Nitric Oxide 13 (2005) 196–203

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Nitric oxide mediated oxidative stress injury in rat skeletal musclesubjected to ischemia/reperfusion as evaluated by chemiluminescence

Karina Zimiani, Flávia Alessandra Guarnier, Helen Cristrina Miranda,Maria Angelica Ehara Watanabe, Rubens Cecchini ¤

Laboratory of Pathophysiology of Free Radicals, University of Londrina, 86051990 Londrina, Brazil

Received 8 June 2005; revised 8 June 2005

Abstract

The involvement of nitric oxide («NO) in oxidative stress in the rat gastrocnemius muscle subjected to ischemia/reperfusion injurywas investigated using a speciWc and sensitive chemiluminescence (CL) method for measurement of both membrane lipid peroxideand total tissue antioxidant capacity (TRAP). In addition, inhibitors of nitric oxide synthase enzymes were used. The CL time-coursecurve increased dramatically after 1, 2, and 4 h of reperfusion, reaching values about 12 times higher than those of both control andischemic rats. Initial velocity (V0) increased from 13.6 cpm mg protein¡1 min¡1 in the ischemic group, to 7341–8524 cpm mg protein¡1 min¡1 following reperfusion. The administration of L-NAME prior to reperfusion signiWcantly reduced(p < 0.007) the time-course of the CL curve, decreasing the V0 value by 51% and preventing antioxidant consumption for 1 h follow-ing reperfusion. No signiWcant change in CL time-course curve and TRAP values were observed with aminoguanidine treatment. Oncontrary, after 4 h following reperfusion, pre treatment with aminoguanidine led to a signiWcant decrease (p < 0.0001) in the time-course of the CL curve, where V0 decreased by 75% and TRAP returned to control levels. No signiWcant change in CL time-coursecurve and TRAP values were observed with L-NAME treatment. When RT-PCR was carried out with an iNOS-speciWc primer, asingle band was detected in RNA extracted from muscle tissue of only the 4 h ischemia/4 h reperfusion group. No bands were foundin either the control, 4 h ischemia or 4 h ischemia/1 h reperfusion groups. Based on these results, we conclude that «NO plays animportant role in oxidative stress injury, possibly via ¡ONOO, in skeletal muscle subjected to ischemia/reperfusion. Our results alsoshow that cNOS isoenzymes are preferentially involved in «NO generation at the beginning of reperfusion and that iNOS isoenzymeplays an important role in reperfusion injury producing «NO later in the process. 2005 Elsevier Inc. All rights reserved.

Keywords: Chemiluminescence; Ischemia/reperfusion; Nitric oxide; Oxidative stress; Skeletal muscle

The clinical and pathophysiological aspects of ische-mia/reperfusion (IR) following injury to skeletal musclehas been studied for a considerable time [1]. Reactiveoxygen (ROS) and nitrogen species (RNS) that havebeen implicated in IR in tissue injury include thehydroxyl radical («OH), hydrogen peroxide (H2O2),superoxide anion O2

«¡, nitric oxide («NO), and peroxyni-trite (ONOO¡) [2,3]. These reactive species may arise

* Corresponding author. Fax: +43 33714267.E-mail address: cecchini@uel.br (R. Cecchini).

1089-8603/$ - see front matter 2005 Elsevier Inc. All rights reserved.doi:10.1016/j.niox.2005.07.002

very early during IR injury, coming from several sourcessuch as the electron transport chain in mitochondria, thexanthine/xanthine oxidase reaction [4,5] and RNS fromcNOS («NO, ONOO¡) [6,7]. Besides these sources ofO2

«¡, the derangement of calcium-dependent cNOS hasbeen suggested to be the main source of O2

«¡ in muscleischemia/reperfusion [8]. Recent studies in LangendorV-perfused hearts showed that the cytochrome P450monooxygenase family appears to play a major role inischemia/reperfusion injury [9]. NOS-independent «NOproduction from NO2

¡ has also been suggested to occur

K. Zimiani et al. / Nitric Oxide 13 (2005) 196–203 197

in reperfusion injury in skeletal muscle [10]. ROS subse-quently promote the release of proinXammatory media-tors from endothelial cells, leading to the expression ofadhesion molecules, increased adherence of neutrophils,a respiratory burst, and eventually again to ROS andRNS generation [11]. Lipid peroxidation represents oneof the most signiWcant processes preceding cell degenera-tion and necrosis. Oxidative muscle damage in ischemiaand reperfusion has been studied for years [1,12]. In ear-lier work, we studied the oxidative damage of lipids inischemia/reperfusion injury in skeletal muscle by a verysensitive chemiluminescence procedure [13,14].

The involvement of «NO in reperfusion injury in skel-etal muscle has been investigated under various condi-tions. However, these studies do not provide clear-cutevidence of an involvement of RNS in early mechanismsof tissue derangement leading to muscle degenerativelesion and eventually necrosis. Also, the role of RNS inIR injury remains controversial and has been reported tobe either deleterious or beneWcial.

A 1-h period of ischemia followed by 20 min of reper-fusion in human vastus lateralis muscle was shown tocause an increase in «NO under ischemic conditions butnot after reperfusion [15]. However, lipid peroxidationmeasured by thiobarbituric reactive substance levelsincreased only after reperfusion in the latter study. ThisproWle was also seen in rabbit skeletal muscle perfusionshowing a rapid increase in «NO after onset of ischemia,with a subsequent decrease following reperfusion. In thismodel, perfusion with the «NO donor, S-nitroso humanserum albumin, prevented vessel constriction after reper-fusion, decreased interstitial edema, and preserved mito-chondrial function. Oxidative stress measured byglutathione status was shown to be prevented by the«NO donor [6]. «NO blood levels in rat subjected to ische-mia and reperfusion in the rectus femoris muscle,showed a signiWcant increase 5 and 30 min followingreperfusion. Concomitantly, there also occurred anincrease in O2

«¡ generation [7]. Mice over-expressingiNOS display complex cardiac pathophysiologicalchanges and elevated ONOO¡ and O2

«¡ generation, sug-gesting the involvement of «NO via ONOO¡ generationin the mechanism of cardiac damage [16]. In contrast, ina cardiac I/R model with 30 min of ischemia followed by30 min reperfusion, various functional derangementsand increased alkyl and alkoxyl radical generation wereobserved during the reperfusion period. However, treat-ment with L-NAME modiWed neither the physiologicalparameter nor the release of spin adducts, indicatingthat «NO may not be implicated in the oxidative stress[17]. In other studies, treatment with L-NAME 30 minprior to reperfusion for 24 h, preceded by 2 h of ischemia,signiWcantly reduced rat gastrocnemius muscle damageassessed by the nitroblue tetrazolium dye viabilitymethod [18]. However, skeletal muscle necrosis was notprevented when L-NAME was infused 5 min prior to

reperfusion for 24 h, preceded by 4 h of ischemia [19].Mouse gastrocnemius muscle subjected to 3 h of ische-mia followed by 4 h of reperfusion, was found to showsevere interstitial edema and muscle tissue damage. Inaddition, there was either increased immunoreactivityfor the three isoforms of NOS or increased generation ofO2

«¡ that was inhibited by L-NAME [20]. Treatment ofmice and rats with L-NAME, 30 min prior to tourniquetrelease reduced loss of gastrocnemius muscle viabilityevaluated by the nitroblue tetrazolium dye method [18].In heart, L-NAME was shown to reduce the infarct areasigniWcantly [21]. In contrast, it has been suggested that«NO may protect against ischemia/injury reperfusion.The nitric oxide donor, SIN-1 (linsidomine), improvesthe survival of myocutaneous Xaps and decreases neu-trophil accumulation after 6 h of ischemia followed by18 h of reperfusion. These latter results suggest theinvolvement of «NO in protection against ischemia/reperfusion injury [22]. However, this Wnding is contro-versial since it is known that SIN-1 is a very eYcientgenerator of O2

«¡ and «NO, thereby supplying ONOO¡

[23,24]. L-Arginine treatment signiWcantly reduced thenecrosis area in rat rectus femoris muscle in a model of4 h of ischemia followed by a 24 h reperfusion period[19]. Also, L-arginine treatment prevented microvesselconstriction and decreased muscular reperfusion edema[8]. L-Arginine was also shown to protect the myocar-dium against ischemia/reperfusion injury [21].

In view of this conXicting scenario of the «NO role inreperfusion injury and since there is no clear evidencerelating «NO to oxidative stress injury in skeletal muscle,we investigated in the present study the involvement of«NO in the mechanism of tissue oxidative damage. Weused either NOS inhibitors and RT-PCR of iNOS andspeciWc and sensitive chemiluminescence methods, whichallowed us to study both peroxidative damage and totalantioxidant levels.

Methods

In vivo ischemia/reperfusion model

Adult male Wistar rats, weighing 200–250 g, were usedfor the study, and they were obtained from the AnimalHouse of the Biological Sciences Center at the Universid-ade Estadual de Londrina where they were bred. The ani-mals were transferred to the Animal House of theDepartment of Pathological Sciences and kept in polyeth-ylene cages with a stainless steel cover, under controlledtemperature and ventilation and a 12-h photoperiod.During the course of the experiments, the animals hadfree access to water and commercial food. After the ratswere anesthetized in an ether chamber, the left hindlimbwas exsanguinated using an Esmarch bandage [25], and arubber tourniquet was applied to the limb root. After 4 h,

198 K. Zimiani et al. / Nitric Oxide 13 (2005) 196–203

the tourniquet was removed and the limb reperfused for1, 2 or 4 h. Sham control animals were anesthetized with-out induced ischemia and further handled in the samemanner as the experimental ones. Sham and experimentalgroups were anesthetized with sodium pentabarbitone(i.p.), 50 mg/kg. The ischemic/reperfused and contralateralgastrocnemius muscles were excised and kept frozen inliquid nitrogen until experimental analysis. L-NAME(20 mg/kg) or aminoguanidine (50 mg/kg) were injectedintraperitonially 1 h prior to tourniquet removal and thegastrocnemius muscle excised after 1 and 4 h of reperfu-sion. Experimental groups consisted of 5 animals each,and control groups of 8 animals.

Preparation of muscle homogenates

Muscles were placed on ice and homogenized Wvetimes for 30-s periods at 60-s intervals in an Ultraturraxhomogenizer, containing 100 mg of tissue per 10 ml of30 mM KH2PO4/K2HPO4 buVer and 120 mM KCl, atpH 7.4. This homogenate was used for the tert-butylhydroperoxide-stimulated chemiluminescence assay.Protein was determined according to Lowry [26] as mod-iWed by Miller [27]. The supernatant of the homogenatewas obtained by centrifugation at 11,000g for 15 min in arefrigerated centrifuge, and used for the TRAP assay.

Measurement of tert-butyl hydroperoxide-initiated chemiluminescence of muscle homogenates

Reaction mixtures were placed in 20-ml scintillationvials (low potassium glass), containing the following atthe respective Wnal concentrations: total muscle homoge-nate from control, ischemic and reperfused tissue (0.4–1.2 mg/ml protein), 30 mM KH2PO4/K2HPO4 buVer, pH7.4, 120 mM KCl, and 3 mM tert-butyl hydroperoxide, ina total volume of 2 ml. The tert-butyl hydroperoxide-ini-tiated chemiluminescence reaction was measured in aBeckman LS 6000 liquid scintillation counter set to theout-of-coincidence mode with a response range of 300–620 nm for 80 min. The vials were kept in the dark up tothe moment of assay which was carried out in a darkroom at 30 °C [28,29]. Results are expressed in counts perminute per milligram protein (cpm/mg protein). V0 val-ues were obtained by linear regression of the ascendingpart of the chemiluminescence curve.

Measurement of the total antioxidant capacity of muscle

Total antioxidant capacity of muscle homogenatewas measured by chemiluminescence, in a reactionmedium containing 20�M 2,2�-azo-bis(2-amindinopro-pane) and 200�M luminol. The addition of 100–200 �Lof supernatant decreased the chemiluminescence tobasal levels for a period (induction time) proportional tothe antioxidant content of the sample until reaching the

chemiluminescence regeneration level. The system wascalibrated with trolox. Total antioxidant capacity ofmuscle (TRAP) levels were expressed in �mol of troloxequivalents [30].

Isolation of total RNA and RT-PCR

Preparation of RNARNA was extracted from the total homogenate of the

gastrocnemius muscles of animals [31].

RT-PCR and primer conditionscDNA synthesis—RNA (between 20 and 40 ng) from

cells was reverse transcribed and ampliWed using Gene-Amp Kit for RT-PCR (Perkin-Elmer). Reverse tran-scription was carried out with 50 pmol of the oligo(dT)primer using 50 U of cloned murine leukemia virus(MuLV) reverse transcriptase (Perkin-Elmer; GeneAmpRNA PCR kit, part number N808-0017) at 42 °C for60 min in a thermocycler (PCR Sprint ThermoHybaidfrom Biosystems). Reactions were performed in the pres-ence of 50 mM Tris–HCl, pH 8.3, 75 mM KCl, 3 mMMgCl2, and 200 �M dNTP.

�-Actin primers�1r (sense 5� TGT GAT GGT GGG TAT GGG

TCA G 3�) and �2r (antisense 5� TTT AAT GTC ACGCAC GAT TTC C 3�) (GenBank Accession No.NM_031144) served as the actin primers.

DetectionPCR conditions for primers were: 94 °C for 1 min fol-

lowed by 35 cycles of 94 °C for 30 s, 58 °C for 30 s, 72 °Cfor 1 min, and Wnally, 72 °C for 10 min using a thermocy-cler (PCR-Sprint Hybaid). A PCR product of 514 bp for�-actin was detected in agarose gels (1.5%).

iNOS primersiNOS1r (sense 5� ATG GAA CAG TAT AAG GCA

AAC ACC 3�) and iNOS2r (antisense 5� GTT TCTGGT CGA TGT CAT GAG CAA AGG 3�) (GenBankAccession No. NM_012611) served as the iNOS primers.

DetectionPCR conditions for primers were: 94 °C for 5min fol-

lowed by 35 cycles of 94 °C for 1min, 55 °C for 1 min,72 °C for 1 min, and Wnally, 72°C for 10 min using a ther-mocycler (PCR-Sprint Hybaid). The Wnal 10�l of the PCRproduct was mixed with 10�l of loading buVer (total vol-ume, 20�l) and 0.02% xylene cyanol. A PCR product of220 bp for iNOS was detected in agarose gels (1.5%).

Statistical analysis

The results are shown as means § SEM. Data wereevaluated using a non-paired Student’s t test or one-way

K. Zimiani et al. / Nitric Oxide 13 (2005) 196–203 199

analysis of variance (ANOVA) for repeated measureswith the Tukey–Kramer multiple comparison procedure.Correlation analysis was used to determine V0. A diVer-ence of p < 0.05 was considered signiWcant.

Results

Tert-butyl hydroperoxide-initiated chemiluminescence in muscle homogenates

Higher chemiluminescence (CL) yields correspond toa preceding peroxidative attack of tissue by ROS. Whengastrocnemius muscle homogenates obtained from rats4 h after ischemia were added to tert-butyl hydroperox-ide, no change in the CL curve was noted compared tocontrol rats. The CL value of both control and ischemiagroups reached a maximal emission of 10,000 cpm/mgprotein. However, after 1 (IR1), 2 (IR2), and 4 h (IR4) ofreperfusion, the rate at which CL increased was dramati-cally greater, with similar levels of intensity, reaching amaximal rate of emission of approximately 120,000 cpm/mg protein (Fig. 1). The CL shown by the contralaterallimbs of all rats subjected to ischemia and reperfusionwas not signiWcantly diVerent from the CL obtained forcontrol muscles (data not shown). The kinetics analysisperformed by linear regression in the ascending part ofthe curve showed an initial velocity V0 between 7300 and8600 cpm mg protein¡1 min¡1 after 1–4 h of reperfusioncompared to a value of no more than 300 for either con-trol or ischemia only group (Table 1). Administration(i.p.) of L-NAME 1 h before reperfusion (IR1LN) alteredsigniWcantly (p < 0.007) the CL time-course curve evalu-

Fig. 1. Time-course of hydroperoxide-initiated chemiluminescence intotal muscle homogenate of control rats after 4 h ischemia (I) and 1(IR1), 2 (IR2) or 4 h (IR4) after reperfusion. Data from the curveswere compared by the Tukey–Kramer multiple comparison procedure.*p < 0.0001 compared to controls. +p < 0.0001 compared to ischemicrats.

0 10 20 30 40 50 60 70 800

20.000

40.000

60.000

80.000

100.000

120.000

140.000

+

+

+

*

*

*

Che

milu

mun

esce

nce

(cpm

/ mg

prot

ein)

Time (minutes)

Control Isq IR1 IR2 IR4

ated 1 h after reperfusion (Fig. 2) compared to the groupsubjected to 1 h of reperfusion. Also, V0 was reducedfrom 8524 § 149 cpm mg protein¡1 min¡1 in the IR1group to 4337 § 101 in IR1LN (49% reduction) (Table1). Treatment with aminoguanidine (i.p.) 1 h beforereperfusion did not lower signiWcantly the CL rate after1 h of reperfusion (IR1AM) (Fig. 2). V0 decreased to4091 cpm mg protein¡1 min¡1 which was a 52% reductionrelative to the 1-h reperfused group (Table 1). Whenevaluated 4 h after reperfusion (IR4AM), the CL rate foraminoguanidine-treated rats showed a very signiWcantreduction (p < 0.0001) (Fig. 3) and V0 was reduced to1989 § 91.4 cpm mg protein¡1 min¡1, with a 75% reduc-tion in relation to the 4-h reperfused group (Table 1).Treatment with L-NAME (i.p.) 1 h before reperfusiondid not reduce signiWcantly the CL rate after 4 h of

Table 1Initial velocity (V0) of tert-butyl hydroperoxide-initiated chemilumi-nescence in total homogenate of rat muscle subjected to ischemia/reperfusion

V0 values were obtained using the linear increasing part of the chemi-luminescence curve by linear regression. r is the value of correlationcoeYcient.

Groups V0 (cpm mg protein¡1 min¡1) r % reduction

Control 253 § 42.3 0.717 —Ischemia 13.6 § 4.14 0.875 —IR1 8524 § 149 0.996 —IR2 7341 § 1029.9 0.981 —IR4 7781 § 1019.0 0.983 —IR1AM 4091 § 78.0 0.996 52IR1LN 4337 § 101.0 0.995 49IR4AM 1989 § 91.4 0.996 75IR4LN 4524 § 101.6 0.994 41

Fig. 2. EVect of L-NAME and aminoguanidine on the time-course ofhydroperoxide-initiated chemiluminescence in total homogenates ofrat muscle subjected to 4 h ischemia and after 1 h of reperfusion. Datafrom the curves were compared by a non-paired Student’s t test. L-NAME (LN, 20 mg/kg) and aminoguanidine (AM, 50 mg/kg) wereadministered 1 h prior to the beginning of reperfusion. *p < 0.0001 inrelation to controls; +p < 0.0001 in relation to ischemic rats;**p < 0.007 compared to IR1.

0 10 20 30 40 50 60 70 800

20000

40000

60000

80000

100000

120000

**C

hem

ilum

ines

cenc

e (c

pm/ m

g pr

otei

n)

+*

Time (minutes)

Control Isq IR1 IR1Amino IR1LNAME

200 K. Zimiani et al. / Nitric Oxide 13 (2005) 196–203

reperfusion (IR4LN) (Fig. 3), although V0 decreased to4524 cpm mg protein¡1 min¡1 (Table 1).

Total antioxidant capacity of muscle

Fig. 4 shows the total antioxidant capacity of muscle(TRAP) values of both ischemia/reperfusion groups andgroups pre-treated with L-NAME or aminoguanidine.Control values were 35 § 1.8 �M trolox equivalents; amarked decrease in antioxidant capacity was observed atthe end of a 4-h period of ischemia (14 § 0.6 �M,p < 0.001). In addition, a signiWcant reduction was seenafter 1 h reperfusion (11 § 0.7 �M, p < 0.02), which wasreversed by treatment with L-NAME 1 h prior to reper-fusion to 19 § 2.0 �M (73% increase). Treatment withaminoguanidine 1 h before reperfusion, did not increasethe TRAP value observed 1 h after reperfusion. How-ever, 4 h after reperfusion, a dramatic increase in thisvalue to 40 § 5.2�M (344% increase) compared to thatof 4-h control reperfusion (9 § 1.0 �M) was observed.L-NAME treatment did not protect against antioxidantcapacity reduction after 4 h reperfusion.

RT-PCR of iNOS

RNA extracted from muscle tissue of control animalssubjected to 4 h of ischemia, 4 h of ischemia/1 h of reper-fusion, and 4 h of ischemia/4 h of reperfusion was reversetranscribed to cDNA. To determine the integrity of theRNA samples, we utilized �-actin-speciWc oligonucleo-tide primers to amplify a speciWc sequence by PCR. Ineach of the muscle RNA samples, we observed only the

Fig. 3. EVect of L-NAME and aminoguanidine on the time-course ofhydroperoxide-initiated chemiluminescence in total homogenates ofrat muscle subjected to 4 h ischemia and after 4 h of reperfusion. Datafrom the curves were compared by a non-paired Student’s t test. L-NAME (LN, 20 mg/kg) and aminoguanidine (AM, 50 mg/kg) wereadministered 1 h prior to the beginning of reperfusion. *p < 0.0001 inrelation to controls; +p < 0.0001 in relation to ischemic rats; **p < 0.01compared to IR4.

0 10 20 30 40 50 60 70 800

20000

40000

60000

80000

100000

120000

140000+*

**

Che

milu

min

esce

nce

(cpm

/ mg

prot

ein)

Time (minutes)

Control Isq IR4 IR4Amino IR4LNAME

expected band, indicating that these RNAs were ofacceptable integrity for ampliWcation (Fig. 5A).

When iNOS-speciWc primer was used, a single bandwas detected in the RNA prepared from muscle homog-enate only in the 4 h ischemia/4 h reperfusion group(Fig. 5B).

Discussion

One of the most important early events in celldegeneration leading to necrosis is the lipid peroxidationprocess that occurs mainly in the cell membrane. In

Fig. 4. EVect of L-NAME and aminoguanidine on total antioxidantcapacity (TRAP) in homogenate supernatant of rat muscles subjectedto 4 h ischemia and afterward 1 h or 4 h of reperfusion. Groups werecompared by a non-paired Student’s t test. L-NAME (20 mg/kg) andaminoguanidine (50 mg/kg) were administered 1 h prior to reperfusion.*p < 0.001 in relation to control; **p < 0.02 compared to ischemic;+p < 0.007 in relation to IR1; ++p < 0.0004 compared to IR4.

C I IR1 IR1AM IR1LN IR4 IR4AM IR4LN0

5

10

15

20

25

30

35

40

45

50

*

++

*

+*

*****TR

AP

(uM

Tro

lox)

Groups

Fig. 5. Expression of mRNA in homogenate of gastrocnemius musclefrom Wistar rats after ischemia/reperfusion. (A) Integrity of RNAsamples. RT-PCR of �-actin mRNA of muscle tissue from treated ratsand control group demonstrated only a 514-bp product, indicating theviability of RNA. (B) Expression of mRNA for iNOS of muscle tissuefrom Wistar rats. Lane 1: ladder, 100 bp; lane 2, controls; lane 3, 4 hischemia; lane 4, 4 h ischemia/1 h reperfusion; lane 5, 4 h ischemia/4 hreperfusion.

K. Zimiani et al. / Nitric Oxide 13 (2005) 196–203 201

addition, lipid peroxidation represents the most frequentevent resulting from free radical attack of biologicalstructures. The present study examined the possibleinvolvement of «NO in oxidative stress in the gastrocne-mius muscle of rats subjected to ischemia and reperfu-sion injury. Tert-butyl hydroperoxide-initiatedchemiluminescence was used to evaluate the integrity ofnon-enzymatic antioxidant defense mechanisms[28,29,32]. In addition, we carried out a kinetic analysisof the ascending part of the chemiluminescence curveunder the assumption that variation in V0 (initial veloc-ity) values depends on the level of preexisting lipid per-oxide in the tissue [14], and also taking into account thevery fast overall rate constant of the lipoperoxidativepropagation reaction of about 109 M¡1 s¡1 [33]. To avoidside reactions and to reproduce every emission measure-ment as best possible, we optimized the reaction toobtain an emission rate curve as close as possible to thatdescribing simple mono-exponential decay after maxi-mal emission has been obtained [34]. Zamburlini et al.[34] used puriWed lipid hydroperoxide or plasma LDLlipid hydroperoxide and found that the emissionobtained was proportional to the lipid hydroperoxidecontent of the sample. A possible relationship betweenchemiluminescence and tissue damage has been demon-strated [14,29,32,35]. In the present study, using homoge-nates from rat hindlimbs subjected to ischemia for 4 h,no variation in the chemiluminescence curve wasobserved when compared to the control. However, after1 h of reperfusion, the chemiluminescence level increaseddramatically more than 10 times. No further increasewas observed after 2 and 4 h of reperfusion. Kineticsrevealed very high V0 values after reperfusion (rangingbetween 7300 and 8600 cpm mg protein¡1 min¡1),compared to both ischemia and control groups whichshowed V0 values of less than 300 cpm mgprotein¡1 min¡1. These results clearly reveal that reperfu-sion elicits a very aggressive and fast free radical attackof the tissue inducing consumption of antioxidantdefense and promoting increased levels of tissue lipidperoxides and tissue damage. A relationship between CLand lipid peroxidation has been demonstrated [36], aswell as a positive correlation between CL and tissuedamage [35]. In agreement, total muscle antioxidantcapacity (TRAP) was found to be signiWcantly reduced 1and 4 h following reperfusion. Previous studies from ourlaboratory demonstrated progressive muscle damageevaluated by histopathologic inspection, histochemistry,and ultrastructural analysis, which was accompanied byan increase in chemiluminescence up to 72 h of reperfu-sion [14].

Studies carried out over the last years have investi-gated the eVect of «NO on several biochemical, physio-logical and structural parameters in skeletal musclesubjected to ischemia and reperfusion [8,10,18–20]. How-ever, these studies have not provided clear evidence

about RNS involvement in either oxidative stress orlipid peroxidation and the early mechanism of tissuederangement leading to muscle damage and eventuallyto necrosis. Also, the role of RNS in IR injury remainscontroversial; it has been reported to be either deleteri-ous or beneWcial. Few studies have dealt speciWcally withthe involvement of «NO in oxidative stress injury in skel-etal muscle subjected to ischemia/reperfusion. Thesestudies have used isolated procedures such as malonal-dehyde or GSH measurements. For instance, gastrocne-mius muscle subjected to 2 h ischemia followed by 3 hreperfusion in rats did not show any variation with orwithout L-NAME pre-treatment relative to control,regarding MDA or GSH levels [37]. Glutathione ratiosincreased signiWcantly in gastrocnemius muscle after2.5 h of ischemia followed by 2 h reperfusion. Treatmentwith S-NO-HSA, an NO donor, has been shown toreduce the glutathione ratio to control levels [6]. It is wellknown that MDA measurement is prone to artifact andmust not be used as the sole indicator of lipid peroxida-tion [38]. Therefore, we used a more sensitive and speciWcmethod for measuring lipid peroxidative damage. Thedata presented here clearly show that «NO plays animportant role in skeletal muscle damage due to oxida-tive stress. Treatment of rats with L-NAME, a partiallyselective inhibitor of cNOS (cNOS/iNOS IC50 ratio D 11)[39] signiWcantly reduced the rate of chemiluminescenceincrease to 49% of the V0 value of gastrocnemius muscleevaluated 1 h after reperfusion compared to a 1 h reper-fusion in the absence of L-NAME treatment. Over thesame time period, total antioxidant capacity increasedby 73%. Treatment with aminoguanidine, a partiallyselective inhibitor of iNOS (iNOS/cNOS IC50 ratio D 11)[39], on the contrary, did not reduce signiWcantly thechemiluminescence rate and did not increase the tissueantioxidant capacity. It appears therefore, that constitu-tive NOS plays a more signiWcant role in the oxidativestress damage occurring at the beginning of reperfusionthan inducible NOS. Considering both the increasedchemiluminescence emission curve and the dramatic V0increase after 1 h reperfusion and since treatment with L-NAME reduced this value signiWcantly, we suggest that«NO plays a very important role in the peroxidative dam-age to muscle tissue. In addition, taking into account thelow reactivity of «NO, it may be postulated that the morereactive species ONOO¡ is involved in this process.Therefore, «NO at a high concentration must be formedtogether with O2

«¡ generation, and this seems to be thecase. In the rabbit hindlimb model there was a signiWcantincrease in «NO determined in the wall of the femoralartery by porphyrin microsensors during ischemia withsubsequent reduction, accompanied by an additionaldecrease at the beginning of reperfusion [6]. In the rathindlimb subjected to 4 h of ischemia, there was a highyield of «NO in perfused blood after 15 min of reperfu-sion, followed by an exponential decay during 60 min of

202 K. Zimiani et al. / Nitric Oxide 13 (2005) 196–203

reperfusion, occurring simultaneously with high O2«¡

formation [7]. Human vastus lateralis muscle subjectedto 60 min ischemia and 20 min reperfusion, showed highlevels of «NO after ischemia, which decreased to controllevels after reperfusion [15]. Mice gastrocnemius musclesubjected to 3 h of ischemia and 4 h of reperfusion,showed severe edema and increased eNOS and nNOSactivities. In addition, non-enzymatic generation of «NOwas seen in ischemic tissue, which produced levels higherthan that generated by NOS [40]. One hour after reper-fusion, increased generation of O2

«¡ was also observedboth in the cell membrane and cytoplasm, and was alsoinhibited by L-NAME, suggesting the involvement ofcNOS in O2

«¡ formation [20]. High generation of «NO byNOS in rabbit skeletal muscle reperfusion leads toreduced L-arginine levels and high O«¡ yield. Treatmentwith L-arginine prevents excessive release of O«¡ and pre-serves «NO concentration [8]. Similar results have beenobtained with L-arginine infusion in rat skin Xaps [41]and rat skeletal muscle [19]. Several brain studies haveconWrmed that NOS, under conditions of low concentra-tions of L-arginine and with oxygen as the electronacceptor, gives rise to O2

«¡ formation [42,43]. This condi-tion can be created in reperfused tissue after onset ofischemia. Low L-arginine concentration, high «NO for-mation and a shift of the NOS activity from synthesis of«NO to NADPH oxidase activity yielding O2

«¡, leads to amixture of «NO and O2

«¡ species which subsequently canreact with each other at a rate limited by diVusion(6.7 £ 109 M¡1 s¡1), to produce ONOO¡ [17]. There aremany other sources of O«¡, but during I/R 70% of O«¡ isproduced by NOS [8]. ONOO¡ is a powerful oxidizingcytotoxic species which approaches «OH in potency andleads to initiation of lipid peroxidation [24]. Our resultsstrongly suggest that «NO through the formation ofONOO¡ is involved in membrane lipid peroxidativedamage during reperfusion very early following theonset of ischemia.

Reactive species and breakdown products in musclepromote the release of proinXammatory factors fromendothelial cells and other sources [11]. In response toinXammatory cytokines or endotoxins, there is an upreg-ulation of iNOS associated with increased NOS activity[16]. The cremaster muscle of rats subjected to 5 h ofischemia followed by 90 min of reperfusion, leads toincreased generation of «NO from iNOS, and the selec-tive iNOS inhibitor 1400 W reduces the negative eVectsof ischemia and reperfusion on vessel diameter and mus-cle blood Xow [44] . Considering our results discussedabove, we suggest that the inhibition of iNOS withaminoguanidine, a partially selective iNOS inhibitor,reduces oxidative stress after prolonged reperfusion. Ourresults revealed a marked reduction in the chemilumi-nescence rate in gastrocnemius, with a V0 reduction of75% and total antioxidant capacity (TRAP) increase of344% in the group treated with aminoguanidine after 4 h

of reprefusion. The RT-PCR analysis using an iNOS-speciWc primer, revealed a single band detected in theRNA prepared from muscle tissue homogenate only inthe 4 h ischemia/4 h reperfusion group. This suggests aninvolvement of iNOS in the lipoperoxidative damagemechanism following a 4 h reperfusion with no apparentinvolvement up to 1 h of reperfusion, since no band wasdetected at that time.

Based on our results, we conclude that «NO plays aimportant role in oxidative stress injury via lipid perox-ide production and antioxidant consumption in skeletalmuscle subjected to ischemia/reperfusion, probably viaONOO¡ formation. Our results show as well that cNOSisoenzymes are preferentially involved at the beginningof reperfusion and that the iNOS isoenzyme plays animportant role in reperfusion injury in the later stages ofthe process.

Acknowledgments

We are grateful to the Coordenação de Aperfeiçoa-mento de Pessoal de Nível Superior—CAPES for pro-viding grant support. We thank the laboratorytechnician Jesus Antonio Vargas of the Department ofPathological Sciences—University of Londrina, for hishelp during this study. Dr. Albert Leyva assisted withEnglish language editing of this paper.

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