Monoglyceride lipase-like enzymatic activity is responsible for hydrolysis of 2-arachidonoylglycerol in rat cerebellar membranes

Abstract

2-Arachidonoylglycerol (2-AG) is an endogenous cannabinoid that binds to CB1 and CB2 cannabinoid receptors, inducing cannabimimetic effects. However, the cannabimimetic effects of 2-AG are weak in vivo due to its rapid enzymatic hydrolysis. The enzymatic hydrolysis of 2-AG has been proposed to mainly occur by monoglyceride lipase (monoacylglycerol lipase). Fatty acid amide hydrolase (FAAH), the enzyme responsible for the hydrolysis of N-arachidonoylethanolamide (AEA), is also able to hydrolyse 2-AG. In the present study, we investigated the hydrolysis of endocannabinoids in rat cerebellar membranes and observed that enzymatic activity towards 2-AG was 50-fold higher than that towards AEA. Furthermore, various inhibitors for 2-AG hydrolase activity were studied in rat cerebellar membranes. 2-AG hydrolysis was inhibited by methyl arachidonylfluorophosphonate, hexadecylsulphonyl fluoride and phenylmethylsulphonyl fluoride with ic₅₀ values of 2.2 nM, 241 nM and 155 μM, respectively. Potent FAAH inhibitors, such as OL-53 and URB597, did not inhibit the hydrolysis of 2-AG, suggesting that 2-AG is inactivated in rat cerebellar membranes by an enzyme distinct of FAAH. The observation that the hydrolysis of 1(3)-AG and 2-AG occurred at equal rates supports the role of MGL in 2-AG inactivation. This enzyme assay provides a useful method for future inhibition studies of 2-AG degrading enzyme(s) in brain membrane preparation having considerably higher MGL-like activity when compared to FAAH activity.

Introduction

N-Arachidonoylethanolamide (AEA) and 2-arachidonoylglycerol (2-AG) are presently considered to be the most important endogenous ligands for central cannabinoid receptor (CB1) and peripheral cannabinoid receptor (CB2) [1], [2], [3]. The endocannabinoids are produced by neurons “on demand,” act near the site of their synthesis and, as is typical for neuromodulators, they are effectively metabolized to ensure rapid signal inactivation [1], [2], [3]. AEA has remained in the spotlight of endocannabinoid research, although increasing number of observations support an even more important role for 2-AG in the endocannabinoid system. 2-AG is present in the rat brain in amounts 170–1000 times greater than AEA [3], [4], and 2-AG acts as a potent and full-efficacy agonist at both CB1 and CB2 receptors [4], [5], [6], [7], [8]. In contrast, AEA is clearly less potent and acts only as a partial agonist at the CB receptors [3], [7], [8]. The biological inactivation of AEA is generally believed to occur via cellular uptake through facilitated diffusion [9], [10], [11], followed by intracellular enzymatic hydrolysis to arachidonic acid and ethanolamine by the membrane-bound fatty acid amide hydrolase (FAAH) [9], [12], [13], [14]. However, the existence of any AEA transporter has been questioned [15]. FAAH has a broad substrate specificity, and it is also able to hydrolyse other fatty ethanolamides and fatty esters such as 2-AG [16]. In fact, the specific activity of FAAH for 2-AG has been reported to be even higher when compared to AEA [17], [18], [19]. In addition, it is well known that also other enzymes such as lipases and esterases are able to hydrolyse 2-AG [20], [21]. Recent evidence contradicts the physiological role of FAAH in 2-AG inactivation. Goparaju et al. [22] reported that the 2-AG hydrolase activity of porcine brain can be separated from FAAH. Beltramo and Piomelli [23] demonstrated that a known FAAH inhibitor does not inhibit 2-AG hydrolysis in human astrocytoma cells, indicating that 2-AG hydrolysis may depend on another hydrolase for inactivation. Furthermore, 2-AG hydrolysis is preserved in FAAH-deficient mice, which cannot hydrolyse AEA [24]. The most plausible enzyme responsible for 2-AG hydrolysis in vivo remains monoglyceride lipase (MGL, EC 3.1.1.23). This enzyme was reported over 20 years ago to catalyze the hydrolysis of monoacylglycerols that contain an arachidonoyl group in the sn-2 position [25]. MGL is a serine hydrolase that specifically hydrolyses 2- and 1(3)-ester bonds of monoglycerides to fatty acid and glycerol [20], preferring the hydrolysis of sn-2-arachidonoylglycerol to sn-2-oleoyl- [25], [26] or sn-2-palmitoylglycerol [27]. Additionally, a recent report by Dinh et al. [28] supports the observation that 2-AG is mainly hydrolyzed by MGL in intact neurons. However, the relative importance of these degradative pathways in 2-AG inactivation remains elusive.

Inhibitors of endocannabinoid hydrolases could offer a rational therapeutic approach in treating certain disease states, where higher endocannabinoid activity would be beneficial. An advantage of such enzyme inhibition over direct cannabinoid agonists could result in higher selectivity, as it would increase activity of endocannabinoid system only at sites where on-going production of endocannabinoids is taking place.

In our previous CB1 receptor activation studies (Savinainen et al. [7]), we reported that rat cerebellar membrane preparation was able to hydrolyse 2-AG, although being almost totally devoid of FAAH activity. In this study, the hydrolyzing activity of rat cerebellar membrane preparation towards 2-AG and other endocannabinoids was examined in more detail, in order to develop a validated method for studies on novel inhibitors of 2-AG degrading enzymes. Additionally, we studied various FAAH inhibitors as inhibitors for the monoglyceride lipase-like activity found in the cerebellar preparation.