British Journal of Pharmacology (2007) 150, 641–651 & 2007 Nature Publishing Group All rights reserved 0007–1188/07 $30.00 www.brjpharmacol.org

RESEARCH PAPER Endothelium-dependent metabolism by endocannabinoid hydrolases and cyclooxygenases limits vasorelaxation to and 2-arachidonoylglycerol

W-SV Ho and MD Randall

School of Biomedical Sciences, University of Nottingham Medical School, Queen’s Medical Centre, Nottingham, UK

Background and purpose: The endocannabinoids, N-arachidonoylethanolamide (anandamide) and 2-arachidonoylglycerol (2-AG) are rapidly degraded by fatty acid amide hydrolase (FAAH) and (MGL). Whilst these lipid mediators are known to modulate vascular tone, the extent to which they are inactivated via local metabolism in the vasculature remains unclear. Experimental approach: In rat isolated small mesenteric arteries, the regulatory role of FAAH, MGL and cyclooxygenase (COX) in relaxant responses to anandamide and 2-AG was evaluated by using inhibitors of these enzymes. Relaxations to non- hydrolysable analogues of endocannabinoids and arachidonic acid were also examined. Key results: Relaxation to anandamide but not 2-AG was potentiated by the selective FAAH inhibitor, URB597 (1 mM). In contrast, MAFP (10 mM; an inhibitor of FAAH and MGL) enhanced responses to both anandamide and 2-AG. Inhibition of COX-1 by indomethacin (10 mM) potentiated relaxations to 2-AG, whereas inhibition of COX-2 by nimesulide (10 mM) potentiated anandamide-induced relaxation. With the exception of MAFP, effects of FAAH and COX inhibitors were dependent on the endothelium. Relaxation to and noladin ether, the non-hydrolysable analogues of anandamide and 2-AG respectively, were insensitive to the enzyme inhibitors. Conclusion and implications: This study shows that local activity of FAAH, MGL and COX, which is present largely in the endothelium, limits the vasodilator action of endocannabinoids in rat small mesenteric arteries. Despite the differential roles played by these enzymes on relaxation to anandamide versus 2-AG, our results suggest that inhibitors of these enzymes enhance the vascular impact of endocannabinoids. British Journal of Pharmacology (2007) 150, 641–651. doi:10.1038/sj.bjp.0707141; published online 22 January 2007

Keywords: anandamide; 2-arachidonoylglycerol; fatty acid amide hydrolase; monoacylglycerolipase; cyclooxygenase; rat mesenteric artery; endothelium Abbreviations: MAFP, methyl arachidonyl fluorophosphonate; URB597, 30-carbamoyl-biphenyl-3-yl-cyclohexylcarbamate

Introduction

Endocannabinoids, in particular the prototypical endocan- analogue, methanandamide, often act via activation of the nabinoid, N-arachidonoylethanolamide (anandamide), have (CB)1 or the transient receptor poten- been shown to induce vasodilatation, modulate regional tial vanilloid 1 (TRPV1) receptor, or both (Lake et al., 1997; blood flow and arterial blood pressure and reduce heart rate Gebremedhin et al., 1999; Zygmunt et al., 1999; Ralevic et al., (Randall et al., 1996; Lake et al., 1997; Gardiner et al., 2002; 2000; Gardiner et al., 2002; Ho and Hiley, 2003). In the rat Batkai et al., 2004). Although the precise mechanisms of mesenteric artery, relaxations to anandamide and metha- action may depend on the vascular regions, species and state nandamide are largely mediated by TRPV1 receptors on of anaesthesia, anandamide and its metabolically stable perivascular sensory nerves (Zygmunt et al., 1999; Ralevic et al., 2000; Ho and Hiley, 2003). In addition, activation of

CB1 receptors, a novel endothelial 2 þ þ Correspondence: Dr W-S Vanessa Ho, School of Biomedical Sciences, coupled to Ca -activated K channels and/or direct University of Nottingham Medical School, Queen’s Medical Centre, inhibition of voltage-dependent calcium entry might also Nottingham NG7 2UH, UK. play a role (Ho and Hiley, 2003; Offertaler et al., 2003; E-mail: [email protected] Received 2 November 2006; revised 29 November 2006; accepted 30 O’Sullivan et al., 2004). However, in comparison with November 2006; published online 22 January 2007 anandamide, little is known about the vasorelaxant effects Endocannabinoid vasorelaxation and metabolism 642 W-SV Ho and MD Randall of the other major endocannabinoid, 2-arachidonoylglycerol clinically, to enhance endocannabinoid activity. However, it (2-AG) despite observations that tissue content of 2-AG is should be pointed out that other metabolic pathways might often more than 200-fold higher than that of anandamide also play a role in the metabolism of endocannabinoids. (e.g., hippocampus: Makara et al., 2005; heart: Pacher et al., In particular, cyclooxygenases (both COX-1 and COX-2 2005; cerebral artery: Rademacher et al., 2005). Several isoforms) could act on arachidonic acid subsequent to the studies have shown that 2-AG induces hypotension (Me- hydrolysis of endocannabinoids (Grainger and Boachie- choulam et al., 1998; Jarai et al., 2000) and vasorelaxation Ansah, 2001; Wahn et al., 2005); both anandamide and (Kagota et al., 2001; Ho and Hiley, 2004; Gauthier et al., 2-AG are potential substrates for COX-2 (Yu et al., 1997;

2005). By activating the CB1 receptor, 2-AG and its non- Kozak et al., 2000). This raises the possibility that a hydrolysable analogue noladin ether could reduce blood combined application of endocannabinoid hydrolases and pressure (Jarai et al., 2000). In mesenteric arteries, relaxant COX inhibitors could further increase the life-span of responses to 2-AG, which are mimicked by noladin ether, endocannabinoids. þ might involve CB1 receptors and K channels (Kagota et al., Depending on the vascular regions and species, effects 2001; Ho and Hiley, 2004). However, there have been of endocannabinoids on local vascular control could be conflicting reports regarding its action as a vasodilator regulated or mediated or both, by metabolism. This is (Wagner et al., 1999; Zygmunt et al., 1999; Kagota et al., possibly in part due to differences in the expression of 2001; Ho and Hiley, 2004), perhaps owing to its greater metabolizing enzymes. Where degradation is likely to susceptibility to degradation than anandamide (Jarai et al., regulate, rather than mediate, anandamide relaxations 2000; Gauthier et al., 2005). (Gebremedhin et al., 1999; Zygmunt et al., 1999; Ho and It is now recognized that endocannabinoids are taken up Hiley, 2003), the extent to which endocannabinoids are by various cell types, followed by rapid metabolism by inactivated via local metabolism in the vasculature, remains intracellular enzymes. Catabolism of both anandamide and unclear. Importantly, the potential role of FAAH- and MGL- 2-AG occurs via hydrolysis to arachidonic acid, and ethano- mediated hydrolysis in the vascular wall has not been lamine and glycerol, respectively. Anandamide hydrolysis is systematically studied and compared. In this study, we have mediated primarily by fatty acid amide hydrolase (FAAH; investigated the effects of known inhibitors of FAAH and Cravatt et al., 1996, 2001). Activity of this serine hydrolase MGL on the vasorelaxant responses to anandamide and has been found, for example, in blood vessels, heart, liver 2-AG in rat isolated small mesenteric arteries. In addition, the and brain (Deutsch and Chin, 1993; Desarnaud et al., 1995; involvement of the two isoforms of COX was also examined. Pratt et al., 1998). Development of effective inhibitors has enabled the characterization of FAAH and its role in endocannabinoid signalling. For instance, the selective and Methods potent FAAH inhibitor 30-carbamoyl-biphenyl-3-yl-cyclohex- ylcarbamate (URB597) has been shown to increase tissue Myograph studies content of anandamide, but not 2-AG, in the heart and brain Male Wistar rats (200–300 g; Charles River UK Ltd, Kent, UK) (Kathuria et al., 2003; Batkai et al., 2004). Application of were stunned by a blow to the back of their neck and killed URB597 alone has also been shown to lower blood pressure by cervical dislocation. All animal care and use was in in anaesthetized, hypertensive rats (Batkai et al., 2004). 2-AG accordance with the UK Animal (Scientific Procedures) Act might also serve as a substrate for FAAH (Goparaju et al., 1986. The third-order branches of the superior mesenteric 1998); indeed systemic and local treatment with FAAH artery were removed and cleaned of adherent tissue. inhibitors has been shown to elevate 2-AG content (Bifulco Segments (2 mm in length) were mounted in a Mulvany– et al., 2004; de Lago et al., 2005; Maione et al., 2006). Halpern type wire myograph (Model 610M, Danish Myo Moreover, a distinct serine hydrolase called the monoacyl- Technology, Aarhus, Denmark) and maintained at 371Cin glycerol lipase (MGL) also plays an important role in the gassed (95% O2/5% CO2) Krebs–Henseleit solution of the hydrolysis of 2-AG, especially in the brain (Karlsson et al., following composition (mM): NaCl 118, KCl 4.7, MgSO4 1.2, 2001; Cravatt and Lichtman, 2002; Dinh et al., 2002; KH2PO4 1.2, NaHCO3 25, CaCl2 2, D-glucose 10, as described Kathuria et al., 2003). As for FAAH, MGL appears to have previously (Ho and Hiley, 2003). Vessels were equilibrated a relatively ubiquitous tissue distribution; for example, its and set to a basal tension of 2–2.5 mN. The integrity of the activity has been found in the heart, macrophages, adipose endothelium was assessed by precontracting the vessel with tissue and brain (Tornqvist and Belfrage, 1976; Di Marzo 10 mM methoxamine (an a1 adrenoceptor agonist), followed et al., 1999; Dinh et al., 2002). Since the identification of by relaxation with 10 mM carbachol (a muscarinic receptor MGL, its sensitivity to some of the known serine hydrolase agonist); vessels showing relaxations of 490% were desig- inhibitors has been investigated (see Ho and Hillard, 2005 for nated as endothelium-intact. When endothelium was not review). Notably, the compound methyl arachidonyl fluoro- required, it was removed by rubbing the intima with a phosphonate (MAFP), which is commonly used to inhibit human hair; carbachol-induced relaxation of less than 10% FAAH, has also been found to be a potent inhibitor of MGL indicated successful removal. activity in cytosolic and membrane fractions of the brain (Goparaju et al., 1999; Dinh et al., 2002; Saario et al., 2004). The crucial role of FAAH and MGL in the inactivation of Experimental protocols anandamide and 2-AG suggests that inhibitors of these After the test for endothelial integrity, vessels were left for enzymes could be utilized experimentally and, perhaps also 30 min and then precontracted with 10 mM methoxamine.

British Journal of Pharmacology (2007) 150 641–651 Endocannabinoid vasorelaxation and metabolism W-SV Ho and MD Randall 643

This was followed by construction of a cumulative concen- UK) and URB754 (Cayman Chemical) were dissolved in tration–relaxation curve to a cannabinoid or arachidonic 100% dimethyl sulphoxide. acid. Each vessel was exposed only to one relaxant agent. In this study, most experiments were performed in matched vessels; effects of putative inhibitors or endothelial removal Results were compared with the control responses obtained in separate vessels of the same rat. Relaxation to anandamide and 2-AG To investigate the role of metabolism in the relaxant Anandamide induced concentration-dependent relaxation 7 responses to endocannabinoids, inhibitors of FAAH of rat isolated mesenteric arteries (pEC50 ¼ 6.7 0.1, 7 (URB597, arachidonyl trifluoromethyl ketone (ATFMK), Emax ¼ 101 1%, n ¼ 5). Removal of the endothelium caused MAFP), MGL (ATFMK, MAFP, 6-methyl-2-[(4-methylphenyl)- a significant rightward shift in the concentration–response amino]-4H-3,1-benzoxazin-one (URB754) and COX (indo- curve to anandamide (pEC50 ¼ 6.570.1, Emax ¼ 10171%, methacin, flurbiprofen and nimesulide) were used. One n ¼ 5; Po0.01). or more of these enzyme inhibitors were added to the Another endocannabinoid, 2-AG also caused vasorelaxa- myograph bath 30 or 45 min before, and were kept present tion in mesenteric arteries, albeit with lower potency than 7 7 during construction of the concentration–response curve of anandamide (pEC50 ¼ 5.5 0.1, Emax ¼ 75 10%, n ¼ 5). an endocannabinoid or its metabolically stable analogue Endothelial removal significantly reduced the potency and (methanandamide and noladin ether). In a separate set of the maximal effect (at 10 mM) of 2-AG-induced relaxations 7 7 experiments, effects of MAFP and COX inhibitors on the (pEC50 ¼ 5.4 0.1, Emax ¼ 41 11%, n ¼ 5; Po0.001). relaxant responses to arachidonic acid, a likely breakdown product of the endocannabinoids, were also examined. Incubation of the mesenteric arteries with MAFP or ATFMK Effects of FAAH and MGL inhibitors on relaxation to anandamide often reduced the contractions to methoxamine, possibly The compound URB597 is an irreversible, selective inhibitor owing to inhibition of cytosolic phospholipase A2 (LaBelle of FAAH and displays no activity at MGL (Kathuria et al., and Polyak, 1998). Therefore, an increased concentration (up 2003). It inhibits FAAH with IC50 values as low as 0.5 nM to 30 mM) of methoxamine was used to precontract vessels to (Kathuria et al., 2003) and 1 mM URB597 has been shown to obtain a similar level of tone to that evoked in the absence enhance the endogenous anandamide content in rat brain of MAFP and ATFMK. The tension generated in the test for slices (Makara et al., 2005). Here, in endothelium-intact endothelial integrity was 11.370.5 mN, as compared with vessels, the presence of URB597 (1 mM) potentiated the 10.170.5 mN (40 vessels) in the presence of MAFP. Tension relaxant responses to anandamide, resulting in a leftward was 10.670.8 mN in the endothelial test, as compared with displacement in the concentration–response curve (control, 7 7 9.370.8 mN in the presence of ATFMK (22 vessels). pEC50 ¼ 6.7 0.1, Emax ¼ 101 1%, n ¼ 4; with URB597, 7 7 pEC50 ¼ 7.2 0.1, Emax ¼ 98 4%, n ¼ 4; Po0.01; Figure 1a). However, URB597 had no significant effect on anandamide Data and statistical analysis relaxations in endothelium-denuded vessels (with URB597, 7 7 All relaxation responses are expressed as percentage relaxa- pEC50 ¼ 6.7 0.2, Emax ¼ 101 1%, n ¼ 5; Figure 1b). tion of the tone induced by methoxamine. Values are given In another set of experiments, the effects of other FAAH as mean 7s.e.m. and n represents the number of animals inhibitors, namely MAFP and ATFMK which could also used. Emax represents the maximum effect and pEC50 the inhibit MGL (see Ho and Hillard, 2005 for review) were negative logarithm of the concentration of relaxant giving tested. In membrane preparations, the enzymes MAFP and half the maximal relaxation; these values were determined ATFMK have been shown to inhibit FAAH at nanomolar directly from individual log concentration–response curves. concentrations (De Petrocellis et al., 1997; Deutsch et al., Statistical comparisons of concentration–response curves 1997). However, in view of the large variation in their were made by two-way analysis of variance (Prism 4, potency at MGL (inhibition occurs at concentrations from GraphPad Software Inc.) of the whole data set. P-values of low nM to mM; Ho and Hillard, 2005), they were used at 10 mM less than 0.05 were taken as statistically significant. to ensure inhibition of both FAAH and MGL in isolated vessels. In endothelium-intact mesenteric arteries, the presence of MAFP (10 mM) potentiated the relaxations 7 7 Drugs to anandamide (control, pEC50 ¼ 6.7 0.2, Emax ¼ 100 2, 7 7 Methoxamine and carbachol (Sigma Chemical Co., Poole, n ¼ 5; with MAFP, pEC50 ¼ 7.2 0.1, Emax ¼ 100 1%, n ¼ 5; UK) were dissolved in deionized water. Anandamide Po0.01). Similar results were also obtained with 10 mM 7 (N-arachidonoylethanolamide) and R-( þ )-methananamide ATFMK (with endothelium, control, pEC50 ¼ 6.9 0.2, 7 7 (Tocris Bioscience, Bristol, UK) were supplied in Tocrisolve Emax ¼ 100 1%, n ¼ 5; with ATFMK, pEC50 ¼ 7.6 0.1, 7 100 (1:4 soya:water emulsion) and diluted with deionized Emax ¼ 99 1%, n ¼ 5; Po0.001). It was noted that MAFP, water. 2-AG, noladin ether (2-AG ether; Tocris) and arachi- ATFMK or URB597 induced a similar leftward displacement donic acid (Cayman Chemical, Ann Arbor, MI, USA) were of anandamide concentration–response curves, with about supplied in 100% ethanol and diluted with deionized water. threefold increase in the potency of anandamide. URB597 (Cayman Chemical), MAFP (Tocris), nimesulide and The presence of 1 mM URB597 had no significant relaxant indomethacin and (7)-flurbiprofen (Sigma) were dissolved effect per se (data not shown). However, incubation of vessels in 100% ethanol. ATFMK (Alexis Corporation, Nottingham, with ATFMK and especially MAFP reduced methoxamine-

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0 -8 -7 -6 -5 log (methanandamide) Figure 1 Effects of FAAH inhibitors on anandamide-induced relaxation in mesenteric arteries. Relaxation was elicited by anandamide (a, b)or methanandamide (c) alone, or in the presence of 1 mM URB597 in endothelium-intact or -denuded vessels. Values are shown as means and vertical lines represent s.e.m.; n ¼ 4–7. The results with URB597 for endothelium-intact mesenteric arteries were also seen with two other less selective FAAH inhibitors, MAFP (10 mM) and ATFMK (10 mM) (data not shown). induced contractions (data not shown; see Methods). This Effects of FAAH and MGL inhibitors on relaxation to 2-AG is possibly owing to their additional inhibitory effect on The selective FAAH inhibitor URB597 (1 mM) had no effect cytosolic phospholipase A2 and thereby reduces methoxa- on 2-AG induced relaxation in endothelium-intact vessels 7 7 mine-induced production of contractile mediators (LaBelle (control, pEC50 ¼ 5.4 0.1, Emax ¼ 89 4%, n ¼ 4; with and Polyak, 1998), and/or an increase in the vascular content URB597, pEC50 ¼ 5.570.1, Emax ¼ 9871%, n ¼ 4; Figure 2a). of vasodilator endocannabinoids. In contrast, the presence of MAFP (10 mM) enhanced 2-AG 7 The putative MGL inhibitor URB754 (3 mM; Makara et al., relaxations (with endothelium, control, pEC50 ¼ 5.6 0.1, 7 7 2005) had no effect on relaxations induced by anandamide Emax ¼ 85 8%, n ¼ 5; with MAFP, pEC50 ¼ 5.9 0.2, 7 (data not shown). Emax ¼ 100 1%, n ¼ 5; Po0.001; Figure 2b). On the other hand, ATFMK appeared to modify 2-AG relaxations in a more complex fashion. In the presence of 10 mM ATFMK, relaxations tended to be potentiated at lower Effects of URB597 on relaxation to methanandamide concentrations but inhibited at higher concentrations of 7 Methanandamide, a metabolically stable analogue of anand- 2-AG (with endothelium, control, pEC50 ¼ 5.6 0.1, 7 7 amide (Abadji et al., 1994), induced endothelium-dependent Emax ¼ 89 6%, n ¼ 7; with ATFMK, pEC50 ¼ 6.1 0.2, 7 relaxation in mesenteric arteries (with endothelium, Emax ¼ 81 8%, n ¼ 7; Figure 2c). Based on two-way analysis 7 7 pEC50 ¼ 6.5 0.2, Emax ¼ 93 3%, n ¼ 6; without endothe- of variance of the concentration–response curves, there was 7 7 lium, pEC50 ¼ 5.9 0.2, Emax ¼ 91 6%, n ¼ 5; Po0.001). The a significant interaction (Po0.05) between the ATFMK presence of URB597 (1 mM) had no effect on relaxations to treatment and 2-AG concentrations. A similar tendency 7 7 methanandamide (control, pEC50 ¼ 6.9 0.2, Emax ¼ 96 3%, was observed in endothelium-denuded vessels (control, 7 7 7 7 n ¼ 7; with URB597, pEC50 ¼ 6.9 0.2, Emax ¼ 94 4%, n ¼ 7; pEC50 ¼ 5.4 0.2, Emax ¼ 62 11%, n ¼ 5; with ATFMK, 7 7 Figure 1c). pEC50 ¼ 5.5 0.2, Emax ¼ 48 11%, n ¼ 5; Figure 2d).

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0 0 -7 -6 -5 -7 -6 -5 log (2-AG) log (2-AG) Figure 2 Effects of FAAH inhibitors on 2-AG-induced relaxation in mesenteric arteries. Relaxation was elicited by 2-AG alone, or in the presence of 1 mM URB597 (a), 10 mM MAFP (b)or10mM ATFMK (c, d) in endothelium-intact or -denuded vessels. Values are shown as means and vertical lines represent s.e.m. n ¼ 4–7.

7 7 In contrast, URB754 (3 mM) applied either alone or in and URB597, pEC50 ¼ 7.4 0.2, Emax ¼ 102 1%, n ¼ 7; combination with URB597 (1 mM) had no effect on the 2-AG- Po0.001; Figure 3d, c.f. Figure 1a). In contrast, the relaxant induced relaxation (data not shown). effects of methanandamide were unaffected by nimesulide 7 7 (control, pEC50 ¼ 6.5 0.3, Emax ¼ 92 4, n ¼ 6; with nimesu- 7 7 lide, pEC50 ¼ 6.4 03, Emax ¼ 93 2%, n ¼ 6). Effects of COX inhibitors on relaxation to anandamide The COX inhibitor indomethacin (10 mM) had no significant effect on anandamide-induced relaxation, either in the Effects of COX inhibitors on relaxation to 2-AG 7 presence (with indomethacin, pEC50 ¼ 6.9 0.1, Relaxations to 2-AG were significantly potentiated by 10 mM 7 7 Emax ¼ 98 3%, n ¼ 4; Figure 3a) or absence of the endothe- indomethacin (with endothelium, control, pEC50 ¼ 5.5 0.1, 7 7 7 7 lium (control, pEC50 ¼ 6.5 0.1, Emax ¼ 101 1%, n ¼ 5; with Emax ¼ 78 5%, n ¼ 7; with indomethacin, pEC50 ¼ 5.9 0.1%, indomethacin, pEC50 ¼ 6.570.2, Emax ¼ 10071%, n ¼ 4). Emax ¼ 8675%, n ¼ 6; Po0.05; Figure 4a). However, in In endothelium-intact vessels, the selective COX-2 inhi- endothelium-denuded vessels, indomethacin had no signifi- 7 7 bitor nimesulide (10 mM; Warner et al., 1999) caused a small, cant effect (control, pEC50 ¼ 5.4 0.1%, Emax ¼ 51 14%, 7 7 but significant, potentiation of anandamide-induced relaxa- n ¼ 5; with indomethacin, pEC50 ¼ 5.3 0.1, Emax ¼ 62 13%, 7 7 tion (control, pEC50 ¼ 6.6 0.1, Emax ¼ 97 2%, n ¼ 5; with n ¼ 5; Figure 4b). 7 7 nimesulide, pEC50 ¼ 6.9 0.2, Emax ¼ 102 1%, n ¼ 5; The COX inhibitor flurbiprofen, which displays a greater Po0.01; Figure 3b). In the absence of endothelium, nime- preference for COX-1 over COX-2 isoform than indometha- sulide had no effect on anandamide relaxations (control, cin (Warner et al., 1999), was also used. The presence of 7 7 pEC50 ¼ 6.6 0.2, Emax ¼ 98 2%, n ¼ 5; with nimesulide, flurbiprofen (10 mM) also caused a leftward displacement 7 7 pEC50 ¼ 6.5 0.2, Emax ¼ 100 1%, n ¼ 5; Figure 3c). Interest- of the concentration–response curve to 2-AG (with endothe- ingly, the combined treatment of the FAAH inhibitor, lium, control, pEC50 ¼ 5.570.1, Emax ¼ 8278%, n ¼ 4; with 7 7 URB597 and nimesulide had similar potentiation effects flurbiprofen, pEC50 ¼ 5.9 0.2, Emax ¼ 95 2%, n ¼ 5; compared to those of URB597 alone (control, Po0.01; Figure 4c). However, nimesulide (10 mM) had 7 7 pEC50 ¼ 7.0 0.1, Emax ¼ 100 2%, n ¼ 6; with nimesulide no effect on 2-AG-induced relaxation (with endothelium,

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0 0 -8 -7 -6 -5 -8 -7 -6 -5 log (anandamide) log (anandamide) Figure 3 Effects of COX inhibitors on anandamide-induced relaxation in mesenteric arteries. Relaxation was elicited by anandamide alone, or in the presence of 10 mM indomethacin (a), 10 mM nimesulide (b, c)or10mM nimesulide plus 1 mM URB597 (d) in endothelium-intact or -denuded vessels. Values are shown as means and vertical lines represent s.e.m. n ¼ 4–6.

7 7 7 7 control, pEC50 ¼ 5.4 0.1, Emax ¼ 96 2%, n ¼ 5; with nime- methacin, pEC50 ¼ 6.0 0.2, Emax ¼ 94 5, n ¼ 4), or MAFP 7 7 7 7 sulide, pEC50 ¼ 5.6 0.3, Emax ¼ 81 10%, n ¼ 5; Figure 4d). (control, pEC50 ¼ 6.3 0.2, Emax ¼ 99 1%, n ¼ 5; with 10 mM 7 7 Interestingly, the combined treatment of indomethacin MAFP, pEC50 ¼ 6.6 0.2, Emax ¼ 90 2%, n ¼ 5). and the putative MGL inhibitor, MAFP (10 mM each) greatly enhanced relaxations to 2-AG; an apparent additive effect of 7 the two inhibitors was observed (control, pEC50 ¼ 5.4 0.1, Effects of COX inhibitors and MAFP on relaxation to arachidonic Emax ¼ 77715%, n ¼ 5; with indomethacin and MAFP, pEC50 ¼ acid 7 7 6.3 0.3, Emax ¼ 100 1%, n ¼ 5; Po0.001; Figure 5a; cf. Figures For comparison, vasoactive effects of arachidonic acid, the 2b and 4a). In endothelium-denuded vessels, MAFP also caused common product from hydrolysis of both anandamide and a significant potentiation effect on 2-AG relaxations (control, 2-AG were also examined. In endothelium-intact mesenteric 7 7 pEC50 ¼ 5.3 0.1, Emax ¼ 63 15%, n ¼ 4; with indomethacin arteries, arachidonic acid caused concentration-dependent 7 7 7 7 and MAFP, pEC50 ¼ 5.4 0.1, Emax ¼ 92 4%, n ¼ 4; Po0.001; relaxation (pEC50 ¼ 5.7 0.2, Emax ¼ 99 2%; n ¼ 4; Figure 6a). Figure 5b). Removal of the endothelium greatly reduced arachidonic acid 7 7 relaxations (pEC50 ¼ 4.8 0.2, Emax ¼ 85 6%; n ¼ 4; Po0.001; Figure 6a). Effects of indomethacin and MAFP on relaxation to noladin ether Indomethacin (10 mM; Po0.01), but not nimesulide The non-hydrolysable analogue of 2-AG, noladin ether (10 mM), significantly enhanced arachidonic acid-induced 7 7 (Sugiura et al., 1999) induced endothelium-dependent relaxation (control, pEC50 ¼ 5.7 0.1, Emax ¼ 98 1%; n ¼ 7; 7 7 relaxation in the mesenteric arteries (with endothelium, with indomethacin, pEC50 ¼ 6.5 0.2, Emax ¼ 99 1%; n ¼ 6; 7 7 7 7 pEC50 ¼ 6.0 0.2, Emax ¼ 90 5%, n ¼ 5; without endothe- with nimesulide, pEC50 ¼ 6.0 0.3, Emax ¼ 99 1%; n ¼ 5; lium, pEC50 ¼ 5.370.1, Emax ¼ 55714%, n ¼ 5; Po0.001). Figure 6b). On the other hand, relaxations to arachidonic Unlike the case for 2-AG, relaxations induced by noladin acid were not affected by treatment with 10 mM MAFP 7 7 ether were not affected by either indomethacin (control, (control, pEC50 ¼ 5.7 0.2, Emax ¼ 97 2%; n ¼ 4; with MAFP, 7 7 7 7 pEC50 ¼ 6.2 0.2, Emax ¼ 94 4%, n ¼ 4; with 10 mM indo- pEC50 ¼ 5.9 0.2, Emax ¼ 89 4%; n ¼ 4).

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0 0 -7 -6 -5 -7 -6 -5 log (2-AG) log (2-AG) Figure 4 Effects of COX inhibitors on 2-AG-induced relaxation in mesenteric arteries. Relaxation was elicited by 2-AG alone, or in the presence of 10 mM indomethacin (a, b), 10 mM flurbiprofen (c)or10mM nimesulide (d) in endothelium-intact or -denuded vessels. Values are shown as means and vertical lines represent s.e.m. n ¼ 4–7.

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0 0 -7 -6 -5 -7 -6 -5 log (2-AG) log (2-AG)

Figure 5 Effects of the combined treatment of indomethacin (10 mM) and MAFP (10 mM) on relaxation to 2-AG in endothelium-intact (a) and -denuded (b) mesenteric arteries. Values are shown as means and vertical lines represent s.e.m. n ¼ 4–5.

Discussion and conclusions a more important role in the metabolism of 2-AG in this vascular preparation. The present study suggests that in the rat small mesenteric The primary route for anandamide catabolism is FAAH- artery, relaxant responses to anandamide and 2-AG are mediated hydrolysis into arachidonic acid and ethanolamine limited by hydrolysis to arachidonic acid and subsequent (Cravatt et al., 1996, 2001). Indeed, we found that the production of COX metabolites. For anandamide, catabo- selective FAAH inhibitor, URB597 significantly potentiated lism by FAAH and COX-2 play a significant role in its anandamide-induced relaxation in rat mesenteric arteries. inactivation. In contrast, MGL-like activity and COX-1 play Two other FAAH inhibitors, ATFMK and MAFP, which are

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abcontrol (with endothelium) with endothelium + indomethacin without endothelium + nimesulide

100 100

80 80

60 60

40 40 % Relaxation % Relaxation 20 20

0 0 -7 -6 -5 -4 -7 -6 -5 -4 log (arachidonic acid) log (arachidonic acid)

Figure 6 Effects of endothelium removal (a), indomethacin (10 mM; b) and nimesulide (10 mM; b) on relaxation to arachidonic acid in mesenteric arteries. Values are shown as means and vertical lines represent s.e.m. n ¼ 4–6.

structurally distinct from URB597 (Deutsch et al., 1997; membrane and cytosolic fractions of the brain, MAFP

Kathuria et al., 2003), similarly potentiated anandamide inhibits MGL with an IC50 as low as 2 nM (Goparaju et al., relaxations. These results agree with the initial findings of 1999; Saario et al., 2004), which is similar to IC50 values others, that phenylmethylsulphonyl fluoride, a nonspecific found for FAAH inhibition in enzyme assays (De Petrocellis serine hydrolase inhibitor, enhances mesenteric relaxations et al., 1997; Deutsch et al., 1997). Thus, the observed to anandamide (White and Hiley, 1997; Mendizabal et al., differential effects of MAFP and URB597 on 2-AG relaxations 2001). Moreover, methanandamide, the metabolically stable could suggest the involvement of MGL. It was noted that analogue of anandamide (Abadji et al., 1994), induced MAFP is also known to inhibit cytosolic phospholipase A2 similar mesenteric relaxations that were insensitive to (Lio et al., 1996), which by unknown mechanisms, could also URB597. Together, our results point to a regulatory role for contribute to the relaxant responses to 2-AG. However, this FAAH-mediated hydrolysis of anandamide in the vascular seems unlikely based on the pharmacological profile of wall. It has been shown previously that FAAH are expressed relaxations induced by 2-AG, noladin ether and arachidonic and functional in endothelial cells of bovine coronary artery acid. First, ATFMK is also an inhibitor of cytosolic phospho-

(Pratt et al., 1998) and mouse cerebral microcirculation lipase A2 (Street et al., 1993) but it only tended to potentiate (Chen et al., 2004). Here, URB597 had no significant effect relaxations to lower concentrations (p1 mM) of 2-AG. One on anandamide relaxations in endothelium-denuded vessels, possible explanation is that ATFMK is less effective than suggesting that FAAH activity largely resides in the endothe- MAFP at reducing MGL activity, as has been shown in the lium of rat small mesenteric arteries. brain (Goparaju et al., 1999; Dinh et al., 2002; Saario et al., The other major endocannabinoid 2-AG is also liable to 2004). Second, noladin ether, a metabolically stable analo- degradation. In fact, 2-AG might be more susceptible to gue of 2-AG, mimicked the endothelium-dependent mesen- degradation than anandamide both in vitro and in vivo (Pratt teric relaxation to 2-AG, but its effects were not affected by et al., 1998; Jarai et al., 2000; Savinainen et al., 2001; MAFP. Third, MAFP had no effect on arachidonic acid- Gauthier et al., 2005). Recent evidence suggests that 2-AG induced relaxation. This argues against the possibility that is primarily hydrolysed by MGL to arachidonic acid and inhibition of cytosolic phospholipase A2 by MAFP somehow glycerol (Cravatt and Lichtman, 2002; Dinh et al., 2002; potentiated responses to the hydrolysis product of 2-AG, Kathuria et al., 2003). Nevertheless, as 2-AG could also serve arachidonic acid. Taken together, the present results are as a substrate for FAAH (Goparaju et al., 1998), FAAH- consistent with 2-AG catabolism via MGL-like activity in the mediated hydrolysis might also play a role in the inactiva- vascular wall, although involvement of other esterases tion of 2-AG (Bifulco et al., 2004; de Lago et al., 2005; Maione cannot be ruled out. Given that the potentiation effect of et al., 2006). In this study, the lack of effect of URB597 on MAFP was observed in vessels with and without endothe- 2-AG relaxations indicates that FAAH has little impact on lium, MGL activity is probably present in both endothelial 2-AG metabolism in the isolated mesenteric preparation. and smooth muscle cells. In an attempt to characterize Interestingly, however, MAFP significantly potentiated further the MGL-like activity in the mesenteric artery responses to 2-AG in endothelium-intact and -denuded pharmacologically, we also tested the effects of URB754, vessels. We propose that this potentiation occurs as a result which has recently been suggested to act as a selective of the inhibition of MGL by MAFP. A number of studies have inhibitor of MGL with no activity towards FAAH (Makara also shown that MAFP is a combined FAAH and MGL et al., 2005). We found that URB754 had no detectable effect inhibitor (Di Marzo et al., 1999; Goparaju et al., 1999; Dinh on relaxation to 2-AG. This may seem contradictory to our et al., 2002; Saario et al., 2004); it probably acts by targeting proposal that MGL activity (MAFP-sensitive) limits the the arachidonyl substrate site of the two enzymes. In relaxant effects of 2-AG. However, during the course of this

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ab anandamide 2-AG MGL - URB597 - MAFP FAAH & other esterases MAFP (ATFMK) (ec) (ec & vsm) ATFMK arachidonic acid arachidonic acid COX-2 (ec) COX-2 - COX-1 - indomethacin nimesulide (ec) (ec) flurbiprofen prostamides prostanoids prostanoids (contractile > relaxing agents) (contractile > relaxing agents) Figure 7 Schematic diagrams showing the proposed metabolic pathways for anandamide (a) and 2-AG (b) in rat small mesenteric arteries. The postulated locations (in parentheses) and inhibitors of the metabolizing enzymes are also shown. FAAH, fatty acid amide hydrolase; MGL, monacylglycerol lipase; COX, cyclooxygenase; ec, endothelium; vsm, vascular smooth muscle.

study, other researchers have independently found that the results were interpreted as evidence for a more important commercially available URB754 is ineffective in inhibiting 2- role for COX-1 compared to COX-2 in regulating relaxations AG hydrolysis and thus its ability to target MGL has been elicited by 2-AG. Alternatively, simultaneous inhibition of questioned (e.g. Saario et al., 2006). both COX isoforms (by indomethacin and flurbiprofen at An increasing number of reports indicate that metabolism 10 mM) was required to potentiate 2-AG relaxations. However, of endocannabinoid by COX might be implicated in the the lack of effect of nimesulide alone indicates that COX-2 cardiovascular actions of endocannabinoids (Jarai et al., metabolism plays a very small part, if any, in 2-AG 2000; Gauthier et al., 2005; Wahn et al., 2005). Therefore, degradation. The effect of indomethacin was absent in in this study, we further examined the role of COX-1 and denuded vessels and thus points to COX-1 metabolism of COX-2 in the relaxation to endocannabinoids. The COX 2-AG in the endothelium. Given that COX-2 but not COX-1 inhibitor, indomethacin had no significant effect on relaxa- could directly oxidize 2-AG (Kozak et al., 2000), COX-1 tions to anandamide, consistent with results from previous metabolism is presumed to occur downstream of 2-AG studies (Ho and Hiley, 2003; O’Sullivan et al., 2004). hydrolysis (Figure 7b). Interestingly, relaxant responses to Interestingly, selective inhibition of COX-2 with nimesulide arachidonic acid were also potentiated by indomethacin, resulted in a small but significant enhancement in ananda- suggesting that COX-1-derived contractile metabolites mide-induced relaxation in endothelium-intact vessels. might be produced from 2-AG. This might, at least partly, Nimesulide did not cause additional potentiation when co- explain the enhanced 2-AG relaxations in the presence of applied with the FAAH inhibitor URB597, so it is likely that COX-1 inhibitors (Figure 7b). Indeed, relaxations to noladin the metabolism mediated by COX-2 occurs downstream of ether were unaffected by indomethacin. At present, the anandamide hydrolysis (Figure 7a). Nevertheless, it remains mechanisms underlying the differential effects of indo- possible that COX-2 catalyses a direct oxidation reaction methacin (presumed COX-1 inhibition) and nimesulide with anandamide producing prostamides (Yu et al., 1997; (COX-2 inhibition) on the relaxations to anandamide and Figure 7a). This might contribute to the small inhibitory 2-AG remain unclear. It is possible that 2-AG hydrolysis, effect of nimesulide on anandamide relaxations, as the mediated by MGL and perhaps other esterases, is strongly putative prostamides are inactive at prostanoid EP and FP coupled with COX-1 activity, which might also explain the receptors and hence likely to display no vasorelaxant activity finding that 2-AG relaxations were greatly enhanced by the (Matias et al., 2004). Recently, Chen et al. (2005) showed that combined treatment of MAFP and indomethacin. Hypothe- prolonged treatment with anandamide and methananda- tically, such functional coupling can be achieved by some mide (after X1 h incubation) increases COX-2 expression in form of physical association between the enzymes, so that mouse cerebral endothelial cells. However, our observations arachidonic acid produced from MGL-mediated hydrolysis that these had much faster relaxant responses is in close proximity to COX. (minutes), and that methanandamide induced a nimesulide- To conclude, the present study provides pharmacological insensitive relaxation, argue against a significant contribu- evidence suggesting that, in the rat small mesenteric artery, tion by this pathway to the mesenteric relaxations. It is hydrolysis via FAAH and MGL-like activity limits the noteworthy that, although the role for COX-2 (inducible) vasodilator actions of anandamide and 2-AG, respectively. as compared to COX-1 (constitutively active) in vascular For 2-AG, a combined inhibition of MGL and COX could control under physiological conditions is still undefined, the further enhance its vascular impact. Our results also indicate detection of basal COX-2 activity in rat mesenteric arteries is that the endothelium represents a major metabolic barrier in consistent with recent findings that COX-2 is expressed in the regulation of endocannabinoid actions, in as much as it mesenteric arteries of healthy mice (Guo et al., 2005), rabbits determines the presence of significant FAAH, MGL, COX-1 (Zhang et al., 2005) and humans (Tabernero et al., 2003). and COX-2 activity, although MGL-mediated hydrolysis of In contrast to anandamide, indomethacin and the more 2-AG might also occur in the mesenteric smooth muscle COX-1-selective inhibitor, flurbiprofen (Warner et al., 1999) (Figure 7). In support of this proposal, it has recently been but not nimesulide potentiated 2-AG relaxations. These found that the vascular content of both anandamide and

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