Identification of a Novel Endocannabinoid-Hydrolyzing Enzyme Expressed by Microglial Cells

Abstract

The endocannabinoids (eCBs) anandamide and 2-arachidonoyl glycerol (2-AG) are inactivated by a two-step mechanism. First, they are carried into cells, and then anandamide is hydrolyzed by fatty acid amide hydrolase (FAAH) and 2-AG by monoacylglycerol lipase (MGL). Here we provide evidence for a previously undescribed MGL activity expressed by microglial cells. We found that the mouse microglial cell line BV-2 does not express MGL mRNA and yet efficiently hydrolyzes 2-AG. URB597 (3Ј-carbamoyl-biphenyl-3-ylcyclohexylcarbamate) reduces this hydrolysis by 50%, suggesting the involvement of FAAH. The remaining activity is blocked by classic MGL inhibitors [[1,1-biphenyl]-3-yl-carbamic acid, cyclohexyl ester (URB602) and MAFP (methylarachidonyl fluorophosphate)] and is unaffected by inhibitors of COXs (cyclooxygenases), LOXs (lipooxygenases), and DGLs (diacylglycerol lipases), indicating the involvement of a novel MGL activity. Accordingly, URB602 leads to selective accumulation of 2-AG in intact BV-2 cells. Although MGL expressed in neurons is equally distributed between the cytosolic, mitochondrial, and nuclear fractions, the novel MGL activity expressed by BV-2 cells is enriched in mitochondrial and nuclear fractions. A screen for novel inhibitors of eCB hydrolysis identified several compounds that differentially block MGL, FAAH, and the novel MGL activity. Finally, we provide evidence for expression of the novel MGL by mouse primary microglia in culture. Our results suggest the presence of a novel, pharmacologically distinct, MGL activity that controls 2-AG levels in microglia.

Implication of the anti-inflammatory bioactive lipid prostaglandin D2-glycerol ester in the control of macrophage activation and inflammation by ABHD6

“…Indeed, until recently, data pointed to MAGL as the primary enzyme responsible for 2-AG hydrolysis, at least in the brain (13,14). The role of ABHD6 in 2-AG hydrolysis was first put forth in BV2 cells that did not express MAGL (16) and subsequently in neurons (17,18). Here, we also show that the J774 and RAW264.7 macrophage cell lines do not express MAGL and that ABHD6 inhibition leads to increased 2-AG levels in these cells and reduces their activation.…”

“…Because of this control of brain 2-AG levels, inhibition of MAGL leads to increased central levels of 2-AG and therefore to undesirable psychotropic side effects due to activation of the CB 1 cannabinoid receptor (15). More recently, other enzymes have been implicated in 2-AG hydrolysis, such as the newly annotated enzyme α/β-hydrolase domain 6 (ABHD6) (16)(17)(18)(19). Here, we demonstrate the anti-inflammatory effects of inhibiting ABHD6 and thus increasing 2-AG levels, without the side effects of MAGL inhibition.…”

The synthesis and biological evaluation of para-substituted phenolic N-alkyl carbamates as endocannabinoid hydrolyzing enzyme inhibitors

“…The presumption that electronwithdrawing groups in para-position on the phenyl ring would increase the electrophilicity of the carbonyl carbon prompted us to prepare a series of N-alkyl carbamates with various electron-withdrawing and electron-donating substituents (Table 4). Methoxy carbonyl (30)(31)(32)(33), nitro (34)(35)(36), cyano (37 and 38), and bromine (39) were selected as electron-withdrawing (EWG), and methoxy (40) and methyl (41) as electron-donating groups (EDG). The neutral effect was evaluated with the unsubstituted O-phenyl carbamates 42 and 43.…”

“…However, the main enzyme responsible for the inactivation of 2-AG in the brain has been suggested to be MGL (EC 3.1.1.23) [9,33,37], although other hydrolytic activities may also contribute to the metabolism [34][35][36][37]. MGL is a serine hydrolase, which hydrolyzes medium-and long-chain fatty acid esters such as 2-AG and its analogue 2-oleoylglycerol (2-OG) [9,38].…”

Role of CB1 cannabinoid receptors on GABAergic neurons in brain aging

“…Astrocytes and microglial cells can synthesize endocannabinoids because they express enzymes involved in the synthesis (12,13) and degradation (13,14) of endocannabinoids. In the central nervous system, cannabinoid type 1 (CB1) receptors are found primarily on neurons and at a marginal level, on glial cells (15).…”