ANANDAMIDE, AN ENDOGENOUS CANNABIMIMETIC EICOSANOID, BINDS TO THE CLONED HUMAN CANNABINOID RECEPTOR AND STIMULATES RECEPTOR-MEDIATED SIGNAL-TRANSDUCTION

Arachidonylethanolamide (anandamide), a candidate endogenous cannabino id ligand, has recently been isolated from porcine brain and displayed cannabinoid-like binding activity to synaptosomal membrane preparatio ns and mimicked cannabinoid-induced inhibition of the twitch response in isolated murine vas deferens. In this study, anandamide and several congeners were evaluated as cannabinoid agonists by examining their a bility to bind to the cloned cannabinoid receptor, inhibit forskolin-s timulated cAMP accumulation, inhibit N-type calcium channels, and stim ulate one or more functional second messenger responses. Synthetic ana ndamide, and all but one congener, competed for [H-3]CP55,940 binding to plasma membranes prepared from L cells expressing the rat cannabino id receptor. The ability of anandamide to activate receptor-mediated s ignal transduction was evaluated in Chinese hamster ovary (CHO) cells expressing the human cannabinoid receptor (HCR, termed CHO-HCR cells) and compared to control CHO cells expressing the muscarinic m5 recepto r (CHOm5 cells). Anandamide inhibited forskolin-stimulated cAMP accumu lation in CHO-HCR cells, but not in CHOm5 cells, and this response was blocked with pertussis toxin. N-type calcium channels were inhibited by anandamide and several active congeners in N18 neuroblastoma cells. Anandamide stimulated arachidonic acid and intracellular calcium rele ase in both CHOm5 and CHO-HCR cells and had no effect on the release o f inositol phosphates or phosphatidylethanol, generated after activati on of phospholipase C and D, respectively. Anandamide appears to exhib it the essential criteria required to be classified as a cannabinoid/a nandamide receptor agonist and shares similar nonreceptor effects on a rachidonic acid and intracellular calcium release as other cannabinoid agonists.