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Uma Anand,1 Barbara Pacchetti,2 Praveen Anand,3 Mikael Hans Sodergren1,2 1Medical Cannabis Research Group, Department of Surgery and Cancer, Imperial College London, London, W12 0HS, UK; 2Curaleaf International Limited, London, EC2A 2EW, UK; 3Professor of Clinical Neurology, Department of Brain Sciences, Imperial College London, London, W12 0HS, UKCorrespondence: Uma Anand, Medical Cannabis Research Group, Department of Surgery and Cancer, Imperial College London, Hammersmith Campus, Du Cane Road, London, W12 0HS, UK, Tel +44 020 3313 2362, Fax +44 020 3313 3363, Email u.anand@imperial.ac.ukBackground: The endocannabinoid 2-Arachidonyl glycerol (2-AG) exerts dose-related anti-nociceptive effects, which are potentiated by the related but inactive 2-palmitoyl glycerol (2-PG) and 2-linoleoyl glycerol (2-LG). This potentiation of analgesia and other in vivo measures was described as the “entourage effect”. We investigated this effect on TRPV1 signalling in cultured dorsal root ganglion (DRG) nociceptors.Methods: Adult rat DRG neurons were cultured in medium containing NGF and GDNF at 37°C. 48 h later cultures were loaded with 2 μM Fura2AM for calcium imaging, and treated with 2-AG, 2-PG and 2-LG, individually or combined, for 5 min, followed by 1 μMol capsaicin. The amplitude and latency of capsaicin responses were measured (N=3– 7 rats, controls N=16), and analysed.Results: In controls, 1 μMol capsaicin elicited immediate calcium influx in a subset of neurons, with average latency of 1.27 ± 0.2 s and amplitude of 0.15 ± 0.01 Units. 2-AG (10– 100 μMol) elicited calcium influx in some neurons. In the presence of 2-AG (0.001– 100 μMol), capsaicin responses were markedly delayed in 64% neurons by up to 320 s (P< 0.001). 2-PG increased capsaicin response latency at 0.1 nMol-100 μMol (P< 0.001), in 60% neurons, as did 2-LG at 0.1– 100 μMol (P< 0.001), in 76% neurons. Increased capsaicin response latency due to 2-AG and 2-PG was sensitive to the CB2 but not to the CB1 receptor antagonist. Combined application of 1 μMol 2-AG, 5 μMol 2-PG and 10 μMol 2-LG, also resulted in significantly increased capsaicin response latency up to 281.5 ± 41.5 s (P< 0.001), in 96% neurons, that was partially restored by the CB2, but not the CB1 antagonist.Conclusion: 2-AG, 2-LG and 2-PG significantly delayed TRPV1 signalling in the majority of capsaicin-sensitive DRG neurons, that was markedly increased following combined application. Further studies of these endocannabinoids are required to identify the underlying mechanisms.Keywords: entourage effect, endocannabinoids, DRG neurons, nociception, analgesia |
