Effects of inhibition of cyclooxygenase 2 on levels of endocannabinoids in the carrageenan model of inflammatory pain

Ian Robinson, Denise Richardson, Maulik Jhaveri, David Barrett, David Kendall, Victoria Chapman. University of Nottingham, Nottinghan, United Kingdom.

Cannabinoids and endocannabinoids (ECs) have anti-nociceptive effects following the activation of cannabinoid CB1 receptors, which are present on primary afferent fibres as well as spinal and supraspinal neurones. Metabolism is a key limiting factor in the anti-nociceptive effects of ECs. The ECs are primarily metabolised by fatty acid amide hydrolase (FAAH) but are also substrates for the inducible cyclooxygenase 2 (COX2) enzyme (Kozak et al., 2002). Systemic administration of COX2 inhibitors attenuates carrageenan-induced inflammatory hyperalgesia. The aim of the present study was to determine the effects of local hind paw injection of the selective COX2 inhibitor, nimesulide, on levels of ECs in the hindpaw in the carrageenan model of inflammatory pain.

Male Sprague-Dawley rats, weighing 240-260g, were group-housed (4 per cage), food and water were available ad libitum with a 12 hour light/dark cycle. Rats received intraplantar injection of nimesulide (50μg in 50μl) or vehicle (50μl 3% Tween 80 in saline) 30 minutes prior to intraplantar injection of 2% carrageenan or saline (100μl). At three hours post carrageenan / saline injection, rats were killed and hindpaw tissue was dissected and stored at -80oC. ECs were extracted and purified from hindpaw tissue using an ethyl acetate/hexane solvent extraction, followed by a solid phase extraction (SPE) protocol. Extracts were then analysed by liquid chromatography coupled tandem mass spectrometry (Richardson et al., 2007). Data were analysed using Mann-Whitney t-tests and are expressed as mean ± SEM (n=7-14).

Intraplantar injection of nimesulide in saline-treated rats resulted in a significant (p<0.05) increase in levels of N-arachidonoylethanolamine, AEA (121.3±18.41 pmol/g), N-oleoylethanolamine, OEA, (0.84±0.14 nmol/g) and 2-arachidonoylglycerol, 2-AG, (4.12±0.77 nmol/g), whereas levels of N-palmitoylethanolamine (PEA, 13.15±1.18nmol/g) were decreased in the ipsilateral hindpaw, compared to vehicle controls (AEA: 30.14±2.76 pmol/g and OEA: 0.4±0.04, 2AG: 1.92±0.36 and PEA: 22.09±2.36 nmol/g respectively). These data demonstrate that COX2 metabolises ECs in vivo. Intraplantar injection of nimesulide in carrageenan-treated animals resulted in a significant (p<0.05) increase in levels of AEA (16.68±2.6 pmol/g) and PEA (12.11±1.16 nmol/g) in the ipsilateral hindpaw, compared to vehicle controls (AEA 9.38±2.0 pmol/g, PEA 7.78±1.12nmol/g). Thus, levels of AEA and PEA were decreased following hindpaw inflammation, but under these conditions COX2 appears to metabolise both AEA and PEA. These data suggest that the effects of the COX2 inhibitor nimesulide on nociceptive processing may be, in part, due to elevated levels of ECs.

Kozak et al. (2002) J Biol Chem 277, 44877-85

Richardson et al. (2007) Anal Biochem 360, 216-26