ANANDAMIDE AMIDOHYDROLASE ACTIVITY IN RAT-BRAIN MICROSOMES - IDENTIFICATION AND PARTIAL CHARACTERIZATION

An amidohydrolase activity present in rat brain microsomes catalyzes t he hydrolysis of N-arachidonoyl-[H-3]ethanolamine ([H-3]anandamide), a n endogenous cannabimimetic substance, forming [H-3]ethanolamine and a rachidonic acid. Amidohydrolase activity is maximal at pH 6 and 8, is independent of divalent cations, has an apparent K-m for [H-3]anandami de of 12.7 +/- 1.8 mu M, and has a V-max of 5630 +/- 200 pmol/min/mg o f protein. Phenylmethylsulfonyl fluoride, a serine protease inhibitor, and p-bromophenacyl bromide, a histidine-alkylating reagent, inhibit the activity, whereas N-ethyhnaleimide and various nonselective peptid ase inhibitors (EDTA, o-phenanthroline, bacitracin) have no effect. Br ain amidohydrolase activity exhibits high substrate specificity for [H -3]anandamide; N-gamma-linolenoyl-, N-homo-gamma-linolenoyl-, and N-11 ,14-eicosadienoyl- are hydrolyzed at markedly slower rates. Moreover, N-11-eicosaenoyl- and N-palmitoyl-[H-3]ethanolamine are not hydrolyzed . [H-3]Anandamide hydrolysis is inhibited competitively by nonradioact ive anandamide and by other N-acylethanolamines with the following ran k order of potency: anandamide > N-linoleoyl- = N-cis-linolenoyl- = N- gamma-linolenoyl- = N-homo-gamma-linolenoyl- > N-11,14-eicosadienoyl- > N-oleoyl- > N-docosahexaenoyl- > N-docosatetraenoyl > N-linoelaidoyl - > N-eicosaenoyl- > N-palmitoyl greater than or equal to N-elaidoyl- = N-eicosanoyl-ethanolamine = no effect. Amidohydrolase activity is hi gh in liver and brain and low in heart, kidney, intestine, stomach, lu ng, spleen, and skeletal muscle. Within the central nervous system, hi ghest activity is found in globus pallidus and hippocampus, two region s rich in cannabinoid receptors, and lowest activity is found in brain stem and medulla, where cannabinoid receptors are sparse. The results, showing that brain amidohydrolase activity is selective for anandamid e and enriched in areas of the central nervous system with high densit y of cannabinoid receptors, suggest that this activity may participate in the inactivation of anandamide at its sites of action.