Inhibition of nitric oxide production in RAW264.7 macrophages by cannabinoids and palmitoylethanolamide

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Abstract

We have investigated the inhibition of lipopolysaccharide stimulated nitric oxide production in RAW264.7 macrophages by the cannabinoids and the putative cannabinoid CB2-like receptor ligand, palmitoylethanolamide. (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate ((+)-WIN55212) and, to a lesser extent (−)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxy-propyl)cyclohexan-1-ol (CP55940), significantly inhibited lipopolysaccharide stimulated nitric oxide production. The level of inhibition was found to be dependent on the concentration of lipopolysaccharide used to induce nitric oxide production. Palmitoylethanolamide significantly inhibited nitric oxide production induced by lipopolysaccharide. The inhibition of nitric oxide production by (+)-WIN55212 but not palmitoylethanolamide was significantly attenuated in the presence of the cannabinoid CB2 receptor antagonist, N-[(1S)-endo-1,3,3-trimethyl bicyclo [2.2.1] heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR144528). (+)-WIN55212 produced a pertussis toxin-sensitive parallel rightward shift in the log concentration–response curve for lipopolysaccharide, causing a fivefold increase in the EC50 value for lipopolysaccharide with no change in the Emax value. (−)-WIN55212 had no effect on the log concentration–response curve for lipopolysaccharide. Palmitoylethanolamide did not produce a rightward shift in the lipopolysaccharide concentration–response curve. However, it did produce a pertussis toxin-insensitive reduction in the Emax value. The results suggest that the inhibition of lipopolysaccharide mediated nitric oxide release by (+)-WIN55212 in murine macrophages is mediated by cannabinoid CB2 receptors. In contrast, the inhibition by palmitoylethanolamide does not appear to be mediated by cannabinoid receptors.

Introduction

Cannabinoid CB2 receptor expression has been identified in a range of immunological cells including B and T cells, monocytes and macrophages Bouaboula et al., 1993, Galiègue et al., 1995. It has been suggested that cannabinoid CB2 receptors may have an immunomodulatory role. These receptors have been shown to modulate cytokine production, humoral response, proliferative responses, microbiocidal activity and antigen processing in a number of immune cell types (Klein et al., 1998). Most of these in vitro studies have implied an immunosuppressive action of cannabinoids Luo et al., 1992, Klein et al., 1991, Fischer-Stenger et al., 1993 however, in some studies, enhancing effects have been observed Derocq et al., 1995, Zhu et al., 1994.

Nitric oxide has a dual role as (1) a proinflammatory mediator in the immune system with both antiviral (Lowenstein et al., 1996) and antibacterial (Nathan and Hibbs, 1991) actions and (2) a neurotransmitter in the central nervous system, affecting memory and learning (Dawson and Snyder, 1994). The murine macrophage cell line RAW264.7 expresses mRNA for CB2 but not CB1 cannabinoid receptors Jeon et al., 1996, Waksman et al., 1999. Reports have shown that the psychoactive cannabinoid (−)-Δ9-tetrahydrocannabinol inhibits nitric oxide production by murine macrophages and RAW264.7 macrophages while the inactive isomer, (+)-Δ9-tetrahydrocannabinol, only weakly inhibits nitric oxide release (Coffey et al., 1996). In addition, in these cells (−)-Δ9-tetrahydrocannabinol appears to inhibit both forskolin-stimulated cyclic AMP production and inducible nitric oxide synthase (iNOS) transcription (Jeon et al., 1996). However, these studies do not directly demonstrate the involvement of cannabinoid CB2 receptors, as they did not involve the use of cannabinoid receptor antagonists. Moreover, although (−)-Δ9-tetrahydrocannabinol has affinity for both cannabinoid CB1 and CB2 receptors, it has particularly low efficacy at cannabinoid CB2 receptors, to the extent that it can behave as an antagonist at these sites in some preparations Bayewitch et al., 1996, Bouaboula et al., 1999.

The purpose of this investigation was three-fold. Firstly, to further investigate whether the effects of cannabinoids on lipopolysaccharide induced nitric oxide production in RAW264.7 macrophages could be directly linked with the cannabinoid CB2 receptor. Secondly, to address the need for a sensitive assay for cannabinoid CB2 receptor agonists and antagonists in a native system. Thirdly, to investigate any functional interaction of palmitoylethanolamide with cannabinoid CB2 receptors. There is some degree of confusion as to the ability of this compound to interact with cannabinoid CB2 receptors. Palmitoylethanolamide reduces inflammatory responses (Mazzari et al., 1996) and has potent analgesic actions, which are attenuated by the cannabinoid CB2 receptor antagonist, N-[(1S)-endo-1,3,3-trimethyl bicyclo [2.2.1] heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR144528) Jaggar et al., 1998, Calignano et al., 1998. However, although palmitoylethanolamide has been proposed as an endogenous cannabinoid CB2 receptor ligand (Facci et al., 1995), it does not appear to displace the high affinity cannabinoid ligand [3H]CP55940 from membranes of Chinese Hamster Ovary (CHO) cells transfected with human cannabinoid CB2 receptors (Showalter et al., 1996) or from cannabinoid CB2 receptors on spleen or mast cell membranes (Ross et al., 1999a). Thus, we felt it appropriate to investigate whether palmitoylethanolamide interacts with the cannabinoid CB2 receptor in macrophages.

In order to investigate whether inhibition of lipopolysaccharide induced nitric oxide release from macrophages is linked to the cannabinoid CB2 receptor we used a number of approaches. A comparison was made of the actions of the cannabinoid receptor agonist (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate ((+)-WIN55212), which has slightly higher affinity for the cannabinoid CB2 receptor, with its inactive isomer (−)-WIN55212 (Pertwee, 1997). We have made use of the high affinity, cannabinoid CB2 selective antagonist SR144528 (Rinaldi-Carmona et al., 1998). In addition, we investigated the effects of pretreatment of the cells with the Gαi/Gαo receptor uncoupling agent, pertussis toxin on the action of these compounds.

Section snippets

Drugs and chemicals

CP55940 [(−)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexan-1-ol] was obtained from Pfizer, WIN55212 from Research Biochemicals International, palmitoylethanolamide from Tocris and SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride] and SR144528 from Sanofi Recherche. Roswell Park Memorial Institute Medium (RPMI), Dulbecco's Modified Eagles Medium (DMEM)/f-12 Ham, penicillin/streptomycin,

Effect of cannabinoids and palmitoylethanolamide on single concentrations of lipopolysaccharide

Fig. 1 shows the actions of various compounds incubated simultaneously with either 200 or 20 ng ml−1 lipopolysaccharide. (+)-WIN55212 (Fig. 1a) significantly inhibited nitric oxide production induced by 200 ng ml−1 lipopolysaccharide at 10 μM and by 20 ng ml−1 lipopolysaccharide at 1 and 10 μM. The % inhibition by (+)-WIN55212 at both 1 and 10 μM was significantly greater in the presence of the lower concentration of lipopolysaccharide (P<0.01, one way ANOVA followed by Tukey's multiple

Discussion

An inhibitory effect of (−)-Δ9tetrahydrocannabinol on lipopolysaccharide mediated nitric oxide release in murine macrophage cell line RAW264.7 has been observed by others, however these experiments did not directly implicate cannabinoid CB2 receptors Jeon et al., 1996, Coffey et al., 1996. The data presented here demonstrate that this effect is also produced by the synthetic cannabinoid (+)-WIN55212. It was possible to attenuate the inhibition of lipopolysaccharide stimulated nitric oxide

Acknowledgements

This work was supported by grants 039538 and 047980 from the Wellcome Trust and from Pfizer (to RAR and RGP). We thank Pfizer for their assistance in setting up the nitric oxide assay, GlaxoWellcome for the human cannabinoid CB2 transfected cells and Sanofi Recherche for SR141716A and SR144528.

References (31)

  • M. Bouaboula et al.

    Cannabinoid-receptor expression in human leukocytes

    Eur. J. Biochem.

    (1993)
  • M. Bouaboula et al.

    Gi protein modulation induced by a selective inverse agonist for the peripheral cannabinoid receptor CB2: implication for intracellular signalization cross-regulation

    Mol. Pharmacol.

    (1999)
  • A. Calignano et al.

    Control of pain initiation by endogenous cannabinoids

    Nature

    (1998)
  • T.M. Dawson et al.

    Gases as neurotransmitters: Nitric Oxide and carbon monoxide in the brain

    J. Neurosci.

    (1994)
  • L. Facci et al.

    Mast cells express a peripheral cannabinoid receptor with differential sensitivity to anandamide and palmitoylethanolamide

    Proc. Nat. Acad. Sci. U.S.A.

    (1995)
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