Endocannabinoids and related N-acylethanolamines: biological activities and metabolism

Abstract The plant Cannabis sativa contains cannabinoids represented by Δ9-tetrahydrocannabinol, which exert psychoactivity and immunomodulation through cannabinoid CB1 and CB2 receptors, respectively, in animal tissues. Arachidonoylethanolamide (also referred to as anandamide) and 2-arachidonoylglycerol (2-AG) are well known as two major endogenous agonists of these receptors (termed “endocannabinoids”) and show various cannabimimetic bioactivities. However, only 2-AG is a full agonist for CB1 and CB2 and mediates retrograde signals at the synapse, strongly suggesting that 2-AG is physiologically more important than anandamide. The metabolic pathways of these two endocannabinoids are completely different. 2-AG is mostly produced from inositol phospholipids via diacylglycerol by phospholipase C and diacylglycerol lipase and then degraded by monoacylglycerol lipase. On the other hand, anandamide is concomitantly produced with larger amounts of other N-acylethanolamines via N-acyl-phosphatidylethanolamines (NAPEs). Although this pathway consists of calcium-dependent N-acyltransferase and NAPE-hydrolyzing phospholipase D, recent studies revealed the involvement of several new enzymes. Quantitatively major N-acylethanolamines include palmitoylethanolamide and oleoylethanolamide, which do not bind to cannabinoid receptors but exert anti-inflammatory, analgesic, and anorexic effects through receptors such as peroxisome proliferator-activated receptor α. The biosynthesis of these non-endocannabinoid N-acylethanolamines rather than anandamide may be the primary significance of this pathway. Here, we provide an overview of the biological activities and metabolisms of endocannabinoids (2-AG and anandamide) and non-endocannabinoid N-acylethanolamines

N-cyclohexanecarbonylpentadecylamine: a selective inhibitor of the acid amidase hydrolysing N-acylethanolamines, as a tool to distinguish acid amidase from fatty acid amide hydrolase

Anandamide (N-arachidonoylethanolamine) and other bioactive N-acylethanolamines are degraded to their corresponding fatty acids and ethanolamine. This hydrolysis is mostly attributed to catalysis by FAAH (fatty acid amide hydrolase), which exhibits an alkaline pH optimum. In addition, we have identified another amidase which catalyses the same reaction exclusively at acidic pH values [Ueda. Yamanaka and Yamamoto (2001) J. Biol. Chem. 276, 35552-35557]. In attempts to find selective inhibitors of this acid amidase, we screened various derivatives of palmitic acid, 1-hexadecanol. and 1-pentadecylamine with N-palmitoylethanolamine as substrate. Here we show that N-cyclohexanecarbonyl-pentadecylamine inhibits the acid amidase from rat lung with an IC50 of 4.5 muM, without inhibiting FAAH at concentrations Lip to 100 muM. The inhibition was reversible and non-competitive. This compound also inhibited the acid amidase in intact alveolar macrophages. With the aid of this inhibitor, it was revealed that rat basophilic leukaemia cells possess the acid amidase as well as FAAH. Thus the inhibitor may be a useful tool to distinguish the acid amidase from FAAH in various tissues and cells and to elucidate the physiological role of the enzyme

A second N-acylethanolamine hydrolase in mammalian tissues

It is widely accepted that fatty acid amide hydrolase (FAAH) plays a central role in the hydrolysis of anandamide. However, we found a second N-acylethanolamine hydrolase in animal tissues which hydrolyzed anandamide at acidic pH. This "acid amidase" was first detected with the particulate fraction of human megakaryoblastic CMK cells, and was solubilized by freezing and thawing without detergent. The enzyme was distinguish able from FAAH in terms of (1) the optimal activity at pH 5, (2) stimulation by dithiothreitol, (3) low sensitivity to two FAAH inhibitors (methyl arachidonyl fluorophosphonate and phenylmethylsulfonyl fluoride), and (4) high content in lung, spleen and macrophages of rat. The acid amidase purified from rat lung was the most active with N-palmitoylethanolamine among various long-chain N-acylethanolamines. To develop specific inhibitors for this enzyme, we screened various analogues of N-palmitoylethanolamine. Among the tested compounds, N-cyclohexanecarbonylpentadecylamine was the most potent inhibitor which does-dependently inhibited the enzyme with an IC50 value of 4.5 mu M without inhibiting FAAH at concentrations LIP to 100 mu M. The inhibitor was a useful toot to distinguish the acid amidase from FAAH with rat basophilic leukemia (RBL-1) cells that express both the enzymes. (c) 2005 Flsevier Ltd. All rights reserved

Esters, retroesters, and a retroamide of palmitic acid: pool for the first selective inhibitors of N-palmitoylethanolamine-selective acid amidase.

Cyclohexyl hexadecanoate, hexadecyl propionate, and N-(3-hydroxypropionyl)pentadecanamide, respectively ester, retroester, and retroamide derivatives of N-palmitoylethanolamine, represent the first selective inhibitors of "N-palmitoylethanolamine hydrolase" described so far. These compounds are devoid of affinity for CB(1) and CB(2) receptors and characterized by high percentages of inhibition of N-palmitoylethanolamine-selective acid amidase (84.0, 70.5, and 76.7% inhibition at 100 microM, respectively) with much lower inhibitory effect on either fatty acid amide hydrolase or the uptake of anandamide