Comparison of effects of anandamide at recombinant and endogenous rat vanilloid receptors

Abstract

Background. Anandamide, an endogenous lipid, activates both cannabinoid (CB1) and vanilloid (VRI) receptors, both of which are co-expressed in rat dorsal root ganglion (DRG) cells. Activation of either receptor results in analgesia but the relative contribution of CB1 and VRI in anandamide-induced analgesia remains controversial. Here we compare the in vitro pharmacology of recombinant and enclogenous VRI receptors using calcium imaging, in clonal and DRG cells, respectively. We also consider the contribution of CB1 and VRI receptors to anandamide-induced analgesia. Methods. Using a Flurometric Imaging Plate Reader (FLIPR™), calcium imaging has been used to study the effects of several vanilloid and cannabinoid ligands in rat VRI-transfected HEK293 (rVRI-HEK) cells and in DRG cells. The effect of pre-exposure of several vanilloid and cannabinoids has also been compared in DRG cells. Results. The VRI agonists; capsaicin, olvanil, (N-(4-hydroxyphenyl-arachinoylamide) (AM404) and anandamide caused a concentration-dependent increase in intracellular calcium concentration ([Ca2+]i), with similar temporal profiles in both rVRI-HEK and DRG cells, and potency (pEC50) values of 8.25 (SEM 0.11), 8.37 (0.04), 6.96 (0.06), 5.85 (0.01) and 7.45 (0.10), 7.55 (0.07), 6.10 (0.13), approximately 5′.5, respectively. These responses were inhibited by the VRI antagonist capsazepine (I μM). In contrast, application of synthetic cannabinoid antagonists failed to inhibit the anandamide-induced increase in (Ca2+]1. Reapplication of VRI agonists significantly inhibited a subsequent challenge to either capsaicin or anandamide in either cell type, whilst pre-exposure to cannabinoid agonists were without effect. Conclusion. Here we provide evidence that the pharmacology of recombinant rVRI receptors is similar to those enclogenously expressed in DRG cells. Moreover, we have shown that VRI, but not CB1, receptors are involved in anandamide-induced responses in dorsal root primary neurones in vitro. Therefore, the analgesic properties of anandamide are likely to be mediated, at least in part, by VRI activation in DRG cells in vivo.