Expression of cannabinoid receptors in knee synovial tissue from patients with osteoarthritis and rheumatoid arthris

Kurian, N1, Liaque, M.L1, Garle, M.J1, Reeve, A.J2, Kendall, D.A1, Scammell, B3 & Chapman, V1.( 1School of Biomedical Sciences, University of Nottingham, Nottingham, U.K. 2Pain Department, Neurology and GI CEDD, GlaxoSmithKline, Harlow, U.K. 3Division of Orthopaedic and Accident Surgery, Nottingham University Hospitals, Queen’s Medical Centre, Nottingham, U.K.)

Cannabinoid CB1 and CB2 receptors are Gα i-coupled GPCRs expressed predominantly in central and peripheral neurons and immune cells respectively. Recent studies have demonstrated the therapeutic potential of cannabis-based drugs in inflammatory diseases (Croxford et al., 2005). The aim of this study was to provide evidence for expression of cannabinoid receptors and the endocannabinoid metabolising enzyme fatty acid amide hydrolase (FAAH) in synovial tissue from patients with endstage osteoarthritis (OA) and rheumatoid arthritis (RA). The expression of cannabinoid receptors in synovial tissue was assessed using Western blot analysis. Approximately 8μg (CB1 receptor) or 27μg (CB2 receptor) of protein was electrophoresed. Immunoblots were probed with anti-CB1 (Calbiochem), anti-CB2 (Cayman) or monoclonal β-actin antibody (Sigma) and visualised by enhanced chemiluminescence detection. Expression of CB1 and CB2 receptor protein was calculated as a percentage of expression of the internal control, β-actin. For determining FAAH activity, membrane fragments were prepared from synovial tissue. The FAAH activity of each sample was measured by monitoring the release of [3H]-ethanolamine after incubation of homogenates with radiolabelled anandamide ([3H]-AEA; Holt et al., 2005). FAAH activity in synovial tissue was expressed in pmol AEA hydrolysed min-1 mg-1protein. Data are represented as means ± s.e.m. of n number of patients from three separate experiments.

Expression of CB1 (OA: 59 ± 4%, RA: 67 ± 12% of β-actin expression, n=4) and CB2 (OA: 33 ± 3%, RA: 51 ± 14% of β-actin expression, n=4) receptor protein in human synovial tissue were demonstrated by Western blot analysis. Preabsorbing with antigenic peptide abolished the identified bands. CHO-K1 cells recombinantly expressing either the human CB2 receptor or human CB1 receptor protein were used as a positive control. There were no significant differences between receptor expressions in synovial tissue from OA versus RA patients. FAAH activity was detected in membrane preparations from knee synovial tissue. However, there were no significant differences in FAAH activity in tissue from OA (2.8 ± 0.4 pmolesmg-1min-1, n=5) and RA (1.6 ± 0.4 pmolesmg-1min-1, n=4) patients. The enzyme activity in synovial tissues was far lower than in rat liver (988 ± 84 pmolesmg-1min-1) previously demonstrated to be rich in FAAH activity (DeBank et al. 2005). FAAH enzyme activity measured was markedly reduced in the presence of the selective FAAH inhibitor, URB597 (1μM; Mor et al., 2004). In summary, we report the presence of CB1 and CB2 receptors and FAAH in the knee synovium of patients with endstage OA and RA. This supports the possible development of cannabinoid-based drugs for the treatment of arthritic diseases.

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