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The nitric oxide–cGMP signaling pathway plays asignificant role in tolerance to the analgesiceffect of morphine
Ercan Ozdemir, Ihsan Bagcivan, Nedim Durmus, Ahmet Altun, and Sinan Gursoy
Abstract: Although the phenomenon of opioid tolerance has been widely investigated, neither opioid nor nonopioid mech-anisms are completely understood. The aim of the present study was to investigate the role of the nitric oxide (NO)–cyclicguanosine monophosphate (cGMP) pathway in the development of morphine-induced analgesia tolerance. The study wascarried out on male Wistar albino rats (weighing 180–210 g; n = 126). To develop morphine tolerance, animals were givenmorphine (50 mg/kg; s.c.) once daily for 3 days. After the last dose of morphine was injected on day 4, morphinetolerance was evaluated. The analgesic effects of 3-(5’-hydroxymethyl-2’-furyl)-1-benzylindazole (YC-1), BAY 41-2272,S-nitroso-N-acetylpenicillamine (SNAP), NG-nitro-L-arginine methyl ester (L-NAME), and morphine were considered at 15or 30 min intervals (0, 15, 30, 60, 90, and 120 min) by tail-flick and hot-plate analgesia tests (n = 6 in each study group).The results showed that YC-1 and BAY 41-2272, a NO-independent activator of soluble guanylate cyclase (sGC), signifi-cantly increased the development and expression of morphine tolerance, and L-NAME, a NO synthase (NOS) inhibitor,significantly decreased the development of morphine tolerance. In conclusion, these data demonstrate that the nitric oxide–cGMP signal pathway plays a pivotal role in developing tolerance to the analgesic effect of morphine.
Key words: antinociception, morphine, nitric oxide, sGC activators, YC-1, BAY 41-2272.
Resume : Le phenomene de tolerance aux opioıdes a fait l’objet de nombreuses etudes, mais les mecanismes opioıdes etnon opioıdes ne sont pas entierement elucides. La presente etude a eu pour but d’examiner le role de la voie monoxyded’azote (NO) guanosine monophosphate cyclique (GMPc) dans le developpement de la tolerance analgesique induite par lamorphine. On a mene l’etude sur des rats males albinos Wistar (poids entre 180 et 210 g; n=126). Pour developper la tole-rance, on a administre la morphine (50 mg/kg ; s.c.) aux rats une fois par jour pendant 3 jours. On a evalue la toleranceapres la derniere injection de morphine le jour 4. On a examine les effets analgesiques de 3-(5’-hydroxymethyl-2’-furyl)-1-benzylindazole (YC-1), de BAY 41-2272, de S-nitroso-N-acetylpenicillamine (SNAP), de NG-nitro-L-arginine methyl ester(L-NAME) et de morphine a 15 ou 30 minutes d’intervalle (0, 15, 30, 60, 90 et 120 min) au moyen des tests du retrait dela queue et de la plaque chaude (n = 6 chez chaque groupe). Les resultats ont montre que YC-1 et BAY 41-2272, un acti-vateur de la guanylate cyclase soluble (GCs) independant de NO, ont augmente de maniere significative le developpementet l’expression de la tolerance a la morphine, et que L-NAME, un inhibiteur de la NO synthase (NOS), a diminue de faconmarquee le developpement de la tolerance a la morphine. Ces resultats demontrent que la voie de signalisation monoxyded’azote-GMPc joue un role important dans la tolerance a l’effet analgesique de la morphine.
Mots-cles : antinociception, morphine, monoxyde d’azote, activateurs de la GCs, YC-1, BAY 41-2272.
[Traduit par la Redaction]
Introduction
Morphine is an opiate analgesic commonly used for thetreatment of severe and chronic pain. The long-term efficacyof morphine is often limited by the development of toler-ance to the analgesic effect. Despite extensive research ef-forts in the area of opioid tolerance, the exact mechanismsby which this phenomenon occurs remain largely unknown.Recent evidence suggests that nitric oxide (NO), an endoge-nous molecule implicated in the regulation of a number of
physiologic and pathogenic processes, is involved in the de-velopment of morphine tolerance (Babey et al. 1994; Hein-zen and Pollack 2004; Joharchi and Jorjani 2007).
Several studies have implicated the N-methyl-D-aspartate(NMDA)/NO system in the development of morphine toler-ance (Zarrindast et al. 2002; Ozek et al. 2003; Heinzen andPollack 2004). Much of the in vivo research evaluating theinterplay between morphine and NO has focused on the ef-fects of NO synthase (NOS) inhibitors, NMDA receptor an-tagonists, or exogenous administration of L-arginine on the
Received 20 August 2010. Accepted 10 November 2010. Published on the NRC Research Press Web site at cjpp.nrc.ca on 14 January2011.
E. Ozdemir.1 Department of Physiology, Cumhuriyet University School of Medicine, 58140 Sivas, Turkey.I. Bagcivan, N. Durmus, and A. Altun. Department of Pharmacology, Cumhuriyet University School of Medicine, 58140 Sivas, Turkey.S. Gursoy. Department of Anesthesiology and Reanimation, Cumhuriyet University School of Medicine, 58140 Sivas, Turkey.
1Corresponding author (e-mail: [email protected]).
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Can. J. Physiol. Pharmacol. 89: 89–95 (2011) doi:10.1139/Y10-109 Published by NRC Research Press
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pharmacodynamics of morphine. For example, it has beendemonstrated that coadministration of morphine withNMDA receptor antagonists or non-isoform-specific NOSinhibitors, both of which reduce NO concentrations, resultedin attenuated development of antinociceptive tolerance(Bhargava et al. 1998; Bilsky et al. 1996). Furthermore, pre-treatment with or coadministration of L-arginine, which in-creases NO concentrations, resulted in the development offunctional tolerance to the analgesic effects of morphine(Babey et al. 1994; Bhargava et al. 1997; Liu et al. 2006).
Brignola et al. (1994) reported that L-arginine, a nitric ox-ide precursor, reduces the antinociceptive effect of mor-phine, whereas the constitutive nitric oxide synthase(cNOS) inhibitors potentiate the morphine analgesia in thetail-flick test (Przewłocki et al. 1993; Machelska et al.1997). He suggested that inhibition of the spinal NOS poten-tiates the m, d, and, to a lesser extent, k opioid receptor-mediated spinal antinociception in both acute and prolongedpain. Furthermore, cNOS inhibitors were demonstrated to at-tenuate the tolerance developed to the analgesic effect ofmorphine (Majeed et al. 1994; Bhargava and Zhao 1996).Through considerable evidence that NO modulates synaptictransmission in both the central and peripheral nervous sys-tem (Meller and Gebhart 1993), it has been suggested thatNO is involved in nociceptive processes either in the periph-ery or within the spinal cord (Haley et al. 1992). Enhancingmorphine antinociception after inhibition of NO synthesis inrats (Przewłocki et al. 1993) and blocking morphine toler-ance in mice indicate a selective action of NO in the mech-anisms of m receptor-mediated tolerance and dependence(Kolesnikov et al. 1993; Majeed et al. 1994). Accordingly,administration of aminoguanidine, a selective induced nitricoxide synthase (iNOS) inhibitor (Salerno et al. 2002), mark-edly improved the antinociceptive effect of morphine(Abdel-Zaher et al. 2006) and attenuated the increase in uri-nary nitrite concentration in diabetic mice (Grover et al.2000).
While a link between morphine antinociceptive tolerancedevelopment and NO production clearly exists, informationregarding the effects of the NO system on morphine antino-ciceptive tolerance is less definitive. Based on previous re-sults, the aim of this study was to investigate a possibleinvolvement of the nitric oxide–cGMP signaling pathway intolerance to the analgesic effect of morphine in rats.
Materials and methods
AnimalsMale adult Wistar albino rats weighing 180–210 g were
used (n = 126). Animals were housed in a room with an am-bient temperature of 22 ± 3 8C, a 12 h light : 12 h darkcycle and free access to water and food. Animals were ac-climatized to laboratory conditions before the test. All ex-periments were carried out blind between 0900 and 1700 h(n = 6 in each experimental group). The experimental proto-cols were approved by the Cumhuriyet University AnimalEthics Committee.
Drug administration3-(5’-hydroxymethyl-2’-furyl)-1-benzylindazole (YC-1, NO-
independent activator of sGC), S-nitroso-N-acetylpenicillamine
(SNAP, NO donor), NG-nitro-L-arginine methyl ester(L-NAME, NOS inhibitor), 2-[1-[(2-fluorophenyl) methyl]-1H-pyrazolo[3,4-b] pyridin-3-yl]-5(4-morpholinyl)-4,6-pyrimi-dinediamine (BAY 41-2272, NO-independent activator ofsGC; Sigma-Aldrich, St. Louis, Mo.), and morphine sulphate(Cumhuriyet University Hospital, Turkey) were dissolved inphysiological saline. Solutions were freshly prepared on thedays of the experiments. Subcutaneous (s.c.) morphine (5 mg/kg), intraperitoneal (i.p.) YC-1 (10 mmol/kg), SNAP (30 mg/kg), L-NAME (40 mg/kg), and BAY 41-2272 (10 mg/kg) wereadministered before the analgesia tests.
Induction of morphine toleranceThe animals were rendered tolerant to morphine using the
method from a previous study on the induction of morphinetolerance (Zarrindast et al. 2002). Tolerance was induced onday 1 (after challenge dose of morphine testing) through day3 by administering morphine (50 mg/kg; s.c.) once daily. Onday 4, tail-flick and hot-plate tests were done for each rat toaverage them as a baseline latency, then a challenge dose ofmorphine (5 mg/kg; s.c.) was injected; 15 min after mor-phine injection, other tail-flick and hot-plate tests were car-ried out, to average them so that the the post-drug latencyfor each rat could be found and the development of toler-ance to morphine evaluated.
Antinociceptive assay
Hot-plate testIn this test, animals were individually placed on a hot
plate (May AHP 0603 Analgesic Hot-plate Commat, Tur-key) with the temperature adjusted to 55 ± 3 8C. The latencyto the first sign of paw-licking or jump response to avoid theheat was taken as an index of the pain threshold; the cutofftime was 30 s, to avoid damage to the paw. The antinocicep-tive response on the hot plate is considered to result from acombination of central and peripheral mechanisms (Kanaanet al. 1996).
Tail-flick testWe used a standardized tail-flick apparatus (May TF 0703
Tail-flick Unit, Commat, Turkey) to evaluate thermal noci-ception. The radiant heat source was focused on the distalportion of the tail at 3 cm after administration of the vehicleand study drugs. Following vehicle or compound administra-tion, tail-flick latencies (TFL) were obtained. The infraredintensity was adjusted so that basal TFL occurred at 2.8 ±0.4 s. Animals with a baseline TFL below 2.4 or above3.2 s were excluded from further testing. The cutoff latencywas set at 15 s to avoid tissue damage. Any animal not re-sponding after 15 s was excluded from the study. The alge-sic response in the tail-flick test is generally attributed tocentral mechanisms (Kanaan et al. 1996; Ramabadran et al.1989).
Experimental protocolsTolerance was induced on day 1 through day 3 by admin-
istering morphine (50 mg/kg; s.c.) once daily. On day 4, toevaluate the effects of YC-1, BAY 41-2272, SNAP, and L-NAME on development or expression of morphine toler-ance, morphine-tolerant animals received YC-1 (10 mmol/
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kg; i.p.), SNAP (30 mg/kg; i.p.), BAY 41-2272 (10 mg/kg;i.p.), and L-NAME (40 mg/kg; i.p.) 15 min after the lastdose of morphine. In the morphine-treated rats, after induc-tion of morphine tolerance, analgesic response to the chal-lenge dose (5 mg/kg; s.c.) was determined on day 4 at 15or 30 min intervals after the morphine injection.
The analgesic effects of YC-1, SNAP, BAY 41-2272, andmorphine were considered at 15 or 30 min intervals (0, 15,30, 60, 90, and 120 min) by tail-flick and hot-plate test inrats (n = 6 in each group) on day 1. In the saline-treatedgroup, the animals received saline (10 mL/kg) instead ofmorphine during the induction session. In addition to this,all of the drugs were given in that volume i.p.
Data analysisTo calculate the percent of maximal antinociceptive ef-
fects (% MPE), tail-withdrawal latencies (tail-flick) and lickand (or) escape latencies (hot-plate) were converted to per-cent antinociceptive effect using the following equation:
% MPE ¼ ½ðtest latency� baselineÞ=ðcutoff � baselineÞ� � 100
The baseline latency was measured for each rat. The base-line latencies were approximately 2.4–3.2 s in all rats. Ani-mals with a baseline latency below 2.4 or above 3.2 s wereexcluded from further testing. The % MPE was calculatedseparately for each rat based on single baseline score.
Statistical analysisThe effect of antinociception was measured and the mean
of % MPEs in all groups was calculated. All experimentalresults were expressed as mean ± SEM (standard error ofmean). The data were analysed by analysis of variance, fol-
Fig. 1. Effects of 3-(5’-hydroxymethyl-2’-furyl)-1-benzylindazole(YC-1), BAY 41-2272, S-nitroso-N-acetylpenicillamine (SNAP),and NG-nitro-L-arginine methyl ester (L-NAME) on the develop-ment of morphine tolerance. (A) Effects of YC-1, BAY 41-2272,SNAP, morphine, and L-NAME in the tail-flick test; (B) effects ofYC-1, BAY 41-2272, SNAP, morphine, and L-NAME in the hot-plate test. Pretreatment of morphine-tolerant animals with YC-1,BAY 41-2272, and SNAP significantly decreased the percent ofmaximal possible effects (% MPE; increased tolerance to mor-phine) in the YC-1, SNAP, and BAY 41-2272 treatment groups inboth the tail-flick (p < 0.05; Fig. 1A) and hot-plate tests (p < 0.05;Fig. 1B). However, pretreatment of animals with L-NAME signifi-cantly increased % MPE (decreased tolerance to morphine) in boththe tail-flick (p < 0.01; Fig. 1A) and hot-plate tests (p < 0.01; Fig.1B). Each point represents the mean ± SEM of % MPE for 6 rats.*, p < 0.01; **, p < 0.05 compared with the saline-treated group.
Fig. 2. Effect of 3-(5’-hydroxymethyl-2’-furyl)-1-benzylindazole(YC-1) on morphine analgesia. (A) Effect of YC-1 (10 mmol/kg;i.p.) in the tail-flick test; (B) effect of YC-1 in the hot-plate test.YC-1 in combination with morphine produced a significant de-crease in percent of maximal possible effects (% MPE) in both thetail-flick (p < 0.05; Fig. 2A) and hot-plate assays (p < 0.05; Fig.2B) as compared with the morphine-treated rats. The maximum %MPE was observed at 60 min after administration of morphine, viathe tail-flick and hot-plate tests. In addition to this, YC-1 alone de-monstrated no antinociceptive effect in analgesia tests. Each pointrepresents the mean ± SEM of % MPE for 6 rats. *, p < 0.01; **,p < 0.05 compared with the saline-treated group.
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lowed by Tukey’s test, using the computer program SPSS(ver. 15.0 for Windows). In all cases, the criterion for statis-tical significance was p < 0.05.
Results
Effects of YC-1, BAY 41-2272, SNAP, and L-NAME onthe development of morphine tolerance
Pretreatment of animals with YC-1, BAY 41-2272, andSNAP significantly increased the development of toleranceto morphine antinociceptive effect, as indicated by the de-crease of % MPE in the YC-1, SNAP, and BAY 41-2272treatment groups in both the tail-flick (16.6 ± 2.1; 18.0 ±2.3; 15.41 ± 1.8, respectively, p < 0.05; Fig. 1A) and hot-plate tests (30.0 ± 3.1; 31.5 ± 2.6; 30.4 ± 3.8, respectively,p < 0.05; Fig. 1B). However, pretreatment of animals withL-NAME (NOS inhibitor) significantly decreased the devel-opment of tolerance to morphine antinociceptive effect inboth the tail-flick (p < 0.01; Fig. 1A) and hot-plate tests(p < 0.01; Fig. 1B) compared with other groups. The peak
value of this group was observed at 60 min after administra-tion of L-NAME in both the tail-flick and hot-plate tests(64.2 ± 3.1 and 69.3 ± 3.5, respectively). These findingsdemonstrated that the NO-independent sGC activators con-tributed to the development of morphine antinociceptive tol-erance.
Effects of YC-1, BAY 41-2272, SNAP, and L-NAME onmorphine analgesia
YC-1, BAY 41-2272, and SNAP in combination withmorphine produced a significant decrease in % MPE inboth the tail-flick (39.3 ± 3.4; 39.9 ± 3.6; 43.6 ± 2.9, respec-tively, p < 0.05; Fig. 2A, Fig. 3A, Fig. 4A) and hot-plate as-says (44.3 ± 2.4; 29.7 ± 3.1; 45.5 ± 3.2, respectively, p <0.05; Fig. 2B, Fig. 3B, Fig. 4B) as compared with the mor-phine-treated rats. The maximum % MPE was observed at60 min after administration of morphine, according to thetail-flick and hot-plate tests in all groups of rats. YC-1,BAY 41-2272, and SNAP alone demonstrated no antinoci-ceptive effect in both the tail-flick (p > 0.05; Fig. 2A,
Fig. 3. Effect of BAY 41-2272 on morphine analgesia. (A) Effectof BAY 41-2272 (10 mg/kg i.p.) in the tail-flick test; (B) effect ofBAY 41-2272 in the hot-plate test. BAY 41-2272 in combinationwith morphine produced a significant decrease in percent of maxi-mal possible effects (% MPE) in both the tail-flick (p < 0.05; Fig.3A) and hot-plate assays (p < 0.05; Fig. 3B) as compared with themorphine-treated rats. BAY 41-2272 alone demonstrated no antino-ciceptive effect in both the tail-flick and hot-plate tests. Each pointrepresents the mean ± SEM of % MPE for 6 rats. *, p < 0.01; **,p < 0.05 compared with the saline-treated group.
Fig. 4. Effect of SNAP on morphine analgesia. (A) Effect of S-nitroso-N-acetylpenicillamine (SNAP; 30 mmol/kg; i.p.) in the tail-flick test; (B) effect of SNAP in the hot-plate test. When adminis-tered with morphine, SNAP produced a significant decrease in per-cent of maximal possible effects (% MPE) in both the tail-flick (p <0.05; Fig. 4A) and hot-plate assays (p < 0.05; Fig. 4B) as comparedwith the morphine-treated rats. However, SNAP alone demonstratedno antinociceptive effect in the analgesia test. Each point representsthe mean ± SEM of % MPE for 6 rats. *, p < 0.01; **, p < 0.05compared with the saline-treated group.
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Fig. 3A, Fig. 4A, respectively) and hot-plate tests (p > 0.05;Fig. 2B, Fig. 3B, Fig. 4B, respectively).
The antinociceptive effects of different doses of morphineTo determine the effective morphine dose, we measured
the antinociceptive responses for the 3 different doses ofmorphine (2.5, 5, and 7.5 mg/kg; s.c.) at 15/30 min intervalsby tail-flick and hot-plate test. The maximum % MPE wasobserved at 60 min after administration of a 5 mg/kg doseof morphine (36.6 ± 3.8 for the tail-flick test and 59.5 ± 6.5for the hot-plate test; Table 1). The % MPE produced bymorphine (5 mg/kg) was significantly higher than in theother groups (2.5 mg/kg morphine and saline group) in boththe tail-flick (p < 0.05) and hot-plate tests (p < 0.05) in rats.
DiscussionThe mechanisms underlying the tolerance to the antinoci-
ceptive action of opioids are not clear and need to be furtherexplored. The phenomenon of opioid tolerance may involvechanges in several opioid and nonopioid systems. Many re-searchers have suggested that different mechanisms partici-pate in the development of morphine tolerance (Nayebi etal. 2009; Hama et al. 2006; Morgan et al. 2009). One ofthese mechanisms is the NO signaling pathway (Heinzenand Pollack 2004).
NO has been implicated as a biological messenger mole-cule in the central nervous system (Moncada et al. 1989;Garthwaite 1991; Toda et al. 2009). NO is derived fromone of two equivalent guanidino nitrogens of the aminoacid L-arginine by the enzyme NOS, yielding NO and L-citrulline as a co-product. NOS is among the largest andmost complicated of enzymes, and as many as 8 isoformsof NOS have been identified from neurons, macrophages,and endothelial cells (Nathan and Xie 1994; Murad 1994).Activation of NOS and release of NO stimulates the solubleguanylyl cyclase (sGC), which results in an increase in cy-clic GMP levels within the target cell (Deguchi 1977; Bredtand Snyder 1992). In the present study, we found that YC-1,BAY 41-2272 (NO-independent sGC activators), and SNAP(NO donor), when given along with the morphine pretreat-ment, increase the development of morphine antinociceptivetolerance. On the other hand, Xu et al. (1998) suggested thatmethylene blue and LY-83,583 (sGC inhibitors) decreasedthe development of morphine tolerance. These results sug-
gest that the cyclic GMP system may participate in the me-diation of the morphine tolerance. There may be both cyclicGMP-dependent and -independent mechanisms involved inthe effect of NO on the development of tolerance. Indeed,NO has been demonstrated to modulate certain neuronalproteins through a cyclic GMP-independent process. For ex-ample, Hess et al. (1994) reported that exogenous and endo-genously generated NO resulted in the modification ofcysteine residues on neuronal proteins. In particular, expo-sure of synaptosomes to NO inhibited subsequent thiol-linked ADP-ribosylation of the heterotrimeric G protein bypertussis toxin.
However, our results demonstrated that pretreatment ofanimals with L-NAME (NOS inhibitor) significantly de-creased the development of tolerance to morphine antinoci-ceptive effect in the analgesia tests. These results stronglysuggest that inhibition of NO production resulted in an in-hibition of morphine antinociceptive tolerance. This findingis in line with studies reported by others that systemic ad-ministration of NOS inhibitors attenuates the developmentof tolerance to systemic morphine administration (Santa-marta et al. 2005; Kolesnikov et al. 1993; Majeed et al.1994). Recently, administration of L-NAME has also beenreported to have little effect in attenuating tolerance to mor-phine in spinal sites (Dunbar and Yaksh 1996). Taken to-gether, these studies suggest that NO at the supraspinal butnot the spinal site may play an important role in the media-tion of morphine antinociceptive tolerance. Studies by otherssuggest that L-NAME exhibits antinociceptive activity in themouse (Moore et al. 1991; Malmberg and Yaksh 1993). Inthose studies, the antinociceptive activity of L-NAME wasdemonstrated in the formalin-induced paw-licking test, aswell as the acetic acid-induced writhing test and hot-platetest after L-NAME had been administered by i.p. injection.In addition, L-NAME produced antinociception in theformalin-induced paw-licking test after oral administration(Moore et al. 1991). Przewłocki et al. (1993) reported thatL-NAME potentiated morphine-induced antinociception.Based on these findings, it is logical to suspect that the re-versal effect of L-NAME on morphine-induced antinocicep-tion after the development of tolerance might be caused bythe additive or synergistic actions between the possible L-NAME-induced antinociceptive effect and morphine-induced antinociceptive effect.
Table 1. The antinociceptive effects of different doses of morphine.
Time (min) 0 15 30 60 90
Tail-flickSaline 4.1±1.2 4.3±0.8 3.9±0.7 4.2±1.0 4.4±1.4Morphine (2.5 mg/kg) 4.0±1.3 8.7±1.9 12.1±3.2 15.4±3.5 9.4±1.8Morphine (5 mg/kg) 3.0±0.9 14.6±4.2 26.8±5.4 36.6±3.8** 24.4±6.2Morphine (7.5 mg/kg) 3.6±1.0 13.7±5.1 25.7±6.3 29.5±2.8* 22.8±4.5
Hot-plateSaline 5.6±1.5 5.1±1.1 4.9±0.9 5.5±1.6 4.9±0.8Morphine (2.5 mg/kg) 8.5±1.8 9.5±5.2 18.9±7.2 23.9±4.6 19.8±5.6Morphine (5 mg/kg) 9.1±1.7 24.8±8.3 44.6±5.3 59.5±6.5** 39.1±6.0Morphine (7.5 mg/kg) 8.6±1.6 21.0±5.0 40.4±7.2 41.5±4.3 27.8±8.0
Note: Data are mean ± SEM. *, p < 0.05; **, p < 0.01 as compared with its saline group (n = 6 ineach group).
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Other studies with L-arginine further support the involve-ment of NO in morphine tolerance (Kielstein et al. 2007).L-Arginine, but not D-arginine, when given with morphine,appears to accelerate tolerance to systemic morphine (Babeyet al. 1994). L-Arginine is the natural substrate for NOS. Ad-ministration of L-arginine may increase the formation of NOand, thereby, possibly enhance the rate of development ofmorphine antinociceptive tolerance.
In conclusion, we found that activation of the NO–cyclicGMP system by the NO-independent activators increased thedevelopment of morphine antinociceptive tolerance. In addi-tion to this, our results demonstrated that pretreatment of an-imals with L-NAME (NOS inhibitor) significantly decreasedthe development of tolerance to morphine antinociceptiveeffect in the analgesia tests. Considering these findings, wecan suggest that the nitric oxide–cGMP signaling pathwayplays a pivotal role in developing tolerance to the analgesiceffect of morphine.
AcknowledgementsThis study was supported by Cumhuriyet University Sci-
entific Research Project (T-329, CUBAP, Sivas, Turkey).
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