Nitric oxide reverses endotoxin-induced inflammatory hyperalgesia via inhibition of prostacyclin...

Pharmacological Research 53 (2006) 177–192

Nitric oxide reverses endotoxin-induced inflammatory hyperalgesia viainhibition of prostacyclin production in mice

B. Tunctana,∗, E. Ozverena, B. Korkmaza, C.K. Buharalioglua,L. Tamerb, U. Degirmencib, U. Atik b

a Department of Pharmacology, Faculty of Pharmacy, Yenisehir Campus, Mersin University,Mersin 33169, Turkey

b Department of Biochemistry, Faculty of Medicine, Mersin University, Mersin, Turkey

Accepted 14 October 2005

Abstract

We examined whether nitric oxide (NO), derived from constitutive NO synthase (NOS) and/or inducible NOS (iNOS), could contribute toendotoxin-induced inflammatory hyperalgesia via interacting with nuclear factor-�B (NF-�B), inducible cyclooxygenase (COX-2) and/or polyADP-ribose synthase (PARS). Injection of endotoxin (10 mg kg−1, i.p.) to mice elicited hyperalgesia, determined by hot plate test, which is prevented byN r( evented byNo ease inh xin-inducedi .©

K

1

tiNdtiltcsmfp

inderliel.s,

ate

herallit-y or

ya-

enansin-nical

-1

ought

1d

O precursor (l-arginine), cNOS/iNOS inhibitor (NG-nitro-l-arginine methyl ester;l-NAME), NF-�B inhibitor (N-acetylserotonin), COX inhibitoindomethacin), COX-2 inhibitor (DFU) and PARS inhibitor (3-aminobenzamide). Endotoxin caused a decrease in serum nitrite levels pr-acetylserotonin,l-arginine, indomethacin, DFU or 3-aminobenzamide. Endotoxin increased serum 6-keto-PGF1� levels diminished byl-argininer aminoguanidine (iNOS inhibitor).l-Arginine,l-NAME, aminoguanidine, DFU or 3-aminobenzamide prevented endotoxin-induced decreart, lungs, kidneys and brain nitrite and malonedialdehyde levels and myeloperoxidase activity. In conclusion, NO reverses endoto

nflammatory hyperalgesia via inhibition of prostacyclin production, and also contributes to the analgesic effect of NF-�B, COX or PARS inhibitors2005 Elsevier Ltd. All rights reserved.

eywords: Mice; Endotoxin; Inflammatory hyperalgesia; Nitric oxide synthase; Cyclooxygenase

. Introduction

NO is synthesized froml-arginine by three isoforms ofhe enzyme NO synthetase (NOS). Of the three establishedsoenzymes of NOS, neuronal NOS (nNOS) and endothelialOS (eNOS) are constitutive (cNOS), low-output and calcium-ependent enzymes, whose physiological function is signal

ransduction. The third form of NOS, inducible NOS (iNOS),s constitutively expressed only in the selected tissues such asung epithelium, and more typically it is synthesized in responseo inflammatory mediators by macrophages and inflammatoryells. NO produced enzymatically in numerous tissues is con-idered as an important intracellular and intercellular messengerolecule in the peripheral and central nervous systems and

unctions in a variety of physiological and pathophysiologicalrocesses (for reviews, see[1–7]).

∗ Corresponding author. Tel.: +90 324 3410605; fax: +90 324 3410605.E-mail address: [email protected] (B. Tunctan).

There is increasing evidence indicating a role for NOthe development, maintenance and mechanisms that unmechanical (e.g. by pressure to the tail or paw)[8,9], chemica(e.g. by acetic acid orp-benzoquinone)[10,11]and thermal (e.gby noxious heat)[8,12,13]hyperalgesia in rodents (for reviewsee[14,15]). l-Arginine/NO/cyclic guanosine monophosph(cGMP) pathway appears to act as a pro-nociceptive[10,13]as well as an anti-nociceptive at supraspinal and peripsites[10]. However, there are contradictory reports in theerature concerning the role of NO as an anti-inflammatorpro-inflammatory agent (for reviews, see[2,3,6,15–18]). Sev-eral studies have suggested thatl-arginine/NO/cGMP pathwais involved in the modulation of pain perception in inflammtory pain models induced by formalin, zymosan or carrage[13,19–21]administered either centrally or peripherally, agle dose of endotoxin also decreases thermal or mechahyperalgesia associated with inflammation in rodents[22–30].Many mediators including NO, prostaglandins, interleukin�(IL-1�), IL-6, tumor necrosis factor-� (TNF-�), metallopro-teinases, nerve growth factor or endogenous opioids are th

043-6618/$ – see front matter © 2005 Elsevier Ltd. All rights reserved.oi:10.1016/j.phrs.2005.10.009

178 B. Tunctan et al. / Pharmacological Research 53 (2006) 177–192

to be major contributers to the endotoxin-induced hyperalgesia.In models of acute and chronic inflammation, it is likely thatNO from eNOS plays a role in the early stages of inflamma-tion by inhibiting leucocyte activation and platelet aggregation,inducing vasodilatation, reducing the microvascular fluid leak-age and quenching of superoxide radicals. Indeed, inhibitors ofcNOS and/or iNOS activity reduce the severity of inflamma-tion and support a role for NO in the pathophysiology asso-ciated with various models of inflammation (for reviews, see[2,3,6,15–18]). However, so far no report has addressed themodulation of endotoxin-induced inflammatory hyperalgesia byl-arginine/NO pathway.

Under inflammatory conditions, both iNOS and induciblecyclooxygenase (COX-2) expression are up-regulated by pro-inflammatory cytokines, indicating increased levels of both NOand prostaglandins (for reviews, see[31,32]). It is well knownthat prostaglandins, synthesized both in the peripherally andcentrally by COX isoforms, play a key role in sensitisationof nociceptors and nociceptive processing. Since both NO andprostaglandins are involved in inflammatory process, it is notsurprising that there are interactions between NOS and COXpathways. Indeed, there have been numerous reports of regula-tory interactions between the NO and prostaglandin biosyntheticpathways (for reviews, see[1,33]). Therefore, pro- or anti-inflammatory effects of NO may depend on the quite complexeffects on the biosynthesis of eicosanoids during inflammation,b

t hiche F-i pro-t lud-im cept Feihe ayo -dN wn tio press -2,a stimu usc[ t ofe nflamm s[ tfor-w omot mei , hepab d thN reg-u ons ulus[

During inflammatory processes, the presence of largeamounts of activated leukocytes results in elevated levels of reac-tive oxygen species, such as superoxide, peroxynitrite, hydroxylradical and hydrogen peroxide and NO. PARS activation, mostlikely due to peroxynitrite production, is also responsible forthe cellular energetic derangement and ultimately cell deathobserved during endotoxemia (for review, see[3]). In addition tothe direct cytotoxic pathway regulated by DNA injury and PARSactivation, it appears that PARS plays an important role in regu-lating the expression of a variety of genes including iNOS. Thelatter pathway may be crucial for the anti-inflammatory effectof either pharmacological inhibition of PARS or deletion of itsgene, since NF-�B pathway is a key element for the production ofmany other inflammatory products, such as TNF-�, IL-6, COXmetabolites and other oxidant species and adhesion molecules.

Lipid peroxidation is also one of the basic mechanismsinvolved in cell and tissue damage. The regulation of non-enzymatic and enzymatic lipid peroxidation reactions by NOreveals novel non-cGMP-dependent reactivities for NO. NO andits metabolites may regulate enzymatic lipid peroxidation andthe production of inflammatory and vasoactive eicosanoids byCOX and lipoxygenase[38]. High reactivity of NO with radicalsmay be beneficial in vivo, such as scavenging peroxyl radical andinhibiting lipid peroxidation.

Thus, the exact role of NO derived from cNOS or iNOSin central perception of pain during inflammatory hyperalge-s tudy,w OSc ral-g -m thisi atedw ecto fil-t agei

2

2

ighto houtt g tot y ofP ersinU tan-d od andwT iron-m houtt y oft d 12 hd ly1 ereu tioni

eing able to either activate or inhibit COX.The transcription factor nuclear factor�-B (NF-�B) activa-

ion is supposed to be one of the principal mechanisms by wndotoxin induces its cellular process. Once activated, N�B

nitiates the transcription of numerous genes coding foreins involved in inflammatory and immune responses, incng pro-inflammatory cytokines (TNF-� and IL-1�), adhesion

olecules, chemokines (e.g. IL-8), receptors (e.g. IL-2 reors) and enzymes (e.g. iNOS and COX-2) (for review, seet al. [18]). NO has also major interactions with this pathwf gene expression controlled by NF-�B in inflammatory conitions. Both activation and inhibition of NF-�B activity byO have also been described. In vitro, NO has been sho

nhibit NF-�B activation in many cell types[34]. The inhibitionf NF-�B by NO has been associated with a reduced exion of pro-inflammatory mediators (cytokines, iNOS, COXdhesion molecules and metalloproteinases) by immunolated cells such as endothelial and vascular smooth m

35]. In vivo, provision of exogenous NO or enhancemenndogenous NO formation has been reported to decrease iation by inhibiting NF-�B activation in various condition

36]. The above-described effects of NO are not straighard, as some studies have suggested that NO can pr

he activation of NF-�B and increase the expression of sonflammatory response proteins in cells such as neuronstocytes, lung epithelial cells and macrophages[1]. On theasis of these contrasted results, it has been proposeO may exert both pro- and anti-inflammatory effects bylating NF-�B either positively or negatively, dependingpecies, timing, cell type and the kind of inflammatory stim37].

-l

o

-

-le

-

te

-

at

ia induced by endotoxin needs to be clarified. In this se examined whether NO derived from cNOS and/or iNould contribute to endotoxin-induced inflammatory hypeesia via inhibition of prostacyclin (PGI2) production. Furtherore, to gain more insight into the mechanisms involved in

nflammatory hyperalgesia model in mice, we also investighether NO and PGI2 could contribute to the analgesic efff NF-�B, COX or PARS inhibitors as well as neutrophil in

ration, oxidative stress, lipid peroxidation and cellular damn kidney, heart, lung and brain tissues.

. Materials and methods

.1. Animals

Male and female Balb/c albino mice, with an average wef 30 g (±10) were used in all experiments performed throug

his study. All animal experiments were carried out accordinhe guidelines of the International Association for the Studain. The protocol was approved by ethics committee of Mniversity School of Medicine. Animals were housed in sard transparent cages (20 per cage) with free access to foater under environmental controlled conditions at 24–25◦C.hey were synchronised by maintenance of controlled envental conditions for at least 2 weeks prior to and throug

he duration of the experiments. The circadian rhythmicithe animals were entrained by a standardised 12 h light anark (lights on at 0900) with a light intensity of approximate00 lx. Automatic timer controlled cool fluorescent bulbs wsed to provide lighting. Visualisation and drug administra

n the dark were supplied by photo-safe red bulbs.

B. Tunctan et al. / Pharmacological Research 53 (2006) 177–192 179

2.2. Materials

Endotoxin (lipopolysaccharide,Escherichia coli O111:B4),N-acetylserotonin,l-arginine,NG-nitro-l-arginine methyl ester(l-NAME), aminoguanidine, 3-aminobenzamide, indometha-cin, sodium nitrite, bovine serum albumin (BSA) and Bradfordreagent were purchased from Sigma Chemical Co. (St. Louis,U.S.A.). Dimethylsulphoxide (DMSO) was obtained fromRiedel-de-Haen (Germany). Other chemicals were obtainedfrom Merck (Darmstadt, Germany). (5,5-Dimethyl-3-(3-florophenyl)-4-(4-methylsulphonyl-2(5H)-furanon) (DFU) waskindly provided by Merck Frosst Canada Inc. (Quebec, Canada).All drugs were prepared daily. Endotoxin,l-arginine,l-NAME,aminoguanidine and 3-aminobenzamide were dissolved insaline;N-acetylserotonin and DFU were dissolved in DMSO(40%, v/v and 70%, v/v, respectively), and indomethacin wasdissolved in sodium bicarbonate (5%, w/v).

2.3. Induction of inflammatory hyperalgesia

In this study, the dosages, timing of injections and protocolsfollowed for the observation of the effects of drugs on nocicep-tion and NOS, NF-�B, COX or PARS activities were based onpreviously established characteristics of each of these drugs. Wehave previously shown that a gradual increase in serum nitritelevels was observed 12 h after intraperitoneal (i.p.) injectionso ap uanid rs,ii 98[ eB butn ofi m-m giT esiam singhm9 n ofp jec-t eachg pre-v ithin6 nt oN esice NOps itoro itor( lec-t bi-nl it-i esia

in mice. Indomethacin (100 mg kg−1, i.p.) [39,40] was usedat a dose preventing the effects of endotoxin on NO andprostaglandin production. The doses of aminoguanidine (0.1,1 or 10 mg kg−1, i.p.) [39,40], N-acetylserotonin (0.1, 1, 10 or100 mg kg−1, i.p.)[43], DFU (0.1, 1 or 10 mg kg−1, i.p.)[41] and3-aminobenzamide (0.1, 1, 10 or 100 mg kg−1, i.p.) [44] wereselected according to the previous in vivo studies with endotoxinor carrageenan and findings of the present study preventing theendotoxin-induced hyperalgesia and decrease in NO produc-tion. All animals were examined by the hot plate test 6 h afterdrug administration as described above. None of the treatmentscaused mortality for 6 h. Mice were sacrificed by the cervical dis-location method after the hot plate test, and blood, lungs, heart,kidneys and brain were collected. Sera were obtained from bloodsamples by centrifugation at 12,100× g for 15 min and stored at−20◦C until analyzed for the measurement of nitrite and 6-keto-prostaglandin F1� (6-keto-PGF1�) levels. The lungs, heart, kid-neys and brain were homogenized in PBS (1.5 ml) (NaCl 8.0 g/l;KCl 0.2 g/l; Na2HPO4 1.4 g/l and KH2PO4 0.2 g/l; pH 7.4). Celldebris was removed by centrifugation at 12,100× g for 20 minand then supernatants were removed and stored at−20◦C untilanalyzed for the measurement of protein, nitrite, malonedi-aldehyde (MDA) and 6-keto-PGF1� levels or myeloperoxidase(MPO) activity.

2.4. Measurement of tissue protein content

eter-m stan-d emS erp oplater dardc ngingf

2

sueh ethodb tivity[ llm (1%s ed edt ure,a eader.S itritec enr

2

-u s-s rer’s

f endotoxin (10 mg kg−1; sublethal dose) at 0900 and reachedeak by 15 h decreased by selective iNOS inhibitor, aminogine (100 mg kg−1, i.p.), and non-selective COX inhibito

ndomethacin (100 mg kg−1, i.p.) and diclophenac (10 mg kg−1,.p.), but not selective COX-2 inhibitors, DFU and NS 339–41]. Endotoxin-induced 6-keto-PGF1� and thromboxan2 (TxB2) levels were also decreased by indomethacin,ot aminoguanidine[40]. Therefore, to induce the activities

NOS, NF-�B, COX-2 and/or PARS enzymes leading to inflaatory hyperalgesia, mice were given endotoxin (10 mg k−1,

.p.) at 0900. Control group received saline (10 ml kg−1, i.p.).o characterize time course of endotoxin-induced hyperalgice were tested for central perception of pain sensitivity uot plate method as described in detail previously[42]. Briefly,ice were individually put on a heated plate (55± 0.2◦C) (AHP601, Commat Ltd., Turkey) and the latency of the first sigaw licking or jumping was recorded 0, 3 and 6 h after in

ions of saline or endotoxin. The latencies recorded inroup were averaged for each time interval. In order toent tissue damage, animals that were not responding w0 s were removed from the hot plate. To test the involvemeOS, NF-�B, COX and PARS enzymes in mediating the algffect of endotoxin, separate groups of mice treated with arecursor (l-arginine), a cNOS/iNOS inhibitor (l-NAME), aelective iNOS inhibitor (aminoguanidine), a selective inhibf NF-�B (N-acetylserotonin), a non-selective COX inhibindomethacin), a selective COX-2 inhibitor (DFU) and a seive PARS inhibitor (3-aminobenzamide) alone or in comation with endotoxin.l-Arginine (1 mg kg−1, i.p.) [10] and-NAME (75 mg kg−1, i.p.) [10,12]were used at doses exhib

ng analgesia in chemically- and/or thermally-induced alg

-

,

f

The protein content in the tissue homogenates was dined according to Coomassie blue method using BSA forard[45]. Briefly, Bradford reagent (200�l) was added to thixture of tissue homogenate (5�l) and distilled water (795�l).amples (100�l) were then pipetted into 96 well microtitlates and absorbance was measured at 620 nm with a micreader (Organo Teknika Microwell System, Holland). Stanurves were also constructed using BSA concentrations rarom 0 to 50�g/ml.

.5. Measurement of serum and tissue nitrite levels

Nitrite (stable product of NO) levels in the sera and tisomogenates were measured by using the diazotization mased on the Griess reaction as an index for NOS ac

40,41]. Briefly, samples (50�l) were pipetted into 96 weicrotiter plates and an equal volume of Griess reagent

ulphanylamide (25�l) and 0.1%N-1-naphtylethylenediaminihydrochloride (25�l) in 2.5% ortophosphoric acid) was add

o each well. After incubation for 10 min at room temperatbsorbance was measured at 540 nm with a microplate rtandard curves were also constructed using sodium noncentrations ranging from 0.25 to 50�M. Serum and tissuitrite levels were expressed as�M or mmol mg−1 protein−1,espectively.

.6. Measurement of serum 6-keto-PGF1α levels

As an index for COX activity, 6-keto-PGF1� (stable prodct of PGI2) concentrations[46] were measured in the tiue homogenates by ELISA according to the manufactu


instructions in the 6-keto-PGF1� assay kit (Cayman Chemi-cal Co., Ann Arbor, MI). Serum 6-keto-PGF1� levels wereexpressed as pg ml−1.

2.7. Measurement of tissue MPO activity

MPO is a haem-containing enzyme within the azurophil gran-ules of neutrophils and MPO activity was measured as a simplequantitative method of detecting leukosequestration. The deter-mination of MPO activity as an index of neutrophil infiltrationin tissue homogenates depends on the fact that oxidized hydro-gen peroxide reduceso-dianozidine. Reducedo-diazidine wasmeasured at 410 nm by spectrophotometer[47]. Tissue MPOactivity was expressed as U mg−1 protein−1.

2.8. Measurement of tissue MDA levels

As an index of oxidative stress, lipid peroxidation and cellulardamage, the levels of MDA in tissue homogenates were deter-mined by thiobarbituric acid reaction according to Yagi[48]. Themethod depends on the measurement of the pink color producedby interaction of the barbituric acid with MDA caused by lipidperoxidation. The colored reaction 1,1,3,3-tetraethoxypropanewas used as the primary standard. Tissue MDA levels wereexpressed as mmol mg−1 protein−1.

2

r-i ere

analyzed by one-way ANOVA with Student–Newman–Keulsmultiple comparisons post test, or Student’st- or Mann–WhitneyU-tests, when necessary, using GraphPad Prism version 3.00 forWindows (Graph Pad Software, San Diego, California U.S.A.,http://www.graphpad.com). P-value of <0.05 is considered sta-tistically significant.

3. Results

3.1. Endotoxin-induced hyperalgesia is associated withdecreased systemic NO production

In order to induce inflammatory hyperalgesia, endotoxin(10 mg kg−1, i.p.) was injected to mice. Afterwards, to char-acterize time course of hyperalgesia, the animals were tested forpain sensitivity to thermal stimulus 0, 3 and 6 h after injectionof endotoxin. Endotoxin caused a significantly decrease in hotplate latency (P = 0.0068,F = 5.76) when compared to baselinevalue and 3 h after drug injection (P-values were 0.0073 and0.0480, respectively) (Fig. 1A). No hyperalgesia was detectedin saline-treated mice during 6 h (P = 0.2358,F = 1.51). Thehot plate latencies recorded in endotoxin-injected mice weresignificantly lower than control group 3 and 6 h after injec-tion (P-values were 0.0047 and 0.0002, respectively). Theseresults provide evidence that systemic administration of endo-t minei c NOp xper-i uallyi d

F ecre ontrol mice.P e as anel B:S after obtainedfs

.9. Statistical analysis

Results were expressed as the mean± S.E.M. for each expemental group.n refers to the number of animals used. Data w

ig. 1. Endotoxin (10 mg kg−1, i.p.) caused hyperalgesia associated with danel A: Thermal hyperalgesia in (©) saline- or (�) endotoxin-injected micerum nitrite levels in (©) saline- or (�) endotoxin-injected mice 0, 3 or 6 h

rom blood samples used for the measurement of nitrite levels. The results arealine-treated group at 0 h.©P < 0.05 vs. saline-treated group at 3 h.+P < 0.05 vs. sa

oxin causes hyperalgesia 3 and 6 h after injection. To deterf thermal stimulus or endotoxin causes a change in systemiroduction, serum levels of nitrite were measured after the e

ments at each time point. Serum nitrite levels were gradncreased in saline- (P = 0.0006,F = 9.70) or endotoxin-treate

ased serum nitrite levels in Balb/c mice as compared to saline-injected cdetermined by the hot plate test 0, 3 or 6 h after drug administration. P

drug administration. Mice were sacrificed after the hot plate test and sera�
expressed as the mean± S.E.M from 8 to 14 mice per treatment group.P < 0.05 vs.
line-treated group at 6 h.�P < 0.05 vs. endotoxin-treated group at 0 h.

http://www.graphpad.com/


mice (P = 0.0048,F = 6.41) (Fig. 1B). The increase in nitritelevels in saline-treated group was significant when compared tobaseline value and 3 h after drug injection (P-values were 0.0381and 0.0005, respectively). From these studies, it appears thatsystemic NO production is increased by thermal stimulus. Onthe other hand, systemic NO production in endotoxin-injectedmice was significantly higher than baseline value (P = 0.0019)and lower than control group (P = 0.0220) 6 h after injec-tion. These results are consistent with the fact that endotoxin-induced hyperalgesia is associated with decreased systemic NOproduction.

3.2. Effect of NO precursor or cNOS/iNOS inhibitors on theendotoxin-induced hyperalgesia and decrease in systemicNO production

In order to establish the contribution of cNOS and/or iNOSto the endotoxin-induced hyperalgesia and decrease in systemicNO production, the NO precursor,l-arginine, cNOS/iNOSinhibitor, l-NAME, or selective iNOS inhibitor, aminoguani-dine, were injected to the animals alone or in combination withendotoxin. Serum nitrite levels were also measured after thehot plate test.l-Arginine at 1 mg kg−1 dose orl-NAME at75 mg kg−1 dose prevented the endotoxin-induced hyperalgesia6 h after drug injection (P < 0.05) (Fig. 2A). On the other hand,

aminoguanidine at 0.1, 1 or 10 mg kg−1 doses, had no effecton the endotoxin-induced hyperalgesia (P > 0.05) (Fig. 2A).These results are consistent with the fact that administrationof NO precursor or inhibitor of cNOS/iNOS, but not iNOS, pre-vents the endotoxin-induced hyperalgesia. Endotoxin-induceddecrease in serum nitrite levels was also prevented byl-arginine(P < 0.05), whilel-arginine had no effect of the levels of saline-treated group (P > 0.05) (Fig. 2B). l-NAME alone or in combi-nation with endotoxin did not change the nitrite levels inducedby saline or endotoxin (P > 0.05) (Fig. 2B). Aminoguanidineat 1 mg kg−1 dose caused a further decrease in nitrite lev-els induced by saline or endotoxin (P < 0.05) (Fig. 2B). Thenitrite levels induced by aminoguanidine at 10 mg kg−1 dosewhen injected with endotoxin were lower than control group(P < 0.05). Aminoguanidine alone had no effect on the serumnitrite levels induced by saline (P > 0.05). These results demon-strate that administration of NO precursor, but not inhibitors ofcNOS/iNOS, prevents the endotoxin-induced decrease in sys-temic NO production.

3.3. Effect of NF-κB inhibitor on the endotoxin-inducedhyperalgesia and decrease in systemic NO production

In order to evaluate the contribution of NF-�B to theendotoxin-induced hyperalgesia and decrease in systemic NO

Fsllot(

ig. 2. Effect of the nitric oxide (NO) precursor,l-arginine (1 mg kg−1, i.p.), the celective inducible NOS (iNOS) inhibitor, aminoguanidine (0.1, 1 or 10 mg kg−1, i.evels in Balb/c mice. Panel A: Thermal hyperalgesia in drug-injected mice asevels in drug-injected mice 6 h after drug administration. Mice were sacrificed af nitrite levels. The results are expressed as the mean± S.E.M from 3 to 14 mice p

reated group.�P < 0.05 vs. aminoguanidine (0.1 mg kg−1)-treated group.�P < 0.0510 mg kg−1)-treated group.

onstitutive NO synthase (cNOS) inhibitor,l-NAME (75 mg kg−1, i.p.) or thep.) in the endotoxin (10 mg kg−1, i.p.)-induced hyperalgesia and serum nitritedetermined by the hot plate test 6 h after drug administration. Panel B: Serum nitritefter the hot plate test and sera obtained from blood samples used for the measurementer treatment group.+P < 0.05 vs. saline-treated group.* P < 0.05 vs. endotoxin-vs. aminoguanidine (1 mg kg−1)-treated group.�P < 0.05 vs. aminoguanidine


Fig. 3. Effect of the selective inhibitor of nuclear factor-�B (NF-�B), N-acetylserotonin (0.1, 1, 10 or 100 mg kg−1, i.p.) in the endotoxin (10 mg kg−1, i.p.)-inducedhyperalgesia and serum nitrite levels in Balb/c mice. Panel A: Thermal hyperalgesia in drug-injected mice as determined by the hot plate test 6 h afterdrugadministration. Panel B: Serum nitrite levels in drug-injected mice 6 h after drug administration. Mice were sacrificed after the hot plate test and sera obtained fromblood samples used for the measurement of nitrite levels. The results are expressed as the mean± S.E.M. from 6 to 14 mice per treatment group.+P < 0.05 vs. saline-treated group.* P < 0.05 vs. endotoxin-treated group.�P < 0.05 vs.N-acetylserotonin (0.1 mg kg−1)-treated group.�P < 0.05 vs.N-acetylserotonin (1 mg kg−1)-treatedgroup.

production, the selective inhibitor of NF-�B, N-acetylserotonin,was injected to the animals alone or in combination withendotoxin. Serum nitrite levels were also measured after thehot plate test.N-acetylserotonin at 100 mg kg−1 dose pre-vented the endotoxin-induced hyperalgesia 6 h after druginjection (P < 0.05) (Fig. 3A). The other doses ofN-acetylserotonin (0.1, 1 or 10 mg kg−1) were not effective toprevent the endotoxin-induced hyperalgesia (P > 0.05). Theseresults demonstrate that increased activity of NF-�B con-tributes to the endotoxin-induced hyperalgesia. Moreover,endotoxin-induced decrease in serum nitrite levels was also pre-vented byN-acetylserotonin at 100 mg kg−1 dose (P < 0.05)(Fig. 3B). On the other hand,N-acetylserotonin at 0.1 or1 mg kg−1 doses caused a further decrease in the endotoxin-induced serum nitrite levels (P < 0.05).N-acetylserotonin alonehad no effect on the serum nitrite levels induced by saline(P > 0.05). Thus, these results show that inhibition of NF-�B by N-acetylserotonin at 100 mg kg−1 dose prevents theendotoxin-induced hyperalgesia and decrease in systemic NOproduction.

3.4. Effect of COX-1/COX-2 inhibitors on theendotoxin-induced hyperalgesia and decrease in systemicNO production

In order to evaluate the contribution of COX-1 and/orCOX-2 enzymes to the endotoxin-induced hyperalgesia anddecrease in systemic NO production, the non-selective COX-1inhibitor, indomethacin, or selective COX-2 inhibitor, DFU,were injected to the animals alone or in combination withendotoxin. Serum nitrite levels were also measured after thehot plate test. Indomethacin, at 100 mg kg−1 dose, or DFU at0.1, 1 and 10 mg kg−1 doses prevented the endotoxin-inducedhyperalgesia 6 h after drug injection (P < 0.05) (Fig. 4A).The effect of solvent of DFU, DMSO (70%, v/v), on hotplate latency (17.75± 0.55 s, n = 6) was not different fromcontrol group (21.19± 1.61 s,n = 14) (P > 0.05). These resultsdemonstrate that increased activity of COX-1 and/or COX-2contributes to the endotoxin-induced hyperalgesia. Further-more, endotoxin-induced decrease in serum nitrite levels wasprevented by indomethacin or DFU at 100 or 1 mg kg−1 doses,


Fig. 4. Effect of the non-selective cyclooxygenase (COX) inhibitor, indomethacin (100 mg kg−1, i.p.) or the selective inducible COX (COX-2) inhibitor, DFU (0.1,1 or 10 mg kg−1, i.p.) in the endotoxin (10 mg/ kg, i.p.)-induced hyperalgesia and serum nitrite levels in Balb/c mice. Panel A: Thermal hyperalgesia in drug-injectedmice as determined by the hot plate test 6 h after drug administration. Panel B: Serum nitrite levels in drug-injected mice 6 h after drug administration. Mice weresacrificed after the hot plate test and sera obtained from blood samples used for the measurement of nitrite levels. The results are expressed as the mean± S.E.M.from 3 to 14 mice per treatment group.+P < 0.05 vs. saline-treated group.* P < 0.05 vs. endotoxin-treated group.

respectively (P < 0.05) (Fig. 4B). DFU at 0.1, 1 or 10 mg kg−1

doses alone had no effect of the serum nitrite levels inducedby saline (P > 0.05). These results provide evidence that theendotoxin-induced decrease in systemic NO production can beprevented by inhibition of COX-1 and/or COX-2.

3.5. Effect PARS inhibitor on the endotoxin-inducedhyperalgesia and decrease in systemic NO production

In order to determine the contribution of PARS to theendotoxin-induced hyperalgesia and decrease in systemic NOproduction, the selective PARS inhibitor, 3-aminobenzamidewas injected to the animals alone or in combination with endo-toxin. Serum nitrite levels were also measured after the hotplate test. 3-Aminobenzamide at 0.1, 1, 10 and 100 mg kg−1

doses prevented the endotoxin-induced hyperalgesia 6 h afterdrug injection (P < 0.05) (Fig. 5A). Antinociceptive effect of3-aminobenzamide at 100 mg kg−1 dose injected with endo-toxin was also higher than the control group (P < 0.05). Theseresults demonstrate that increased activity of PARS contributesto the endotoxin-induced hyperalgesia. Moreover, endotoxin-induced decrease in serum nitrite levels was prevented by3-aminobenzamide at 0.1 or 100 mg kg−1 doses (P < 0.05)(Fig. 5B). 3-Aminobenzamide alone had no effect on theserum nitrite levels induced by saline (P > 0.05). These resultsp ARS

activity to the endotoxin-induced decrease in systemic NOproduction.

3.6. Effect of NF-κB, cNOS/iNOS, COX-2 or PARSinhibitors on the endotoxin-induced increase in systemic6-keto-PGF1α production

In order to evaluate if endotoxin causes a change in sys-temic PGI2 production, serum levels of 6-keto-PGF1� weremeasured after the experiments. Endotoxin caused an increasein serum 6-keto-PGF1� levels after 6 h drug injection (P < 0.05)(Fig. 6). To investigate the contribution of NO or NF-�B,cNOS/iNOS, COX-2 or PARS activity to the endotoxin-inducedincrease in systemic 6-keto-PGF1� production, the NO precur-sor or the selective inhibitors of these enzymes were used atthe doses that prevented the endotoxin-induced hyperalgesiaand/or increase in systemic NO production. The endotoxin-induced increase in serum 6-keto-PGF1� levels was inhibitedby l-arginine or aminoguanidine at 1 mg kg−1 doses (P < 0.05)(Fig. 6). DFU (1 mg kg−1), 3-aminobenzamide (0.1 mg kg−1),N-aceytlserotonin (100 mg kg−1) or l-NAME (75 mg kg−1) hadno effect on the endotoxin-induced increase in serum 6-keto-PGF1� levels (P > 0.05) (Fig. 6). These results are consistentwith the fact that administration of NO precursor or inhibitor ofiNOS prevents the endotoxin-induced increase in systemic PGI2p
rovide evidence to support contribution of increased P roduction.


Fig. 5. Effect of the selective polyADP-ribose synthase (PARS) inhibitor, 3-aminobenzamide (0.1, 1, 10 or 100 mg kg−1, i.p.) in the endotoxin (10 mg kg−1, i.p.)-induced hyperalgesia and serum nitrite levels in Balb/c mice. Panel A: Thermal hyperalgesia in drug-injected mice as determined by the hot plate test6 h afterdrug administration. Panel B: Serum nitrite levels in drug-injected mice 6 h after drug administration. Mice were sacrificed after the hot plate test and sera obtainedfrom blood samples used for the measurement of nitrite levels. The results are expressed as the mean± S.E.M. from 5 to 14 mice per treatment group.+P < 0.05vs. saline-treated group.* P < 0.05 vs. endotoxin-treated group.�P < 0.05 vs. 3-aminobenzamide (10 mg kg−1)-treated group.©P < 0.05 vs. 3-aminobenzamide(100 mg kg−1)-treated group.

3.7. Effect of NO or NF-κB, cNOS/iNOS, COX-2 or PARSinhibitors on the endotoxin-induced tissue nitrite levels

In order to determine if endotoxin changes production ofNO in kidney, heart, lung and brain, levels of nitrite in the tis-sues were measured after the experiments. Endotoxin causeddecreased levels of nitrite in heart (Fig. 7B), lung (Fig. 7C)and brain (Fig. 7D) (P < 0.05), but not in kidney (P > 0.05)(Fig. 7A). To investigate the contribution of NO or NF-�B,cNOS/iNOS, COX-2 or PARS activity to the endotoxin-inducedlevels of tissue nitrite levels, the NO precursor or the selec-tive inhibitors of these enzymes were used at the doses thatprevented the endotoxin-induced hyperalgesia and/or decreasein systemic NO production.l-Arginine (1 mg kg−1), l-NAME(75 mg kg−1), aminoguanidine (1 mg kg−1), DFU (1 mg kg−1)or 3-aminobenzamide (0.1 mg kg−1), but notN-acetylserotonin(100 mg kg−1), caused increased levels of nitrite levels inthe kidneys from saline or endotoxin-treated mice (P < 0.05)(Fig. 7A). l-Arginine, l-NAME, aminoguanidine, DFU or 3-aminobenzamide, but notN-acetylserotonin, reversed the effectof endotoxin on heart (Fig. 7B) and lung (Fig. 7C) nitrite levels(P < 0.05). In brain,l-arginine and all of these inhibitors pre-vented the endotoxin-induced decrease in nitrite levels (P < 0.05)

(Fig. 7D). These results demonstrate that administration of NOprecursor or inhibition of NF-�B, cNOS/iNOS, COX-2 or PARSprevents the endotoxin-induced decrease in tissue nitrite levels.

3.8. Effect of NO or NF-κB, cNOS/iNOS, COX-2 or PARSinhibitors on the endotoxin-induced decrease in tissue MPOactivity

In order to determine if endotoxin causes neutrophil infil-tration in kidney, heart, lung and brain, activity of MPO inthe tissues were measured after the experiments. Endotoxindecreased MPO activity in all of these tissues (Fig. 8) (P < 0.05).To investigate the contribution of NO or NF-�B, cNOS/iNOS,COX-2 or PARS activity to the endotoxin-induced decreasein kidney MPO activity,l-arginine or the selective inhibitorsof these enzymes were used at the doses that prevented theendotoxin-induced hyperalgesia and/or decrease in systemicNO production. In kidney,N-acetylserotonin (100 mg kg−1),l-NAME (75 mg kg−1), aminoguanidine (1 mg kg−1), DFU(1 mg kg−1) or 3-aminobenzamide (0.1 mg kg−1), but not l-arginine (1 mg kg−1), prevented the effect of endotoxin onMPO activity (Fig. 8A) (P < 0.05). l-Arginine, l-NAME,aminoguanidine, DFU (1 mg kg−1) or 3-aminobenzamide


Fig. 6. Effect of the selective inhibitor of nuclear factor-�B (NF-�B), N-acetylserotonin (100 mg kg−1, i.p.), nitric oxide (NO) precursor,l-arginine(1 mg kg−1, i.p.), the constitutive NO synthase (cNOS) inhibitor,l-NAME(75 mg kg−1, i.p.), the selective inducible NOS (iNOS) inhibitor, aminoguani-dine (1 mg kg−1, i.p.), the selective inducible cyclooxygenase (COX-2) inhibitor,DFU (1 mg kg−1, i.p.), or the selective polyADP-ribose synthase (PARS)inhibitor, 3-aminobenzamide (0.1 mg kg−1, i.p.), in the endotoxin (10 mg kg−1,i.p.)-induced increase in serum 6-keto-PGF1� levels in Balb/c mice. Drug-injected mice were sacrificed 6 h after following by the hot plate test and seraobtained from blood samples used for the measurement of 6-keto-PGF1� levels.The results are expressed as the mean± S.E.M. from 3 to 5 mice per treatmentgroup.+P < 0.05 vs. saline-treated group.* P < 0.05 vs. endotoxin-treated group.

(0.1 mg kg−1), but notN-acetylserotonin, reversed the effect ofendotoxin on MPO activity in heart (Fig. 8B) and lung (Fig. 8C)(P < 0.05). In brain,l-arginine and all of these inhibitors pre-vented the effect of endotoxin on MPO activity (Fig. 8D)(P < 0.05). These results provide evidence that NO or inhi-bition of NF-�B, cNOS/iNOS, COX-2 or PARS prevents theendotoxin-induced neutrophil infiltration in these tissues.

3.9. Effect of NO or NF-κB, cNOS/iNOS, COX-2 or PARSinhibitors on the endotoxin-induced decrease in tissue MDAlevels

In order to determine effect of endotoxin on oxidativestress, lipid peroxidation and cellular damage in kidney, heartlung and brain, MDA levels in the tissues were measuredafter the experiments. Endotoxin caused a decrease in MDAlevels in all of these tissues (Fig. 9) (P < 0.05). To investi-gate the contribution of NO or NF-�B, cNOS/iNOS, COX-2or PARS activity to the endotoxin-induced decrease in tis-sue MDA levels, the NO precursor or the selective inhibitorsof these enzymes were used at the doses that prevented tendotoxin-induced hyperalgesia and/or increase in systemiNO production. In kidney,N-acetylserotonin (100 mg kg−1),l-NAME (75 mg kg−1), aminoguanidine (1 mg kg−1), DFU(1 mg kg−1) or 3-aminobenzamide (0.1 mg kg−1), but not l-a −1 A

levels (P < 0.05) (Fig. 9A). l-Arginine,l-NAME, aminoguani-dine, DFU or 3-aminobenzamide, but notN-acetylserotonin,reversed the effect of endotoxin on heart (Fig. 9B) and lung(Fig. 9C) MDA levels (P < 0.05). In brain,l-arginine or all ofthese inhibitors prevented the effect of endotoxin on MDA lev-els (Fig. 9D) (P < 0.05). Therefore, from these results it appearsthat NO or inhibition of NF-�B, cNOS/iNOS, COX-2 or PARSprevents the endotoxin-induced oxidative damage, lipid perox-idation and cellular damage.

4. Discussion

These results in the endotoxin-induced inflammatory hyper-algesia determined by the hot plate test in mice showed that:(1) thermal stimulus increases systemic NO production; (2)systemic administration of endotoxin increases sensitivity tothermal stimulus associated with decreased systemic NO pro-duction; (3) blockade of cNOS, but not iNOS, prevents theendotoxin-induced inflammatory hyperalgesia; (4) increasedactivity of NF-�B, COX-1 and/or COX-2 or PARS also con-tributes to the endotoxin-induced inflammatory hyperalgesia;(5) increased NO production contributes to the analgesic effectsof inhibitors of NF-�B, COX-1 and/or COX-2 or PARS; (6) NOderived from cNOS and/or iNOS reverses the endotoxin-inducedinflammatory hyperalgesia via inhibition of PGI2 production;and (7) endotoxin decreases tissue neutrophil infiltration asw agep lve-m cedi1

cen-t hotp sc tingc threed tiono ani andc thatt OSa howc icalc yo dianvw tralp ailyfl sen-s

ern-i orya ef sen-s velso ctso
rginine (1 mg kg ), prevented the effect of endotoxin on MD
,

hec

ell as oxidative stress, lipid peroxidation and cellular damrevented by NO. Taking our results into consideration, invoent of NO derived from cNOS/iNOS to the endotoxin-indu

nflammatory hyperalgesia via interacting with NF-�B, COX-/COX-2 or PARS can be proposed as shown inFig. 10.

The previous studies have shown that NO is involved inral perception of pain to thermal stimulus determined bylate or tail flick tests[8,12,13]. In our study, thermal stimuluaused an increase in levels of serum nitrite without affecentral perception of pain when the test was repeated atifferent time points suggesting a time-dependent fluctuaf systemic NO production. Indeed, NO is well known as

mportant constituent of the mechanism of biological clocksircadian rhythms[49]. We and others have demonstratedhe levels of blood and urine nitrite/nitrate as well as the cNnd/or iNOS activity in brain, kidney, testis, lung and aorta sircadian rhythmicity under physiological or pathophysiologonditions in mice and rats[40,50–52]. As previously shown bur group, basal pain sensitivity also has a significant circaariation in mice determined by hot plate test[12]. Althoughe did not investigate the chronorhythmic pattern of cenerception of pain in this study, it can be concluded that ductuations in systemic NO production, but not basal painitivity, still occur in mice exposed to thermal stimulus.

There are also contradictory reports in the literature concng role of NO as an anti-inflammatory or pro-inflammatgent (for reviews, see[2,3,6,15–18]). In the present study, w

ound that i.p. injection of endotoxin gradually increaseditivity to thermal stimulus associated with decreased lef nitrite in serum, heart, lung and brain. All of these effef endotoxin were prevented by the NO precursor,l-arginine.


Fig. 7. Effect of the selective inhibitor of nuclear factor-�B (NF-�B), N-acetylserotonin (100 mg kg−1, i.p.), nitric oxide (NO) precursor,l-arginine (1 mg kg−1,i.p.), the constitutive NO synthase (cNOS) inhibitor,l-NAME (75 mg kg−1, i.p.), the selective inducible NOS (iNOS) inhibitor, aminoguanidine (1 mg kg−1, i.p.),the selective inducible cyclooxygenase (COX-2) inhibitor, DFU (1 mg kg−1, i.p.), or the selective polyADP-ribose synthase (PARS) inhibitor, 3-aminobenzamide(0.1 mg kg−1, i.p.), in the endotoxin (10 mg kg−1, i.p.)-induced levels of kidney (panel A), heart (panel B), lung (panel C) or brain (panel D) nitrite in Balb/cmice. Drug-injected mice were sacrificed 6 h after following by the hot plate test and kidneys, heart, lungs and brain were collected. The supernatantsfrom organhomogenates were used for the measurement of nitrite levels. The results are expressed as the mean± S.E.M. from 5 to 11 mice per treatment group.+P < 0.05 vs.saline-treated group.* P < 0.05 vs. endotoxin-treated group.

Moreover, inhibition of cNOS byl-NAME also prevented theendotoxin-induced inflammatory hyperalgesia. The antinoci-ceptive effect ofl-NAME was associated with increased levelsof nitrite in heart, lung and brain, but not serum. On the otherhand, selective inhibition of iNOS by aminoguanidine did notreversed the hyperalgesic effect of endotoxin. A further decreasein the endotoxin-induced levels of serum nitrite was observedfollowing injection of aminoguanidine, whereas it caused

increased levels of nitrite in heart, lung and brain. In the presentstudy, thermal stimulus also caused an increase in systemic NOproduction without changing hot plate latency in saline-injectedmice. Moreover,l-arginine,l-NAME or aminoguanidine alonehad no effect on hot plate latency and basal levels of serum nitrite.Therefore, these data provide convincing evidence that NO playsa role in the central perception of pain associated with inflam-mation induced by endotoxin. Our results also suggest that NO


Fig. 8. Effect of the selective inhibitor of nuclear factor-�B (NF-�B), N-acetylserotonin (100 mg kg−1, i.p.), nitric oxide (NO) precursor,l-arginine (1 mg kg−1,i.p.), the constitutive NO synthase (cNOS) inhibitor,l-NAME (75 mg kg−1, i.p.), the selective inducible NOS (iNOS) inhibitor, aminoguanidine (1 mg kg−1, i.p.),the selective inducible cyclooxygenase (COX-2) inhibitor, DFU (1 mg kg−1, i.p.), or the selective polyADP-ribose synthase (PARS) inhibitor, 3-aminobenzamide(0.1 mg kg−1, i.p.), in the endotoxin (10 mg kg−1, i.p.)-induced decrease in kidney (panel A), heart (panel B), lung (panel C) or brain (panel D) myeloperoxidase(MPO) activity in Balb/c mice. Drug-injected mice were sacrificed 6 h after following by the hot plate test and kidneys, heart, lungs and brain were collected. Thesupernatants from organ homogenates were used for the measurement of MPO activity. The results are expressed as the mean± S.E.M. from 6 to 11 mice pertreatment group.+P < 0.05 vs. saline-treated group.* P < 0.05 vs. endotoxin-treated group.

produced froml-arginine by cNOS and/or iNOS, expressed in,at least, kidney, heart, lung and brain, acts as an antinociceptivemediator in this inflammatory hyperalgesia model in mice. Onthe other hand, NO derived from cNOS, expressed in tissues,including spinal cord, but not in kidney, heart, lung and brain,appears to contribute to hyperalgesic effect of endotoxin.

Prostaglandins, synthesized both in the peripherally and cen-trally by COX isoforms, play a key role in nociceptive processingas well as endotoxin-induced hyperalgesia (for review, see[32]).In the present study, endotoxin caused an increase in systemicPGI2 production as measured by serum 6-keto-PGF1� levels.

A selective COX-2 inhibitor, DFU, however, did not preventthe endotoxin-induced increase in serum 6-keto-PGF1� levels.We have previously shown that morning or evening injectionof endotoxin at 10 mg kg−1 dose to mice results in increasedserum concentrations of 6-keto-PGF1� and thromboxane B2inhibited by 100 mg kg−1 indomethacin without preventing themortality [40]. In another study, the same dose of endotoxin didnot alter the brain, kidney and lung content of 6-keto-PGF1�

[41]. These findings suggest that prostaglandin production inorgan level remains unchanged during endotoxemia to preventfrom the deleterious effects of eicosanoids overproduced by


Fig. 9. Effect of the selective inhibitor of nuclear factor-�B (NF-�B), N-acetylserotonin (100 mg kg−1, i.p.), nitric oxide (NO) donor,l-arginine (1 mg kg−1, i.p.), theconstitutive NO synthase (cNOS) inhibitor,l-NAME (75 mg kg−1, i.p.), the selective inducible NOS (iNOS) inhibitor, aminoguanidine (1 mg kg−1, i.p.), the selectiveinducible cyclooxygenase (COX-2) inhibitor, DFU (1 mg kg−1, i.p.), or the selective polyADP-ribose synthase (PARS) inhibitor, 3-aminobenzamide (0.1 mg kg−1,i.p.), in the endotoxin (10 mg kg−1, i.p.)-induced decrease in kidney (panel A), heart (panel B), lung (panel C) or brain (panel D) malondialdehyde (MDA) levels inBalb/c mice. Drug-injected mice were sacrificed 6 h after following by the hot plate test and kidneys, heart, lungs and brain were collected. The supernatants fromorgan homogenates were used for the measurement of MDA levels. The results are expressed as the mean± S.E.M. from 6 to 11 mice per treatment group.+P < 0.05vs. saline-treated group.* P < 0.05 vs. endotoxin-treated group.

COX enzymes. In this study, the endotoxin-induced inflamma-tory hyperalgesia and decreased levels of serum nitrite wereassociated with increased levels of serum PGF1� prevented byl-arginine or aminoguanidine. Since under inflammatory condi-tions, both iNOS and COX-2 expression are up-regulated by pro-inflammatory cytokines, indicating increased levels of both NOand prostaglandins (for reviews, see[31,32]), it is not surprisingthat there are interactions between iNOS and COX-2 pathways.Several studies using NOS inhibitors and NO scavengers have

demonstrated that NO stimulates the formation of prostaglandinsin in vitro [53,54]and in vivo[8,20,55–58]. On the other hand,NOS inhibitors also act to increase COX-2 expression andprostaglandin synthesis in response to cytokine stimulation andthe addition of NO donors reverses the effect suggesting NO alsoinhibits COX-2 activity[59–61]. NO has also shown to activatesCOX-1, but inhibits COX-2-derived prostaglandin productionin vitro [62]. Furthermore, some studies suggest that there is nointeraction between NO and prostaglandin synthesis[63–65].


Fig. 10. Proposed mechanism for the involvement of nitric oxide (NO) derivedfrom constitutive NOS (cNOS) and/or inducible NOS (iNOS) to the endotoxin-induced inflammatory hyperalgesia via interacting with nuclear factor-�B (NF-�B), constitutive cyclooxygenase (COX-1) and/or inducible COX (COX-2)or polyADP-ribose synthase (PARS). Increased activity of NF-�B, COX-1/COX-2 or PARS and decreased production of NO by cNOS/iNOS areresponsible for the endotoxin-induced inflammatory hyperalgesia associatedwith decrease in neutrophil infiltration, oxidative stress, lipid peroxidationand cellular damage. It should be noted that NO reverses the endotoxin-induced inflammatory hyperalgesia via inhibition of PGI2 production. Fur-thermore, any pharmacological approach which increases NO productionwould inhibit PGI2 production and prevents these effects of endotoxin. Theinvolvement of cNOS/iNOS, NF-�B, COX-1/COX-2 and PARS enzymes wasshown by using their inhibitors,l-NAME/aminoguanidine,N-acetylserotonin,indomethacin/DFU and 3-aminobenzamide, respectively. The role of NO wasalso determined by using its precursor,l-arginine.

It has been shown that prostaglandins inhibit[66–71], increase[39–41,69,71–74]or do not affect[53] NO production by iNOSin several in vitro and in vivo models. This wide variability inthe observations may result from the use of different cell linesand tissues, as well as differences in methods of cell activation or in inflammatory models. To insure that NO derived fromcNOS and/or iNOS reduces endotoxin-induced inflammatoryhyperalgesia via inhibition of PGI2 production, indomethacinand DFU were used as positive controls. Indomethacin or DFUalone had no effect on hot plate latency and basal levels of serumnitrite. Our results with selective or non-selective COX inhibitorsuggest that inhibition of COX-1 and/or COX-2 prevents theendototoxin-induced inflammatory hyperalgesia by increasingsystemic and tissue NO production. Selective COX-2 inhibitionalso caused an increased production of nitrite in kidney, heartlung and brain without affecting the endotoxin-induced levelsof serum 6-keto-PGF1�. Therefore, it can be concluded that NOmediates to the analgesic effects of selective or non-selectivCOX inhibitors. The findings are also consistent with the stud-ies in endotoxemic mice[39–41]showing that PGI2 produced byCOX-2 and, in part by COX-1, appear to increase the systemicoverproduction of NO by cNOS and/or iNOS whereas tissueNO production appears to be decreased by PGI2 derived fromCOX-2 in this inflammatory hyperalgesia model in mice. There-fore, it can be concluded that NO derived from cNOS and/oriNOS reduces endotoxin-induced inflammatory hyperalgesia via

inhibition of PGI2 production. Our results with endotoxin andl-arginine also suggest that NO inhibits COX activity and sys-temic PGI2 production during inflammatory process. On theother hand, selective inhibition of iNOS activity by aminoguani-dine causes a decrease in systemic PGI2 production suggestingthe increasing effect of NO on COX activity.

NO has major interactions with this pathway of gene expres-sion controlled by NF-�B in inflammatory conditions. Bothactivation and inhibition of NF-�B activity by NO have also beendescribed (for review, see[18]). In this study, the endotoxin-induced inflammatory hyperalgesia associated with decreasedlevels of serum nitrite was prevented byN-acetylserotonin.Selective NF-�B inhibition also caused an increased productionof nitrite in brain without affecting the endotoxin-induced levelsof kidney, heart and lung nitrite and serum 6-keto-PGF1�. N-acetylserotonin alone had no effect on hot plate latency and basallevels of serum nitrite. These results suggest that increased activ-ity of NF-�B contributes to the endotoxin-induced inflammatoryhyperalgesia. In consistent with previous studies[36], it is alsopossible that NO may cause inhibitory effect on NF-�B. On theother hand, in contrast to previous results, our data suggest thatinhibition of NF-�B pathway prevents the endototoxin-inducedinflammatory hyperalgesia by increasing systemic and brain NOproduction.

Considering the potential importance of PARS activationduring NO-mediated apoptosis and the role of peroxynitrite,w adeo ide,i revi-o localor eralr ide,h ioni l.[ g 3-a , didn andr d bye toryh nitritew hibi-t ney,h cedl .1,1 ncya midea outa inhi-b toryh ction.

onn ns auset llulare sev-e ide

-

,

e

e also investigated the effect of pharmacological blockf PARS activity by a selective inhibitor, 3-aminobenzam

n endotoxin-induced inflammatory hyperalgesia. It has pusly been shown that 3-aminobenzamide decreases ther systemic inflammatory response in endotoxin-[75] or car-ageenan[44,76]-induced inflammation. There are also seveports showing that PARS inhibitors, e.g. 3-aminobenzamave inhibitory effect on iNOS induction and NO product

n in vitro [77,78] and in vivo[76,79–81]studies. Wray et a82] have also been shown that PARS inhibitors, includinminobenzamide, nicotinamide, 1,5-dihydroxyisoquinolineot reduce the circulatory failure, the renal dysfunctionise in lactate levels, or the overproduction of NO causendotoxin. In this study, the endotoxin-induced inflammayperalgesia associated with decreased levels of serumas prevented by 3-aminobenzamide. Selective PARS in

ion also caused an increased production of nitrite in kideart, lung and brain without affecting the endotoxin-indu

evels of serum 6-keto-PGF1�. 3-Aminobenzamide alone at 00 or 100 mg kg−1 doses had no effect on hot plate latend basal levels of serum nitrite. However, 3-aminobenzat 1 mg kg−1 dose increased the basal pain sensitivity withffecting levels of serum nitrite. These results suggest thatition of PARS prevents the endototoxin-induced inflammayperalgesia by increasing systemic and tissue NO produ

There are conflicting reports showing the effect of NOeutrophil infiltration and lipid peroxidation[83,84]. It has beehown that excessive NO and especially peroxynitrite may cissue damage, e.g. through lipid peroxidation and/or cenergy depletion induced by activation of PARS. There areral reports showing the inhibitory effect of 3-aminobenzam


on lipid peroxidation[80,81]. Requintina and Oxenkrug[43]demonstrated that lipid peroxidation, as measured by MDA + 4-hydroxyalkenals levels in the brain and liver but not kid-ney homogenates in mice was increased after administrationof endotoxin and a single dose ofN-acetylserotonin simul-taneously injected with endotoxin markedly protected micefrom the lethal effect of endotoxin in mice. In this study,N-acetylserotonin,l-arginine,l-NAME, aminoguanidine, DFU or3-aminobenzamide at doses that prevent the endotoxin-induceddecrease in systemic NO production, reversed the endotoxin-induced decrease in the MPO activity or the MDA levels inkidney, heart, lung or brain. Therefore, it can be concluded thatendotoxin decreases tissue neutrophil infiltration and lipid per-oxidation prevented by NO. These results are consistent withthe observation that NO behaves as a potent prooxidant[83,84].Moreover, NO does not seem to have a role as a proinflammatorymediator in this inflammatory hyperalgesia model in mice.

In conclusion, NO reverses endotoxin-induced inflamma-tory hyperalgesia via inhibition of PGI2 production, andalso contributes to the analgesic effect of NF-�B, COX orPARS inhibitors. Furthermore, consideration of the interactionbetween NO and NF-�B, COX-2 or PARS pathways may helpin the design of new therapeutic strategies to patients withinflammatory hyperalgesia. The anti-nociceptive effect ofl-arginine/NO/cGMP pathway enhances the importance of NOfor treatment of systemic inflammatory disorders with pain.

A

key(

R

aniand

er-acol

andhem

istol

on-nts

ti-

emica

ideino

ffectrats.

[ -uced

[11] Larson AA, Kovacs KJ, Cooper JC, Kitto KF. Transient changes inthe synthesis of nitric oxide result in long-term as well as short-term changes in acetic acid-induced writhing in mice. Pain 2000;86:103–11.

[12] Guney HZ, Gorgun CZ, Tunctan B, Uludag O, Hodoglugil U, AbaciogluN, et al. Circadian-rhythm-dependent effects ofl-NG-nitroargininemethyl ester (l-NAME) on morphine-induced analgesia. Chronobiol Int1998;15:283–9.

[13] de Moura RS, Rios AA, Santos EJ, Nascimento AB, de Castro ResendeA, Neto ML, et al. Role of the NO-cGMP pathway in the systemicantinociceptive effect of clonidine in rats and mice. Pharmacol BiochemBehav 2004;78:247–53.

[14] Luo ZD, Cizkova D. The role of nitric oxide in nociception. Curr RevPain 2000;4:459–66.

[15] Vanegas H. To the descending pain-control system in rats, inflammation-induced primary and secondary hyperalgesia are two different things.Neurosci Lett 2004;361:225–8.

[16] Blantz RC, Munger K. Role of nitric oxide in inflammatory conditions.Nephron 2002;90:373–8.

[17] Tunctan B, Altug S. The use of nitric oxide synthase inhibitors in inflam-matory diseases: a novel class of anti-inflammatory agents. Curr MedChem: Anti-Inflammatory Anti-Allergy Agents 2004;3:271–301.

[18] Feihl F, Oddo M, Waeber B, Liaudet L. Inhibitors of nitrogen oxidespecies production in animal models of inflammation and future direc-tions for therapy in inflammatory disorders. Curr Med Chem Anti-Inflammatory Anti-Allergy Agents 2004;3:239–59.

[19] Tegeder I, Del Turco D, Schmidtko A, Sausbier M, Feil R, HofmannF, et al. Reduced inflammatory hyperalgesia with preservation of acutethermal nociception in mice lacking cGMP-dependent protein kinase I.Proc Natl Acad Sci USA 2004;101:3253–7.

[20] Dudhgaonkar SP, Kumar D, Naik A, Devi AR, Bawankule DU, Tandanase-acol

[ if-asescience

[ lnooratorycology

[ bedianlan-Res

[ ucedRes

[ al.pon-41.

[ l ofmice.

[ mod-n Res

[ ur SJ,uced

Neu-

[ ffectdmin-Pain

[ edianuced

cknowledgement

This work was supported by Novartis, Istanbul, Tur2002–2004).

eferences

[1] Laroux FS, Pavlick KP, Hines IN, Kawachi S, Harada H, BharwS, et al. Role of nitric oxide in inflammation. Acta Physiol Sc2001;173:113–8.

[2] Guzik TJ, Korbut R, Adamek-Guzik TJ. Nitric oxide and supoxide in inflammation and immune regulation. Physiol Pharm2003;54:469–87.

[3] Cuzzocrea C. Effect of inhibitors of nitric oxide in animal modelsfuture directions for therapy in inflammatory disorders. Curr Med CAnti-Inflammatory Anti-Allergy Agents 2004;3:261–70.

[4] Govers R, Oess S. To NO or not to NO: ’where?’ is the question. HHistopathol 2004;19:585–605.

[5] Fleming I, Busse R. The physiology of nitric oxide: control and csequences. Curr Med Chem Anti-Inflammatory Anti-Allergy Age2004;3:189–205.

[6] Wu CC. Nitric oxide and inflammation. Curr Med Chem: AnInflammatory Anti-Allergy Agents 2004;3:217–22.

[7] Dedon PC, Tannenbaum SR. Reactive nitrogen species in the chbiology of inflammation. Arch Biochem Biophys 2004;423:12–22.

[8] Park YH, Shin CY, Lee TS, Huh IH, Sohn UD. The role of nitric oxand prostaglandin E2 on the hyperalgesia induced by excitatory amacids in rats. J Pharm Pharmacol 2000;52:431–6.

[9] Chen SR, Pan HL. Spinal nitric oxide contributes to the analgesic eof intrathecal [d-pen2, d-pen5]-enkephalin in normal and diabeticAnesthesiology 2003;98:217–22.

10] Abacioglu N, Tunctan B, Akbulut E, C¸ akici I. Participation of the components of L-arginine/nitric oxide/cGMP cascade by chemically-indabdominal constriction in the mouse. Life Sci 2000;67:1127–37.

l

SK. Interaction of inducible nitric oxide synthase and cyclooxygen2 inhibitors in formalin-induced nociception in mice. Eur J Pharm2004;492:117–22.

21] Tao F, Tao YX, Zhao C, Dore S, Liaw WJ, Raja SN, et al. Dferential roles of neuronal and endothelial nitric oxide synthduring carrageenan-induced inflammatory hyperalgesia. Neuros2004;128:421–30.

22] Kanaan SA, Safieh-Garabedian B, Haddad JJ, Atweh SF, AbdeAM, Jabbur SJ, et al. Effects of various analgesic and anti-inflammdrugs on endotoxin-induced hyperalgesia in rats and mice. Pharma1997;54:285–97.

23] Saade NE, Abou Jaoude PG, Saadeh FA, Hamoui S, Safieh-GaraB, Kanaan SA, et al. Fos-like immunoreactivity induced by intraptar injection of endotoxin and its reduction by morphine. Brain1997;769:57–65.

24] Cahill CM, Dray A, Coderre TJ. Priming enhances endotoxin-indthermal hyperalgesia and mechanical allodynia in rats. Brain1998;808:13–22.

25] Shavit Y, Cohen E, Gagin R, Avitsur R, Pollak Y, Chaikin G, etEffects of prenatal morphine exposure on NK cytotoxicity and ressiveness to LPS in rats. Pharmacol Biochem Behav 1998;59:835–

26] Raghavendra V, Agrewala JN, Kulkarni SK. Melatonin reversalipopolysacharides-induced thermal and behavioral hyperalgesia inEur J Pharmacol 2000;395:15–21.

27] Kawashima N, Fugate J, Kusnecov AW. Immunological challengeulates brain orphanin FQ/nociceptin and nociceptive behavior. Brai2002;949:71–8.

28] Safieh-Garabedian B, Poole S, Haddad JJ, Massaad CA, JabbSaade NE. The role of the sympathetic efferents in endotoxin-indlocalized inflammatory hyperalgesia and cytokine upregulation.ropharmacology 2002;42:864–72.

29] Kehl LJ, Kovacs KJ, Larson AA. Tolerance develops to the eof lipopolysaccharides on movement-evoked hyperalgesia when aistered chronically by a systemic but not an intrathecal route.2004;111:104–15.

30] Talhouk RS, Saade NE, Mouneimne G, Masaad CA, Safieh-GarabB. Growth hormone releasing hormone reverses endotoxin-ind


localized inflammatory hyperalgesia without reducing the upregulatedcytokines, nerve growth factor and gelatinase activity. Prog Neuropsy-chopharmacol Biol Psychiatry 2004;28:625–31.

[31] Perez-Sala D, Lamas S. Regulation of cyclooxygenase-2 expression bynitric oxide in cells. Antioxid Redox Signal 2001;3:231–48.

[32] Turini ME, DuBois RN. Cyclooxygenase-2: a therapeutic target. AnnuRev Med 2002;53:35–57.

[33] Goodwin DC, Landino LM, Marnett LJ. Effects of nitric oxide andnitric oxide-derived species on prostaglandin endoperoxide synthase andprostaglandin biosynthesis. FASEB J 1999;13:1121–36.

[34] Katsuyama K, Shichiri M, Marumo F, Hirata Y. NO inhibits cytokine-induced iNOS expression and NF-kappaB activation by interfering withphosphorylation and degradation of IkappaB-alpha. Arterioscler ThrombVasc Biol 1998;18:1796–802.

[35] Peng HB, Spiecker M, Liao JK. Inducible nitric oxide: an autoreg-ulatory feedback inhibitor of vascular inflammation. J Immunol1998;161:1970–6.

[36] Kang JL, Park W, Pack IS, Lee HS, Kim MJ, Lim CM, et al.Inhaled nitric oxide attenuates acute lung injury via inhibition ofnuclear factor-kappa B and inflammation. J Appl Physiol 2002;92:795–801.

[37] Skidgel RA, Gao XP, Brovkovych V, Rahman A, Jho D, Predescu S, etal. Nitric oxide stimulates macrophage inflammatory protein-2 expres-sion in sepsis. J Immunol 2002;169:2093–101.

[38] Bloodsworth A, O’Donnell VB, Freeman BA. Nitric oxide regulationof free radical- and enzyme-mediated lipid and lipoprotein oxidation.Arterioscler Thromb Vasc Biol 2000;20:1707–15.

[39] Tunctan B, Uludag O, Altug S, Abacioglu N. Effects of nitric oxide syn-thase inhibition in lipopolysaccharide-induced sepsis in mice. PharmacolRes 1998;38:405–11.

[40] Tunctan B, Altug S, Uludag O, Abacioglu N. Time-dependent vari-eRes

[ ofin a

[ s in

[ ridero-

Sci

[ aboDP-ation

[ ofdye

[ G,clinActa

[ rk:

[ In:ork:

[ theInt

[ al.biol

[ licgenic

[52] Tunctan B, Weigl Y, Dotan A, Peleg L, Zengil H, Ashkenazi I, et al.Circadian variation of nitric oxide synthase activity in mouse tissue.Chronobiol Int 2002;19:393–4.

[53] Salvemini D, Misko TP, Masferrer JL, Seibert K, Currie MG, NeedlemanP. Nitric oxide activates cyclooxygenase enzymes. Proc Natl Acad SciUSA 1993;90:7240–4.

[54] Watkins DN, Garlepp MJ, Thompson PJ. Regulation of the induciblecyclo-oxygenase pathway in human cultured airway epithelial (A549)cells by nitric oxide. Br J Pharmacol 1997;121:1482–8.

[55] Sautebin L, Di Rosa M. Nitric oxide modulates prostacyclin biosynthe-sis in the lung of endotoxin-treated rats. Eur J Pharmacol 1994;262:193–6.

[56] Salvemini D, Settle SL, Masferrer JL, Seibert K, Currie MG. Regulationof prostaglandin production by nitric oxide; an in vivo analysis. Br JPharmacol 1995;114:1171–8.

[57] Soler M, Camacho M, Molins-Pujol AM, Vila L. Effect of an imidazo-lineoxyl nitric oxide on prostaglandin synthesis in experimental shock:possible role of nitrogen dioxide in prostacyclin synthase inactivation. JInfect Dis 2001;183:105–12.

[58] Toriyabe M, Omote K, Kawamata T, Namiki A. Contribution of inter-action between nitric oxide and cyclooxygenases to the productionof prostaglandins in carrageenan-induced inflammation. Anesthesiology2004;101:983–90.

[59] Swierkosz TA, Mitchell JA, Warner TD, Botting RM, Vane JR. Co-induction of nitric oxide synthase and cyclo-oxygenase: interactionsbetween nitric oxide and prostanoids. Br J Pharmacol 1995;114:1335–42.

[60] Habib A, Bernard C, Lebret M, Creminon C, Esposito B, Tedgui A, etal. Regulation of the expression of cyclooxygenase-2 by nitric oxide inrat peritoneal macrophages. J Immunol 1997;158:3845–51.

[61] Amin AR, Vyas P, Aattur M, Leszczynska-Piziak J, Patel I, Weissmannible

[ etOX-nol

[ itricstratein Hophys

[ A.and

eukot

[ rmstionsacol

[ itricJ774.

[ meds and

[ or-path-de168–

[ en-95;49:

[ andts

ations in serum nitrite, 6-keto-prostaglandin F1� and thromboxanB2 levels induced by lipopolysaccharide in mice. Biol Rhythm2000;31:499–514.

41] Tunctan B, Altug S, Uludag O, Demirkay B, Abacioglu N. Effectscyclooxygenase inhibitors on nitric oxide production and survivalmice model of sepsis. Pharmacol Res 2003;1145:1–12.

42] Turner RA. Analgesics. In: Turner RA, editor. Screening methodpharmacology. New York: Academic Press; 1965. p. 100–32.

43] Requintina PJ, Oxenkrug GF. Differential effects of lipopolysacchaon lipid peroxidation in F344N, SHR rats and BALB/c mice, and ptection of melatonin and NAS against its toxicity. Ann NY Acad2003;993:325–33.

44] Cuzzocrea S, Zingarelli B, Gilad E, Hake P, Salzman AL, SzC. Protective effects of 3-aminobenzamide, an inhibitor of poly(Aribose)synthase in a carrageenan-induced model of local inflammEur J Pharmacol 1998;342:67–76.

45] Bradford MM. A rapid and sensitive method for the quantitationmicrogram quantities of protein utilizing the principle of protein-binding. Anal Biochem 1976;72:248–54.

46] Rosenkranz B, Fischer C, Reimann I, Weimer KE, BeckFrolich JC. Identification of the major metabolite of prostacyand 6-ketoprostaglandin F1 alpha in man. Biochim Biophys1980;619:207–13.

47] Golowich SP, Kaplan SD. Methods in enzymology, vol. II. New YoAcademic Press Inc.; 1955.

48] Yagi K. Lipid peroxides and related radicals in clinical medicine.Armstrong D, editor. Free radicals in diagnostic medicine. New YPlenum Press; 1994. p. 1–15.

49] Golombek DA, Agostino PV, Plano SA, Ferreyra GA. Signaling inmammalian circadian clock: the NO/cGMP pathway. Neurochem2004;45:929–36.

50] Uludag O, Tunctan B, Guney HZ, Uluoglu C, Altug S, Zengil H, etTemporal variation in serum nitrite levels in rats and mice. ChronoInt 1999;16:527–32.

51] Globig S, Witte K, Lemmer B. Urinary excretion of nitric oxide, cycGMP, and catecholamines during rest and activity period in transhypertensive rats. Chronobiol Int 1999;16:305–14.

.

G, et al. The mode of action of aspirin-like drugs: effect on inducnitric oxide synthase. Proc Natl Acad Sci USA 1997;92:7926–30.

62] Clancy R, Varenika B, Huang W, Ballou L, Attur M, Amin AR,al. Nitric oxide synthase/COX cross-talk: nitric oxide activates C1 but inhibits COX-2-derived prostaglandin production. J Immu2000;165:1582–7.

63] Curtis JF, Reddy NG, Mason RP, Kalyanaraman B, Eling TE. Noxide: a prostaglandin H synthase 1 and 2 reducing cosubthat does not stimulate cyclooxygenase activity or prostaglandsynthase expression in murine macrophages. Arch Biochem Bi1996;335:369–76.

64] Boquet M, Cebral E, Motta A, Beron de Astrada M, Gimeno MRelationship between mouse uterine contractility, nitric oxideprostaglandin production in early pregnancy. Prostaglandins LEssent Fatty Acids 1998;59:163–7.

65] Hamilton LC, Warner TD. Interactions between inducible isofoof nitric oxide synthase and cyclo-oxygenase in vivo: investigausing the selective inhibitors, 1400W and celecoxib. Br J Pharm1998;125:335–40.

66] Marotta P, Sautebin L, Di Rosa M. Modulation of the induction of noxide synthase by eicosanoids in the murine macrophage cell lineBr J Pharmacol 1992;107:640–1.

67] Raddasi K, Petit JF, Lemaire G. LPS-induced activation of primurine peritoneal macrophages is modulated by prostaglandincyclic nucleotides. Cell Immunol 1993;149:50–64.

68] Tetsuka T, Daphna-Iken D, Srivastava SK, Baier LD, Maine JD, Mrison AR. Cross-talk between cyclooxygenase and nitric oxideways: prostaglandin E2 negatively modulates induction of nitric oxisynthase by interleukin 1. Proc Nat Acad Sci USA 1994;91:1272.

69] Milano S, Arcoleo F, Dieli M, D’Agostino R, D’Agostino P, DNucci G, et al. Prostaglandin E2 regulates inducible nitric oxide sythase in the murine macrophage cell line J774. Prostaglandins 19105–15.

70] Pang L, Hoult JR. Repression of inducible nitric oxide synthasecyclooxygenase-2 by prostaglandin E2 and other cyclic AMP stimulanin J774 macrophages. Biochem Pharmacol 1997;53:493–500.


[71] Guhring H, Hamza M, Sergejeva M, Ates M, Kotalla CE, Ledent C, etal. Role for endocannabinoids in indomethacin-induced spinal antinoci-ception. Eur J Pharmacol 2002;454:153–63.

[72] Gaillard T, Mulsch A, Klein H, Decker K. Regulation by prostaglandinE2 of cytokine-elicited nitric oxide synthesis in rat liver macrophages.Biol Chem 1992;373:897–902.

[73] Hinz B, Brune K, Rau T, Pahl A. Flurbiprofen enantiomers inhibitinducible nitric oxide synthase expression in RAW 264.7 macrophages.Pharm Res 2001;18:151–6.

[74] Tunctan B, Yaghini FA, Estes A, Malik KU. Prostaglandins inhibitcytochrome P450 4A activity and contribute to endotoxin-inducedhypotension in rats via nitric oxide production. FASEB 2004;18:A1034–5.

[75] Szabo A, Salzman AL, Szabo C. Poly (ADP-ribose) synthetase activationmediates pulmonary microvascular and intestinal mucosal dysfunction inendotoxin shock. Life Sci 1998;63:2133–9.

[76] Szabo C, Lim LH, Cuzzocrea S, Getting SJ, Zingarelli B, Flower RJ,et al. Inhibition of poly(ADP-ribose)synthase exerts anti-inflammatoryeffects and inhibits neutrophil recruitment. J Exp Med 1997;186:1041–9.

[77] Szabo C, Zingarelli B, Salzman AL. Role of poly-ADP ribosyltrans-ferase activation in the vascular contractile and energetic failure elicitedby exogenous and endogenous nitric oxide and peroxynitrite. Circ Res1996;78:1051–63.

[78] Ibuki Y, Mizuno S, Goto R. Gamma-irradiation-induced DNA damageenhances NO production via NF-kappaB activation in RAW264.7 cells.Biochim Biophys Acta 2003;1593:159–67.

[79] Jijon HB, Churchill T, Malfair D, Wessler A, Jewell LD, Parsons HG, etal. Inhibition of poly(ADP-ribose) polymerase attenuates inflammationin a model of chronic colitis. Am J Physiol Gastrointest Liver Physiol2000;279:G641–51.

[80] Ozdulger A, Cinel I, Unlu A, Cinel L, Mavioglu I, Tamer L, etal. Poly(ADP-ribose) synthetase inhibition prevents lipopolysaccharide-induced peroxynitrite mediated damage in diaphragm. Pharmacol Res2002;46:67–73.

[81] Kiefmann R, Heckel K, Doerger M, Schenkat S, Kupatt C, StoeckelhuberM, et al. Role of PARP on iNOS pathway during endotoxin-inducedacute lung injury. Intensive Care Med 2004;30:1421–31.

[82] Wray GM, Hinds CJ, Thiemermann C. Effects of inhibitors ofpoly(ADP-ribose) synthetase activity on hypotension and multiple organdysfunction caused by endotoxin. Shock 1998;10:13–9.

[83] Hogg N, Kalyanaraman B. Nitric oxide and lipid peroxidation. BiochimBiophys Acta 1999;1411:378–84.

[84] d’Ischia M, Palumbo A, Buzzo F. Interactions of nitric oxide with lipidperoxidation products under aerobic conditions: inhibitory effects on theformation of malondialdehyde and related thiobarbituric acid-reactivesubstances. Nitric Oxide: Biol Chem 2000;4:4–14.

Nitric oxide reverses endotoxin-induced inflammatory hyperalgesia via inhibition of prostacyclin...

Documents

Transcript of Nitric oxide reverses endotoxin-induced inflammatory hyperalgesia via inhibition of prostacyclin...