Melatonin inhibits the expression of the inducible isoform of nitricoxide synthase and nuclear factor kappa B activation in rat skeletalmuscle

Introduction

Unaccustomed exercise has been demonstrated to increasethe generation of reactive oxygen and nitrogen species in

biological tissues that increase metabolic rate and oxygenconsumption, such as skeletal muscle and myocardium[1, 2]. These species include nitric oxide (NO), which

mediates physiological vasodilatation, protects againstcytokine-induced damage and is a major immunosuppres-sive factor for T-cell immunity [3]. However, the activationof the inducible isoform of nitric oxide synthase (iNOS)

may produce NO in amounts high enough to stimulateinflammatory processes [4]. During inflammation, NO andits metabolites, such as peroxynitrite, are potentially

cytotoxic and capable of injuring the invading pathogensand eliminating altered cells [5]. Indiscriminated destructionof cells and tissues by NO and its reactive nitrogen

intermediates may play a significant role in the pathologyof many inflammatory conditions [4]. Thus, inhibition ofthe iNOS pathway is a rationale approach for attenuation

of inflammation and suppression of NO production may bean effective therapeutic strategy for preventing inflamma-tory reactions and diseases [6].The process of iNOS expression involves different signal

transduction pathways, including nuclear translocation ofthe transcription factor nuclear factor kappa B (NF-jB) [7].NF-jB belongs to the Rel family of transcriptional activa-

tor proteins and is present in the cytoplasm in an inactivestate, bound with the inhibitory IjB subunit proteins.NF-jB is activated by a variety of external stimulants, such

as H2O2, proinflammatory cytokines or phorbol esters, viaphosphorylation of IjB by IjB kinases (IKK). Phosphory-lation of IjB sets the stage for the dissociation and nucleartranslocation of NF-jB, which binds to the corresponding

DNA sequence of the target genes, including iNOS [8].Different authors have shown that acute exercise transientlystimulates NF-jB signalling in skeletal muscle [1, 9, 10],

and that this up-regulates the expression of iNOS [11, 12].Melatonin (N-acetyl-5-methoxytryptamine), a lipophylic

indole amine derived from tryptophan, was thought for

years to be produced exclusively in the pineal gland as ahormone, but more recently it was detected in many othertissues [13]. Melatonin, the major product of the pinealgland, plays a fundamental role in the neuroimmuno-

endocrine system [14, 15], but also functions as a potentantioxidant that scavenges hydroxyl free radicals and manyrelated reactants [16, 17]. The potency of melatonin in

breaking the lipid peroxidation chain reaction is 10 timeshigher than that of vitamin C and a-tocopherol [18]. Thefree radicals known to be scavenged by melatonin include

in addition to the highly toxic hydroxyl radicals, alsoperoxynitrite anion and hypochlorous acid that contributeto the inflammatory response and associated tissue destruc-

tion [19]. Melatonin is not toxic even in high doses, and an

Abstract: This study investigated whether the induction of inducible nitric

oxide synthase (iNOS) produced by acute exercise in rat skeletal muscle

could be prevented by melatonin and whether iNOS down-regulation was

related to inhibition of nuclear factor kappaB (NF-jB) activation. Male

Wistar rats received melatonin i.p. at a dose of 1.0 mg/kg body weight

30 min before being exercised for 60 min on a treadmill at a speed of 25 m/

min and a 10% slope. Exercise caused a significant induction of iNOS protein

levels and a marked activation of NF-jB that were significantly prevented in

rats treated with melatonin. Exercise also resulted in increased IjB kinasea(IKKa) and phosphorylated IjBa protein levels, whereas IjBa content

decreased. These effects were blocked by melatonin administration. The

increase in the muscle concentration of thiobarbituric acid reactive

substances and in the oxidized/reduced glutathione ratio induced by exercise

was partially prevented by melatonin. Our data indicate that melatonin has

potent protective effects against damage caused by acute exercise in rat

muscle, preventing oxidative stress, NF-jB activation and iNOS over-

expression. These findings support the view that melatonin treatment, by

abolishing the IKK/NF-jB signal transduction pathway, might block the

production of noxious mediators involved in the inflammatory process.

Marıa Alonso, Pilar S. Colladoand Javier Gonzalez-Gallego

Department of Physiology, University of Leon,

Leon, Spain

Key words: exercise, melatonin, nitric oxide,

nuclear factor kappaB, oxidative stress

Address reprint requests to Javier Gonzalez-

Gallego, Department of Physiology, University

of Leon, 24071 Leon, Spain.

E-mail: dfijgg@unileon.es

Received December 9, 2005;

accepted January 26, 2006.

J. Pineal Res. 2006; 41:8–14Doi:10.1111/j.1600-079X.2006.00323.x

� 2006 Blackwell Munksgaard

Journal of Pineal Research

8

LD50 has not been established for any animal [20]. Itsprotective role has been reported in various experimentalmodels of tissue damage by reducing oxidative stress and

lipid peroxidation [21–23], and a number of studies havedemonstrated that by inhibiting NO production or influ-encing the activation of NF-jB, melatonin exerts importantanti-inflammatory actions [24]. However, its antioxidant

and anti-inflammatory properties in an exercise contexthave not been investigated.

A single bout of exercise has been reported to activate

NF-jB binding in rat muscles [9, 10]. Recent data suggestthat the NF-jB signalling pathway can be activated in aredox-sensitive manner during muscular contraction, pre-

sumably because of increased oxidant production [1]. Thepresent study was designed to investigate whether theinduction of iNOS in muscle produced by acute exercisecould be prevented by melatonin and whether iNOS down-

regulation was related with inhibition of NF-jB activation.

Materials and methods

Animals and experimental procedures

All experiments were performed in accordance with theGuiding Principles for Research Involving Animals (NAS)[25]. Male Wistar rats (200 ± 10 g) were housed in a

controlled environment with a 12:12 hr light–dark cycleand were provided with rat chow (Panlab, Barcelona,Spain) and water ad libitum. The lighting schedule wasregulated so that lights were on from 08.00 to 20.00 hr and

the room temperature was maintained at 22�C.Animals were randomly divided into four groups: a

control group (C), a melatonin-treated control group (CM),

an acute exercise group (E), and a melatonin-treatedexercise group (EM). All animals were accustomed torunning on a motor-driven treadmill designed for rats

(model LI8706, Letica, Barcelona, Spain), beginning at8 m/min for 10 min the week previous to the exerciseperformance [26]. On the day of the experiment, E and EMrats carried out a single bout of acute exercise on the

treadmill consisting in 60 min at 25 m/min and a 10% slopestarting at 9:00 hr in the morning. Thirty minutes beforestarting the exercise, melatonin was injected intraperiton-

eally at a dose of 1 mg/kg body weight dissolved in 0.5 mLof 0.9% saline containing 0.5% of ethanol (group EM) [27].Rats in the group E were treated with an equal volume of

0.9% saline containing 0.5% of ethanol.After 2 hr of recovery, rats were anaesthetized with

sodium pentobarbital (50 mg/kg i.p.). Blood was rapidly

drawn from the carotid artery and plasma prepared forlater analysis and stored at )80�C. The skeletal muscle deepvastus lateralis (DVL) was rapidly removed from both legsand frozen in liquid nitrogen. The entire surgical procedure

took <10 min.Hundred milligrams of frozen muscle tissue from each rat

was homogenized using a glass-teflon homogenizer in 1 mL

ice-cold homogenization buffer (Tris 10 mm, sucrose0.25 m, EDTA 5 mm, NaCl 50 mm, sodium phosphate30 mm, NaF 50 mm, sodium orthovanadate 100 lm, DTT

1 mm, PMSF 1 mm and protease inhibition cocktail, pH7.4) and centrifuged at 9000 g for 15 min. The resultant

supernatant (cytosolic fraction) was harvested, the proteincontent determined (Lowry) and kept at )80�C for furtherdeterminations. All the procedures described bellow were

performed in this cytosolic fraction.

Biochemical markers of oxidative stress

Oxidised and reduced glutathione analysis was performedby the method of Hissin and Hilf (1976) [28], based inthe glutathione-specific reaction to optaldehyde, which

leads to the generation of a fluorescent compound thatbecomes excited at 350 nm and has a peak of fluorescentemission at 420 nm. The variations in GSSG/GSH ratio

represent a good indicative of the oxidative status, sothat increases in this parameter suggest an oxidative stresssituation.The amount of aldehydic products generated by lipid

peroxidation was quantified by measuring the concentra-tion of thiobarbituric acid reactive substances (TBARS)[29]. For this purpose, a final amount of 3 mg per sample

was assayed. The samples were incubated at 90�C for30 min after adding 500 lL of 0.37% thiobarbituric acid in15% trichloroacetic acid, then centrifuged at 4�C at 2000 g

for 15 min. Spectrophotometric absorbance was deter-mined in the supernatant at 535 nm.

Western blot

Samples of cytosolic fraction containing 20–75 lg ofprotein were separated by sodium dodecyl sulphate–poly-

acrylamide gel electrophoresis (SDS–PAGE) (9–14% acryl-amide) and transferred to nitrocellulose. Nonspecificbinding was blocked by preincubation of the nitrocellulose

in phosphate-buffered saline containing 5% bovine serumalbumin for 1 hr. The nitrocellulose was then incubatedovernight at 4�C with polyclonal and monoclonal-specific

antibodies against rat iNOS (130 kDa), IjBa (36 kDa)(Santa Cruz Biotechnology, Santa Cruz, CA, USA),phospho-IjBa (40 kDa) and IKKa (85 kDa) (Cell Signa-ling, Danvers, MA, USA) obtained in goat (polyclonal) and

mouse (monoclonal). Bound primary antibody was detec-ted with HRP-conjugated antigoat or antimouse antibody(DAKO, Glostrup, Denmark) and blots were developed

using an enhanced chemiluminescence detection system(ECL kit; Amersham Pharmacia, Buckingham, UK). Thedensity of the specific bands was quantified with an imaging

densitometer (Scion Image, Maryland, MA, USA).

Electrophoretic mobility shift assays (EMSA)

Nuclear extracts were prepared from skeletal musclevastolateral as described previously [30] in order tostudy the activation of the transcription factor NF-jB.A commercial double-stranded oligonucleotide (5¢-AGTT-GAGGGGACTTTCCCAGGC-3¢) purchased fromPromega (Madison, WI, USA) was briefly end-labelled by

T4 polynucleotide kinase [30]. The DNA-binding reactionwas performed by mixing 10 lg of nuclear extract inincubation buffer (10 mm Tris–HCl pH 7.5, 40 mm NaCl,

1 mm EDTA and 4% glycerol) and 1 lg poly (dI-dC). After15 min on ice, the labelled oligonucleotide (30 000 c.p.m.)

Melatonin and exercise

9

was added and the mixture incubated 20 min at roomtemperature. For the competition assay, the nuclear extract(10 lg) was preincubated with homologous unlabelled NF-

jB oligonucleotide for 5 min on ice, followed by theaddition of c-32P end-labelled NF-jB probe. For competi-tion studies, 3.5 pmol of unlabelled NF-jB oligonucleotide(competitor) or 3.5 pmol of labelled NF-jB oligonucleotide

mutate (noncompetitor) were mixed 15 min before theincubation with the labelled oligonucleotide. All sampleswere electrophoresed through a 6% polyacrimide gel for

90 min at 150 V. The gel was then dried and autoradio-graphed at )70�C with intensifying screens on hyperfilm(Kodak, Madrid, Spain) for a whole week. The density of

the specific bands was quantified with an imaging densi-tometer (Scion Image).

Statistical analysis

All data are reported as mean ± S.E. of the mean(S.E.M.). For statistical comparisons, a one-way ANOVA

was utilized with a Newman–Keuls test to show significancebetween groups. P-values <0.05 were considered to besignificant. An spss v. 13.0 statistical software (Chicago, IL,

USA) was used.

Results

The muscle concentration of TBARS significantly increasedin exercised rats (+47%), while values did not significantlydiffer from the controls in exercised rats receiving melato-

nin. Exercise also induced a significant increase in bothGSSG concentration (+114%) and in the GSSG/GSHratio (+185%) that were partially prevented by melatonin

(Table 1).Fig. 1 illustrates the effects of exercise and melatonin on

iNOS expression measured by Western blot. Exercise

coursed with a significant induction of iNOS protein levelsin the DVL muscle (+126%). Melatonin administrationabrogated this effect (Fig. 1).To study the effects on NF-jB-binding activity, muscle

nuclear extracts were studied by EMSA. As shown inFig. 2, exercise induced a marked activation of NF-jB(+186%) that was significantly prevented in rats treated

with melatonin.As it has been well documented that activation of NF-jB

correlates with rapid proteolytic degradation of IjBa [1],

we assessed protein levels of the nonphoshorylated andphosphorylated forms of IjBa. Exercise resulted in asignificant decrease of the nonphosphorylated form

()53%), while phosphorylated IjBa protein level was

markedly increased (+119%). These effects were blockedby melatonin administration (Fig. 3).

As IjBa phosphorylation is controlled by IKK in mostcells, we measured the content of IKK in rat DVL muscle.Protein level of IKKa was up-regulated in exercised rats(+79%), and this effect was absent in animals receiving

melatonin (Fig. 4).

Discussion

During inflammation, cells respond to cytokines by expres-sing iNOS [31]. In this situation, NO may react with

reactive oxygen species (ROS) such as the superoxideradical to yield the highly reactive oxidant species peroxy-nitrite, leading to more aggressive oxidative and nitrosative

stress [4, 32]. Our experiments confirm the exercise-medi-ated induction of iNOS expression that has been previously

Table 1. Effect of exercise and melatoninon thiobarbituric acid reactive substances(TBARS), reduced glutathione (GSH),oxidized glutathione (GSSG) and GSSG/GSH ratio in rat deep vastus lateralismuscle

C CM E EM

TBARS (nmol/mg prot) 4.3 ± 0.3 4.3 ± 0.4 6.3 ± 0.4* 3.2 ± 0.3*#GSH (lmol/g muscle) 3.2 ± 0.2 3.4 ± 0.3 2.9 ± 0.1 2.7 ± 0.1GSSG (nmol/g muscle) 298.3 ± 28.2 309.9 ± 7.1 663.4 ± 25.8* 577.6 ± 26.1*#GSSG/GSH 9.2 ± 1.3 9.1 ± 1.1 26.4 ± 1.9* 23.7 ± 1.6*#

Data are shown as the mean ± S.E.M. for eight animals. C, untreated control group; CM,melatonin-treated control group; E, exercised group; EM, melatonin-treated exercisedgroup. *P < 0.05 significantly different from C; #P < 0.05 significantly different from E.

Fig. 1. Effect of exercise and melatonin on Western blot analysis ofinducible nitric oxide synthase (iNOS) protein in rat deep vastuslateralis (DVL) muscle. Total cellular protein was separated on12% sodium dodecyl sulphate–polyacrylamide gel electrophoresis(SDS–PAGE) and blotted with anti-iNOS antibodies. (A) A rep-resentative Western blot photograph. (B) Mean values ± S.E.M.,n ¼ 8. See Table 1 legend for group definitions. *Different from C,P < 0.05; #different from E, P < 0.05.

Alonso et al.

10

described in muscle cells by different authors [12], and

demonstrate that melatonin inhibits the induction of iNOSprotein. Results differ from the recent report that micro-vascular protection from reperfusion injury in skeletalmuscle is attributable to its free radical scavenging activity,

but not to its anti-inflammatory effect, because in thisexperimental situation melatonin is not able to inhibit theenhanced iNOS expression [33]. However, in murine

macrophages treated with lipopolysaccharide (LPS) [7],liver and lung of rat with multiple organ dysfunctionsyndrome [34] or liver of rats treated with thioacetamide

[35], melatonin has been shown to inhibit iNOS expression.

Treatment with melatonin has also been shown to reducenitrotirosine formation and the development of inflamma-tion associated with spinal cord trauma [36].One pathway by which NO can contribute to organ injury

is activation of inflammatory cascades through the NF-jB,resulting in inflammation manifested by cytokine expression[6]. It has been previously reported that activation of NF-jBis essential for the activation of iNOS expression in ratgastrocnemius muscle [12] and skeletal muscle myocites [11].In addition, a linear correlation has been observed between

NF-jB activation and iNOS expression in skeletal muscle ofpatients with chronic heart failure [37], and administrationof the NF-jB inhibitor pyrrolidine dithiocarbamate down-

regulates iNOS transcription and affords protection against

Fig. 2. Effect of exercise and melatonin on nuclear factor jBactivation. Specific binding was verified by addition of unlabelled(cold) oligonucleotide (competitor, C)) or labelled oligonucleotidemutate (noncompetitor, C+). (A) A representative Electrophoreticmobility shift assays. (B) n ¼ 8. See Table 1 legend for groupdefinitions. *Different from C, P < 0.05; #different from E,P < 0.05.

Fig. 3. Effect of exercise and melatonin on Western blot analysis ofIjBa and phosphorylated IjBa (p-IjBa) in rat DVL muscle. Totalcellular protein was separated on 12% SDS–PAGE and blottedwith antibodies against IjBa or phosphor-IjBa antibodies. (A) Arepresentative Western blot photograph. (B) n ¼ 8. See Table 1legend for group definitions. *Different from C, P < 0.05; #dif-ferent from E, P < 0.05.

Melatonin and exercise

11

ischaemia/reperfusion injury in rat cremaster muscle [38].Therefore, we tested whether the inhibitory effect by

melatonin of iNOS expression may be mediated, at least inpart, by inhibition of NF-jB activation. NF-jB may be animportant signal orchestrating the inflammatory response

and, when activated, translocates into the nucleus, where itbinds to the promoter region of target genes and results inthe subsequent activation of inflammatory mediators, withup-regulation of pro-inflammatory cytokines, such as

tumour necrosis factor a, and induction of iNOS gene [39].Because of the ubiquitous role in the pathogenesis ofinflammatory gene expression, NF-jB is a current target for

treating inflammatory diseases and inhibition of NF-jBactivation may be of therapeutic benefit in various types ofinflammation. Under normal physiological conditions, NF-

jB forms a complex with its inhibitors, the IjBs, and ismaintained in the cytosol in this inactive state. Activation ofNF-jB, in turn, is induced by phosphorylation of IjB, inresponse to diverse stimuli including ROS, which leads to itsdegradation and results in unmasking of nuclear localiza-tion signals that allow NF-jB to be translocated into the cellnucleus [40]. NF-jB can be freed from its inhibitors through

the direct action of protein kinases, termed the IkappaBkinases (IKK) that form a complex consisting of thecatalytic subunits IKKa and IKKb and the regulatory

subunit IKKc [41]. Activation of the IKK complex leads tothe phosphorylation of the IjBs, thus targeting them forpolyubiquitination and degradation by the 26S proteosome

complex. Freed from its inhibitor, NF-jB enters the nucleusand transactivates NF-jB-responsive genes [42, 43]. It has

been previously reported that an acute bout of exercise inrats increases NF-jB activation [9] and, recently, it has beenfound that exercise causes a local activation of IKK [10],

and results in a decrease of the cytosolic content of IjBwhereas phospho-IjB increases [1]. These data were con-firmed in the current study, revealing that the activation ofIKKa, the major IKK band detected in rat skeletal muscle

[1], and proteolysis of IjBa were coincident with activationand nuclear translocation of NF-jB, and this was accom-panied by iNOS gene up-regulation.

In exercised rats, melatonin decreased IjBa degradationby inhibiting up-regulation of IKKa and effects on theIKK/IjB cascade would, in turn, contribute to inhibition of

NF-jB activation. Inhibitory effects of melatonin onNF-jB-binding activity have been previously found inseveral tissues and different experimental systems, such asHELa S3 cells [44], rat spleen [45], cultured macrophages [7]

or thioacetamide-treated liver [35], this inhibitory effectcontributing to the reduction of inflammatory injury [46].The melatonin-induced suppression of the release of NF-jBwas accompanied by down-regulation of the expression ofiNOS, confirming results in other experimental models [47–49] and supporting the previous report that elevations of

NO and nitrotyrosine levels are reduced by administrationof antioxidants in parallel to an inhibition of NF-jBactivation [50].

Nuclear factor kappaB is activated in response to oxida-tive stress in a variety of cells [51]. Following acute exercise,the mitochondrial electron transport chain and othersources such as cytosolic xanthine oxidase, peroxisomal

catalase and NADPH oxidase [52–54] can generate ROSgeneration over resting levels [55], and this may result inoxidative injury, including tissue lipid peroxidation, enzyme

inactivation or changes in glutathione status [56–59]. Ourdata support these findings and confirm, by the increase inboth TBARS and the GSSG/GSH ratio, that a bout of acute

exercise results in the presence of oxidative stress in the DVLmuscle. It has been proposed that an ROS can directlyactivate NF-jB by activating IKK [60] or by degrading or

modifying IjB in the cytoplasmic NF-jB-IjB complex [61].Although we did not directly measure ROS generation, thefact that melatonin reduced oxidative stress, NF-jB bindingand IjBa phosphorylation in exercised rats suggests that

melatonin is able to influence the redox-sensitive steps of theNF-jB in skeletal muscle cells. The decrease in TBARSconcentration induced by melatonin in comparison to

control animals is difficult to explain, but could be becauseof different reasons, such as changes in gene expression ofantioxidant enzymes [62]. It has been recently proposed that

decreased activation of NF-jB, likely resulting from thereduction of local oxidative stress, may play a role in theamelioration of the inflammatory response to carbontetrachloride [63]. Previous studies have shown that mela-

tonin is able to attenuate oxidative stress, iNOS inductionand NF-jB-binding activity in thioacetamide-treated ratliver [35], and there is recent evidence that prevention of

ROS production by allopurinol abolishes up-regulation ofiNOS and NF-jB activation in muscle of exercised rats [12].Our data support the suggestion that oxidative stress is the

initial change induced by acute exercise, followed byactivation of NF-jB-dependent pathways.

Fig. 4. Effect of exercise and melatonin on Western blot analysis ofIjB kinasea (IKKa) protein in rat DVL muscle. Total cellularprotein was separated on 12% SDS–PAGE and blotted with anti-IKKa antibody. (A) A representative Western blot photograph. (B)n ¼ 8. See Table 1 legend for group definitions. *Different from C,P < 0.05; #different from E, P < 0.05.

Alonso et al.

12

Our data indicate that administration of melatonin haspotent protective effects against damage caused by acuteexercise in rat muscle, preventing oxidative stress, NF-jBactivation and iNOS over-expression. Although the causalrelationship between the events here described remains tobe established, melatonin treatment, by abolishing theIKK/NF-jB signal transduction pathway, might block the

skeletal muscle production of noxious mediators involvedin the inflammatory process. Results support previoussuggestions that melatonin may be useful in the therapy of

conditions associated with local or systemic inflammation,but further studies should be required to identify the anti-inflammatory potential of melatonin in the clinical setting.

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