The activity of anandamide at vanilloid VR1 receptors requires facilitatedtransport across the cell membrane and is limited by intracellular metabolism

The endogenous ligand of CB1 cannabinoid receptors, anandamide, is also a f ull agonist at vanilloid VR1 receptors for capsaicin and resiniferatoxin, t hereby causing an increase in cytosolic Ca2+ concentration in human VR1-ove rexpressing (hVR1-HEK) cells. Two selective inhibitors of anandamide facili tated transport into cells, VDM11 and VDM13, and two inhibitors of anandami de enzymatic hydrolysis, phenylmethylsulfonyl fluoride and methylarachidono yl fluorophosphonate, inhibited and enhanced, respectively, the VR1-mediate d effect of anandamide, but not of resiniferatoxin or capsaicin, The nitric oxide donor, sodium nitroprusside, known to stimulate anandamide transport , enhanced anandamide effect on the cytosolic Ca2+ concentration. According ly, hVR1-HEK cells contain an anandamide membrane transporter inhibited by VDM11 and VDM13 and activated by sodium nitroprusside, and an anandamide hy drolase activity sensitive to phenylmethylsulfonyl fluoride and methylarach idonoyl fluorophosphonate, and a fatty acid amide hydrolase transcript. The se findings suggest the following. (i) Anandamide activates VR1 receptors b y acting at an intracellular site. (ii) Degradation by fatty acid amide hyd rolase limits anandamide activity on VR1; and (iii) the anandamide membrane transporter inhibitors can be used to distinguish between CB1 or VR1 recep tor-mediated actions of anandamide. By contrast, the CB, receptor antagonis t SR141716A inhibited also the VR1-mediated effect of anandamide and capsai cin on cytosolic Ca2+ concentration, although at concentrations higher than those required for CB1 antagonism.