).

Therefore, the enhanced formation of superoxideradicals and of peroxynitrite in GTN-treated plate-lets can lead to oxidative cell damage and can thuscontribute to an increase in platelet activity ob-served in washed ex vivo platelets obtained from theblood of GTN-tolerant animals (15).

FIG. 6. Reactive oxygen species (ROS) formed in ex vivo plate-lets measured as the rate of formation of nitroxyl radicals CP in

Experiments with Bloodsuspensions of platelets (100,000 cells/ml) after the addition ofGTN (0.5 mM), GTN (0.5 mM) / 500 U/ml SOD, hydralazine (1

It is known that blood plasma is rich in ascorbatemM) / GTN (0.5 mM). Data are means SE, n Å 5. Ω, control;(0.01–0.1 mM). Therefore, for the experiments with

…, GTN,

, GTN / SOD; h, hydralazine / GTN.

blood we used CP-H which is more resistant to ascor-bate than TEMPONE-H (6). The results concerning

of nitrate tolerance via an inactivation of enzymes the formation of reactive oxygen species were mea-involved in the enzymatic release of NO from GTN sured as the rate of CP formation in blood followingand an NO-induced accumulation of cGMP that pro- the addition of GTN (final concentration 0.5 mM),vides NO-induced vasodilation. GTN (0.5 mM) / SOD (500 U/ml), hydralazine (1

mM) / GTN (0.5 mM) and are depicted in Fig. 7. It

Experiments with Platelets

It is known that intracellular concentrations ofascorbate in platelets are about 0.5 mM, which ismuch greater than those in smooth muscle and endo-thelial cells (18). Therefore, for the experiments withplatelets we used CP-H which is more resistant toascorbate than TEMPONE-H (6). The rate of forma-tion of reactive oxygen species was measured as therate of CP formation in ex vivo platelets (100,000cells per 1 ml) following the addition of 0.5 mM GTNor 0.5 mM GTN / 500 U/ml SOD or 1 mM hydral-azine / 0.5 mM GTN (Fig.6). It was found that theaddition of 0.5 mM GTN increased the formationrate of reactive oxygen species up to 365% (from 4to 14.6 nmol/min). The addition of 500 U/ml SODdecreased the CP formation. The addition of DMSO

FIG. 7. Reactive oxygen species (ROS) formed in whole blood(final concentration 1%) reduced CP formation fromcontaining citrate measured as the rate of formation of nitroxyl365 to 278% (data not shown). Therefore, GTN me-radical CP after the addition of GTN (0.5 mM), GTN (0.5 mM)/

tabolism in smooth muscle cells increases the forma- 500 U/ml SOD, hydralazine (1 mM) / GTN (0.5 mM). Data aretion of superoxide radicals which partially react with means SE, n Å 4. Ω, control; …, GTN;

, GTN / SOD; h,

hydralazine / GTN.released NO yielding peroxynitrite.

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430 DIKALOV ET AL.

was found that the addition of 0.5 mM GTN aug- the rate of release of NO derived from nitrate dueto enzymatic reactions in vascular cells. We supposemented the formation of reactive oxygen species up

to 182% (from 17 to 31 nmol/min). The addition of that GTN induces superoxide radical formationmainly extracellularly because the addition of SOD500 U/ml SOD reduced the CP formation from 182

to 164%. The addition of DMSO (final concentration significantly reduced the superoxide-mediated for-mation of the nitroxyl radicals CP or TEMPONE.1%) decreased the formation of CP from 182 to 173%

(data not shown). Therefore, GTN metabolism in NADH-oxidases contribute to the enhanced forma-tion of superoxide radicals in vascular cells duringplatelets and erythrocytes increases the formation

of different reactive oxygen species including super- treatment by GTN. During GTN metabolism in vas-cular cells a significant part of the superoxide radi-oxide radicals and peroxynitrite.

It is interesting that in our experiments the addi- cals react with NO and peroxynitrite is formed.The hydroxylamines TEMPONE-H and CP-H cantion of hydralazine (1 mM) to the blood before GTN

treatment reduced the formation of CP during GTN be used for the quantitative determination of super-oxide radicals and of peroxynitrite in biologicaltreatment from 182 to 132%. This effect of hydral-

azine can be attributed to the inactivation of NADH- systems.oxidases of blood cells in the same way as in washedex vivo platelets and in isolated EC.

ACKNOWLEDGMENTSTherefore, the GTN-induced formation of superox-ide radicals and of peroxynitrite in blood under GTN The authors are grateful to the German Heart Foundationtherapy in vivo can lead to oxidative damage of pro- (Frankfurt/Main, Germany) and to the Russian Foundation of

Fundamental Researches (Grant 95-04-12506) for financial sup-teins, to oxidation of thiols, and to increased lipidport.peroxidation.

REFERENCESCONCLUSION

1. Darley-Usmar, V. M., Wiseman, H., and Halliwell, B. (1995).It was found that in vitro CP-H and TEMPONE-Nitric oxide and oxygen radicals: A question of balance. FEBSH react directly with peroxynitrite and the productsLett. 369, 131–135.are nitroxyl radicals. Both TEMPONE-H and CP-H

2. Villa, L. M., Salas, E., Darley-Usmar, V. M., Radomski,can be used for the quantitative determination ofM. W., and Moncada, S. (1994). Peroxynitrite induces both

superoxide radicals and peroxynitrite in different vasodilation and impaired vascular relaxation in the isolatedchemical and biological systems. The rate of reduc- perfused rat heart. Proc. Natl. Acad. Sci. USA 91, 12383–tion of CP by ascorbate was 66-fold slower than the 12387.

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pared to TEMPONE-H (LD50 in rats 2800 and 800 R. M. (1995). A practical method for preparing peroxynitritemg/kg respectively for CP and TEMPONE (19)). solutions of low ionic strength and free of hydrogen peroxide.

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