The endogenous lipid anandamide is a full agonist at the human vanilloid receptor (hVR1)

Abstract

The endogenous cannabinoid anandamide was identified as an agonist for the recombinant human VR1 (hVR1) by screening a large array of bioactive substances using a FLIPR-based calcium assay. Further electrophysiological studies showed that anandamide (10 or 100 microM) and capsaicin (1 microM) produced similar inward currents in hVR1 transfected, but not in parental, HEK293 cells. These currents were abolished by capsazepine (1 microM). In the FLIPR anandamide and capsaicin were full agonists at hVR1, with pEC(50) values of 5. 94+/-0.06 (n=5) and 7.13+/-0.11 (n=8) respectively. The response to anandamide was inhibited by capsazepine (pK(B) of 7.40+/-0.02, n=6), but not by the cannabinoid receptor antagonists AM630 or AM281. Furthermore, pretreatment with capsaicin desensitized the anandamide-induced calcium response and vice versa. In conclusion, this study has demonstrated for the first time that anandamide acts as a full agonist at the human VR1.

Figure 1

Anandamide-gated currents in hVR1-HEK293 cells and rat DRG neurons are capsazepine sensitive. (A) Application of anandamide (10 μM; grey bar) or capsaicin (1 μM; open bar) led to the appearance of inward currents in cells voltage clamped at −70 mV. These currents were blocked completely by co-application of capsazepine (1 μM; solid bar). Data are representative traces, typical of n=7–9. (B) Application of anandamide (100 μM; shaded bar) led to the appearance of inward currents in DRG neurons voltage clamped at −70 mV. These cells were also shown to be sensitive to capsaicin (1 μM; open bar). (C) Anandamide-gated currents were blocked by capsazepine (1 μM; solid bar). Data are representative traces, typical of n=3.

Figure 2

Anandamide-induced Ca²⁺ responses are concentration-dependent [Ca²⁺]_i was monitored using Fluo-3AM in hVR1-HEK293 cells before and after the addition of capsaicin (100 pM–10 μM), anandamide (100 pM–10 μM) or methanandamide (100 pM–10 μM) at pH 6.4 or 7.4. Responses were measured as peak increase in fluorescence minus basal, expressed relative to the maximum capsaicin response and are given as mean±s.e.mean, where n=3–8.