title: Metabolism of 2-acylglycerol in rabbit and human platelets. Involvement of monoacylglycerol lipase and fatty acid amide hydrolase.
abstract: The endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide (N-arachidonoylethanolamine, AEA) are produced by neurons and other cells, including platelets, in a stimulus-dependent manner and act as signaling molecules; they are then inactivated through transport into cells followed by enzymatic degradation. A number of studies showed that monoacylglycerol lipase (MAGL) plays an important role in the degradation of 2-AG. In this study we investigated the enzymatic degradation of 2-acylglycerols in rabbit platelets and we characterized the responsible enzyme(s). [(3)H]2-AG and [(3)H]2-oleoylglycerol (2-OG) were both metabolized to [(3)H]glycerol and the respective fatty acid in a time and protein concentration-dependent manner, apparently by the action of MAGL activity. In the presence of the specific fatty acid amide hydrolase (FAAH) inhibitors URB597 and AM374, though, 2-OG hydrolysis was inhibited up to 55% in a concentration-dependent manner (IC(50) = 129.8 nM and 20.9 nM respectively). These results indicate the involvement of both MAGL and FAAH on 2-acylglycerol hydrolysis. MAGL was further characterized in the presence of URB597 and it was found that 2-monoacylglycerols were hydrolyzed in a time, pH and protein concentration-dependent manner and hydrolysis followed Michaelis-Menten kinetics, with an apparent K(M) of 0.11 microM and V(max) of 1.32 nmol/min*mg protein. Subcellular fractionation of platelet homogenate showed that MAGL activity was present in both the cytosolic and membrane fractions. In conclusion, the endocannabinoid 2-AG, as well as other 2-acylglycerols, are substrates of both FAAH and MAGL; the latter was characterized for the first time in platelets. In human platelets, under the same experimental conditions, the hydrolysis of 2-acylglycerols was higher and MAGL activity showed a different sensitivity against the inhibitors mentioned above. Finally, immunoblot analysis revealed the presence of MAGL, both in rabbit and human platelets, with a molecular mass of approximately 33 kDa.
authors.affiliation: Department of Chemistry, University of Athens, Panepistimioupolis, 15771 Athens, Greece.
authors.forename: Eleni
authors.initials: E
authors.lastname: Gkini
authors.forename: Dimitris
authors.initials: D
authors.lastname: Anagnostopoulos
authors.forename: Mary
authors.initials: M
authors.lastname: Mavri-Vavayianni
authors.forename: Athanasia
authors.initials: A
authors.lastname: Siafaka-Kapadai
mesh_terms: D000581:Amidohydrolases / Q000037:antagonists & inhibitors / Q000097:blood*; D000818:Animals; D001095:Arachidonic Acids / Q000097:blood*; D001549:Benzamides / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D002219:Carbamates / Q000494:pharmacology; D063388:Endocannabinoids; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005989:Glycerides / Q000097:blood*; D005990:Glycerol / Q000097:blood; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D015151:Immunoblotting; D008994:Monoacylglycerol Lipases / Q000037:antagonists & inhibitors / Q000097:blood*; D010168:Palmitates / Q000494:pharmacology; D011817:Rabbits; D013347:Subcellular Fractions / Q000201:enzymology; D014316:Tritium; D000099252:Fatty Acid Amide Hydrolases
chemical_list: D000581:Amidohydrolases; D001095:Arachidonic Acids; D001549:Benzamides; D002219:Carbamates; D063388:Endocannabinoids; D004791:Enzyme Inhibitors; D005989:Glycerides; D005990:Glycerol; D008994:Monoacylglycerol Lipases; D010168:Palmitates; D014316:Tritium; D000099252:Fatty Acid Amide Hydrolases; C104963:AM 374; C500528:cyclohexyl carbamic acid 3'-carbamoylbiphenyl-3-yl ester; C094503:glyceryl 2-arachidonate; C505247:2-oleoylglycerol