Anandamide amidase

Once inside the cell, anandamide is hydrolyzed by a specific hydrolase, anandamide amidase (AEAase) or fatty acid amidohydrolase (FAAH) (Desarnaud, 1995 Deutsch, 1993). This enzyme is membrane associated and shows significant specificity for anandamide (Desarnaud, 1995 Lang, 1999). 

There is some evidence that in cells with low anandamide amidase activity, such as platelets and neutrophils, anandamide is inactivated by an oxidative pathway involving 12(5)-lipoxygenase (Edgemond, 1998). Metabolism of anandamide by enzymes of the arachidonic acid cascade... 

Figure 6 Anandamide metabolism NAPE, N-arachidonylphosphatidyl-ethanol-amides PLD, phospholipase D AEA, anandamide AC, anandamide carrier protein AT, anandamide transporter AEAase, anandamide amidase AA, arachidonic acid.

Anandamide amidase recognizes and hydrolyzes 2-AG (Goparaju, 1999 Di Marzo, 1999 Lang, 1999) however, there is evidence for the existence of another specific hydrolase [monoacylglycerol (MAG) lipase] that hydrolyzes 2-AG (D. Piomelli and A. Makriyannis, 2000, personal communication). In addition to this pathway, 2-AG diffuses rapidly into the cell membrane where it could be either hydrolyzed to arachidonic acid and glycerol or esterified back to phosphoglycerides (Di Marzo, 1999b). 

Arachidonyl ethanolamide (anandamide) is a naturally-occurring brain constituent that acts as a CBl and CB2 agonist and exhibits pharmacological activity in mice comparable to cannahinoids (Fride and Mechoulam (1993), Crawley et al. (1993) and Smith et al. (1994)). Anandamide is cleaved in vivo by anandamide amidase. Thus, inhibitors of anandamide amidase have the effect of indirectly stimulating the CBl and CB2 receptors by increasing in vivo levels of anandamide. In addition to acting at the CBl and CB2 receptors, cannahinoids also affect cellular membranes, thereby producing undesirable side effects... 

The assays (see above) for arachidonoyl ethanolamide hydrolysis (anandamide amidase) employed H-anandamide as the substrate... 

Interestingly, the enzymatic activities found for anandamide amidase may be related to those reported in the literature for the hydrolysis of other fatty acid amides. Bachur and Udenfriend (1966) and Schmid and colleagues (1990) described a rat liver microsomal enzyme that hydrolyzed fatty acid ethanolamides. Natarajan and colleagues (1984) described activity in a dog brain microsomal fraction that hydrolyzed... 

PUTATIVE TRANSITION-STATE INHIBITORS OF ANANDAMIDE AMIDASE... 

To further explore the question of the mechanism of anandamide amidase inhibition, analogs of anandamide were synthesized (Koutek et al. 1994). These anandamide analogs (figure 6) represent three classes of putative transition-state inhibitors trifluoromethyl ketone (4), -keto ester (3), and -keto amide derivatives (2). The general strategy of this study was based upon the h q)othesis that polarized carbonyls, such as those in trifluoromethyl ketones and -keto carboxylate derivatives, may form stabilized hydrates or enzyme adducts that mimic the tetrahedral... 

Very recently the authors developed a novel anandamide amidase inhibitor, AM374, whose potency in vitro and in neuroblastoma cells significantly exceeds that of other compounds developed to date as well as PMSF. In intact neuroblastoma cells, AM374 was found to dramatically increase the level of undegraded anandamide 5 5-fold at 10 nM. Interestingly, its affinity for the CBl receptor was approximately tenfold weaker than anandamide (Deutsch and Makriyannis, unpublished data). 

Deutsch DG, Omeir R, Arreaza G, Salehani D, Prestwich GD, Huang Z, Howlett A (1997b) Methyl arachidonyl fluorophosphonate a potent irreversible inhibitor of anandamide amidase. Biochem Pharmacol 53 255-260... 

Lang W, Qin C, HiU WA, Lin S, Khanolkar AD, Makriyannis A (1996) High-performance hquid chromatographic determination of anandamide amidase activity in rat brain microsomes. Anal Biochem 238 40-45... 

Bilfinger TV, Salzet M, Fimiani C, Deutsch DG, Tramu G, Stefano GB (1998) Pharmacological evidence for anandamide amidase in human cardiac and vascular tissues. Int J Cardiol 64 815-22... 

Bilfinger, TV., Salzet, M., Fimiani, C., Deutsch, A., Tramu, G., and Stefano, G.B. (1998). Pharmacological evidence for anandamide amidase in human cardiac vascular tissues. International Journal of Cardiology, 64 (Suppl. 1) S15-S22. 

Stefano GB, Rialas CM, Deutsch DG, Salzet M. (1998) Anandamide amidase inhibition enhances anandamide-stimulated nitric oxide release in invertebrate neural tissues. Brain Research 793 341-345. 

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