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Evidence for the involvement of the nitric oxide–cGMP pathway in the antinociception of morphine in the formalin test

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Abstract

The effect of inhibition of nitric oxide synthesis and guanylate cyclase on the peripheral antinociceptive effect of morphine was assessed by using the formalin test in the rat. Saline, NG-monomethyl-l-arginine, a nitric oxide synthesis inhibitor (50 μg) and methylene blue, a guanylate cyclase inhibitor (500 μg), did not exhibit any antinociceptive activity. However, morphine (10 μg) produced a significant antinociceptive effect in phases 2a and 2b, which was reduced by pretreatment with either NG-monomethyl-l-arginine or methylene blue. These results suggest that the local administration of morphine induces antinociception by the activation of the l-arginine–nitric oxide–cGMP pathway.

Introduction

In addition to the supraspinal and spinal sites of action of morphine, it has been found that opiates cause peripheral antinociception (Ferreira and Nakamura, 1979b; Ferreira, 1983). This observation was further confirmed by several authors (Stein, 1993). The molecular mechanism underlying the peripheral antinociceptive action of opiates, however, is still a matter of debate. Because inflammatory hyperalgesia was thought to be due to an increased neuronal content of Ca2+/cAMP (Ferreira and Nakamura, 1979a) and this notion was supported by the in vitro studies of Collier and Roy (1974), it was suggested that the peripheral antinociceptive effect of opiates was associated with inhibition of activation of adenylate cyclase (Ferreira and Nakamura, 1979b). These results have been extended by several observations of Levine and Taiwo (1989)and Taiwo and Levine (1992). Recently, however, we proposed that peripheral opiate antinociception was due to the activation of the l-arginine–nitric oxide–cGMP pathway. This proposal was based on the observation that local administration of l-arginine produces antinociception in rats with carrageenin-induced hyperalgesia, the effect being blocked by nitric oxide synthesis inhibitors and methylene blue (Duarte et al., 1990). In prostaglandin- and carrageenin-induced hyperalgesia the local administration of opiates or non-enzymatic nitric oxide donors also produces antinociception. While pretreatment of the paws with methylene blue inhibit the action of morphine and of the nitric oxide donor, the nitric oxide synthase inhibitor only inhibited opiate analgesia (Duarte et al., 1990; Ferreira et al., 1991). The effect of morphine was potentiated by specific inhibitors of cGMP phosphodiesterase (MY5445), and intraplantar injections of dibutyryl–cGMP caused antinociception (Ferreira and Nakamura, 1979a). The finding that morphine stimulates cGMP formation (Minneman and Iversen, 1976) is in line with our suggestion.

There are, however, several observations indicating that the arginine–nitric oxide–cGMP pathway plays a hyperalgesic rather than a peripheral antinociceptive effect. In fact, it has been reported that either intraplantar (Haley et al., 1992) or systemic administration of NG-nitro-l-arginine methyl ester (l-NAME), but not d-NAME, produces dose-dependent antinociception in the second phase of the formalin test. The nociceptive or inflammatory role of the arginine–nitric oxide pathway has been described for bradykinin, substance P, carrageenin and dextran (Kawabata et al., 1994). The simplest explanation for these apparent contradictions may lie in the fact that the arginine–nitric oxide–cGMP pathway may have different roles depending on the different groups of nociceptive stimuli involved in the participation of different types of primary sensory neurons. Moreover, some agents that are assumed to be specific inhibitors of nitric oxide synthase may have additional effects. Intraplantar l-NAME, for example, causes antinociception in both formalin (Haley et al., 1992; Malmberg and Yaksh, 1993) and paw mechanical hyperalgesia tests. l-NAME, however, is a rather peculiar agent since it seems to block, and even to induce if given chronically, nitric oxide synthase and guanylate cyclase in neural tissue (Miller et al., 1996). In the present study, we reinvestigated, using the behavioral nociceptive response induced by formalin in the rat, the peripheral effects of morphine, NG-monomethyl-l-arginine (l-NMMA, a nitric oxide synthesis inhibitor), and methylene blue (an inhibitor of the soluble guanylate cyclase) and of their joint administration.

Section snippets

Animals

Male Wistar rats weighing 130–180 g with free access to food and water were used. All experiments were carried out according to the IASP guidelines on the use of animals in pain research.

Nociception test

Rats were placed in an open plexiglas observation chamber for 30 min to allow them to accommodate to their surroundings, then they were removed for formalin administration. The right hind paw of the rat was injected with 50 μl of dilute formalin (1%–5%), using a 30-gauge needle. The animal was then returned to

Results

Local administration of 10 μg of morphine, given 20 min before formalin (1%), produced a significant reduction (P<0.05) in the number of flinches in the second phase, 60 and 50% for phase 2a (5–40 min) and 2b (40–60 min), respectively, when compared to saline (Fig. 1Fig. 2). This effect was not observed in the first phase (0–5 min, Fig. 1, Fig. 2) or when morphine was administered in the contralateral paw or when a higher concentration (5%) of formalin was injected (data not shown).

Discussion

It is now becoming clear that the results obtained in the formalin test are highly susceptible to the concentration of formalin or agent tested. Low formalin concentrations elicit sub-maximal nociceptive behaviors. This facilitates the detection of the effects of weak analgesics. However, with injection of fixed concentration of formalin, intraplantar injections of l-arginine either enhance (low doses 0.1–1 μg per paw) or diminish (10 μg per paw) the frequency of the second-phase nociceptive

Acknowledgements

The authors gratefully acknowledge the technical assistance of I.R. dos Santos and S.R. Rosas. Dr. Vinicio Granados-Soto is a postdoctoral fellow from Third World Academy of Sciences South–South Fellowship Program and from the Department of Pharmacology of the Faculty of Medicine of Ribeirão Preto, USP, Brazil. Support from CNPq is gratefully acknowledged.

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