nitric oxide synthase-independent generation of nitric oxide in rat skeletal muscle...

NO

D

*PUU

R

ieoie2ocNmciShddpifim

n

OPE

NITRIC OXIDE: Biology and ChemistryVol. 3, No. 1, pp. 75–84 (1999)Article ID niox.1999.0211, available online at http://www.idealibrary.com on

1CA

itric Oxide Synthase-Independent Generation of Nitricxide in Rat Skeletal Muscle Ischemia-Reperfusion Injury

. A. Lepore,*,1 A. V. Kozlov,† A. G. Stewart,‡ J. V. Hurley,* W. A. Morrison,* and A. Tomasi§

Bernard O’Brien Institute of Microsurgery, St. Vincent’s Hospital, Fitzroy, 3065, Melbourne, Australia; †Institute ofharmacology and Toxicology, University of Veterinary Medicine, Vienna, Austria; ‡Department of Pharmacology,niversity of Melbourne, Melbourne, Australia; and §Dipartimento di Scienze Biochimiche, Patologia Generale,niversita Degli Studi Di Modena, Modena, Italy

eceived October 16, 1998, and in revised form January 26, 1999

It is known that both normal and injured cells ofmpbt

iL

ydiipmhHifLhmNt

iehn

We have used electron paramagnetic resonance tonvestigate the time course of nitric oxide (NO) gen-ration and its susceptibility to inhibitors of nitricxide synthase (NOS) in ischemia–reperfusion (IR)njury to rat skeletal muscle in vivo. Significant lev-ls of muscle nitroso-heme complexes were detected4 h postreperfusion, but not after at 0.05, 3, and 8 hf reperfusion. The levels of muscle nitroso-hemeomplexes were not decreased by the NOS inhibitor-nitro-L-arginine methyl ester as a single dose (30g/kg) prior to reperfusion or as multiple doses

ontinued throughout the reperfusion (total admin-stered, 120 mg/kg) or by the potent NOS inhibitor-methylisothiourea (3 mg/kg). In contrast, nitroso-eme levels were reduced by the glucocorticoidexamethasone (2.5 mg/kg). Muscle necrosis in vitroid not result in the formation of nitroso-heme com-lexes. The finding that reperfusion after ischemia

s necessary for NO formation suggests that an in-lammatory pathway is responsible for NOS-ndependent NO formation in IR injury to skeletal

uscle. © 1999 Academic Press

Key Words: electron paramagnetic resonance;itroso-heme.

1 To whom correspondence should be addressed at Bernard’Brien Institute of Microsurgery, St. Vincent’s Hospital, Victoriaarade, Fitzroy, 3065, Melbourne, Australia. Fax: 061394160926.-mail: [email protected].

089-8603/99 $30.00opyright © 1999 by Academic Pressll rights of reproduction in any form reserved.

any types, including neurons, muscle cells, neutro-hils and macrophages, can generate NO by thereakdown of L-arginine via the nitric oxide syn-hase (NOS)2 pathway (1–3).

Our laboratory has reported that the NOS inhib-tors, nitro-iminoethyl-L-ornithine (L-NIO), N-nitro--arginine methyl ester (L-NAME) and S-meth-lisothiourea (SMT), and also the glucocorticoidexamethasone, improve muscle viability after

schemia–reperfusion (IR) injury to skeletal muscle,ndicating that NOS-dependent NO productionlays a role in this type of injury (4, 5). In cardiacuscle, a similar protective effect of NOS inhibitors

as been reported after short periods of ischemia (6).owever, after prolonged ischemia, Zweier et al.

dentified a second NOS-independent pathway forormation of NO that was only partially inhibited by-NAME, but was more effectively inhibited by oxy-emoglobin, a scavenger of NO irrespective of itsode of production. Radiolabeling indicated thatO was derived by reduction of nitrite, such reduc-

ion being correlated with tissue acidosis (7). NOS-

2 Abbreviations used: NOS, nitric oxide synthase; L-NIO, nitro-minoethyl-L-ornithine; L-NAME, N-nitro-L-arginine methyl-ster; SMT, S-methylisothiourea; IR, ischemia–reperfusion; Hb,emoglobin; Mb, myoglobin; EPR, electron paramagnetic reso-ance; NO3

2/NO22, NOx

2.

75

independent production of NO has also been demon-sh

uanmss

csa

M

D2ca(hrtttlmpttSit

efpficditt

sampled for EPR analysis. Plasma was stored at2

tmswbwcl

mmodpcafrgeaer3

hpl7smfdpwig

ptttc

76 LEPORE ET AL.

trated to occur in neutrophils in vitro (8) and inuman gastric mucosa, oral cavity, and kidney (9).The amount of NO in tissue can be measured by

tilizing the high affinity of NO for hemoglobin (Hb)nd myoglobin (Mb) to form stable paramagneticitroso-heme adducts with a unique electron para-agnetic resonance (EPR) signal (7, 10–19). In this

tudy “nitroso-heme complexes” will be used to de-cribe NO bound to either Hb or Mb.This paper reports a study by EPR of the time

ourse for NO production during IR injury to ratkeletal muscle and the effects of NOS inhibitorsnd dexamethasone on NO generation.

ATERIALS AND METHODS

In vivo ischemia–reperfusion model. Male Sprague–awley rats (specific pathogen free, weighing 200–40 g) were housed in temperature- and humidity-ontrolled rooms with food and water freely avail-ble. Rats were anesthetized using intraperitonealip) sodium pentabarbitone (100 mg/kg) and totalind limb ischemia was applied for 2 h using theubber band tourniquet method (4). The total dura-ion of anesthesia was approximately 3.2 h. Normo-hermic muscle temperature was maintained be-ween 35 and 36°C using a heating blanket andamp (20). Muscle temperature was monitored by

eans of a myocardial temperature probe (22-gaugerobe, Mallincrodt Medical, Inc., St. Louis, MO). Athe end of ischemia the tourniquet was removed andhe limb allowed to reperfuse for 0.05, 3, 8, or 24 h.ham control animals were given anesthetic but no

schemia and were sacrificed at 0, 3, 8, or 24 h afterhe sham procedure.

Circulating blood was sampled, at the end of isch-mia (1 min prior to reperfusion), and within theirst 3 min of reperfusion (0.05 h), or at 3, 8, or 24 hostreperfusion. Different groups of rats were usedor each of the time points except for the end ofschemia and at 0.05 h where blood was sampledonsecutively. Blood was drawn from the vena cava,istal to the kidney directly into a syringe contain-ng heparin. Blood samples were immediately cen-rifuged at 4°C for 5 min at 1500g. The plasma washen aspirated, and the packed red blood cells were

Copyright © 1999 by Academic Press. All right

70°C until analysis for nitrate/nitrite (NOx2).

Ischemic–reperfused muscles (gastrocnemius andibialis anterior) and the matched contralateraluscles were biopsied immediately following the

ampling of blood at each reperfusion time, afterhich the animal was sacrificed. The tibialis wasiopsied, longitudinally, while the gastrocnemiusas biopsied transversely, consistently from the

entral portion. The remainder of the tissue wasyophilized for the analysis of muscle Hb content.

In vitro muscle necrosis model. Gastrocnemiususcles were excised as described above from nor-al untreated rats. Experiments were carried out

n muscles obtained from both hind limbs of fourifferent animals. Muscle was placed in flasks withhosphate buffer (pH 7.4). Three conditions of ne-rosis were investigated: (a) muscle was incubatedt 37°C for 24 h, (b) muscle was maintained hypoxicor the total time of 24 h at 37°C, and (c) muscle wasendered hypoxic for 3 h and then allowed to reoxy-enate for 21 h at 37°C. Hypoxia was carried out byquilibrating the tissue culture flask with nitrogennd placing it in a hypoxia chamber, which was alsoquilibrated with nitrogen. Reoxygenation was car-ied out by transferring the flask to an incubator at7°C equilibrated with 5% CO2.EPR sampling procedure. Red blood cells that

ad been separated from plasma were drawn into arecooled finger dewar mold, frozen by immersion iniquid N2, expelled from the mold, and maintained at7°K until EPR analysis within 7 days. The NOHbtandard solution (13) was sampled in the sameanner. Muscle specimens were tightly packed to

ill the dimensions of the dewar mold and frozen asescribed above. The uniformity of the muscle sam-ling procedure was confirmed, by lyophilizing andeighing the muscle specimens after EPR record-

ngs were completed (tibialis, 45.5 6 2.5 mg, n 5 25,astrocnemius, 41.8 6 1.8 mg, n 5 25).EPR recording of specimens. The specimen was

laced in the liquid N2 finger dewar and EPR spec-ra were recorded at 77°K using a Brucker 200 spec-rometer. The magnetic field was scanned from 3000o 3600 Gauss where the characteristic nitroso-hemeomplex is classically observed (10, 18) using the

s of reproduction in any form reserved.

following settings: receiver gain, 2.00 3 105; modu-l1cwsh

rnft

N(i

oi(megpddwDtLL

(bclH

deum4twt

(0.1 mg/ml) at 36°C for 100 min. Zinc sulfate (20mbatmwddnt

pcit(aotctmnwPr(

R

ogni3rhhaFertjs

77NOS-INDEPENDENT NO GENERATION IN ISCHEMIA–REPERFUSION

ation amplitude, 5.038 G; modulation frequency,00 kHz; signal channel conversion, 163.84 ms; timeonstant, 327.68 ms; sweep time, 167.77 s; micro-ave power, 12.5 mW. The magnetic field was also

canned between 800 and 1800 Gauss where ferri-eme complexes are detectable (10, 11, 16, 21, 22).Quantification of NO signals. EPR spectra were

ecorded as the first derivative of an absorption sig-al. Nitroso-heme complex levels were then quanti-ied by a double integration of the first derivative ofhe spectra (13).

NOHb standard curve. A standard curve ofOHb (mM) was prepared as previously described

13). Quantified EPR measurements were convertednto NO mM (equivalents) using the standard curve.

In vivo administration of compounds. In one setf experiments, a single dose administered by ipnjection, of physiological saline (1 ml/kg), L-NAME30 mg/kg), dexamethasone (2.5 mg/kg), or SMT (3g/kg), was given 30 min prior to the end of isch-

mia. In a second set of experiments animals wereiven an ip injection of L-NAME (30 mg/kg) 30 minrior to the end of ischemia, followed by a 15 mg/kgose every 4 h until 20 h postreperfusion (a totalose of 120 mg/kg). Controls for these experimentsere given saline at the same times (every 4 h).examethasone sulfate was obtained from Labora-

orio Farmacologico (Milanese, s.r.l., Italy).-NAME and SMT were obtained from Sigma (St.ouis, MO).Hb analysis. Lyophilized gastrocnemius muscle

20–30 mg) was homogenized in 1 ml of phosphateuffer (pH 7.4) at 4°C. The homogenate was thenentrifuged at 1400g and the supernatant was ana-yzed for Hb levels using a Sigma Diagnostics Totalb kit (Sigma).Nitrate and nitrite (NOx

2) levels. NOx2 levels were

etermined by a modification of the method of Greent al. (23). Lyophilized muscle specimens, previouslysed for EPR analysis, were weighed and then ho-ogenized in 1 ml of phosphate buffer (pH 7.4) at

°C. The homogenate was centrifuged at 1400g andhe supernatant was divided; 300 ml of supernatantas incubated either with or without nitrate reduc-

ase (0.28 U/ml, Sigma) in the presence of NADPH


g/ml) was then added and the precipitate removedy centrifugation at 11,000g. Finally, the Griess re-ction (23) was carried out on the supernatant andhe absorption measured at 550 nM. For the deter-ination of plasma NOx

2 levels, 300 ml of plasmaas used in the place of supernatant and the proce-ure was followed as described above. NOx

2 levelsetermined from a standard curve were expressed inmol/g of dry muscle tissue or as the mM concentra-ion in plasma.

Statistics. Animals whose average ischemic tem-erature fell outside the range of 35–36°C were ex-luded from the data analysis due to the documentednfluence of temperature on muscle necrosis (20) andhe possible influence of temperature on NO levels10, 24). Statistical analysis was carried out by annalysis of variance (ANOVA) followed by Dunnett’sr Tukey’s multiple comparison method to identifyhe treatments, or times, which differed signifi-antly. Linear regression was used to investigaterends over time after reperfusion. Single- andultiple-dose administration of treatments showed

o significant differences; therefore, these groupsere combined for analysis. A significance level of, 0.05 was used throughout. In all cases a loga-

ithmic transformation, using natural logarithmsLn) of the raw data, was used.

ESULTS

Muscle nitroso-heme signal. No detectable levelsf muscle nitroso-heme complexes were found in theastrocnemius or tibialis muscles of sham controlonischemic rats (n 5 7) or in rats subjected to 2 h of

schemia and reperfused for 0.05 h (n 5 8), 3 h (n 5), or 8 h (n 5 3) (Fig. 1a). In contrast, at 24 heperfusion, an intense signal showing the typicalyperfine splitting characteristic of the nitroso-eme complex was present in all the gastrocnemiusnd tibialis muscles of all the rats examined (n 5 9,ig. 1a). The EPR spectra obtained for the contralat-ral limbs of all rats subjected to ischemia andeperfusion were not different from the spectra ob-ained for control muscles taken from rats not sub-ected to IR injury. At lower magnetic fields thepectral feature assigned to ferri-heme complexes


w0p21t

thdn

SmpmtgNo(0

Faresto

Ff(f

78 LEPORE ET AL.

as undetectable in control tibialis muscles or at.05 and 3 h reperfusion. However, ferri-heme com-lexes were observed at 8 h in (n 5 1 of 5) and at4 h in (n 5 5 of 5) tibialis muscles examined (Fig.b, arrow a). The second spectral feature present inhis region was transferrin (Fig. 1b, arrow b).

Administration of saline, L-NAME (single or mul-iple doses), or SMT had no effect on the nitroso-eme signal observed at 24 h post-IR. However,examethasone reduced the magnitude of theitroso-heme signal (Fig. 2a).

IG. 2. (a) EPR spectra showing the effect of saline, L-NAME,t 24 h post-IR. Saline, L-NAME, SMT, or dexamethasone (Deeperfusion. In a second group of animals, multiple doses of salinevery 4 h throughout the reperfusion. All spectra were recorded atpectra using the double integration method and converted into Nibialis and gastrocnemius muscles were analyzed. The NO (mM ef Ln NO (mM equivalents) for the two muscles is plotted in the

IG. 1. (a) EPR spectra showing the time course of the appearanollowed by 0.05, 3, 8, or 24 h of reperfusion. Nonischemic sham cb) EPR spectra showing ferri-heme complexes detectable in rateature present was transferrin (arrow b). Nonischemic sham con


Quantification of nitroso-heme levels (Fig. 2b).ince the responses of the tibialis and gastrocne-ius muscles to the treatments followed a similar

attern with no evidence of interaction betweenuscle type and treatment (F5,19 5 0.36, P 5 0.867),

he levels of nitroso-heme complexes for tibialis andastrocnemius muscles were averaged for each rat.itroso-heme complex levels following either saline

r L-NAME, as single (n 5 4, 4) or multiple dosesn 5 5, 5), were not significantly different (F1,19 5

.00, P 5 1.00) (Fig. 2b). Hence, the data for the two

or dexamethasone on muscle nitroso-heme complexes detectableadministered as a single dose by ip injection 30 min prior to

AME were commenced 30 min prior to reperfusion and continued(b) Muscle nitroso-heme levels were quantified from the recordedequivalents) by using a standard curve of NOHb (mM). Both the

ents) were then expressed as natural logarithms (Ln). The mean

itroso-heme complexes in rat skeletal muscle after 2 h of ischemiamuscle is represented by con. All spectra were recorded at 77°K.l muscle at 24 h postreperfusion (arrow a). The second spectraluscle (con) is also shown. Spectra were recorded at 77°K.

SMT,x) wasor L-N77°K.O (mMquivalfigure.

ce of nontrolskeletatrol m


sLls4(cnrrl(

s(rdstHtwe

significantly different from those in nonischemicc

NfmfdwaPvt

etihafnti

pevh

N0382222

c2map

FartamL

80 LEPORE ET AL.

aline groups were combined and similarly for-NAME. A significant difference in nitroso-heme

evels was found between the four treatment groups,aline, SMT, L-NAME, and dexamethasone (F3,21 5.67, P 5 0.012). Neither L-NAME (n 5 9) nor SMTn 5 3) decreased the levels of muscle nitroso-hemeomplexes. However, the mean level of muscleitroso-heme complexes in dexamethasone-treatedats (n 5 4) was 66% less than that in saline-treatedats (Dunnett’s method, P 5 0.065) and wasess than that detected for any other treatmentFig. 2b).

Muscle Hb levels (Fig. 3). Hb levels increasedignificantly during reperfusion in untreated groups0.05, 3, 8, or 24 h saline; linear regression analysis,5 0.57, P 5 0.021). Hb levels for single or multipleoses of saline or L-NAME were not different. Aignificant difference was found among the time andreatments groups (F7,29 5 5.10, P 5 0.001). Muscleb levels at 24 h for saline-, L-NAME-, or SMT-

reated rats were significantly higher comparedith nonischemic controls. However, muscle Hb lev-ls in dexamethasone-treated rats at 24 h were not

IG. 3. Muscle Hb levels in nonischemic sham control (con)nimals and after 2 h of ischemia followed by 0.05, 3, 8, or 24 h ofeperfusion. Muscle Hb levels at 24 h of reperfusion for animalsreated with saline, L-NAME, SMT, or dexamethasone (Dex) arelso shown. Hb levels per gram of dry muscle tissue were deter-ined using a standard curve of Hb and are expressed as the then (Hb/g) dry wt.


ontrols or saline-treated rats (Tukey’s method).Muscle and circulating NOx

2 levels. (Table 1).Ox

2 levels in the tibialis did not differ significantlyrom those in the gastrocnemius muscle. Hence, theean of the NOx

2 levels in the two muscles was usedor the analysis. NOx

2 levels for single and multipleoses of saline or L-NAME were not different. Thereere no significant differences in muscle NOx

2 levelsmong the time and treatment groups (F7,35 5 0.91,5 0.507). Samples derived from plasma did not re-

eal any significant changes in NOx2 levels among the

ime and treatment groups (F6,33 5 0.92, P 5 0.495).Circulating nitroso-heme levels. Circulating lev-

ls of nitroso-heme complexes were undetectable inhe venous blood of control nonischemic animals. Inschemic–reperfused animals, a characteristic nitroso-eme signal was detectable in only 7 of 15 animalst 1 min prior to reperfusion and within 3 min reper-usion. The magnitude of these perireperfusion sig-als was not different (F1,6 5 3.30, P 5 0.119) (spec-ra not shown). Nitroso-heme signal was undetectablen circulating blood at 3, 8, or 24 h of reperfusion.

In vitro muscle necrosis. Nitroso-heme com-lexes were not detected in muscles that had beenxcised and allowed to become necrotic for 24 h initro (n 5 4) under conditions of (a) normoxia, (b)ypoxia, or (c) hypoxia followed by reoxygenation.

TABLE I

NOx2 Levels in Muscle and Plasma

IR time andtreatment

Muscle NOx2

Ln (nmol/g)Plasma NOx

2

Ln (mM)

onischemic control 6.12 6 0.16 (n 5 5) 2.05 6 0.59 (n 5 4).05 h 6.74 6 0.05 (n 5 4) 2.88 6 0.41 (n 5 7).0 h 6.52 6 0.55 (n 5 3) Not measured.0 h 6.56 6 0.48 (n 5 3) 1.11 6 1.13 (n 5 3)4 h saline 6.11 6 0.19 (n 5 12) 2.66 6 0.26 (n 5 11)4 h L-NAME 6.03 6 0.07 (n 5 9) 2.66 6 0.46 (n 5 8)4 h SMT 6.75 6 0.39 (n 5 3) 3.01 6 0.04 (n 5 3)4 h Dex 6.40 6 0.53 (n 5 4) 2.18 6 0.24 (n 5 4)

Note. NOx2 levels for muscle and plasma in nonischemic

ontrol animals and after 2 h ischemia followed by 0.05, 3, 8, or4 h reperfusion are shown. The effects of the different treat-ents on NOx

2 levels at 24 h are also shown. Muscle NOx2 levels

re expressed as Ln (nmol/g) of dry muscle tissue or Ln (mM) inlasma.


DISCUSSION

M2ptao(pmi(hrimpTspiitrlsSp(cHtNsbep

tcwbmps

band tourniquet model in rat hind limb ischemia(

dotupmnchaactfctsNbi

iftmFlcShetgdiaspNocbL


NOS-independent NO generation in muscle.uscle nitroso-heme complexes were detectable at

4 h post-IR but not earlier. Consistent with theresence of nitroso-heme complexes and/or NO washe detection also of ferri-heme complexes in musclet 24 h post-IR. Ferri-heme is the oxidation productf the nitroso-heme complex reaction with oxygen10, 16, 21) or of NO reaction with oxy-heme com-lexes (16, 21, 22). In the previous study using thisodel we have demonstrated increased NOS activ-

ty and NOS messenger RNA at 8 h postreperfusion4). In the present study the levels of muscle nitroso-eme complexes detected at 24 h post-IR were noteduced by treatment with L-NAME, a nonselectivenhibitor of NOS isoforms (25), either as a single or

ultiple dose, or by treatment with SMT, a highlyotent inhibitor selective for inducible NOS (25–28).he compounds were given at a time and dosagehown to be effective in reducing necrosis in ourrevious study (4) and which show evidence of NOSnhibition (28, 29). In the current IR model (4), theres extensive necrosis (>80%) and less than 15% ofhe muscle is being perfused with blood at 24 h ofeperfusion (Hickey, Herbert, and Stewart, unpub-ished observations). This no-reflow phenomenon re-ults in a depletion of ATP and NADPH (30, 31).ince enzymatic formation of NO is absolutely de-endent on NADPH and requires molecular oxygen25), it is highly unlikely that regions of no reflowan produce NO by a NOS-dependent pathway.owever, this dose not preclude the possibility that

he remaining regions of perfused muscle produceO from a NOS-dependent pathway. Not with-

tanding the foregoing arguments, it remains possi-le that L-NAME, even when administered repeat-dly, did not completely inhibit this NOS-dependentathway.Circulating nitroso-heme complexes. The finding

hat nitroso-heme complexes were detectable in cir-ulating blood only at 1 min prior to reperfusion andithin 3 min of reperfusion suggests that circulatinglood was not the source of the signal detectable inuscle at 24 h or vice versa. These circulating

erireperfusion signals may be related to circulatoryhock, which has been associated with the rubber


32).

Muscle Hb levels and lack of correlation withetection of nitroso-heme complexes. The methodf NO detection used relies on the binding of NO tohe heme compounds, Hb, or Mb, and the IR modelsed displays considerable hemorrhage at 24 host-IR. Muscle Hb levels were measured to deter-ine whether these influenced the detection ofitroso-heme complexes. Muscle Hb levels in-reased linearly during reperfusion, yet nitroso-eme complexes were not detectable before 24 h inny of the muscles. Elevated Hb levels were founds early as 3 and 8 h, at which times nitroso-hemeomplexes were undetectable. Thus an increase inhe levels of Hb does not appear to be a crucialactor for the detection of NO as nitroso-hemeomplexes. Since muscle Mb is present throughouthe time course of IR and Mb is a single chainubunit of Hb, it will always be available to bindO. Once formed, both NOMb and NOHb haveeen reported to be stable for several hours evenn the presence of oxygen (16).

Possible mechanisms for the formation of NOS-ndependent NO in muscle. NOS-independent NOormation can occur in several tissues by the reduc-ion of NO2

2 to NO (9). There are three possibleechanisms by which this can occur in IR injury.irst, the decrease in tissue pH occurring with pro-

onged ischemia has been correlated with an in-rease in the formation of NO from 15NO2

2 (7, 33).econd, in addition to acid pH, ischemic heart tissueas been found to contain nonenzymatic reducingquivalents that reduce NO2

2 to NO (33). Third, neu-rophil myeloperoxidase can convert NO2

2 into nitro-en species including nitrogen dioxide (8) which mayimerize and breakdown to NO (34). Our NOx

2 stud-es showed that sufficient NOx

2 was present in bloodnd muscle in all treatment groups to act as a sub-trate for NOS-independent NO production. It ap-ears that the injury to muscle does not increaseOx

2 levels which, in any case, greatly exceed levelsf nitroso-heme complexes. NOx

2 measured in mus-le specimens is likely to be derived from perfusinglood and from local production via NOS-dependent-arginine metabolism (35). NOx

2 in the blood has


been shown to be derived from diet (36) and NOS-d

srnhhig

eshaptvI

LilcmmsrshplmaHtntarfN

C

m

tectable at 24 h postreperfusion but not at earliertfcdgrtmii

A

sRo(y

R

82 LEPORE ET AL.

ependent L-arginine metabolism (37).No-reflow leads to anaerobic metabolism and acido-

is (38). The acidosis resulting from the extensive no-eflow in this model would be expected to result inonenzymatic NO production measured as nitroso-eme complexes in the nonperfused muscle. Nitroso-eme complexes have been previously demonstrated

n the necrotic centers of a variety of tumors and allo-rafts of cardiac or skeletal muscle (16, 39, 40).Our failure to detect nitroso-heme complexes in

xcised muscle allowed to become necrotic and pre-umably acidotic in vitro, either with or withoutypoxia and reoxygenation, suggests that necrosislone is not sufficient to generate nitroso-heme com-lexes. Reperfusion may be required for the forma-ion of the complexes. Neutrophils, which can con-ert NO2

2 into nitrogen species (8), are abundant inR skeletal muscle at 24 h post-IR (4).

Our previous study showed that in contrast to-NAME, dexamethasone was effective in protect-

ng from muscle necrosis, even if administered asate as 8 h postreperfusion (4). These results indi-ate that the protective action of dexamethasone onuscle viability after 8 h of reperfusion was via aechanism other than NOS inhibition. Dexametha-

one has a multifaceted action on the inflammatoryesponse in IR injury (4, 41, 42) and in this modelignificantly reduces neutrophil influx (4). If nitroso-eme complex formation is dependent on polymor-honuclear infiltration then the reduction of theevel of nitroso-heme complexes by dexamethasone

ay be due to its ability to reduce neutrophil influxnd, consequently, myeloperoxidase activity (4).owever, dexamethasone reduced neutrophil infil-

ration of IR muscle by only about 15% (4), whereasitroso-heme levels were reduced by 66% followinghe same dose of dexamethasone. Alternatively ordditionally, dexamethasone may reduce the no-eflow phenomenon (43), improving muscle bloodlow which reduces tissue acidosis and, therefore,Ox

2 reduction to NO.

ONCLUSIONS

We have shown that in a rat skeletal muscleodel of IR injury, nitroso-heme complexes are de-


imes. Nitroso-heme complex formation is not af-ected by NOS inhibitors but is reduced by the glu-ocorticoid dexamethasone. Necrosis alone, pro-uced by in vitro incubation of muscle does notenerate nitroso-heme complexes, indicating thateperfusion and the accompanying acute inflamma-ion may be necessary for nitroso-heme complex for-ation. Both the mechanism of production of NOS-

ndependent NO and the role of this process in IRnjury remain to be explored.

CKNOWLEDGMENTS

This work was supported by research funds from the Univer-ity of Modena (Italy) and from the National Health and Medicalesearch Council (Australia). We thank Eros Melleti (Universityf Modena) for helpful technical suggestions and Dr. Ken SharpeUniversity of Melbourne) for consultation on the statistical anal-sis of the data.

EFERENCES

1. Anngard, E. (1994). Nitric oxide mediator murderer and med-icine. Lancet 343, 1199–2003.

2. Bredt, D. S., and Snyder, S. H. (1992). Nitric oxide: A novelneuronal messenger. Neuron 8, 3–11.

3. Stewart, A. G., Phan, L. H., and Grigoriadis, G. (1994). Phys-iological and pathophysiological roles of nitric oxide. Micro-surgery 15, 693–699.

4. Zhang, B., Knight, K. R., Dowsing, B., Guida, E., Phan , L. H.,Hickey, M. J., Morrison, W. A., and Stewart, A. G. (1997).Timing of administration of dexamethasone or the nitric ox-ide synthase inhibitor, nitro-L-arginine methyl ester, is crit-ical for the effective treatment of ischemia–reperfusion injuryto rat skeletal muscle. Clin. Sci. 93, 167–174.

5. Phan, L. H., Hickey, M. J., Niazi, Z. B. M., and Stewart, A. G.(1994). Nitric oxide synthase inhibitor, nitro-iminoethyl-L-ornithine, reduces ischemia–reperfusion injury in rabbit skel-etal muscle. Microsurgery 15, 703–707.

6. Zweier, J. L., Wang, P., and Kuppusamy, P. (1995). Directmeasurement of nitric oxide generation in the ischemic heartusing electron paramagnetic resonance spectroscopy. J. Biol.Chem. 270, 304–307.

7. Zweier, J. L., Wang, P., Samouilov, A., and Kuppusmay, P.(1995). Enzyme-independent formation of nitric oxide in bio-logical tissues. Nat. Med. 1, 804–809.

8. Eiserich, J. P., Hristova, M., Cross, C. E., Jones, D. A., Free-man, B. A., Halliwell, B., and Van Der Vliet, A. (1998). For-mation of nitric oxide-derived inflammatory oxidants by my-eloperoxidase in neutrophils. Nature 391, 393–397.


9. Weitzberg, E., and Lundberg, J. O. N. (1998). Nonenzymatic

1

1

1

1

1

1

1

1

1

1

2

21. Wink, D. A., Grisham, M. B., Mitchell, J. B., and Ford, P. C.

2

2

2

2

2

2

2

2

3

3

3

3

3

3


nitric oxide production in humans. Nitric Oxide Biol. Chem.2, 1–7.

0. Hall, D. M., Buettner, G. R., Matthes, R. D., and Gisolfi, C. V.(1994). Hyperthermia stimulates nitric oxide formation: Elec-tron paramagnetic resonance detection of NO-Heme in blood.J. App. Physiol. 77, 548–553.

1. Kosaka, H., Sawai, Y., Sakaguchi, H., Kumura, E., Harda, N.,Watanabe, M., and Shiga, T. (1994). ESR spectral transitionby arteriovenous cycle in nitric oxide hemoglobin of cytokine-treated rats. Am. J. Physiol. 266, C1400–C1405.

2. Kosaka, H., Watanabe, M., Yoshihara, N., Harada, N., andShiga, T. (1992). Detection of nitric oxide production inlipopolysaccharide-treated rats by ESR using carbon monox-ide hemoglobin. Biochem. Biophys. Res. Commun. 184, 1119–1124.

3. Kozlov, A., Bini, A., Iannone, A., Zini, I., and Tomasi, A.(1996). Electron paramagnetic resonance characterization ofrat neuronal nitric oxide production ex vivo. Methods Enzy-mol. 268A, 299–236.

4. Kumura, E., Yoshimine, T., Iwatsuki, K., Yamanaka, S.,Tanaka, T., Hayakaw, T., Shiga, T., and Kosaka, H. (1996).Generation of nitric oxide and superoxide during reperfusionafter focal cerebral ischemia in rats. Am. J. Physiol. 270,C748–C752.

5. Lancaster, J. R., Langrehr, J. M., Bergonia, H. A., Murase,N., Simmons, R. L., and Hoffman, R. A. (1992). EPR detectionof heme and non heme iron-containing protein nitrosylationby nitric oxide during rejection of rat heart allograft. J. Biol.Chem. 267, 10994–10998.

6. Symons, M. C. R., Rowland , I. J., Deighton, N., Shorrock, K.,and West, K. P. (1994). Electron spin resonance studies ofnitrosyl hemoglobin in human liver, colon and stomach tis-sues. Free Radical Res. Commun. 21, 197–202.

7. Tanaka, S., Kamiike, T., Ito, S., Nozaki, F., Uchikoshi, F.,Miyata, M., Nakata, S., Shirakura, R., Matsuda, H., Kumura,E., Shiga, T., and Kosaka, H. (1995). Evaluation of nitricoxide during acute rejection after heart transplantation inrats. Transplant. P. 27, 576–577.

8. Westenberger, U., Thanner, S., Ruf, H. H., Gersonde, R. K.,Sutter, G., and Trentz, O. (1990). Formation of free radicalsand nitric oxide derivative of hemoglobin in rats during shocksyndrome. Free Radical Res. Commun. 11, 167–168.

9. Rein, H., Ristau, O., and Scheler, W. (1972). On the influenceof allosteric effectors on the electron paramagnetic spectrumof nitric oxide hemoglobin. FEBS Lett. 24, 24–26.

0. Wilson, Y. T., Lepore, D. A., Riccio, M., Hickey, M. J., Pen-ington , A. J., Hayward, P. G., Hurley, J. V., and Morrison,W. A. (1997). Mild hypothermia protects against ischemia–reperfusion injury in rabbit skeletal muscle. Br. J. Plast.Surg. 50, 343–348.


(1996). Direct and indirect effects of nitric oxide in chem-ical reactions relevant to biology. Methods Enzymol. 268A,12–31.

2. Doyle, M. P., and Hoekstra, J. W. (1981). Oxidation of nitro-gen oxides by bound dioxygen in hemoproteins. J. Inorg.Biochem. 14, 351–385.

3. Green, L. C., Wagner, D. A., Glogowski, J., Skipper, P. L.,Wishnok, J. S., and Tannenbaum, S. R. (1982). Analysis ofnitrate, nitrite, and [15N]nitrate in biological fluids. Anal.Biochem. 126, 131–138.

4. Malyshev, I. Y., Manukhina, E. B., Mikoyan, V. D., Kubrina,L. N., and Vanin, A. F. (1995). Nitric oxide is involved inheat-induced HSP 70 accumulation. FEBS Lett. 370, 159–162.

5. Southan, G. J., and Szabo, C. (1996). Selective pharmacolog-ical inhibition of distinct nitric oxide synthase isoforms. Bio-chem. Pharmacol. 51, 383–394.

6. Southan, G. J., Szabo, C., and Thiemermann, C. (1995). Iso-thioureas: Potent inhibitors of nitric oxide synthases withvariable isoform selectivity. Br. J. Pharmacol. 114, 510–516.

7. Nakene, M., Klinghofer, V., Kuk, J. E., Donnelly, J. L.,Budzik, G. P., Pollock , J. S., Basha, F., and Carter, G. W.(1995). Novel potent and selective inhibitors of inducible ni-tric oxide synthase. Mol. Pharmacol. 47, 831–834.

8. Szabo, C., Southan, G. J., and Thiemermann, C. (1994). Ben-eficial effects and improved survival in rodent models ofseptic shock with S-methylisothiourea sulfate, a potent andselective inhibitor of inducible nitric oxide synthase. Proc.Natl. Acad. Sci. USA 91, 12472–12476.

9. Knox, L. A., Stewart, A. G., Hayward, P. G., and Morrison,W. A. (1994). Nitric oxide synthase inhibitors improve skinflap survival in the rat. Microsurgery 15, 708–711.

0. Hickey, M. J., Knight, K. R., Lepore, D. A., Hurley, J. V., andMorrison, W. A. (1996). Influence of postischemic administra-tion of oxyradical antagonists on ischemic injury to rabbitskeletal muscle. Microsurgery 17, 517–523.

1. Kerrigan, C. L., and Stotland, M. A. (1993). Ischemia–reperfusion injury: A review. Microsurgery 14, 165–175.

2. Ward, P. H., Maldonado, M., and Vivaldi, E. (1992). Oxygen-derived free radicals mediate liver damage in rats subjectedto tourniquet shock. Free Radical Res. Commun. 17, 313–325.

3. Samouilov, A., Kuppusamy, P., and Zweier, J. L. (1998).Evaluation of the magnitude and rate of nitric oxide produc-tion from nitrite in biological systems. Arch. Biochem. Bio-phys. 357, 1–7.

4. Beckman, J. S., and Koppenol, W. H. (1996). Nitric oxide,superoxide, and peroxynitrite: The good, the bad, and theugly. Am. J. Physiol. 271, C1424–C1437.

5. Moncada, S., and Higgs, A. (1993). The L-arginine-nitric oxidepathway. N. Engl. J. Med. 329, 2002–2012.


36. Forman, D., Al-Dabbagh, S., and Doll, R. (1985). Nitrates,

3

3

3

40. Dodd, N., and Silcock, J. (1980). Electron spin resonance

4

4

4

84 LEPORE ET AL.

nitrites and gastric cancer in Great Britain. Nature 313,620–625.

7. Rhodes, P. M., Leone, A. M., Francis, P. L., Struthers, A. D.,and Moncada, S. (1995). The L-arginine: Nitric oxide pathwayis the major source of plasma nitrite in fasted humans. Bio-chem. Biophys. Res. Commun. 209, 590–596.

8. Knight, K. R. (1994). Review of postoperative pharmacologi-cal infusions in ischemic skin flaps. Microsurgery 15, 675–684.

9. Dodd, N. J. F., and Silcock, J. M. (1976). Electron spin resonancestudy of changes during the development of solid Yoshida tu-mour. II. Paramagnetic ions. Br. J. Cancer 34, 556–565.


study of changes in implanted muscle: A model for implantedtumours. Clin. Phys. Physiol. Meas. 1, 229–235.

1. Wright, C. E., Rees, D. D., and Moncada, S. (1992). Protectiveand pathological roles of nitric oxide in endotoxin shock.Cardiovasc. Res. 26, 48–57.

2. McCall, T. B., Palmer, R. M. J., and Moncada, S. (1991).Induction of nitric oxide synthase in rat peritoneal neutro-phils and its inhibition by dexamethasone. Eur. J. Immunol.21, 2523–2527.

3. Dolan, R. W., Kerr, D. C., and Arena, S. (1995). Improvedreflow and viability in reperfused rat island groin flaps usingdexamethasone. Microsurgery 16, 86–89.


nitric oxide synthase-independent generation of nitric oxide in rat skeletal muscle...

Documents