Elsevier

Life Sciences

Volume 78, Issue 7, 11 January 2006, Pages 719-723
Life Sciences

(−)-Linalool inhibits in vitro NO formation: Probable involvement in the antinociceptive activity of this monoterpene compound

https://doi.org/10.1016/j.lfs.2005.05.065Get rights and content

Abstract

Recent studies performed in our laboratory have shown that (−)-linalool, the natural occurring enantiomer in essential oils, possesses anti-inflammatory, antihyperalgesic and antinociceptive effects in different animal models. The antinociceptive and antihyperalgesic effect of (−)-linalool has been ascribed to the stimulation of the cholinergic, opioidergic and dopaminergic systems, to its local anaesthetic activity and to the blockade of N-Methyl-d-aspartate receptors (NMDA). Since nitric oxide (NO) and prostaglandin E2 (PGE2) play an important role in oedema formation and hyperalgesia and nociception development, to investigate the mechanism of these actions of the (−)-linalool, we examined the effects of this compound on lipopolysaccharide (LPS)-induced responses in macrophage cell line J774.A1. Exposure of LPS-stimulated cells to (−)-linalool significantly inhibited nitrite accumulation in the culture medium without inhibiting the LPS-stimulated increase of inducible nitric oxide synthase (iNOS) expression, suggesting that the inhibitory activity of (−)-linalool is mainly due to the iNOS enzyme activity. In contrast, exposure of LPS-stimulated cells to (−)-linalool failed, if not at the highest concentration, both in inhibiting PGE2 release and in inhibiting increase of inducible cyclooxygenase-2 (COX2) expression in the culture medium. Collectively, these results indicate that the reduction of NO production/release is responsible, at least partially, for the molecular mechanisms of (−)-linalool antinociceptive effect, probably through mechanisms where cholinergic and glutamatergic systems are involved.

Introduction

(−)-Linalool is the natural occurring enantiomer of the monoterpene compound commonly found as a major volatile component of the essential oils in several aromatic plant species. It has been shown that (−)-linalool possess anti-inflammatory and antinociceptive activity in several experimental models. In fact, (−)-linalool administration in rats inhibits carrageenan-induced oedema (Peana et al., 2002) and reduces pain responses elicited by different stimulus, i.e., acetic acid-induced writhing (Peana et al., 2003), hot plate, formalin injection (Peana et al., 2004a), hyperalgesia induced by carrageenan, l-glutamate and prostaglandin E2 (PGE2) (Peana et al., 2004b). Antinociceptive effects of (−)-linalool have been related to the positive interference with muscarinic, opioid and dopaminergic transmission, since it was reduced by pre-treatment with the unselective muscarinic receptor antagonist atropine, the opioid receptor antagonist naloxone or the dopamine D2 receptor antagonist sulpiride (Peana et al., 2003, Peana et al., 2004a) and negative modulation of glutamate transmission (Elisabetsky et al., 1999, Silva Brum et al., 2001a, Silva Brum et al., 2001b). Furthermore, the antinociceptive effect of (−)-linalool has complicated pathway; indeed, it cannot be excluded an involvement of ATP-sensitive K+ channels, since glibenclamide, an inhibitor of these channels, has been shown to abolish the antinociceptive effect of (−)-linalool (Peana et al., 2004a). Moreover, this compound possesses local anaesthetic activity (Ghelardini et al., 1999) and antioxidant properties (Celik and Ozkaya, 2002).

The intraplantar injection of carrageenan and formalin cause the production and release of nitric oxide (NO) at the injured side (Omote et al., 2001). In recent years, considerable evidence has accumulated suggesting a role for NO as a mediator of inflammation (Lyons, 1995). NO increases the synthesis/release of pro-inflammatory mediators such as cytokines and reactive oxygen species (Marcinkiewicz et al., 1995) and prostanoids (Sautebin et al., 1995), resulting in promotion of inflammatory reaction. In this way, peripherally released NO contributes to the development of oedema and hyperalgesia in tissue injury and inflammation. In the inflammatory process we can observe the expression of several inducible enzymes that contribute to the release of pro-inflammatory mediators like NO and PGE2 by the inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) respectively (Dudhgaonkar et al., 2004). COX-2 is the inducible form of the enzyme, the synthesis of which is triggered by those cytokines that also induce iNOS (Clancy and Abramson, 1995). The two pathways interact closely and NO can stimulate COX-2 activity by combining with its heme component (Dudhgaonkar et al., 2004). Carrageenan and formalin injection induce expression of iNOS and COX-2.

Since NO and/or PGE2 are related to the transmission of nociceptive stimulus in inflammatory site, in this work we investigate the effect of (−)-linalool on the formation and release of NO and PGE2 in macrophages cell line J774.A1 stimulated by lipopolysaccharide (LPS), a cellular model of inflammation.

Section snippets

Reagents

Escherichia coli lipopolysaccharide (LPS) was obtained from Fluka (Milan, Italy). 3-(4,5-Dimethyl-thiazolyl-2yl) 2,5 diphenyl tetrazolium bromide (MTT), PBS, NaCl, KCl, Na2HPO4, K2HPO4, Tris–HCl, sodium orthovanadate, phenylmethylsulfonylfluoride, bovine serum albumin (BSA) and 6-mercaptopurine (6-MP) were obtained from Sigma Chemicals Co. (Milan, Italy). Kodak X-Omat film, ECL detection system, Hybond polyvinyldene difluoride membrane were from Millipore (USA). Leupeptin, trypsin inhibitor,

Effect of (−)-linalool on NO release and iNOS expression

Stimulation of macrophages J774.A1 with LPS (10 μg/ml) induced the expression of iNOS measurable as protein expression or as NO release. (−)-Linalool added to LPS-stimulated macrophages J774.A1 at concentrations of 10 7, 10 5 or 10 3 M, significantly reduced NO release evaluated as nitrites with respect to the control LPS group (Fig. 1). This inhibition was not very high but it was however concentration related.

The Western blotting analysis for the iNOS expression did not reveal any change in

Discussion

Recent studies have suggested that NO peripherally produced by different NOS isoforms contributes to oedema formation and development of nociception and hyperalgesia (Omote et al., 2001, Rivot et al., 2002). Results of present study show that the antinociceptive activity of (−)-linalool could be related to the inhibition of NO formation/release, but not to the interference in the PGs pathway. Exposure of LPS-stimulated J774.A1 macrophages to (−)-linalool, significantly and in

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