The Role Of Cannabinoid 1 Receptor (CB1) In N-arachidonylethanolamine (Anandamide) Responsiveness Of The Transient Receptor Potential Vanilloid Type 1 Ion Channel (TRPV1) In Cultured Rat Primary Sensory Neurons (PSN)

S Selvarajah1, J Chen1,2, A Varga1, S Brain3, JPM White1, L Urban4, L Buluwela5, I Nagy1. 1Section of Anaesthetics, Pain Medicine and Intensive Care, Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, Chelsea and Westminster Hospital, 369 Fulham Road, London, SW10 9NH, UK, 2Department of Anaesthesiology, Southwest Hospital, Third Military Medical University, Gaotanyan 19 Street, Shapingba, Chongqing 400038, P. R., China, 3Cardiovascular Division, King ’ s College London, 150 Stamford Street, London, SE1 9NH, UK, 4Preclinical Safety Profiling, Novartis Institutes for BioMedical, Research, Inc., 250 Massachusetts Avenue, Cambridge, MA 02139, USA, USA, 5Cancer Cell Biology, Division of Cancer, Department of Surgery and Cancer, Hammersmith Hospital, Du Cane Road, London, W12 0HS, UK

Anandamide is a lipid signalling molecule which is synthesised by a series of cells including a sub-population of nociceptive PSN (nPSN). A major proportion of nPSN which have the ability to synthesise anandamide co-expresses the two main targets of anandamide, TRPV1 and the CB1 receptor. While activation of TRPV1 results in excitation, activation of the CB1 receptor results in inhibition. The net effect of anandamide on nPSN depends on the concentration of anandamide, as well as the post-translational modification state of, and the cross-talk between, TRPV1 and the CB1 receptor. Various aspects of anandamide-evoked effects on nPSN have been assessed in recent years. However, no data on the effects of TRPV1 – CB1 receptor cross-talk on anandamide-induced actions in nPSN are available. Our aim in this study was to ascertain how the TRPV1 – CB1 receptor cross-talk affects anandamide-evoked actions in cultured rat PSN.

PSN cultures were prepared from dorsal root ganglia (DRG) of adult Sprague-Dawley rats, adult C57BL/6 x129SvJ (wild type (WT)) and TRPV1-/- mice. Whole-cell voltage-clamp recordings measurements of changes in intracellular Ca2+ concentrations ([Ca2+]i) and single cell reverse transcriptase polymerase chain reaction (RT-PCR) were used. Anandamide (1-30µM), the selective TRPV1 activator capsaicin (500nM or 1µM) and the selective CB1 receptor antagonist rimonabant (200nM) were applied to the cells through a small plastic tube positioned ˜100µm (in whole-cell recordings) or ˜500µm (in calcium imaging) from the cell(s) from which the measurements were done. All experiments were done at 37ºC unless otherwise stated. Statistical analysis was done by paired or unpaired Student’s t-test, or analysis of variance followed by Fisher’s post-hoc test as appropriate.

Anandamide superfusion to cultured PSN induced concentration-dependent inward currents (1-30µM; n=23) and increase in [Ca2+]i (30µM; n=22 of 94). Anandamide increased [Ca2+]i in PSN isolated from DRG of WT but not in PSN isolated from DRG of TRPV1-/- mice. All anandamide-responsive neurons were also responsive to capsaicin. However, only ˜2/3 of the capsaicin-responsive neurons were responsive to anandamide. Capsaicin-evoked responses were greater in the dual-responsive (DR; n=29 of 39) than in the capsaicin-only-responsive (COR; n=10 of 39) neurons (p<0.01). In DR neurons anandamide evoked smaller responses than capsaicin (p<0.01). Rimonabant did not affect the EC50 in of either type of anandamide-evoked responses (p<0.05). However, rimonabant reduced the Hill co-efficient for the fast-activating, but not for the slowly-activating currents. Further, rimonabant also reduced the amplitude of capsaicin-evoked responses in DR (n=33) but not in COR (n=18) neurons (p<0.05). Single-cell RT-PCR showed that all DR (n=11) and a major proportion of COR (n=8 of 10) neurons express the CB1 receptor. Based on these data we conclude that TRPV1 – CB1 receptor cross-talk may play a major role in controlling anandamide responsiveness of TRPV1, and that cross-talk should be different in DR and COR neurons.