Enhanced Vasorelaxant Effects of the Endocannabinoid-Like Mediator, Oleamide, in Hypertension

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European Journal of Pharmacology 684 (2012) 102–107

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European Journal of Pharmacology

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Cardiovascular Pharmacology Enhanced vasorelaxant effects of the endocannabinoid-like mediator, oleamide, in hypertension

Jamie J. Hopps, William R. Dunn, Michael D. Randall ⁎

Cardiovascular Research Group, School of Biomedical Sciences, University of Nottingham Medical School, Queen's Medical Centre, Nottingham, NG7 2UH, United Kingdom

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Article history: Oleamide is an endocannabinoid-like, fatty acid amide with structural similarities to anandamide. The cardio- Received 30 September 2011 vascular effects of anandamide are enhanced in hypertension and we have now examined how hypertension Received in revised form 8 March 2012 affects responses to oleamide. Vasorelaxant responses to oleamide were signiﬁcantly (Pb0.001) enhanced in Accepted 9 March 2012 aortic rings from spontaneously hypertensive rats (SHRs), such that the maximal relaxation to oleamide was Available online 23 March 2012 40.3±3.5%, compared to 15.7±3.9% in normotensive Wistar Kyoto (WKY) controls. The augmented responses to oleamide in SHR arteries were unaffected by either inhibition of nitric oxide synthase (300 μM Keywords: μ Endocannabinoid-like substance L-NAME) or fatty acid amide hydrolase (1 M URB597) and independent of cannabinoid CB1 receptors or Endothelium the endothelium. The enhanced responses to oleamide were opposed by pre-treatment with capsaicin Oleamide (such that Rmax was reduced to 9.8±1.5%) and this occurred independently of TRPV1 receptor and sensory Hypertension nerve activity, as the TRPV1 antagonist capsazepine (1–5 μM) and the cation channel inhibitor ruthenium Capsaicin red (10 μM) had no effect on the responses to oleamide. However, inhibition of cyclooxygenase (10 μM indo- Cyclooxygenase methacin) enhanced the responses in the WKY aortae, such that the responses were comparable to those in the SHR. The results suggest that the cyclooxygenase pathway has a role in modulating vasorelaxation caused by oleamide in normotensive aortae and that this is lost in hypertension, possibly as an adaptation to the increase in blood pressure. © 2012 Elsevier B.V. Open access under CC BY license.

1. Introduction mediated activity and the enhanced responses to anandamide in hypertension was proposed. The cardiovascular effects of cannabinoids are the subject of grow- Wheal and Randall (2009) examined the vascular effects of ananda- ing interest (Randall et al., 2004). The endocannabinoid, anandamide, mide in SHRs and in rats rendered hypertensive by chronic nitric oxide can have depressor effects (Ho and Gardiner, 2009; Varga et al., 1996; (NO) synthase inhibition with L-NAME. Anandamide caused enhanced Zakrzeska et al., 2010) and its production is upregulated in certain vasorelaxation in aortic rings from SHRs compared to normotensive pathophysiological states (Batkai et al., 2001; Caraceni et al., 2009; WKY control preparations. The increased effects of anandamide were

2010). The CB1 receptor and TRPV1 receptor have both been implicat- attributed to endothelium-dependent mechanisms. Conversely, anan- ed in the hypotensive effects of anandamide (Batkai et al., 2004; Lake damide caused decreased vasorelaxant effects in perfused mesenteric et al., 1997; Li et al., 2003; Wang et al., 2005). Li et al. (2003) showed beds taken from SHRs attributed to decreased NO-dependent vasore- that depressor responses to the stable analogue of anandamide, laxation (Wheal and Randall, 2009). In perfused mesenteric beds, methanandamide, were enhanced in spontaneously hypertensive from rats following chronic NO synthase inhibition, anandamide caused rats (SHRs) compared to normotensive Wistar Kyoto (WKY) controls. enhanced vasorelaxation (Mendizabal et al., 2001; Wheal et al., 2007). Antagonism of transient receptor potential vanilloid receptor type 1 The enhanced responses to anandamide in L-NAME treated rats were (TRPV1) receptors reduced this hypotensive response which was capsaicin-sensitive, suggesting an increase in sensory nerve activity. also associated with an upregulation of calcitonin gene-related pep- This was further supported by the ﬁnding that capsaicin was shown to tide activity in SHRs. Similarly, Wang et al. (2005) described enhanced cause enhanced relaxation in mesenteric beds from rats made hyper- vasorelaxation to anandamide in mesenteric beds in a salt-sensitive tensive by chronic NO inhibition. rat model of hypertension. Increased depressor effects were inhibited Oleamide (cis-9 10 octadecenoamide) is a fatty acid primary amide,

by antagonism of TRPV1 and CB1 receptors, and TRPV1 expression was which was originally derived from sleep-deprived cats (Cravatt et al., upregulated in hypertension. Thus, a link between sensory nerve- 1995) and shares structural similarities with anandamide. In addition, oleamide is sensitive to FAAH degradation, exerts cannabimimetic effects in animal models and has been shown to be a selective agonist for canna- ⁎ Corresponding author. Tel.: +44 115 82 30185; fax: +44 115 82 30141. binoid CB1 receptors in vitro (Boger et al., 2000; Leggett et al., 2004). To E-mail address: [email protected] (M.D. Randall). date, oleamide has been shown to cause vasorelaxation in rat small

mesenteric arteries (Hoi and Hiley, 2006; Sudhahar et al., 2009). Hoi CB1 antagonist (O'Sullivan et al., 2005). Some experiments were carried and Hiley (2006) demonstrated that the vasorelaxation was partly out in the presence of URB597 (1 μM), a fatty acid amide hydrolase endothelium-dependent, capsaicin-sensitive and involved calcium- (FAAH) inhibitor (Ho and Randall, 2007). activated potassium channels. It was postulated that oleamide may act via a novel cannabinoid receptor. Cannabinoid CB1 receptor and TRPV1 re- 2.3. Drugs and reagents ceptor activation have also been implicated in oleamide-induced vasorelaxation in the rat mesenteric resistance arteries (Sudhahar et al., 2009). Oleamide (cis-9 10 octadecenoamide) and AM251 (N-(piperidin- In light of the enhanced cardiovascular effects of anandamide in 1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole- hypertension, the major aim of the current study was to investigate 3-carboxamide) were obtained from Tocris Co. (UK). All other drugs the vasorelaxant effects of oleamide in the SHR model of hypertension. used in this investigation were purchased from Sigma Chemicals Co. (UK). Capsaicin and capsazepine were dissolved in ethanol at stock 2. Materials and methods concentrations of 10 mM. DMSO (Dimethyl sulfoxide) was used to dis- solve stock solutions of oleamide and AM251 at 10 mM and URB597 2.1. Animals (cyclohexylcarbamic acid 3′-carbamoyl-biphenyl-3-yl ester) at 1 mM. G All other drugs were dissolved in distilled water. L-NAME (N -Nitro- – – Male SHR and WKY rats (Charles River UK) (250 350 g; aged 12 L-arginine methyl ester hydrochloride), indomethacin, AM251 and 18 weeks) were used during this investigation. All rats used were capsazepine were incubated for approximately 20 min before pre- housed at the Biomedical Services Unit, University of Nottingham contraction of vessels with methoxamine. Ruthenium red (ammoniated with a 12 h light/dark cycle and in temperature-controlled conditions. ruthenium oxychloride) and niflumic acid were incubated for 30 min, while URB597 was incubated for 10 min before pre-contraction. 2.2. Methods 2.4. Statistical analysis 2.2.1. Aortic rings Rats were stunned by a blow to the back of the head and killed by All responses were expressed as mean data with the associated exsanguination. These procedures are in accordance with UK legisla- standard error of the mean (S.E.M). The GraphPad Prism 5.0 software tion and have been approved by institutional ethical review. This (San Diego, CA) was used to plot mean data as sigmoidal concentra- was followed by the removal of the abdominal aorta. Subsequently tion–response curves. The curves were used to determine potency the aorta was cleared of all connective tissue by blunt dissection and (pEC which is the negative log of the concentration causing 50% of cut into segmental rings (3 mm–5 mm). The aortic rings were placed 50 the maximal relaxation) and maximal response (R ) values. Statis- between two metal wires, with one being fixed and the other attached max tical significance was determined using Student's t-test between two via thread to an isometric transducer and placed in 50 ml organ baths. data sets or one-way ANOVA when comparing multiple data-sets. The organ baths were filled with modified Krebs'–Henseleit buffer solution (NaCl 118, KCl 4.7, MgSO4 1.2, KH2PO4 4.2, NaHCO3 25, 3. Results D-glucose 10, CaCl2 2 (mM)) at 37 °C and gassed (5% CO²/95% O²). The aortic rings were allowed to equilibrate to an optimal baseline tension of 9.8 mN for 1 h. Tension was measured by a Leitica force 3.1. Vascular responses to oleamide in isolated aortae from SHR and WKY transducer coupled to an ADInstruments MacLab recording system. rats

Vessels were pre-contracted with the α1-adrenoceptor agonist, methoxamine (10 μM), to achieve submaximal contraction (ca. 10 mN) Oleamide caused concentration-dependent vasorelaxation of aor- prior to the addition of oleamide (10 nM–10 μM), which was added cu- tic segments from both SHR and WKY rats. Maximal relaxation was ﬁ b mulatively at 5 minute intervals to construct concentration–response signi cantly (P 0.001) greater in SHRs (Rmax =40.3±3.5%, n=6) curves.Toinvestigatetheroleofsensorynerves,somevesselsweresub- when compared to that in aortae from WKY rats (Rmax =15.7±3.9%, jected to pre-treatment with 10 μM capsaicin for 1 h, which desensitises n=6) (Fig. 1). The potency of oleamide was comparable between SHR (pEC50 =6.49±0.55, n=6) and WKY aortae (pEC50 =6.08± the TRPV1 receptors and depletes the neurotransmitters (Zygmunt et al., 1999). Capsaicin pre-treatment was followed by a 20 min wash-out 0.55, n=6). period, prior to methoxamine contraction. Concentration–responses curves to oleamide were also constructed in the presence of capsazepine 3.2. Role of cannabinoid CB1 receptors in the vascular response to (1–5 μM), a TRPV1 antagonist (White et al., 2001). To further analyse oleamide in aortic segments any differences in sensory nerve activity between SHR and WKY rats, concentration–response curves to capsaicin (1 nM–10 μM) were con- The vasorelaxant responses caused by oleamide (Rmax =27.6±3.5%, structed cumulatively. Experiments were also carried out in the presence pEC50 =6.05±0.25, n=8) were unaffected by the presence of the of ruthenium red (10 μM), a blocker of capsaicin-activated cation channels (Harris et al., 2002). The contribution of the endothelium to oleamide-induced responses was assessed by denuding some vessels of endothelial cells. This was achieved by gentle rubbing of the intimal surface with metal forceps. En- dothelial function was examined by addition of carbachol (10 μM) after pre-contraction by methoxamine. Vessels responding to carbachol with less than 20% relaxation of induced tone were deemed to be lacking a functional endothelium. To inhibit nitric oxide synthase some vessels were incubated with L-NAME (300 μM), while in other vessels indomethacin (10 μM) was present to inhibit cyclooxygenase (O'Sullivan et al., 2004). Concentration–response curves to oleamide were also carried out in the presence of niﬂumic acid (10 μM), a selective COX-2 inhibitor (Wheal et al., 2010). In some experiments the role of cannabinoid CB1 Fig. 1. Vasorelaxant responses to oleamide in aortic rings pre-contracted with methox- receptors was examined by incubation of AM251 (1 μM), a cannabinoid amine from SHR and WKY rats. Mean data with bars indicating S.E.M are displayed. 104 J.J. Hopps et al. / European Journal of Pharmacology 684 (2012) 102–107

CB1 receptor antagonist AM251 (1 μM) in SHR preparations (Rmax = 37.3±3.5%, pEC50 =6.32±0.22, n=6). Similarly, oleamide responses (Rmax =13.1±3.2%, pEC50 =5.80±0.40, n=6) were unaffected in aortae from WKY rats in the presence of AM251 (1 μM) (Rmax =15.8± 1.7%, pEC50=5.90±0.21, n=4).

3.3. Effects of endothelial denudation and L-NAME on the vascular responses to oleamide

The vasorelaxant effects of oleamide in both WKY (Rmax =13.1± 3.2%, pEC50=5.80±0.40, n=6) (Fig. 2A) and SHR (Rmax =27.6± 3.5%, pEC50 =6.05±0.25, n=8) (Fig. 2B) arteries were unaffected by Fig. 3. Vasorelaxant responses to oleamide in aortic rings from both SHR and WKY rats endothelial denudation (Rmax =10.8±2.2%, pEC50 =6.20±0.44, n=6 after 1 h pre-treatment with capsaicin (10 μM). Mean data with bars indicating S.E.M are displayed. WKY; Rmax =23.2±5.0%, pEC50 =5.72±0.32, n=9 SHR). The vasorelaxation caused by oleamide in aortae from WKY (Rmax =13.9±4.5%, pEC50 =6.05±0.71, n=5) (Fig. 2C) and SHR (Rmax =40.3±3.5%, pEC50 =6.49±0.22, n=6) (Fig. 2D) rats was unaffected by the pres- 3.5. Effects of the TRPV1 antagonist capsazepine on vasorelaxant ence of L-NAME (300 μM) (Rmax =14.6±1.7%, pEC50 =5.96±0.18, responses to oleamide in aortae from SHR and WKY rats n=6 WKY;Rmax =40.8±3.7%, pEC50=6.14±0.19, n=6 SHR). Vasorelaxation to oleamide in both WKY (Rmax =13.1±3.2%, pEC50 =5.80±0.40 n=6) and SHR (Rmax =27.6±3.5%, pEC50 = 3.4. The effects of capsaicin pre-treatment on vasorelaxation to oleamide 6.05±0.25, n=8) arteries was unaffected by the presence of 1 μM in aortae from SHR and WKY rats capsazepine (Rmax =10.0±1.5%; pEC50 =5.97±0.26, n=6, WKY; Rmax =30.4±3.8%, pEC50 =5.98±0.21, n=7 SHR). Similarly, the Vasorelaxation to oleamide was signiﬁcantly (Pb0.001) reduced in responses to oleamide in WKY (Rmax =6.3±3.7%, pEC50 =5.64± the SHR arteries after pre-treatment with capsaicin for 1 h (Rmax = 0.56, n=7) and SHR arteries (Rmax =43.2±7.1%, pEC50 =6.13± 9.8±1.5%, n=5, SHR) (Fig. 3), compared to the control value given 0.33, n=6) were unaffected by the presence of 5 μM capsazepine above. The potency of oleamide as a vasorelaxant was unaffected. (Rmax =20.3±7.5%, pEC50 =5.34±0.47, n=7 WKY; Rmax =37.7± Pre-treatment with capsaicin caused maximal responses to oleamide 5.5%, pEC50 =6.27±0.35, n=6 SHR). Vasorelaxation to oleamide in aor- in the SHR to become comparable to responses in WKY rats such that tic rings from both WKY (Rmax =6.3±3.7%,pEC50 =5.64±0.56, n=7) relaxation at 10 μM oleamide was 9.5±2.0% (n=5) compared to a and SHRs (Rmax =43.2±7.1, pEC50 =6.13 ±0.33, n= 6) was unaf- relaxation of 7.6±2.8% seen in the WKY arteries (n=6). fected by the presence of 10 μM ruthenium red (Rmax =10.3±2.6%,

Fig. 2. Vasorelaxation of endothelium-denuded aortae from WKY (A) and SHR (B) rats by oleamide. Vasorelaxant responses to oleamide in aortae from WKY (C) and SHR (D) rats in the presence of L-NAME (300 μM). Mean data with bars indicating S.E.M are displayed. J.J. Hopps et al. / European Journal of Pharmacology 684 (2012) 102–107 105

pEC50 5.68±0.30, n=7 WKY; Rmax =42.6±6.3%, pEC50 =5.71±0.23, reported the involvement of CB1 receptors in the vascular responses n=6 SHR). to oleamide (Hoi and Hiley, 2006; Sudhahar et al., 2009). However, the enhanced vasorelaxation to oleamide was unaffected by the pres-

3.6. Vascular responses to capsaicin in isolated aortae from SHR and ence of AM251 and so were independent of CB1 receptor activation. WKY rats A clear ﬁnding from this study is that the enhanced effects of oleamide in SHR aortae are capsaicin-sensitive. This may suggest that the The vasorelaxant responses to capsaicin were comparable in aortic enhanced effects of oleamide are due to an increase in sensory nerve rings from both SHR and WKY rats such that at 10 μM capsaicin vaso- activity in the SHR. Indeed, previous studies have demonstrated in- relaxation in SHR aortae was 30.4±5.3% (n=9) compared to 38.0± creased sensory nerve activity in models of hypertension (Li et al., 9.2% (n=8) (Fig. 4). 2003; Wang et al., 2007). In a previous study, in the NO synthase inhibition model of hypertension, anandamide induced enhanced vasore- 3.7. The effects of cyclooxygenase inhibitors on vasorelaxant responses to laxant responses in mesenteric beds were normalised by capsaicin oleamide in isolated aortae from SHR and WKY rats pre-treatment and so were ascribed to an upregulation of sensory- nerve mediated activity (Wheal and Randall, 2009).

Vasorelaxation to oleamide in SHR arteries (Rmax =40.3±3.5%, The enhanced responses to oleamide were sensitive to capsaicin pEC50 =6.49±0.22, n=6) was unaffected by the presence of pre-treatment and the resulting responses were converted to a simi- 10 μM indomethacin (Rmax =28.7±3.8%, pEC50 =5.90±0.28, n=6) lar magnitude seen in the normotensive controls. This is clearly con- (Fig. 5A). By contrast in aortae from WKY control rats, vasorelaxation sistent with the proposal that the enhanced responses in the SHR to oleamide was signiﬁcantly (Pb0.001) enhanced in the presence of arteries are due to increased sensory nerve activity. However, when 10 μM indomethacin such that relaxation at 10 μM oleamide was additional pharmacological approaches were used, including the 37.3±3.6% (n=7) compared to a relaxation of 15.5±4.5% (n=6) TRPV1 antagonist capsazepine, and the non-selective cation channel at this concentration in the absence of indomethacin (Fig. 5A). The en- blocker, ruthenium red, the enhanced responses were unaffected. hanced responses to oleamide in the presence of indomethacin were This suggests that although the mechanism underlying the enhanced abolished by the removal of the endothelium (Fig. 5B). Vasorelaxation responses to oleamide is capsaicin-sensitive, it does not appear to in- to oleamide in both WKY (Rmax =13.1±6.2%, pEC50 =5.65±0.57, volve TRPV1 channel activation or sensory nerves. Indeed, the vasor- n=6) and SHR (Rmax =43.3±4.4%, pEC50 =6.04±0.18, n=6) aortae elaxant responses to capsaicin alone were comparable between the was unaffected by the COX-2 inhibitor niﬂumic acid (10 μM) (Rmax = hypertensive and normotensive strains. This may suggest that there 22.9±11.3%, pEC50 =5.40±0.58, n=6 WKY; Rmax =41.2±6.6%, is comparable sensory nerve activity between the strains or alterna- pEC50 =5.86±0.27, n=5 SHR). tively that capsaicin is acting via mechanisms distinct from the sensory nerve mediated pathway. Previous studies have demonstrated 3.8. Effects of the FAAH inhibitor, URB597, on vasorelaxant responses to capsaicin-induced vasorelaxation that is also insensitive to TRPV1 oleamide in aortae from SHR and WKY rats antagonism in the rabbit coronary artery (Yeon et al., 2001) and in human and porcine coronary arteries (Gupta et al., 2007). In a sepa-

Vasorelaxation to oleamide in WKY (Rmax = 6.3 ± 3.7%, pEC50 = rate study in arteries from Wistar rats we have also shown that capsa- 5.64±0.56 n=7) (Fig. 6A) and SHR arteries (Rmax =43.2±7.1%; icin inhibits responses to oleamide in a sensory nerve-independent fl pEC50 =6.13±0.32, n=6) (Fig. 6B) was unaffected by the presence of manner, which may involve inhibition of calcium in ux (Hopps et fl URB597 (1 μM) (Rmax =6.3±5.3%, pEC50 =5.60±0.66, n=7 WKY; al., 2012). The inhibitory effects of capsaicin might re ect the actions Rmax =37.3±5.8%, pEC50 =5.87±0.25, n=7 SHR). at additional sites or inhibition of a putative, novel cannabinoid receptor. 4. Discussion Enhanced vasorelaxation in SHR arteries is not unprecedented as we have previously shown augmented responses to anandamide The principal finding of this study is that vasorelaxation to the (Wheal and Randall, 2009). Pomposiello et al. (2001) also reported endocannabinoid-like substance, oleamide is substantially enhanced that vasodilatation to arachidonic acid was enhanced in the renal vas- in the aorta isolated from the SHR model of hypertension and that culature of SHR. This was ascribed to increased production of epox- this may be related to alterations in prostanoid metabolism. yeicoatrienoic acid from arachidonic acid. The aortic endothelium from SHRs is associated with an increased The presence of indomethacin increased the vasorelaxant effects

CB1 receptor expression (Batkai et al., 2004). Therefore our ﬁrst of oleamide in normotensive aortae, such that they were comparable hypothesis was that the enhanced responses to oleamide might be to those from SHR. This clearly highlights the importance of the cyclo-

CB1-dependent (Leggett et al., 2004) Indeed, previous studies have oxygenase pathway in regulating the responses to oleamide. In addition to our ﬁndings, other studies have shown that COX-inhibition can increase the vasorelaxant response to endocannabinoids (Ho and Randall, 2007) and N-oleoylethanolamine, an endocannabinoid-like substance, (Wheal et al., 2010). One possibility for the enhanced responses to oleamide in arteries from normotensive rats is that oleamide or its metabolites are a sub- strate for a COX-pathway and that this is absent in hypertension. Therefore, metabolism of oleamide could act to limit vasorelaxation in aortic rings from normotensive rats and so accounting for the enhanced effects in SHR aortae. Inhibition of the COX-pathway would then remove the metabolic limit on vasorelaxation to oleamide in the WKY and responses are normalised between the two strains. How- ever, this is unlikely as oleamide would not be expected to be a sub- strate for COX as it is monounsaturated, although there is evidence that unsaturated fatty acids may modulate COX activity (Yuan et al., Fig. 4. Vasorelaxation caused by capsaicin in isolated aortae from both SHR and WKY 2009). Alternatively, it is possible that vasoconstrictor prostanoids rats. Mean data with bars indicating S.E.M are displayed. are being released in WKY but not SHR aortae in response to oleamide 106 J.J. Hopps et al. / European Journal of Pharmacology 684 (2012) 102–107

Fig. 5. Vasorelaxation caused by oleamide (A) in aortic rings from both SHR and WKY rats in the presence of 10 μM indomethacin. Responses to oleamide in WKY preparations in the presence of 10 μM indomethacin in endothelium denuded vessels (B). Mean data with bars indicating S.E.M are displayed. and removal of these prostanoids enhances vasorelaxation. In any the removal of the endothelium. In this study, any difference in event, it appears that COX activity in arteries from normotensive rats endothelium-dependent vasorelaxation is unlikely to account for the truncates responses to oleamide and this is absent in the SHR. enhanced effects of oleamide observed in aortic rings from hyperten- One hypothesis to explain the augmented responses which occur sive rats, as endothelial denudation and inhibition of nitric oxide in the SHR and are uncovered in the WKY arteries following COX inhi- synthase did not affect the vascular responses to oleamide. This is in bition could be that oleamide mobilises endogenous arachidonic acid. contrast to oleamide-induced vasorelaxation in small mesenteric The liberated arachidonic acid could then enter the COX pathway. It arteries, which is partly mediated by endothelium-dependent mecha- could be that COX-dependent metabolism of arachidonic to opposing nisms (Hoi and Hiley, 2006; Sudhahar et al., 2009). However, in nor- vasoconstrictor prostanoids occurs in the WKY arteries to limit vasor- motensive controls the potentiation of oleamide responses in the elaxant and that this modulation is absent or impaired in SHR arteries. presence of indomethacin was dependent on a functional endotheli- In this respect we have previously shown that vasorelaxation to N- um. This effect of indomethacin on oleamide-induced vasorelaxation oleoylethanolamine, which cannot be metabolised to arachidonic was not replicated by the presence of niﬂumic acid. At the concentra- acid, is also enhanced by COX inhibition and antagonism of thrombox- tions used during this investigation niﬂumic acid is 100 times more ane receptors (Wheal et al., 2010). This is consistent with increased selective at COX-2 compared to COX-1 (Johnson et al., 1995). There- vasoconstrictor prostanoid activity opposing vasorelaxation. fore, it is more likely COX-1 mediates the potentiating effect of indo- FAAH causes the hydrolysis of oleamide into oleic acid (Cravatt et methacin on the oleamide-induced responses in WKY aortic rings. al., 1996; Deutsch et al., 1997) and has been shown to be necessary The responses to oleamide in the WKY aortae were enhanced by for vasorelaxation of bovine arteries by anandamide (Pratt et al., COX inhibition but this was absent in endothelium denuded vessels, 1998). Coupled to this Batkai et al. (2004) reported an upregulation and this might imply that the COX was endothelial. However, when of myocardial FAAH in SHR rats. However, the enhanced responses the endothelium alone was removed the responses to oleamide in to oleamide in the SHR aorta were not affected by FAAH inhibition. aortae were not enhanced as this would predict. Therefore, the Similarly, responses to oleamide in the normotensive controls were COX-dependent mechanism which appears to modulate responses also unaffected by URB597. This is in agreement with work demon- to oleamide in the WKY but not SHR aorta cannot be localised to strating FAAH-independent vascular responses to oleamide (Hoi and the endothelium. One possibility is that the COX-dependent mecha- Hiley, 2006; Sudhahar et al., 2009). Oleic acid, the product of oleamide nism is throughout the vascular wall (i.e. both endothelial and non- metabolism, also had no vasorelaxant effect in rat small mesenteric endothelial) and that the components synergise, or at least act in par- arteries (Sudhahar et al., 2009). Therefore, it can be concluded that allel. Therefore, removing the endothelium alone does not remove local metabolism via FAAH is not important in mediating responses the COX-dependent mechanism, whereas global COX inhibition to oleamide in either strain of rat. removes these processes. Furthermore, the putative synergy between Wheal and Randall (2009) demonstrated that enhanced vasore- endothelium-dependent modulation and no-endothelial COX is lost laxation to anandamide in aortae from SHR rats was abolished by on removal of the endothelium.

Fig. 6. Vasorelaxant responses to oleamide in the presence of the FAAH inhibitor URB597 (1 μM) in both WKY (A) and SHR (B) arteries. Mean data with bars indicating S.E.M are displayed. J.J. Hopps et al. / European Journal of Pharmacology 684 (2012) 102–107 107

5. Conclusions Hoi, P.M., Hiley, C.R., 2006. Vasorelaxant effects of oleamide in rat small mesenteric artery indicate action at a novel cannabinoid receptor. Br. J. Pharmacol. 147, 560–568. In conclusion, our results demonstrate greatly enhanced vasore- Hopps, J.J., Dunn, W.R., Randall, M.D., 2012. Vasorelaxation to capsaicin and its effects laxant effects of the endocannabinoid-like substance oleamide in aor- on calcium influx in arteries. Eur. J. Pharmacol. 681, 88–93. Johnson, J.L., Wimsatt, J., Buckel, S.D., Dyer, R.D., Maddipati, K.R., 1995. Purification and tae from the SHR model of hypertension. The augmented responses to characterization of prostaglandin H synthase-2 from sheep placental cotyledons. oleamide were abolished by capsaicin pre-treatment but are indepen- Arch. Biochem. Biophys. 324, 26–34. dent of TRPV1 receptor or sensory nerve activity. An important finding Lake, K.D., Martin, B.R., Kunos, G., Varga, K., 1997. 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