ENZYMATIC-SYNTHESIS OF ANANDAMIDE, AN ENDOGENOUS LIGAND FOR THE CANNABINOID RECEPTOR, BY BRAIN MEMBRANES

Anandamide, an endogenous eicosanoid derivative (arachidonoylethanolam ide), binds to the cannabinoid receptor, a member of the G protein-cou pled superfamily. It also inhibits both adenylate cyclase and N-type c alcium channel opening. The enzymatic synthesis of anandamide in bovin e brain tissue was examined by incubating brain membranes with [C-14]e thanolamine and arachidonic acid. Following incubation and extraction into toluene, a radioactive product was identified which had the same R(f) value as authentic anandamide in several thin-layer chromatograph ic systems. When structurally similar fatty acid substrates were compa red, arachidonic acid exhibited the lowest EC(50) and the highest acti vity for enzymatic formation of the corresponding ethanolamides. The c oncentration-response curve of arachidonic acid exhibited a steep slop e, and at higher concentrations arachidonate inhibited enzymatic activ ity. When brain homogenates were separated into subcellular fractions by sucrose density gradient centrifugation, anandamide synthase activi ty was highest in fractions enriched in synaptic vesicles, myelin, and microsomal and synaptosomal membranes. When several areas of brain we re examined, anandamide synthase activity was found to be highest in t he hippocampus, followed by the thalamus, cortex, and striatum, and lo west in the cerebellum, pens, and medulla. The ability of brain tissue to enzymatically synthesize anandamide and the existence of specific receptors for this eicosanoid suggest the presence of anandamide-conta ining (anandaergic) neurons.