European Journal of Pharmacology 684 (2012) 102–107 Contents lists available at SciVerse ScienceDirect European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar 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 article info abstract 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 re- sponses 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 in- crease in blood pressure. © 2012 Elsevier B.V. All rights reserved. 1. Introduction mediated activity and the enhanced responses to anandamide in hy- pertension 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 0014-2999/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2012.03.027 J.J. Hopps et al. / European Journal of Pharmacology 684 (2012) 102–107 103 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 vasore- laxation 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).