urb-597 and 2-oleoylglycerol

Tetrahymena thermophila is a model organism for molecular and cellular biology. Previous studies from our group showed that Tetrahymena contains major components of the endocannabinoid system, such as various endocannabinoids and FAAH. In mammalian cells the endocannabinoid 2-arachidonoylglycerol is inactivated mainly by MAGL. In this study we showed that 2-arachidonoylglycerol and 2-oleoylglycerol are hydrolyzed by the combined actions of MAGL and FAAH. MAGL-like activity was examined in the presence of FAAH specific inhibitors, URB597 or AM374 and showed optimum pH of 8-9, apparent K(M) of 14.1μM and V(max) of 5.8nmol/min×mg. The enzyme was present in membrane bound and cytosolic isoforms; molecular mass was determined at ∼45 and ∼40kDa. MAGL and FAAH could also inactivate endogenous signaling lipids, which might play an important role in Tetrahymena as suggested in mammals. Tetrahymena could be used as a model system for testing drugs targeting enzymes of the endocannabinoid system.

Topics: Amidohydrolases; Arachidonic Acids; Benzamides; Carbamates; Cell Membrane; Cytosol; Endocannabinoids; Enzyme Inhibitors; Glycerides; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Molecular Weight; Monoacylglycerol Lipases; Palmitates; Protozoan Proteins; Tetrahymena thermophila

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.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Benzamides; Blood Platelets; Carbamates; Endocannabinoids; Enzyme Inhibitors; Glycerides; Glycerol; Humans; Hydrogen-Ion Concentration; Hydrolysis; Immunoblotting; Monoacylglycerol Lipases; Palmitates; Rabbits; Subcellular Fractions; Tritium