Involvement of the peroxisome proliferator-activated receptor (PPAR) alpha in vascular response of endocannabinoids in the bovine ophthalmic artery

European Journal of Pharmacology 683 (2012) 197–203

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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Cardiovascular Pharmacology

Involvement of the peroxisome proliferator-activated receptor (PPAR) alpha in vascular response of endocannabinoids in the bovine ophthalmic artery Maria Rosaria Romano, Marcello Diego Lograno ⁎ Department of Pharmacobiology, University of Bari “Aldo Moro”, Via Orabona 4, 70125 Bari, Italy

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Article history: Received 2 February 2012 Received in revised form 14 February 2012 Accepted 26 February 2012 Available online 10 March 2012 Keywords: Anandamide Palmitoylethanolamide Ophthalmic artery Peroxisome proliferators-activated receptor alpha Nitric oxide

a b s t r a c t Endocannabinoids regulate vascular tone in a variety of vascular tissues. This study aimed to investigate the role of peroxisome proliferators-activated receptors (PPARs) in anandamide- and palmitoylethanolamideinduced relaxant responses on the bovine ophthalmic artery and to evaluate the mechanisms involved. The effects of anandamide and palmitoylethanolamide were examined under myographic conditions on arterial rings pharmacologically pre-contracted with 5-HT. Anandamide and palmitoylethanolamide relaxed the ophthalmic artery rings in time- and concentration-dependent manner stimulating the PPAR alpha (PPARα). The vasorelaxation to endocannabinoids was inhibited by PPARα antagonist GW6471 (1 μM), but not the PPAR gamma (PPARγ) antagonist GW9662 (1 μM). Anandamide-induced relaxation was attenuate during the first 60 min by AM251, a selective antagonist of cannabinoid CB1 receptors, and Pertussis toxin, an inhibitor of Gi/o protein; by the contrast, the palmitoylethanolamide-induced vasorelaxation was unaffected by cannabinoid antagonists and Pertussis toxin. Endothelium removal decreases slightly the potency and efficacy to endocannabinoids. The relaxant effect to anandamide and palmitoylethanolamide was inhibited by L-NMMA (300 μM), an inhibitor of nitric oxide synthase, and iberiotoxin (200 nM), a selective blocker of large conductance Ca 2+-activated K+ (BKCa). These data support the view that anandamide and palmitoylethanolamide relax the ophthalmic artery in a time-dependent manner via the transcription factors PPARα suggesting a function for them in the physiological mechanisms of vascular regulation. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Endocannabinoids are described as endogenous agonists of cannabinoid (CB) receptors (Pacher et al., 2006). They are considered unconventional neurotransmitters and are generated on demand in response to a rise in intracellular calcium or metabotropic receptor activation, rather than being stored in vesicles (Cadas et al., 1997). Anandamide was the first endocannabinoid identified (Devane et al., 1992), following numerous other N-acylethanolamines have been found in mammals (Lambert and Fowler, 2005). Emerging evidences suggest that endocannabinoid system is implicated in several physio-pathological processes; their effects are mostly explained via activation of the classical G-protein-coupled CB receptors and related downstream signaling cascades. However, studies carried out in knock-out mice have suggested the existence of new target sites for endocannabinoids such as transient receptor potential vanilloid type 1 (TRPV1) and nuclear receptor peroxisome proliferator-activated receptors (PPARs) (Pistis and Melis, 2010). PPARs are members of the nuclear receptor superfamily, modulate the expression of numerous gene families and are regarded ligand-

⁎ Corresponding author. Tel./fax: + 39 0805442797. E-mail address: [email protected] (M.D. Lograno). 0014-2999/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2012.02.049

activated transcription factors (Touyz and Schiffrin, 2006). The PPAR family comprises three members α, γ, and β/δ, which are mainly involved in the energy homeostasis, in the regulation of metabolism and the modulation of cardiovascular system (Ferrè, 2004). At vascular level, PPARs influence oxidative stress, inflammatory process and cell growth, and appear to be implicated in the vasculoprotective effects. In particular, PPARα and PPARγ are widely expressed in the vascular smooth muscle and endothelial cell where they control the production of nitric oxide and the expression of endothelin1 (Goya et al., 2004; Irukayama-Tomobe et al., 2004; Touyz and Schiffrin, 2006). Recently, it has also been shown that main constituent psychoactive of cannabis Δ 9-tetrahydrocannabinol can induce vasorelaxant responses through activation of PPARγ in different vascular bed (O'Sullivan et al., 2005, 2006). In addition, 2arachidonylglycerol, noladin ether and virodhamine determine an increase of transcriptional PPARα activity (Kozak et al., 2002). Narachidonyl dopamine and synthetic cannabinoids, WIN55212-2 and CP55940, are able to bind to PPARγ (O'Sullivan et al., 2009a). It has been demonstrated that anandamide directly activates some members of PPAR family like PPARα and PPARγ (Bouaboula et al., 2005; Sun et al., 2006). Palmitoylethanolamide that is a structurally similar compound to anandamide activates PPARα transcriptional activity (Lo Verme et al., 2005) and, more importantly, is devoid of cannabinoid-like activity (O'Sullivan, 2007). Numerous evidences

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report that anandamide is a potent modulator of vascular tone and its mechanism of action is multi-factorial and depends on the species and preparation used (O'Sullivan et al., 2005; Romano and Lograno, 2006). In addition, it has been shown that anandamide induces PPARγ-mediated vasorelaxation of rat aorta but not palmitoylethanolamide (O'Sullivan et al., 2009a). Palmitoylethanolamide is categorized as an endocannabinoid-like molecule, able to exert direct vasorelaxation less potent than anandamide (Ho et al., 2008). Therefore, the aim of the present study was to investigate whether anandamide and palmitoylethanolamide can induce a time- and concentration-dependent vasorelaxation through the activation of PPARs in the bovine ophthalmic artery and to examine the involved mechanisms. 2. Materials and methods 2.1. Tissue preparation Experiments were in compliance with the European Community guidelines for the use of experimental animals and were approved by the institutional ethics committee. The technique for isolation and preparation of ophthalmic arterial rings has been performed as described previously by Romano and Lograno (2006). In brief, bovine eye, including the immediate retro-orbital structures, were obtained from a local abattoir within 5 min of slaughter and immediately put in ice-cold oxygenated modified Krebs physiological salt solution of the following composition (mM): NaCl (136.8), KCl (5.4), MgSO4 (0.8), NaHCO3 (12), CaCl2 (2.7), D-glucose (5.0), Naascorbate (0.2), pH 7.4. The main ophthalmic artery running along the optic nerve to the eye was dissected and freed of surrounding connective and adipose tissue. Care was taken not to damage the luminal surface of the preparation. Two adjacent rings were cut from each artery (0.6–1.0 mm in diameter, 2–3 mm in length) and were mounted on fine tungsten wires on a miograph system (Fort 10, WPI, Sarasota, FL, USA) containing modified Krebs physiological salt solution. Tissues were maintained at 37 °C under a tension of 5 mN and gassed with a mixture of 95% O2/5% CO2. Changes in isometric tension were recorded using Chart 5 software. 2.2. Experimental protocols The tissue was allowed to equilibrate for at least 90 min before each experiment. Rings were initially contracted with 5hydroxytryptamine (5-HT) (1 μM) to increase tension. When a stable contraction was maintained, the vasorelaxant effect of a single concentration of endocannabinoid (palmitoylethanolamide or anandamide) or vehicle control on induced tone was assessed as the reduction in tone over time. For each experimental protocol, endocannabinoid-treated and vehicle control experiments were performed in adjacent segments of the same artery. In some experiments, the vascular effects of anandamide and palmitoylethanolamide were investigated in un-contracted vessels. In all vessels, the integrity of the endothelium was assessed by precontracting the vessels with 1 μM 5-HT followed by relaxation with 10 μM carbachol; relaxation greater than 60% were designated as endothelium-intact. In some arterial rings, endothelium was removed by gently rubbing the intimal surface of the vessel with a human hair; carbachol-induced relaxation of 10% indicated successful removal (Romano and Lograno, 2006). To investigate any possible contribution of cannabinoid receptor activation, some experiments were performed in the presence of the cannabinoid CB1 receptor antagonist AM251 (1 μM) or cannabinoid CB2 receptor antagonist AM630 (1 μM), both added 10 min prior pre-contraction. To assess if anandamide and palmitoylethanolamide acts at a Gi/o-protein-coupled receptor, some arterial rings were pre-treated for 45 min with 500 ng/ml Pertussis toxin

(Romano and Lograno, 2006), and the vasorelaxation to anandamide and palmitoylethanolamide or vehicle was investigated. The involvement of transient receptor potential vanilloid 1 (TRPV1) on sensory nerve was investigate by pre-treatment for 30 min with the TRPV1 agonist capsaicin (10 μM) to deplete the sensory nerve of vasoactive neurotransmitters (Zygmunt et al., 1999). To evaluate the functional presence of PPARs in the bovine ophthalmic artery, some experiments were performed with the selective agonist of PPARα WY14643 (1 μM) and the selective agonist PPARγ ciglitazone (1 μM). The contribution of PPAR activation on the endocannabinoid-evoked vasorelaxant effects was investigated in the presence of the selective PPARα antagonist GW6471 (1 μM) or the selective PPARγ antagonist GW9662 (1 μM) both added 10 min prior pre-contraction. In some preparations, the role of endothelium-derived nitric oxide was investigated by utilizing the nitric oxide synthase inhibitor N Gmonomethyl-L-arginine (L-NMMA, 300 μM; Romano and Lograno, 2006). An incubation of 20 min was used for the fatty acid amide hydrolase (FAAH) inhibitor URB597 (1 μM) before determination of anandamide and palmitoylethanolamide responses. Finally, the potassium channel role in the vasorelaxant responses to endocannabinoids was assayed by utilizing iberiotoxin (200 nM), glibenclamide (5 μM), apamin (100 nM) and 4-aminopyridine (1 mM). 2.3. Data analysis All data were expressed as a mean percentage relaxation of 5HT-induced tone, with error bars representing the mean ± S.E.M. Rmax refers to the maximal relaxation achieved. Statistical analysis was performed using Student's t-test and when appropriate analysis of variance (ANOVA), followed by Bonferroni's post hoc test (GraphPad Prism 5.0). In all cases, n = the number of arteries from different animals. All differences were considered as statistically significant when P b 0.05. 2.4. Materials All drugs were purchased from Tocris Bioscience (Bristol, UK) except where indicated. Carbachol chloride, 5-HT creatinine sulfate, iberiotoxin and Pertussis toxin were obtained from Sigma Aldrich (St Louis, MO, USA). URB597 (3′-carbamoyl-biphenyl-3-yl-cyclohexylcarbamate) was obtained from Cayman Chemical (Ann Arbor, MI, USA). 4-aminopyridine, apamin, L-NMMA acetate, carbachol, and Pertussis toxin were dissolved in distilled water. Anandamide was supplied as a water-soluble emulsion and dissolved in distilled water. Capsaicin and glibenclamide were dissolved in ethanol to a stock concentration of 10 mM with further dilutions made daily in Krebs solution. AM251 (N-(piperidin-1-yl)-5-(4-iodophenyl)-1(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide), AM630 (6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl)-methanone), GW6471 ([(2S)-2-[[(1Z)-1-methyl-3-oxo-3[4-(trifluoromethyl) phenyl]-1-propenyl]amino]-3-[4-[2-(5-methyl-2phenyl-4-oxazolyl)ethoxy]phenyl]propyl]-carbamic acid ethyl ester), GW9662 (2-chloro-5-nitro-N-phenylbenzamide), ciglitazone and WY14643 ([[4-chloro-6-[(2,3-dimethylphenyl)amino]-2-pyrimidinyl] thio]acetic acid) were dissolved in dimethylsulphoxide (Sigma) to 10 mM with further dilutions prepared daily in Krebs solution. 3. Results 3.1. Time-dependent vascular response to the anandamide in the bovine ophthalmic artery The anandamide-induced relaxant effect at the concentration 100 nM became significantly different to the vehicle control from 90 min (2 h, Rmax anandamide = 47.8 ± 6.3%, n = 8, *P b 0.05,

M.R. Romano, M.D. Lograno / European Journal of Pharmacology 683 (2012) 197–203

Fig. 1A). At the concentration of 1 μM, the vascular response of anandamide was significantly different to the vehicle control from 45 min (2 h, Rmax anandamide = 68.1 ± 5.9%, n = 8, **P b 0.001, ***P b 0.0001, Fig. 1A). Anandamide 10 μM caused a time-dependent significant relaxation on the bovine ophthalmic artery compared to vehicle control at all times examined (2 h, vehicle control 12.1 ± 6.4% vs Rmax anandamide = 84.2 ± 2.1%, n = 10, ***P b 0.0001, Table 1, Fig. 1A). The endothelial denudation slightly affected the vasorelaxation evoked by anandamide but not significant manner (2 h, anandamide 100 nM, Rmax = 41.8 ± 5.1%, n = 8; anandamide 1 μM, Rmax = 58.3 ± 6.7%, n = 8; anandamide 10 μM, Rmax = 75.6 ± 4.9%, n = 10; Table 1, Fig. 1B). In addition, anandamide (10 μM) showed no effect on vascular tone of ophthalmic artery not contracted pharmacologically (2 h, vehicle control 0.01 ± 0.01 mN vs anandamide 0.03 ± 0.01 mN, n = 6).

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Table 1 Mechanisms involved in the vasorelaxant effects of anandamide.

Anandamide (10 μM) Endothelium-denuded & AM251 (1 μM) & AM630 (1 μM) & PTX (500 ng/ml, 45 min) & Capsaicin (10 μM, 30 min) & GW6471 (1 μM) & AM251 (1 μM) + GW6471 (1 μM) & L-NMMA (300 μM) & URB 597 (1 μM)

1 h% relaxation

2 h% relaxation

58.2 ± 4.2 51.2 ± 3.5 25.3 ± 2.4a 55.6 ± 3.9 22.4 ± 2.1b 55.3 ± 6.6 31.2 ± 1.1b

84.2 ± 2.1 75.6 ± 4.9 79.5 ± 7.7 87.0 ± 6.1 78.1 ± 9.6 81.7 ± 9.8 49.0 ± 3.2b

13.4 ± 4.6c 29.1 ± 3.8c 63.8 ± 5.2

19.3 ± 5.8c 43.9 ± 6.2c 96.0 ± 7.4

Percent relaxation to anandamide after 1 or 2 h in different experimental conditions. Data are expressed as mean ± S.E.M. a (P b 0.05) b(P b 0.001) and c(P b 0.0001) indicate significant difference from control values.

3.2. Time-dependent vascular response to the palmitoylethanolamide in the bovine ophthalmic artery The vasorelaxation evoked by palmitoylethanolamide at the concentration 100 nM was significantly different to the vehicle at 105 and 120 min (2 h, Rmax palmitoylethanolamide = 42.1 ± 4.5%, n = 8, *P b 0.05, Fig. 1C). At the concentration of 1 μM, the vascular effect of palmitoylethanolamide was significantly different from vehicle from 75 min (2 h, Rmax palmitoylethanolamide = 58.8 ± 2.2%, n = 10, *P b 0.05, Fig. 1C). Also palmitoylethanolamide 10 μM caused a significant time-dependent relaxation in arterial rings compared to vehicle control from 60 min (2 h, vehicle control = 11.6 ± 7.7% vs Rmax palmitoylethanolamide = 75.4 ± 5.1%, n = 10, **P b 0.001, Table 2, Fig. 1C). The denudation of endothelium slightly decreased the palmitoylethanolamide-induced vasorelaxation but not significant manner (2 h, palmitoylethanolamide 100 nM, Rmax = 40.1 ± 3.4%, n = 8; palmitoylethanolamide 1 μM, Rmax = 54.8 ± 1.7%, n = 8;

A pre-treatment of arterial rings with AM251 (1 μM), a selective cannabinoid CB1 receptor antagonist, yielded a significant inhibition of vasorelaxant response to anandamide in the first 60 min (1 h, Rmax anadamide = 57.8 ± 4.2%; in the presence of AM251 Rmax anadamide = 25.3 ± 2.4%, P b 0.05; 2 h, Rmax anandamide = 84.2 ± 2.1%; in the presence of AM251 Rmax anandamide = 79.5 ± 7.7%, n = 8;

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3.3. Role of cannabinoid receptors in time-dependent relaxation evoked by endocannabinoids

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palmitoylethanolamide 10 μM, Rmax = 68.5 ± 6.6%, n = 10; Table 2, Fig. 1D). Palmitoylethanolamide, as well as anandamide, showed no effect on vascular tone of ophthalmic artery not contracted pharmacologically (2 h, vehicle control 0.03 ± 0.01 mN vs palmitoylethanolamide 0.02 ± 0.01 mN, n = 6).

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Fig. 1. Anandamide (AEA) and palmitoylethanolamide (PEA) cause time-dependent vasorelaxation in bovine ophthalmic artery pre-contracted with 5-HT (1 μM). (A) Effects of AEA in endothelium-intact arterial rings; (B) effects of AEA in endothelium-denuded arterial rings; (C) effects of PEA in endothelium-intact arterial rings; (D) effects of PEA endothelium-denuded arterial rings, n = 8–10. Values are shown as means and vertical bars represent S.E.M. *P b 0.05, **P b 0.001, ***P b 0.0001 compared to vehicle control.

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Table 2 Mechanisms involved in the vasorelaxant effects of palmitoylethanolamide.

Palmitoylethanolamide (10 μM) Endothelium-denuded & AM251 (1 μM) & AM630 (1 μM) & PTX (500 ng/ml, 45 min) & Capsaicin (10 μM, 30 min) & GW6471 (1 μM) & L-NMMA (300 μM) & URB597 (1 μM)

1 h% relaxation

2 h% relaxation

42.6 ± 1.2 37.7 ± 3.1 38.8 ± 3.4 38.5 ± 4.2 35.6 ± 2.8 37.7 ± 4.8 17.2 ± 3.2a 19.3 ± 6.1a 36.9 ± 2.2

75.4 ± 5.1 65.9 ± 4.2 69.0 ± 3.5 73.8 ± 2.1 76.1 ± 8.8 75.9 ± 9.2 40.9 ± 2.3a 35.6 ± 1.2a 80.9 ± 6.7

Percent relaxation to palmitoylethanolamide after 1 or 2 h in different experimental conditions. Data are expressed as mean ± S.E.M. a (P b 0.05) indicates significant difference from control values.

Table 1, Fig. 2A). A pre-treatment with AM630 (1 μM), a selective cannabinoid CB2 receptor antagonist, did not cause modification of the time-dependent vascular response evoked by anandamide (Table 1, Fig. 2A). On the contrary a pre-treatment of arterial rings with AM251 (1 μM) and AM 630 (1 μM) did not alter the timedependent relaxant response evoked by palmitoylethanolamide (Table 2, Fig. 2B).

3.4. Involvement of PPARs in the time-dependent relaxant effect to endocannabinoids The selective PPARα agonist WY14643 (1 μM) and the selective PPARγ agonist ciglitazone (1 μM) were able to produce significant time-dependent vasorelaxant responses, compared to vehicle in the

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3.5. Signaling pathways involved in the vascular effects of endocannabinoids To investigate the Gi/o protein contribution at the time-dependent vasorelaxation induced by anandamide and palmitoylethanolamide, we have pre-incubated the arterial rings with Pertussis toxin (500 ng/ml) for 45 min. It is remarkable that the vascular response to anandamide has been significantly inhibited only in the first 60 min (1 h, Rmax Pertussis toxin = 22.3 ± 1.7%, n = 6, Table 1, Fig. 5) whereas the vascular response to palmitoylethanolamide was not affected (Table 2). In addition, we have investigated the effects of a pre-treatment of ophthalmic artery with capsaicin (10 μM, 30 min), a TRPV1 receptor agonist, which did not modify the timedependent relaxation induced by endocannabinoids (Tables 1 and 2). In endocannabinoid-induced vasorelaxation, the nitric oxide role was investigated. A pre-incubation with the nitric oxide synthase inhibitor L-NMMA (300 μM) for 30 min in arterial rings with endothelium caused a significant decrease of time-dependent vasorelaxant response evoked by anandamide (2 h, Rmax anandamide =43.9± 6.2%, n =6, Table 1, Fig. 6A) and palmitoylethanolamide (2 h, Rmax palmitoylethanolamide= 35.6 ±1.2%, n= 6, Table 2, Fig. 6B) in the bovine ophthalmic artery. By contrast, L-NMMA (300 μM) did not affect the relaxant responses to endocannabinoids in arterial rings without endothelium (data not shown). The FAAH inhibitor URB597 (1 μM), slightly increased the vasorelaxant response of anandamide (2 h, Rmax anandamide= 96.0 ±7.4%, n = 6, Fig. 7, Table 1) but that palmitoylethanolamide (2 h, Rmax palmitoylethanolamide =80.9± 6.7%, n =6, Table 2).

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bovine ophthalmic artery pre-contracted with 5-HT (1 μM) (2 h, vehicle control 12.3 ± 4.6% vs Rmax WY14643 = 71.7 ± 2.1% and Rmax ciglitazone = 63.2 ± 3.8, n = 6, Fig. 3). It is been intriguing to evaluate the vascular response to anandamide and palmitoylethanolamide in the presence of the selective antagonist of PPARs. The GW6471 (1 μM), a selective PPARα antagonist, significantly inhibited the vasorelaxation induced by anandamide (2 h, Rmax GW6471 = 49.0 ± 3.2%, n = 5, *P b 0.05, **P b 0.001; Table 1, Fig. 4A) and palmitoylethanolamide (2 h, Rmax GW6471 = 40.9 ± 2.3%, n = 6, *P b 0.05; Table 2, Fig. 4B) in a timedependent manner. Moreover, when arterial rings were pretreated with AM251 (1 μM) and GW6471 (1 μM), the vasorelaxant response to anandamide was completely blocked (2 h, Rmax GW6471 & AM251 = 19.3 ± 5.8%, n = 6, ***P b 0.0001; Table 1, Fig. 4A). By contrast, the vasorelaxant to anandamide and palmitoylethanolamide was unaffected by the pre-incubation with GW9662 (1 μM), a selective PPARγ antagonist (Tables 1 and 2).

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M.R. Romano, M.D. Lograno / European Journal of Pharmacology 683 (2012) 197–203

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To test the possible involvement of K+ channels in endocannabinoidevoked vasorelaxation, we pre-incubated vessel preparations with KATP blocker glibenclamide (5 μM), delayed rectifier K+ current blocker 4aminopyridine (1 mM) and the small conductance Ca2+-activated K+ channel blocker apamin (100 nM), which failed to modify the relaxation induced by anandamide and palmitoylethanolamide (Fig. 8A, B). To the contrary, the selective blocker of large conductance Ca2+-activated K+ channel iberiotoxin (200 nM) significantly decreased the vasorelaxant effect of anandamide and palmitoylethanolamide (Pb 0.0001; Fig. 8A, B). Similar findings have been obtained in endothelium-denuded arterial rings; in fact, iberiotoxin (200 nM) significantly reduced the relaxant

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Fig. 4. Effects of GW6471 (1 μM), a selective PPARα antagonist, and GW9662 (1 μM), a selective PPARγ antagonist, on time-dependent relaxation of bovine ophthalmic artery pre-contracted with 5-HT (1 μM). (A) The vasorelaxant responses of AEA in presence of GW6471 (1 μM) alone, GW6471 (1 μM) plus AM251 (1 μM) and GW9662 (1 μM). (B) The vasorelaxant responses of PEA in presence of GW6471 (1 μM) and GW9662 (1 μM), n = 6. Values are shown as means and vertical bars represent S.E.M. *P b 0.05, **P b 0.001, ***P b 0.0001 compared to control.

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Time (min) Fig. 5. Effect of pre-treatment with Pertussis toxin (PTX) (500 ng/ml) on the timedependent vasorelaxation to anandamide (AEA) in bovine ophthalmic artery precontracted with 5-HT (1 μM). n = 6. Values are shown as means and vertical bars represent S.E.M. *P b 0.05, **P b 0.001 compared to control.

Fig. 6. Effects of oxide nitric synthase inhibitor L-NMMA (300 μM) on time-dependent relaxation of bovine ophthalmic artery with endothelium and pre-contracted with 5HT (1 μM) AEA-induced (A) and PEA-induced (B). n = 6. Values are shown as means and vertical bars represent S.E.M. *Pb 0.05, **Pb 0.001, ***P b 0.0001 compared to control.

effect evoked by anandamide and palmitoylethanolamide in arterial rings without endothelium (P b 0.0001; Fig. 8C, D) whereas apamin (100 nM), glibenclamide (5 μM) and 4-AP (1 mM) did not produce any effects (Fig. 8C, D). 4. Discussion The present study shows for the first time that anandamide and palmitoylethanolamide active PPARα in the bovine ophthalmic artery pharmacologically precontracted with 5-HT and suggests that PPARα induction plays an important role to cause time-dependent vasorelaxant responses. This is an agreement with emerging studies revealing that non-CB1/CB2 receptor-mediated responses to cannabinoids occur both in the central nervous system and periphery and the PPAR transcription factors can be considered the potential candidates for cannabinoid responses (Pistis and Melis, 2010). Our findings show that time- and concentration-dependent vasorelaxation to anandamide and palmitoylethanolamide on bovine isolated ophthalmic artery is independent on classical cannabinoid receptors. It is intriguing to point out that previous studies have shown the inability of palmitoylethanolamide to induce relaxation in rat aorta (O'Sullivan et al., 2009a). Presumably, this discrepancy could be due to a different sensibility of palmitoylethanolamide to several tissues and/or species. To evaluate the role of cannabinoid receptors in relaxation to endocannabinoids, the effects of anandamide and palmitoylethanolamide were assayed in the presence of cannabinoid receptor antagonists. The selective cannabinoid CB1 receptor antagonist AM251 inhibited the time-dependent anandamide-induced relaxant response only in the first 60 min. Moreover, during the first 60 min, the vasorelaxation evoked by anandamide was significantly inhibited by Pertussis toxin, suggesting that time-dependent vascular response to anandamide involved a receptor of membrane coupled to Gi/o

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protein such as the cannabinoid CB1 receptor (Turu and Hunyady, 2010). Our previous study has shown the functional presence of cannabinoid CB1 receptors in the bovine ophthalmic artery and that the activation of them by natural and synthesis agonists produced a significant concentration-dependent vasorelaxation (Romano and Lograno, 2006). One could hypothesize that anandamide activates a classical cannabinoid CB1 receptor set on the cellular membrane

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which could trigger intracellular signals that induce the activation of transcription factors such as PPARs. In addition, the selective CB2 receptor antagonist AM630 was insensitive to modify the vasorelaxant action to anandamide. Furthermore, the relaxant response evoked by palmitoylethanolamide has not been modified by the selective CB1 and CB2 receptor antagonists AM251 and AM630 respectively. We have investigated the role of the Pertussis toxin in the vasorelaxation to palmitoylethanolamide. The findings show that Pertussis toxin did not affect the relaxant response to palmitoylethanolamide. This confirmed previous work which demonstrated the inability of palmitoylethanolamide to interact directly with classical CB receptors (Griffin et al., 2000; Ho et al., 2008; Lo Verme et al., 2005). Indeed, anandamide and palmitoylethanolamide are compounds structurally similar even if they seem to have a different pharmacological profile. The endothelial denudation slightly decreases the vasorelaxation induced by anandamide and palmitoylethanolamide supporting the hypothesis that endothelial factors may be necessary but not sufficient as relaxation messengers. The effects of TRPV1 receptors in the relaxant response induced by endocannabinoids have been examined. A pre-incubation with capsaicin to deplete sensorial neurotransmission did not provoke modification to anandamide and palmitoylethanolamide vasorelaxation indicating that endocannabinoid don't establish vascular effects via TRPV1 receptors in the bovine ophthalmic artery. We have evaluated if selective agonists of PPARα and PPARγ, which are primarily expressed in vascular level, could provoke a time-dependent vasorelaxation in the bovine ophthalmic artery precontracted with 5-HT. The selective PPARγ agonist ciglitazone and the selective PPARα agonist WY14643 yielded a significant timedependent vasorelaxation in the bovine ophthalmic artery compared with segments of same artery treated with only vehicle. Hence, the PPAR transcription factors appear to be functionally expressed in the bovine ophthalmic artery such as in other vascular beds (Goya et al., 2004; Touyz and Schiffrin, 2006), since in ophthalmic artery the expression of PPARs has never been studied. To emphasize the possible involvement of PPARs in the vasorelaxant effects time-dependent to endocannabinoids, we have investigated the cannabinoid responses in the presence of selective antagonists of PPARs. The selective PPARα antagonist GW6471

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Fig. 8. Effects of potassium channel blockers on time-dependent relaxation to endocannabinoids in bovine ophthalmic artery pre-contracted with 5-HT (1 μM). (A) AEA effects on arterial rings with endothelium-intact and (B) with endothelium-denuded. (C) PEA effects on arterial rings with endothelium-intact and (D) with endothelium-denuded. n = 6. Values are shown as means and vertical bars represent S.E.M. ***P b 0.0001 compared to control.

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significantly inhibited the relaxant response to anandamide and palmitoylethanolamide. By contrast, the selective PPARγ antagonist GW9662 did not affect the time-dependent vasorelaxation evoked by anandamide and palmitoylethanolamide. Although several studies demonstrated that cannabinoid compounds evoke a time-dependent relaxation in different vascular beds by involving the PPARγ (O'Sullivan et al., 2005, 2009a, 2009b), in this study it is noticeable that time-dependent relaxation induced by endocannabinoids involves the PPARα transcription factors. The role of nitric oxide in the time-dependent vasorelaxation to endocannabinoids was examined. Thus, a pre-treatment with L-NMMA, an inhibitor of nitric oxide synthase, significantly reduced the time-dependent vasorelaxation to endocannabinoids in arterial rings with intact endothelium, but not influenced by the relaxant response to endocannabinoids in arterial rings without endothelium (data not shown). These data are consistent with other studies obtained in a variety of vascular tissues (Ho et al., 2008; O'Sullivan et al., 2005). In addition, the role of the FAAH inhibitor URB597 which slightly increased the time-dependent relaxation to anandamide without modifying the response to palmitoylethanolamide indicating that FAAH was not responsible of vasorelaxation to endocannabinoids has been investigated. In the search for a possible signaling pathway involved in the time-dependent vasorelaxation to endocannabinoids, we have tested the highly selective blocked Ca 2+-activated K + channel iberiotoxin which caused a potent inhibition of time-dependent vasorelaxant effect to endocannabinoids. Moreover, glibenclamide, 4-aminopyridine and apamin did not influence the time-dependent vasorelaxation to endocannabinoids. Hence, the activation of Ca 2+-activated K + channels is involved in relaxation of endocannabinoids in bovine ophthalmic artery. These evidences prompt us that the activation of PPARα leads to a downstream opening of some channels, such as Ca 2+-activated K + channels, which are known to modulate the vasorelaxant effects. Similarly in arterial rings without endothelium, iberiotoxin decreased significantly the time-dependent vasorelaxation to endocannabinoids examined, whereas apamin, glibenclamide and 4aminopyridine failed to modify them. In summary, there are three major goals that we wish to highlight in relation to the data obtained with our experiments: (i) the ability of some endocannabinoids to evoke a time- and concentrationdependent vasorelaxant response in the bovine ophthalmic artery both in the presence and the absence of endothelium; (ii) the involvement of the PPARα in vasorelaxation to anandamide and palmitoylethanolamide; and (iii) vasorelaxation to anandamide was inhibited during the first 60 min from AM251 and Pertussis toxin by emphasizing the role of cannabinoid CB1 receptors to trigger the activation of PPARα. Our results provide the first evidence that anandamide and palmitoylethanolamide are able to activate the PPARα in the bovine ophthalmic artery and to determine a timedependent vasorelaxant response. In addition, the present study shows the important vasorelaxant role of endocannabinoids in ophthalmic artery which raise the supply of oxygen to the retina and prevent ischaemic injury and the ability of endocannabinoids to open a dialog with different receptors coordinating the physiological vascular regulation and expanding the knowledge on the molecular mechanisms involved.

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Acknowledgments We are grateful for the financial support from “Ministero dell'Università e della Ricerca Scientifica e Tecnologica” and for vet help from Vito Masciopinto.

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