L-NAME causes antinociception by stimulation of ... - Semantic Scholar

Research Paper Mediators of Inflammation, 9, 25 –30 (2000 )

NG -NITRO-L-A RG ININE m ethyl es te r (L-NAME ) h as been us ed ex te ns ive ly as a p aradigm atic in h ibitor of NO s yn th as e an d h as bee n s h ow n to cause an tin ocice ption in s e veral e x p erim e ntal m odels. We des cribe h er e h o w L-NAME pr o duced a dos e-dep e nden t an tin ocice ptive effe ct w h en in jected in trap e rito ne ally in the m ous e after ace tic acid in duce d w rith in gs , o r in trap lan tarly in th e rat p aw p r es s ure h yp eralges ia in duced by carragee nin or p ro s taglan din E2. In con tras t ano th er NO s ynth as e in h ibito r, NG -m o nom eth yl-L-ar gin in e (L-NMMA ), h ad n o s ign ifican t effe ct p er s e but in h ibited L-NAME s ys tem ic in duced an tin ocice ption in m ic e an d local in duced an tin ocice ption in th e rat p aw h yp er alges ia tes t. D-NAME h ad no an tino cic ep tive e ffe ct upo n carragee nin -in duced h yp e ralge s ia. Pre tr e atm en t of the p aw s w ith tw o in h ibitor s o f guan ylate cyclas e, m eth yle ne blue (MB) an d 1H-:[1,2,4]-ox adiazo lo-:[4,3-a] quin ox alin –1-o ne (ODQ) abolis h ed th e an tin ocice p tive effe ct of L-NAME. L-Ar gin in e an d the cGMP p h o s p h o dies te ras e in h ibitor, MY 5445 s ignificantly en h an ce d th e L-NAME an tino cic ep tive effect. Th e cen tral an tin ocice p tive effe ct of L-NAME w as blocked by co- adm in is tration of L-NMMA, ODQ and MB. Th e p re s en t s erie s of ex p er im en ts s h ow s th at L-NAME, but no t L-NMMA, h as an an tino cic ep tive effect. It can be s ugge s ted th at L-NAME cause s th e an tino cic ep tive effect by s tim ulation o f th e ar gin in e / NO/ cGMP p ath w ay, s in ce the an tino cic ep tive effe ct of L-NAME can be an tago nized by L-NMMA and abolis h ed by the guanylate cyclas e in h ibitor s (MB and ODQ). In addition , th e NO s ynthas e s ubs trate, L-ar gin in e and th e cGMP p h o s p h odie s teras e in h ibito r, MY 5445 w e re s e en to p oten tiate the effects of L-NAME. Th us, L-NAME us ed alon e, h as lim itation s as a s p e cific in h ibitor o f the ar ginin e-NOcGMP p athw ay an d m ay th er efo r e be a po or p h ar m acological to ol for use in ch aracteris in g p ar ticip ation in p ath op h ys io logical p r oces s es .

L-NAME causes antinociception by stimulation of the arginine-NO-cGMP pathway I. D. G. Duarte1 and S. H. Ferreira2,C A

1

Departamento de Farmacologia, Instituto de Ciencias ˆ Biomedicas ´ –UFMG, CEP 31270 –901, Belo Horizonte, MG, Brazil; and 2 Departamento de Farmacologia, Faculdade de Medicina de Ribeirao ˜ Preto–USP, CEP 14049 –900, Ribeirao ˜ Preto, SP, Brazil

CA

Corresponding Author Te l/fax : (+55 ) 16 6232792 Email: shfe rre [email protected]

Key w o r ds: Carrage enin analgesia, NG-nitro-L-arginine methyl ester (L-NAME), Nitric ox ide (NO), Guanosine 3 9 ,59 -monophophate c GMP, 1H-[1,2,4]-ox adiazolo-[4,3-a] quinox alin–1-one (ODQ), Inducible NO synthase (iNOS), Methyle ne blue (MB), NG-monomethyl-L-arginine (L-NMMA)

Introduction L-NAME (NG-nitro-L-arginine me thyl e ste r ), has be en show n to cause antinocic eption by spinal, supraspinal, loc al (intraplantar ) or systemic administration.1 – 8 As L-NAME is c onside red an spec ific nitric ox ide (NO) synthase inhibitor,2 ,24 the se ex periments w ere taken to supp ort the hypothe sis that stimulation of the arginine /NO/c GMP pathw ay e nhance s nocice ption at various leve ls of the sensory syste m. On the othe r hand, there are several reports indicating that choline rgic or opioidergic stimulation of the

arginine /NO/c GMP pathw ay c ause s ce ntral, spinal or peripheral analge sia,9 –1 2 and some pe riphe ral analgesic s cause antinoc ice ption by stimulation of this pathw ay. 13 –1 6 Furthermore, the central analge sic, arginine , see ms to be associated w ith NO-c GMP stimulation.17 –1 9 Thus there appe ars to be an appare nt contradic tion amongst the various ex pe riments made to asce rtain the role of the arginine /NO/c GMP pathw ay in nocice ption. A gre at de al of information has be en derive d w ith the use of L-NAME as a me thodological tool. How ever L-NAME can be se en to be either analge sic or hype ralgesic in the same te st2 0

ISSN 0962-9351 print/ISSN 1466-1861 online/00/010025-6 © 2000 Taylor & Francis Ltd

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I. D. G. Du a rte a n d S. H. Fe rre ira

w hilst another NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA) doe s not show antinocic eption in the same test as L-NAME, i.e. the formalin te st in mice.2 Re cently, L-NAME has be en reported to stimulate inducible NO synthase (iNOS) gene ex p re ssion.2 1 Be c ause of these apparent contradic tory results, w e e valuate d the possibility that the analge sic effect of L-NAME w as due to stimulation of the arginine /NO/c GMP pathw ay. In the present study L-NAME w as initially assayed in tw o te sts in w hich nocic eption involves an inflammatory stimulus: the acetic acid induc ed w rithings in mic e and in the rat paw pre ssure hyperalgesia te st induc ed by intraplantar administration of c arragee nin. In these te sts L-NAME show ed an antinoc ice ptive effe ct w hich w as significantly inhibited by pre treatme nt of the animals w ith L-NMMA. To furthe r inve stigate the antinoc ice ptive e ffec t of L-NAME and in order to avoid the oedema formation and fac ilitate succ essive injections of drugs, PGE2 instead of c arragee nin w as used to induc e hyp eralge sia in the rat paw pre ssure test. In this te st the peripheral antinocic eptive synergism betw e en L-NAME and the NO synthase substrate, arginine, and the cGMP phosphodie ste rase inhibitor, as w ell the effect of tw o inhibitors of guanylate c GMP activation, me thyle ne blue (MB) and 1H-[1,2,4]-ox adiazolo[4,3-a] quinox alin–1-one (ODQ) w as evaluate d. Finally, since L-NAME has be en show n to cause analgesia by intrac erebrove ntric ular administration,2 w e te ste d L-NAME c o-administration w ith inhibitors of the arginine /NO/c GMP pathw ay, L-NMMA, MB and ODQ.

Materials and Methods Animals The ex perime nts w ere performed on male Wistar rats (150–180 g ) and albino Sw is s mice (22–30 g ). The animals w ere house d unde r natural light, w ith free acce ss to food and w ater. Intrace re broventric ular (i.c.v.) inje ctions in rats w e re made follow ing the me thod described by Cô rre a and Grae ff.22 When single doses of the various drugs w ere used, the y w ere base d on dose resp onse pilot ex p eriments. All ex pe rimental procedures c onformed to the IASP guide line s on the use of animals in pain re search. Rats w ere use d once only. Nociception tests (a ) Writh ing te s t in m ice . This test w as based on the frequenc y of abdominal contortions e voked by an intraperitone al inje ction of 10 ml/kg of 0.6% ac etic acid.8 L-NAME, L-arginine , or vehic le w as injec te d 15 min before ac etic acid administration, and the number of w rithing e vents w ere 26

Mediators of Inflammation · Vol 9 · 2000

counte d for 20 min afte r the noc iceptive challe nge. For antagonism studie s, mice w e re treate d as above, ex c ept that L-NMMA w as administe red 15 min be fore L-NAME. (b ) Hin d pa w hy p e ra lge s ia te s t in ra ts . Our modification of the Randall-Selitto rat paw pressure test w as use d to me asure hyperalge sia.23 In the te st, a p re ssure of 20 mmHg is c ontinuously applie d to the hind paw of the rat until the animal presents a typic al free zing re ac tion (reaction time ). After me asurement of the basal reaction time (c ontrol), hyperalge sia w as induce d eithe r by an intraplantar injection of carrage enin (Cg, 100 m g ) or PGE2 . The inte nsity of hyperalge sia w as quantified as the diffe rence in reaction time (de lta reaction time ) me asure d 3 h after administration of Cg, from the c ontrol reaction time assessed be fore injection of the hyp eralge sic stimulus. The term nocic eption is use d in this paper to de scribe the presenc e of an overt standard behaviour induc ed by the applic ation of a nox ious stimulus in a normal tissue or a non-nox ious stimulus in previously sensitised tissue. The te rm hype ralgesia is used w he n a non-nox ious stimulus causes nocic eption w hen applie d to a se nsitized tissue e ithe r by an inflammatory stimulus like c arragee nin or an hype ralge sic mediator like prostaglandin E2. Drugs Carragee nin (Viscarin ) w as purchased from Marine Colloids, EUA. L-NAME (N-nitro-L-arginine methyl este r, Wellcome , UK) and L-NMMA (NG-monomethylL-arginine ace tate ) w ere purchased from Sigma (St Louis, MO, USA) and me thyle ne blue (MB) w as from Reage n (Brazil ) and ODQ (1H-[1,2,4]-ox adiazolo[4,3-a] quinox alin –1-one ) w as from Tocris Cookson Inc . (St Louis, USA). Statistics All results are presente d as means (SEM of five rats or six to 12 mic e pe r group. Re sults are prese nted as me ans and standard e rrors of the means of groups of at least five animals in e ach group. Diffe re nce s betw e en re sponses w e re evaluate d by ANOVA, follow ed by the Bonferroni t-te st. Results w ith P < 0.05 w ere c onside re d significant.

Results Blockade of L-NAME effect by L-NMMA in two tests of nociception induced by inflammatory stimuli (a) Mic e w rithing te st: nocic eptive be haviour induce d by intrape ritoneal ac etic ac id administration.L-NAME administe re d intrape ritoneally re duc ed the numbe r of abdominal c onstrictions in a dose-de pende nt manner

L-NAME ca u s e s a n tin o ciceptio n by s tim u la tio n o f th e a rg in in e-NO-cGMP pa th w a y

by approx imate ly 40% and 73% at dose s of 30 and 90 mg /kg, respec tively. L-NMMA (10 mg /kg i.p.), significantly blocked the antinoc ic eptive action of L-NAME, but had no effect on its ow n (Fig. 1 ). (b ) Ra t p a w pre s s u re te s t: hy pe ra lg e s ia indu ce d by c a rra g e e n in infla m m a tio n . Intraplantar administration of L-NAME (50 and 300 m g ) produce d a signific ant inhibition of carrage enininduc ed hype ralgesia of up to 40% for the highest dose used (Fig. 2 ). L-NAME-induce d p eriphe ral hyperalgesia w as signific antly inhib ited by pre-treatment of the paw s w ith 100 m g of L-NMMA or 500 m g of MB. Neither MB, nor L-NMMA (up to 500 m g /paw ) had hyp eralgesic effects. Rat paw pressure test: hyperalgesia induced by PGE2 FIG. 1. Inhibition by L-NMMA (10 mg/kg, i.p.) of the antinociceptive effect of intraperitoneal administration of L-NAME on the acetic acid writhing test in mice. The symbols are the mean ± SEM of 6–12 mice/group. *indicates significant differences (P < 0.05) in comparison with the control L-NAME group treated with intraperitoneal saline.

FIG. 2. Antinociceptive effect of intraplantar administration of L-NAME but not of D-NAME on carrageenin-induced hyperalgesia and the blockade of antinociception by local L-NMMA and methylene blue (MB) co-administration. L-NMMA (100 m g/paw), MB (500 m g/paw) or saline (S) were injected 30 min before carrageenin (Cg, 100 m g/paw). L-NAME or D-NAME (50–300 m g/paw) were also injected 30 min before Cg, and the intensity of hyperalgesia was measured 3 h after the hyperalgesic stimulus (see injections diagram). The symbols are the mean + SEM of five rats/ group. *indicates significant differences (P < 0.01) in comparison with the control (saline, O) or treatment with L-NAME, MB, D-NAME.

(a ) An ta go n is m L-NMMA, MB o r ODQ o f L-NAME-ind u c e d a n tin o c ice ptio n . Pilot ex perime nts show ed that doses higher than 200 m g /paw w ere needed to give signific ant antinocic eption. Fig. 3 show s a signific ant antinocice ptive

FIG. 3. Blockade by L-NMMA (LN) and guanylate cyclase inhibitors (MB and ODQ) of the antinociceptive effect of L-NAME on PGE2 induced hyperalgesia. PGE2 was injected at time zero, saline (S) or LN, MB,ODQ, at time 1.5 h and L-NAME or S 2 h after PGE2 . The insert shows the antinociceptive effect of L-NAME (60 = a, 180 = b and 300 m g per paw = c).The bars and symbols are the mean ± SEM of five rats/group. The asterisks mean significant differences (P < 0.05): (a) *in comparison with the control PGE2 ; (b) **in comparison with L-NAME-treated groups. Mediators of Inflammation · Vol 9 · 2000

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I. D. G. Du a rte a n d S. H. Fe rre ira

FIG. 4. Antinociceptive synergism between L-NAME and L-arginine. The measurements were made 3 h after the intraplantar injections of PGE2 (100 ng/paw). L-NMMA, L-Arginine (L-Arg) and L-NAME were injected into the paw 1, 1.5 and 2 h after the PGE2 challenge, respectively. The bars are the mean ± SEM of five rats/group. The asterisks mean significant differences (P < 0.05): (a) *in comparison with the control PGE2 treated either with L-arg, L-NAME, or saline (S); (b) **in comparison with the group which received L-NMMA + L-arg + L-NAME.

FIG. 5. Potentiation of intraplantar injections of L-NAME by the cGMP phosphodiesterase inhibitor, MY 5445. The measurements were made 3 h after the intraplantar injections of PGE2 (100 ng/paw). MY 5445 and (or) L-NAME were injected into the paw 1 and 2 h after PGE2 , respectively. The bars are the mean ± SEM of five rats/group. The asterisk means significant differences (P < 0.05) compared with respective doses of L-NAME. There was no significant differences between the controls groups (C) treated with saline or MY 5445.

effect upon hyp eralgesia induc ed by PGE2 of a dose of 300 m g of L-NAME. Pre treatme nt of the paw s w ith L-NMMA (LN), MB or ODQ prevented the antinocic eptive effe ct of L-NAME. (b ) Arg inine a n d MY 5445 e n ha n c e m e n t o f L-NAME e ffe cts . Figure 4 show s that the assoc iation of L-arginine and L-NAME treatments c ause d a significant antinocic eption compared w ith single tre atments. The re w as no difference among controls groups. L-NMMA significantly inhibited the antinocic eptive effect of the association of L-arginine and L-NAME treatment. The association of the same doses of L-NAME and D-arginine did not cause antinocic eption as c ompare d w ith the single tre atment (data not show n ). Figure 5 show s that MY 5445 enhanc ed, in a doserelated manne r, the antinocic eptive e ffec t of L-NAME. A dose of 180 m g, w hich did not p roduc e antinocic eption in our ex pe riments, p roduc ed an effe ct similar to 300 m g w he n the paw s w e re pretreated w ith MY 5445 (compare w ith Fig. 3 ). (c ) Blo c k a de o f th e intra c e re bro ve n tricu la r a n tin o c iceptive e ffe c t o f L-NAME by c o -tre a tm e n t w ith L-NMMA, MB o r ODQ. L-NAME, w he n administe red i.c .v. at a dose of 300 m g per rat, produc ed pote nt antinocic eption in paw s rendere d hyperalgesic by PGE2 (Fig. 6 ). This L-NAMEinduc ed anti-hype ralgesic e ffec t w as abolishe d by coi.c.v. administration of L-NMMA (300 m g ), MB (400 m g ) and ODQ (8 m g ). Neither L-NMMA, nor MB, 28


FIG. 6. Antinociceptive effect of intracerebroventricular administration of L-NAME (NG -nitro-L-arginine methyl ester) on PGE2 -induced hyperalgesia, and its blockade by i.c.v. L-NMMA (LN, 300 m g,), MB (400 m g) or ODQ (8 m g). These drugs and vehicle were co-injected with L-NAME 2 h after PGE2 (100 ng/paw). The intensity of hyperalgesia was measured 1 h after the i.c.v. injections. The bars are the mean, SEM of five rats/group. *,**mean significant differences (P < 0.01) in comparison with the saline- (Sal) or L-NAME-treated group, respectively.

nor ODQ had hyperalge sia.

any

effe cts

on PGE2

induced

Discussion L-NAME has be en show n by se ve ral laboratorie s 1– 8 to have a pe rip heral and central antinocice ptive action.

L-NAME ca u s e s a n tin o ciceptio n by s tim u la tio n o f th e a rg in in e-NO-cGMP pa th w a y

He re, w e confirm the ir obse rvations using tw o te sts of noc ice ption. In the ac etic acid w rithing te st, systemic administration (i.p .) of L-NAME but not L-NMMA caused an antinocic eptive e ffec t (Fig. 1 ). In addition, w e show ed that L-NMMA inhibite d the antinocic eptive ac tivity of L-NAME in this test as w e ll as in the rat paw p re ssure te st in w hich hype ralgesia w as induce d e ithe r by an inflammatory stimuli like Cg or by an hyperalgesic mediator, PGE2 (Figs 2 –4 ). The D-is omer of NAME show e d no antinoc icep tive ac tivity (Fig. 2 ) in c arragee nin-induc ed hype ralgesia in the rat pressure te sts (Fig. 2 ). It has already be en show n that this is ome r had no antinoc ice ptive effe ct in other te sts .2 Co-inje ction of L-NMMA i.c .v. also inhibited the ce ntral analge sic action of L-NAME (Fig. 6 ). The observed antinocic eptive effe ct of L-NAME appare ntly supports the ide a that the arginine /NO/ cGMP pathw ay c ontributes to nocice ption induce d by inflammatory stimuli, partic ularly bec ause L-NAME is c onside red to be a sele ctive NO synthase inhibitor. 2,2 4 L-arginine -derive d inhibitors, how e ver, have bee n found to have bizarre pharmacologic al e ffec ts. For instance , it has be en de monstrated that L-NAME is a poor inhibitor of L-arginine transport, w hereas L-NMMA and L-NIO substantially inhibit this e ve nt.2 5 Moreove r, L-NAME, but not L-NMMA, is a muscarinic antagonist.26 On the othe r hand, L-NMMA, has be en show n to be have as a partial agonist, sinc e it antagonize s NO synthesis in some tissue s, but stimulate s NO synthesis in is olated arte rial rings.27 Since, in our ex periments, L-NMMA in the doses used did not show any effe ct pe r se, but blocked the antinocic eptive effe ct of L-NAME, w e assume that it may be acting as a NO synthase inhibitor. On the othe r hand, L-NAME may be ac ting either as a substrate for or as an iNOS stimulator. It is know n that during c arragee nin (but not PGE2 ) induc ed hype ralgesia the arginine /NO/c GMP pathw ay is ac tivated.28 The abse nce of activity of the arginine NO cGMP pathw ay in PGE2 -induc ed hyperalge sia is illustrated here by the fac t that neither the NO synthase inhibitors (MB or ODQ) nor the c GMP phosp hodie ste rase inhibitor, MY5445, have any effe ct upon control hype ralgesia (Figs 3 –5 ). Neverthele ss, L-NAME displaye d an antinocic eptive effe ct. Rec ently, L-NAME has bee n described as ac ting as a partial agonist,27 c ausing rapid induction of iNOS ge ne ex pre ssion.21 Stimulation of NO synthesis may ex plain the observed L-NAME antinocic eption, in mode ls like those used in this inve stigation. In these mode ls it has be en previously show n that e ithe r NO donors or drugs w hich stimulate the arginine NO cGMP pathw ay c ause analge sia.9,1 0,13 –18 Thus, the simple st comprehensive ex planation for the fac t that L-NAME antinocic eption w as inhibite d by a NO synthase inhibitor, L-NMMA and w as abolishe d by guanylate cyclase inhibitors, MB or ODQ as w e ll as pote ntiated by e ithe r the NO synthase substrate,

arginine or by the cGMP phosphodieste rase inhibitor MY5445 is that L-NAME is activating iNOS. The fac t that i.c .v. co-inje ctions of MB or ODQ inhibited the ce ntral antinocic eptive e ffec t of L-NAME suggests a similar me chanism of action for the pe rip heral and ce ntral action for this age nt. Finally, it must be pointed out that, in contrast w ith the re sults prese nte d he re , there are several observations indic ating that the intraplantar or syste mic administration of L-NAME has similar e ffec ts to other NO synthase inhibitors in causing antinoc iception.4 ,2 0 This contradic tion may ex plaine d by considering that the ac tivation of the arginine /NO/cGMP pathw ay causes hyp eralgesia or analge sia de pending on the predominant type of fibres involved in the nocice ptive response or depending on the tissue level of NO.2 0 From the the rape utic p oint of view, how e ve r, it se ems that during inflammatory pain in man, the activation of the arginine /NO/cGMP p athw ay causes analgesia. This suggestion is in line w ith the observations that NO donors are either e ffec tive as analgesic s by themselves or in c onjunction w ith other analgesics.2 9 – 32 In conclusion, the pre sent study confirms that L-NAME c ause s analgesia and de monstrated that L-NMMA, another NO synthase inhibitor, significantly blocked both the peripheral and c entral antinocice ptive actions of L-NAME in rats and mic e. L-NAME antinocic eption w as also blocke d by inhibitors of guanilate-c yclase activation and pote ntiated by arginine and by a cGMP phosphodie ste rase inhibitor. These re sults allow us to spe culate that L-NAME causes antinocic eption by ac ting as a partial agonist, thus stimulating iNOS ac tivation in the nocice ptive te sts use d. Furthermore our results draw into que stion the use of L-NAME alone as a me thodological tool to charac te rise the nocic eptive role of the arginineNO-cGMP pathw ay in physiopathological proc esses, in the absenc e of confirmation w ith anothe r NO synthase inhibitor.

Acknowledgements The authors thank I.R. dos Santos for technic al assistanc e. This w ork w as supp orte d by FAPESP (Fundaç a¨ o de Amp aro à Pe squisa do Estado de Sã o Paulo ), FINEP (Financiadora de Estudos e Projetos ), FIPEC (Fundo de Ince ntivo à Pe squisa Té cnic oCie nt´õfica do Banc o do Brasil) and CNPq (Conselho Nac ional de Desenvolvimento Cient´õfico e Te cnoló gic o ).

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