Περίληψη:
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). [ 3H]2-AG and [ 3H]2-oleoylglycerol (2-OG) were both metabolized to [ 3H]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 (Ic50 = 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 KM of 0.11 μM and Vmax 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 ∼ü33 kDa. © 2009 Informa UK Ltd All rights reserved.
Συγγραφείς:
Gkini, E.
Anagnostopoulos, D.
Mavri-Vavayianni, M.
Siafaka-Kapadai, A.
Λέξεις-κλειδιά:
2 arachidonoylglycerol; 2 oleoylglycerol; acylglycerol lipase; cyclohexylcarbamic acid 3' carbamoylbiphenyl 3 yl ester; endocannabinoid; fatty acid amidase; glycerol; palmitylsulfonyl fluoride; unclassified drug; 2 arachidonoylglycerol; 2 oleoylglycerol; 2-arachidonylglycerol; 2-oleoylglycerol; acylglycerol; acylglycerol lipase; amidase; arachidonic acid derivative; benzamide derivative; carbamic acid derivative; cyclohexyl carbamic acid 3' carbamoylbiphenyl 3 yl ester; cyclohexyl carbamic acid 3'-carbamoylbiphenyl-3-yl ester; enzyme inhibitor; fatty acid amidase; fatty-acid amide hydrolase; palmitic acid derivative; palmitylsulfonyl fluoride; tritium, animal cell; animal experiment; article; cell fractionation; comparative study; drug activity; enzymatic degradation; enzyme activity; enzyme analysis; enzyme substrate; human; human cell; human experiment; hydrolysis; IC 50; in vitro study; Michaelis Menten kinetics; molecular weight; nonhuman; normal human; priority journal; rabbit; thrombocyte; animal; blood; drug antagonism; drug effect; enzymology; immunoblotting; metabolism; pH, Oryctolagus cuniculus, Amidohydrolases; Animals; Arachidonic Acids; Benzamides; Blood Platelets; Carbamates; Enzyme Inhibitors; Glycerides; Glycerol; Humans; Hydrogen-Ion Concentration; Hydrolysis; Immunoblotting; Monoacylglycerol Lipases; Palmitates; Rabbits; Subcellular Fractions; Tritium
