A role for endocannabinoids in indomethacin-induced spinal antinociception

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Abstract

Inhibition of prostaglandins synthesis does not completely explain non-steroidal anti-inflammatory drug-induced spinal antinociception. Among other mediators, endocannabinoids are involved in pain modulation. Indomethacin-induced antinociception, in the formalin test performed in spinally microdialysed mice, was reversed by co-administration of the cannabinoid 1 (CB1) antagonist, N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1-H-pyrazole-3-carboxamide (AM-251), but not by co-infusion of prostaglandin E2. Indomethacin was ineffective in CB1 knockout mice. AM-251 also reversed the indomethacin-induced antinociception in a test of inflammatory hyperalgesia to heat. Furthermore, during the formalin test, indomethacin lowered the levels of spinal nitric oxide (NO), which activates cellular reuptake and thus breakdown of endocannabinoids. The pronociceptive effect of an NO donor, 3-methyl-N-nitroso-sydnone-5-imine (RE-2047), was abolished by co-administration of the endocannabinoid transporter blocker N-(4-hydroxyphenyl) arachidonoyl amide (AM-404). Moreover, the antinociceptive activity of the NO synthase inhibitor, N-nitro-l-arginine methyl ester (l-NAME), was reversed by AM-251. Thus we propose that at the spinal level, indomethacin induces a shift of arachidonic acid metabolism towards endocannabinoids synthesis secondary to cyclooxygenase inhibition. In addition, it lowers NO levels with subsequent higher levels of endocannabinoids.

Introduction

Even though non-steroidal anti-inflammatory drugs are widely used for their analgesic effect (Brooks, 1998), the mechanism of this effect is still puzzling. Many observations suggested that non-steroidal anti-inflammatory drugs probably act on targets other than inhibition of the cyclooxygenases, to counteract pain. For example, salicylic acid does not inhibit cyclooxygenase enzyme activity at analgesic concentrations or doses, however, as a pain relieving drug or antipyretic it is equal to acetyl salicylic acid, which itself inhibits cyclooxygenase activity at clinically relevant doses (Brune et al., 1991). At the site of inflammation in periphery, inhibition of prostaglandin synthesis is well established to be the mechanism of non-steroidal anti-inflammatory drugs-induced antinociception, e.g. (Taiwo and Levine, 1990); supplementation of exogenous prostaglandin to peripheral inflamed tissues reversed the antinociceptive effect of indomethacin in the writhing test in mice (Ueno et al., 2001), and in the acutely inflamed joints of cats (Heppelmann et al., 1986). However, at the spinal level, there is only indirect evidence that spinal prostaglandins act as pronociceptive mediators; nociceptive stimuli increase spinal prostaglandin E2 production e.g. Malmberg and Yaksh, 1995, Vetter et al., 2001, and intrathecal (i.t.) injection of cyclooxygenase inhibitors produces a dose dependent antinociceptive effect in the formalin test in rats (Malmberg and Yaksh, 1992a). Prostaglandin E2 unquestionably contributes to spinally mediated allodynia (Minami et al., 1994), but direct evidence for pronociceptive effects of intrathecally applied prostaglandin E2 in the formalin test—which is believed to be a more valid model for clinical pain than other threshold escape models (Tjolsen et al., 1992)—is lacking. There is no evidence in the literature that supplementing prostaglandin E2 diminished an antinociceptive effect of a cyclooxygenase inhibitor at the spinal level. In addition, we have recently shown that at the spinal level, the synthetic cannabinoid R(−)-7-hydroxy-delta-6-tetra-hydrcannabinol-dimethylheptyl (HU-210) induced prostaglandin E2 synthesis simultaneous to its antinociceptive effect (Gühring et al., 2001a).

The discovery of endocannabinoids opened up new insights into nociception; their antinociceptive effects were reported in several pain models e.g. Mechoulam et al., 1995, Pertwee, 2001. Arachidonoyl ethanolamide (anandamide) the endogenous ligand for cannabinoid CB1 receptors was shown to be a metabolite of arachidonic acid (Pestonjamasp and Burstein, 1998). Although it is not clear so far through which pathway arachidonic acid contributes to the synthesis of anandamide or other endocannabinoids (Kuwae et al., 1999), it was shown that arachidonic acid mobilization is a condition favouring increased anandamide synthesis (Pestonjamasp and Burstein, 1998). Moreover, Berger et al. (2001) reported higher levels of biologically active N-acylethanolamines in the brain of piglets fed with long-chain polyunsaturated fatty acids supplemented diet. However, under physiological conditions anandamide is thought to be synthesised via a phospholipase D catalysed hydrolysis of N-arachidonoyl phosphatidylethanolamine (Sugiura et al., 2002).

Recent studies, performed on various cell cultures and tissues, demonstrate that the life span of endocannabinoids is limited by a rapid cellular uptake of endocannabinoids via a membrane transporter with subsequent intracellular degradation (Giuffrida et al., 2001). AM-404 selectively blocks this transporter and protects endocannabinoids from degradation in the cells, whereas nitric oxide (NO) has just an opposite effect Bisogno et al., 2001, Maccarrone et al., 1998, Maccarrone et al., 2000. If there is a tonic release of endocannabinoids, then the endocannabinoid transporter inhibitor, AM-404, should alleviate pain. But so far, this has been tested only in acute nociceptive models providing negative results (Beltramo et al., 2000).

It has been frequently speculated that cyclooxygenase inhibition results in an accumulation of arachidonic acid with subsequent diversion of its metabolism to the lipoxygenase and epoxygenase pathways, e.g. in case of aspirin-induced asthma, postulated years ago (e.g. Szczeklik et al., 1975). However, the possibility of a shift towards endocannabinoids formation was never tested. If an endocannabinoid shift occurs in response to cyclooxygenase inhibition, it might play an important role in indomethacin-induced antinociception. There is so far no evidence for a functional role of the endocannabinoid transporter in pain processing Giuffrida et al., 2001, Pertwee, 2001, whereas the pronociceptive role of NO donors is well established Gühring et al., 2001b, Inoue et al., 1997 without a convincing causal explanation.

In the present work, we investigated the possibility of involvement of endocannabinoids in indomethacin-induced antinociception at the spinal level.

Section snippets

Experimental animals

Animals used for the study were either C57/BL6 mice weighing between 18 and 25 g bred in the Institute of Pharmacology, University of Erlangen-Nürnberg, cannabinoid CB1 receptors wild type (CB1+/+) and cannabinoid CB1 receptor knockout (CB1−/−) mice generated as described (Ledent et al., 1999), or cyclooxygenase wild type, cyclooxygenase 1 knockout (COX1−/−) and cyclooxygenase 2 knockout mice (COX2−/−) (strain numbers: 5002W, 5001M, 5002M, respectively; Taconic, Germantown, NY).

Animals were

Prostaglandin E2 substitution does not reverse the antinociceptive activity of indomethacin

As previously observed, subcutaneous injection of formalin into the hind paw of mice resulted in two phases of pain behaviour, mainly flinching and licking of the injected paw. Results from microdialysed mice were not obviously different from those obtained from non-operated mice. To test the widely assumed pronociceptive role of spinal prostaglandin E2, we perfused it through the spinal microdialysis fibre alone and in combination with indomethacin. The choice of concentrations of

Discussion

It is known that in the presence of a continuous nociceptive afferent barrage into the dorsal horn there is a progressive generation of an arachidonic acid pool available for conversion (Malmberg and Yaksh, 1992b). In the formalin test, but not in the hot plate or tail flick tests, the stimulus-evoked activation of the arachidonic acid cascade can influence the behavioural response (Yaksh and Malmberg, 1994); e.g. it was reported that anandamide (1 fmol, i.t.) completely blocked

Acknowledgements

This study was supported by the Deutsche Forschungsgemeinschaft (SFB 353, A1) and the Egyptian ministry of higher education (predoctoral grant for MH). The authors would like to thank Prof. Klaus Rehse (Pharmaceutical Chemistry, Königin-Luise-Str. 2 and 4, Berlin, Germany) for the generous gift of RE-2047.

References (64)

  • J.L. Hylden et al.

    Intrathecal morphine in mice: a new technique

    Eur. J. Pharmacol.

    (1980)
  • T. Inoue et al.

    Intrathecal administration of a new nitric oxide donor, NOC-18, produces acute thermal hyperalgesia in the rat

    J. Neurol. Sci.

    (1997)
  • T. Kuwae et al.

    Biosynthesis and turnover of anandamide and other N-acylethanolamines in peritoneal macrophages

    FEBS Lett.

    (1999)
  • M. Maccarrone et al.

    Anandamide hydrolysis by human cells in culture and brain

    J. Biol. Chem.

    (1998)
  • M. Maccarrone et al.

    Anandamide uptake by human endothelial cells and its regulation by nitric oxide

    J. Biol. Chem.

    (2000)
  • A.B. Malmberg et al.

    Spinal nitric oxide synthesis inhibition blocks NMDA-induced thermal hyperalgesia and produces antinociception in the formalin test in rats

    Pain

    (1993)
  • R. Mechoulam et al.

    Identification of an endogenous 2-monoglyceride, present in canine gut, that binds to cannabinoid receptors

    Biochem. Pharmacol.

    (1995)
  • S. Milano et al.

    Prostaglandin E2 regulates inducible nitric oxide synthase in the murine macrophage cell line J774

    Prostaglandins

    (1995)
  • T. Minami et al.

    Allodynia evoked by intrathecal administration of prostaglandin E2 to conscious mice

    Pain

    (1994)
  • T.P. Misko et al.

    A fluorometric assay for the measurement of nitrite in biological samples

    Anal. Biochem.

    (1993)
  • L. Pang et al.

    Repression of inducible nitric oxide synthase and cyclooxygenase-2 by prostaglandin E2 and other cyclic AMP stimulants in J774 macrophages

    Biochem. Pharmacol.

    (1997)
  • R.G. Pertwee

    Cannabinoid receptors and pain

    Prog. Neurobiol.

    (2001)
  • V.K. Pestonjamasp et al.

    Anandamide synthesis is induced by arachidonate mobilizing agonists in cells of the immune system

    Biochim. Biophys. Acta

    (1998)
  • J.D. Richardson et al.

    Antihyperalgesic effects of spinal cannabinoids

    Eur. J. Pharmacol.

    (1998)
  • S. Shibuta et al.

    A new nitric oxide donor, NOC-18, exhibits a nociceptive effect in the rat formalin model

    J. Neurol. Sci.

    (1996)
  • F.L. Smith et al.

    Characterization of delta9-tetrahydrocannabinol and anandamide antinociception in nonarthritic and arthritic rats

    Pharmacol. Biochem. Behav.

    (1998)
  • T. Sugiura et al.

    Biosynthesis and degradation of anandamide and 2-arachidonoylglycerol and their possible physiological significance

    Prostaglandins Leukot. Essent. Fatty Acids

    (2002)
  • Y.O. Taiwo et al.

    Effects of cyclooxygenase products of arachidonic acid metabolism on cutaneous nociceptive threshold in the rat

    Brain Res.

    (1990)
  • A. Tjolsen et al.

    The formalin test: an evaluation of the method

    Pain

    (1992)
  • A. Ueno et al.

    Major roles of prostanoid receptors IP and EP(3) in endotoxin-induced enhancement of pain perception

    Biochem. Pharmacol.

    (2001)
  • G. Vetter et al.

    Release of glutamate, nitric oxide and prostaglandin E2 and metabolic activity in the spinal cord of rats following peripheral nociceptive stimulation

    Pain

    (2001)
  • M. Zimmermann

    Ethical guidelines for investigations of experimental pain in conscious animals

    Pain

    (1983)
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