The Endocannabinoid Analgesic Entourage Effect: Investigations in Cultured DRG Neurons

Background: 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 degrees C. 48 h later cultures were loaded with 2 mu M Fura2AM for calcium imaging, and treated with 2-AG, 2-PG and 2-LG, individually or combined, for 5 min, followed by 1 mu Mol capsaicin. The amplitude and latency of capsaicin responses were measured (N=3-7 rats, controls N=16), and analysed. Results: In controls, 1 mu Mol 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 mu Mol) elicited calcium influx in some neurons. In the presence of 2-AG (0.001-100 mu Mol), 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 mu Mol (P< 0.001), in 60% neurons, as did 2-LG at 0.1-100 mu Mol (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 mu Mol 2-AG, 5 mu Mol 2-PG and 10 mu Mol 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.