Effects of two putative monoacylglycerol lipase-selective inhibitors (URB754 & URB602) in the rat mesenteric artery

W.S. Vanessa Ho & Michael D. Randall, School of Biomedical Sciences, University of Nottingham, Queen’s Medical Centre, Nottingham NG7 2UH.

The monoacylglycerol lipase (MGL) has been identified as the primary enzyme for the inactivation of 2-arachidonoyl glycerol (2-AG) via hydrolysis to arachidonic acid and glycerol. 2-AG could also act as a substrate for fatty acid amide hydrolase (FAAH), which is the primary hydrolase for another endocannabinoid, anandamide. Using combined inhibitors of FAAH and MGL, there is evidence suggesting that MGL plays a role in regulating the vascular actions of 2-AG ( see Ho & Randall, 2006; this meeting ). Very recently, a couple of selective MGL inhibitors, namely URB754 ( 6-methyl-2-[(4-methylphenyl)amino]-4H-3,1-benzoxazin-one ) and URB602 ( [1,1’-biphenyl]-3-yl-carbamic acid, cyclohexyl ester) (Hohmann et al. 2005) , have been reported. Here, we have examined the effects of these two inhibitors on vasorelaxation to both 2-AG and anandamide in vitro.

Male Wistar rats (200-300g) were stunned by a blow to the back of the head and killed by cervical dislocation. The third-order branches of the superior mesenteric artery (2mm long) were mounted in a wire myograph and maintained at 37oC in gassed ( 95% O2/5% CO2) Krebs-Henseleit solution (O’Sullivan et al., 2004). In endothelium-intact, methoxamine-precontracted vessels, relaxant responses were determined by cumulative addition of an endocannabinoid or carbachol (a muscarinic receptor agonist). Inhibitors were incubated with vessels for 30 or 45min before determination of a concentration- response curve. Results were expressed as mean±s.e.m. (n≥4 rats) and compared by two-way analysis of variance of the whole data set. P<0.05 was considered statistically significant.

The reportedly MGL-selective inhibitor URB754 ( 3μM ) applied either alone, or in combination with the FAAH inhibitor URB597 ( 3’-carbamoyl biphenyl-3-yl-cyclohexylcarbamate ; 1μM ), had no effect on relaxations to 2-AG (control: pEC50=5.4 ± 0.1 Emax=87 ± 3 % ; + URB754: pEC50=5.4 ± 0.1 Emax=97 ± 2 %; + URB754 & URB597: pEC50=5.5 ± 0.1 Emax=95 ± 2 % ). On the other hand, anandamide-induced relaxation was significantly enhanced by the combined URB597 & URB754 treatment, but not URB754 alone ( control: pEC50=6.7 ± 0.1 Emax=101 ±1% ; + URB754: pEC50=6.7 ± 0.1 Emax=100 ±1%; + URB597 & URB754: pEC50=7.2 ± 0.2 Emax=101 ±1% ). In contrast, the other selective MGL inhibitor, URB602 (100μM ) blunted relaxations to both 2-AG ( % relaxation at 10μM, 39 ± 15 % ) and anandamide ( % relaxation at 3μM, 22 ± 15 % ) . Furthermore, URB602 also significantly attenuated endothelium-dependent relaxations to carbachol ( control: pEC50=6.7 ± 0.2 Emax=98 ± 2 % ; + URB602: pEC50=6.3 ± 0.1 Emax= 88 ± 5% ) and contractions to methoxamine (control: pEC50=5.5 ± 0.1Emax=14 ±1 mN ; + URB602: pEC50=4.7 ± 0.2 Emax=7 ± 2mN ) .

The lack of effect of URB754 herein is consistent with recent doubts over its ability to inhibit MGL. This, together with the possible MGL-independent actions of URB602, suggests that cautious interpretation of results is warranted when URB754 and URB602 are used as pharmacological inhibitors for MGL, at least in the rat mesenteric artery.

O’Sullivan SE et al. (2004). Br J Pharmacol 141: 803-12
Hohmann AG et al (2005). Nature 435: 1108-12.

 This study was supported by the British Heart Foundation (PG/06/064/20985). WSVH is an Anne McLaren Fellow of University of Nottingham.