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Am J Physiol Heart Circ Physiol 282: H2046-H2054, 2002. First published January 24, 2002; doi:10.1152/ajpheart.00497.2001
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Vol. 282, Issue 6, H2046-H2054, June 2002

Anandamide-induced vasorelaxation in rabbit aortic rings has two components: G protein dependent and independent

Somnath Mukhopadhyay, Barry M. Chapnick, and Allyn C. Howlett

Department of Pharmacological and Physiological Science, St. Louis University School of Medicine, St. Louis, Missouri 63104


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The endogenous cannabinoid anandamide (arachidonylethanolamide) produces vasorelaxation in different vascular beds. In the present study, we found that anandamide and a metabolically stable analog, methanandamide, produced dose-dependent (10 nM-10 µM) vasorelaxation of ~80% in a rabbit aortic ring preparation in an endothelium-dependent manner. Non-endothelium-dependent vasorelaxation was observed to be a maximum of 20-22% at >10 µM methanandamide. The efficacious CB1 receptor analogs desacetyllevonantradol (10 µM) and WIN55212-2 (10 µM) failed to produce vasorelaxation; however, the endothelium-dependent vasorelaxation evoked by methanandamide was partially (75%) blocked by the CB1 receptor antagonist SR141716A. The VR1 vanilloid receptor antagonist capsazepine or the calcitonin gene-related peptide (CGRP) antagonist CGRP-(8-37) partially attenuated (25%) the vasorelaxation in endothelium-intact preparations and greatly reduced the response in endothelium-denuded preparations. Pretreatment of aortic rings with NG-nitro-L-arginine methyl ester completely blocked the methanandamide-, capsaicin-, and CGRP-induced vasorelaxation. Pretreatment of aortic rings with pertussis toxin attenuated the methanandamide-induced vasorelaxation in endothelium-intact aortic rings, indicating the involvement of Gi/o proteins in the vasorelaxation; however, pertussis toxin treatment failed to block the endothelium-independent response. Thus, in the rabbit aorta, methanandamide-induced vasorelaxation exhibits two components: 1) in endothelium-intact rings, an SR141716A-sensitive, non-CB1 receptor component that requires pertussis toxin-sensitive G proteins and nitric oxide (NO) production; and 2) in endothelium-denuded rings, a component that is mediated by VR1 vanilloid receptors and possibly by the subsequent release of CGRP that requires NO production but is independent of pertussis toxin-sensitive G proteins.

cannabinoid receptors; vanilloid receptors; pertussis toxin; endothelium; nitric oxide


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CANNABINOID DRUGS are known to produce profound cardiovascular effects in humans and animals (2, 13, 15). Recent findings have demonstrated that some cardiovascular effects are produced by the eicosanoid anandamide (arachidonylethanolamide) and its analogs in various animal models, including conscious or anesthetized normotensive or spontaneously hypertensive rats (19, 20, 39). In anesthetized rats, anandamide produced sequential, triphasic changes consisting of a transient bradycardia and hypotension, followed by a brief pressure increase and finally a relatively long-lasting depressor effect (38). The anandamide-induced prolonged hypotension was blocked by the CB1 antagonist SR141716A (19, 20, 38), suggesting that this effect of anandamide is mediated by the CB1 receptor. The failure of anandamide and other potent cannabinoid receptor agonists to elicit the long-lasting depressor effect in CB1 knockout (-/-) mice provides further support for the involvement of the CB1 receptor in this component of the response (18). Recent studies have suggested that anandamide and other cannabinoid agonists induce hypotension by presynaptic inhibition of norepinephrine (NE) release (23, 26, 39) in a SR141716A-sensitive manner (16, 23). These findings suggest the involvement of a neuronal CB1 receptor signaling mechanism for certain cardiovascular effects of cannabinoid drugs. In contrast, SR141716A failed to attenuate the anandamide-induced activation of both initial bradycardia and subsequent pressure changes (38), suggesting that this particular component of the anandamide response may not involve the CB1 receptor.

In addition to the effect on blood pressure, anandamide evoked vascular smooth muscle relaxation in various endothelium-intact and -denuded arterial preparations (for a review, see Ref. 41). A SR141716A-sensitive, anandamide-induced vasorelaxation that does not respond to classical cannabinoid receptor agonists has been demonstrated by Wagner et al. (40, 41), who proposed that a novel "anandamide receptor" was involved. However, this interpretation is not compatible with the findings of Pratt et al. (31), who proposed that metabolism of anandamide to other eicosanoid products could be responsible for the vasorelaxation.

More recently, the vasorelaxing effects of anandamide (but not classical CB1 agonists) in isolated rat hepatic, rat mesenteric, and guinea pig basilar arterial preparations have been explained by a noncannabinoid receptor mechanism involving the VR1 vanilloid receptor (46). In that study, anandamide-induced vasodilation was shown to be blocked either by the VR1 antagonist capsazepine or the calcitonin gene-related peptide (CGRP) antagonist CGRP-(8-37) but not by SR141716A (46). Results of these studies suggest that anandamide activates VR1 receptors on the perivascular sensory neurons to release CGRP, which would then evoke vascular relaxation.

In the present study, we found that in rabbit arterial ring preparations, methanandamide produced vasorelaxation by two different signal transduction pathways. The major component of the relaxation was attributed to a SR141716A-sensitive non-CB1 receptor-mediated activation of pertussis toxin-sensitive G proteins and involves endothelium-derived nitric oxide (NO) for vasorelaxation. The other component was due to anandamide-activated VR1 receptors and CGRP-mediated vasorelaxation, which involves non-endothelium-derived NO and is independent of pertussis toxin-sensitive G proteins.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents. Methanandamide was purchased from Research Biochemicals (Natick, MA). Desacetyllevonantradol (DALN) was a gift from Pfizer (Groton, CT). WIN55212-2 was obtained from Sigma-RBI (St. Louis, MO). SR141716A was purchased from BioMol (Plymouth Meeting, CA). NE, indomethacin, NG-nitro-L-arginine methyl ester (L-NAME), acetylcholine chloride (ACh), CGRP, CGRP-(8-37), anandamide, and arachidonic acid were obtained from Sigma. Capsaicin and capsazepine were purchased from Tocris (Ballwin, MO). Glyceryl trinitrate (GTN; Nitrostat, 0.4-mg tablets) was a product of Parke-Davis (Ann Arbor, MI). All other chemicals used were of the highest analytic grade and were obtained from Sigma.

Tissue preparation. Male New Zealand White rabbits (2.5-3.5 kg) were anesthetized with ketamine (3 mg/kg im) plus xylazine (1 mg/kg im), followed by pentobarbital sodium (15 mg/kg iv). A cannula was placed into the carotid artery, and heparin (200 U/kg iv) was administered. After 10 min, the animals were exsanguinated. The abdomen was opened by a midline incision, and the aorta was carefully excised and placed in ice-cold Krebs-Ringer bicarbonate (KRB) buffer [containing (in mM) 118.3 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, 0.026 Na-EDTA, and 11.1 glucose; pH 7.4] at 4°C. All connective and perivascular adipose tissues were removed, with caution being taken not to disrupt the endothelial cell lining. Aortas were then refrigerated in KRB buffer for use either the same or the following day. Storage for 24 h did not affect endothelium-dependent vasomotor responses. Transverse vascular rings 3-4 mm long were prepared from the aorta and suspended from an isometric force transducer (Grass FT .03) according to methods previously described (27, 28). Briefly, the rings were mounted horizontally between an L-shaped fixed stainless steel rod and a freely moving stainless steel triangle attached to an isometric force transducer by a S hook in a 25-ml jacketed glass organ bath. Rings were bathed in 20 ml KRB buffer (37°C) and continuously aerated with 95% O2-5% CO2. The KRB buffer also contained indomethacin (10 µM) to inhibit cyclooxygenase activity. The rings were then gradually stretched over a period of 45-60 min until the optimum basal tension (2 g, as predetermined from tension-force studies) was achieved. The tissues were washed with 20 ml of fresh KRB buffer every 15-20 min. After an initial equilibration period of 20 min, rings were contracted with KCl (60 mM) to stabilize and enhance subsequent submaximal contraction (28). The tissues were washed with fresh KRB buffer to remove KCl, and, thereafter, submaximal tone (60-70%) was induced in the rings with NE (100 nM). After the submaximal NE-induced contraction of the rings, the test compounds were added into the incubation medium to achieve the final indicated concentrations in a cumulative manner. When aortic rings were treated with SR141716A, capsazepine, or CGRP-(8-37), these drugs were included for 30 min at 37°C, and anandamide- or methanandamide-induced relaxation was then determined. Data are presented as percent relaxation, calculated as the percentage of the difference between the NE-contracted tone and the basal tone. After each series of additions, the rings were washed several times with fresh KRB buffer and allowed to reequilibrate for 20-30 min.

Measurement of vasorelaxing effects of drugs under endothelium-denuded conditions. For the endothelium-denuded preparation, the endothelium was removed by rubbing the intimal surface of the rings with the roughened shaft of a 23-gauge needle (27, 28). The successful removal of the endothelium was ascertained by the absence or markedly reduced (90%) relaxation produced in response to ACh. These same rings were also tested with the endothelium-independent relaxing agent GTN to assure that the lost response to ACh was due to the loss of the endothelium and not due to damage to the underlying smooth muscle structure. After the removal of the endothelium, the rings were allowed to reequilibrate to basal tension for 30-45 min in KRB buffer, after which the vasorelaxing effects of the indicated drugs were determined.

Treatment with pertussis toxin. To determine the involvement of Gi/o proteins in vasorelaxation, rabbit aortic rings were treated with pertussis toxin (250 ng/ml) or its vehicle for 2 h at 37°C (26). After the incubation period, the rings were washed with KRB buffer and submaximally contracted with NE, and vasomotor responses to agonists and/or antagonists were determined. Experiments were performed such that drug-induced vasorelaxation was determined both before and after pertussis toxin treatment to compare the responses in a paired manner.

Treatment with L-NAME. To determine the role of NO in agonist-induced vasorelaxation, endothelium-intact or -denuded rings were pretreated with L-NAME (30 µM for 45 min at 37°C). The rings were then washed with KRB buffer, allowed to equilibrate for an additional 20 min, and contracted with NE, and vasomotor responses to the test compounds were measured. Agonist-induced vasorelaxation was determined both before and after L-NAME treatment in the same rings to make paired comparisons.

Data analysis. All values are expressed as means ± SE of multiple, separate experiments. Data were analyzed by ANOVA, followed by a Bonferroni post hoc test. The log dose-response curves were analyzed by nonlinear regression analysis of a sigmoidal curve to determine concentrations at half-maximal response and the slope factors (Graphpad Inplot).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Methanandamide-induced vasorelaxation has two components: endothelium-dependent and -independent. Anandamide (100 nM-10 µM) produced a vasorelaxation of NE-contracted aortic rings. The maximal response in endothelium-intact rings was ~60% relaxation (Fig. 1A). Methanandamide, a metabolically stable analog of anandamide (1), also produced a concentration-dependent relaxation of aortic rings (Fig. 1, A and B). No significant differences were observed when relaxations produced by anandamide and methanandamide were compared, suggesting that anandamide degradation is not prevalent under these experimental conditions. In endothelium-intact aortic rings, the maximum relaxation of ~80% was obtained with 20 µM methanandamide and the EC50 was 0.6 µM (Fig. 2B). The onset of relaxation began within 15 s after the addition of methanandamide to the KRB buffer.


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  		Fig. 1.   Vasorelaxant effect of anandamide and methanandamide and cannabinoid receptor agonists on norepinephrine (NE)-contracted rabbit aortic rings. A: anandamide, methanandamide, desacetyllevonantradol (DALN), and WIN55212-2 were applied to rabbit aortic rings at the indicated concentrations, and the percent relaxation was determined. Data are presented as means ± SE of 4 separate aortic rings from different rabbits. B and C: representative polygraph tracings illustrating the vasorelaxant effects of methanandamide on NE-contracted endothelium-intact rabbit aortic ring preparations (B) and those after the endothelium was disrupted (C). Each arrow represents the time of addition, and numbers correspond to the final concentrations of methanandamide. GTN, glyceryl trinitrate.



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  		Fig. 2.   Dose-dependent vasorelaxation of NE-contracted rabbit aortic rings by anandamide (A), methanandamide (B), and acetylcholine (ACh; C) in the presence and absence of SR141716A. Rings were incubated with SR141716A (1 µM) for 30 min before the addition of anandamide, methanandamide, or ACh, which were added to the Krebs-Ringer bicarbonate (KRB) buffer by incrementing the concentration at 2-min intervals. Each point on the graph represents the cumulative final concentration of the vasorelaxant compounds. Data are presented as means ± SE of observations obtained from 6-8 separate aortic rings from different rabbits.

The methanandamide-induced relaxation was markedly attenuated after mechanical denudation of the same ring (compare Fig. 1, C with B; see also Figs. 3B, 4C, and 5B). The non-endothelial-dependent relaxation achieved a maximum of 18-20% at high concentrations of methanandamide (20 µM). Anandamide-induced vasorelaxation was also abrogated (80-85%) after endothelial denudation (data not shown). To control for the integrity of the smooth muscle, addition of the NO donor GTN (0.1 µM) to the endothelium-denuded ring produced almost complete relaxation (95-100%) (Fig. 1C). No significant difference was observed in GTN-induced relaxation before or after endothelium denudation or with a higher concentration of GTN (1 µM) (data not shown).


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  		Fig. 3.   Effect of capsazepine and the calcitonin gene-related peptide (CGRP) antagonist CGRP-(8-37) on methanandamide-induced vasorelaxation of NE-contracted endothelium-intact (A) and endothelium-denuded rabbit aortic rings (B). Capsazepine (3 mM), CGRP-(8-37) (2 mM), or vehicle was added to the KRB buffer 30 min before the addition of methanandamide. Data are presented as means ± SE of 4-6 separate aortic rings from different rabbits. The methanandamide-induced percent relaxation was significantly different (* P < 0.02 and ++P < 0.05) between control and capsazepine- or CGRP-(8-37)-treated rings.



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  		Fig. 4.   Effect of NG-nitro-L-arginine methyl ester (L-NAME) on methanandamide- and capsaicin-induced vasorelaxation of NE-contracted endothelium-intact (A) and endothelium-denuded rabbit aortic rings (B). C: effect of L-NAME on CGRP-induced vasorelaxation of endothelium-intact and endothelium-denuded rabbit aortic rings. Rings were pretreated with L-NAME (30 mM) or its vehicle (water) for 30 min before the contraction with NE, and methanandamide, capsaicin, or CGRP was then added at the indicated cumulative final concentrations. Data are presented as means ± SE of the percent relaxation from 4 separate aortic rings from different rabbits. The percent relaxation was significantly different (* P < 0.02 and ++P < 0.05) between the absence and presence of L-NAME.



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  		Fig. 5.   Effects of pertussis toxin (PTX) on methanandamide-induced vasorelaxation of NE-contracted endothelium-intact (A) and endothelium-denuded rabbit aortic rings (B). Rings were pretreated with PTX (250 ng/ml) for 2 h at 37oC, and methanandamide was then added at the indicated cumulative final concentrations. Data are presented as means ± SE of the percent relaxation from 3 separate aortic rings from different rabbits. A: methanandamide-induced percent relaxation was significantly different (* P < 0.02) between vehicle- and PTX-treated endothelium-intact aortic rings at all the concentrations of methanandamide. B: no significant differences were observed between vehicle- and PTX-treated aortic rings after endothelium denudation.

Chaytor and colleagues (7) reported that in rabbit superior mesenteric arterial preparations, gap junctions are involved in anandamide-induced endothelium-dependent relaxation because the response could be blocked by the gap junction inhibitor 18alpha -glycerrhetinic acid and connexin43 mimetic peptide. We tested for a gap junction involvement in rabbit aortic ring preparations by preincubation of the rabbit aortic rings with the gap junction inhibitor 18alpha -glycerrhetinic acid (50 µM for 1 h). This treatment did not block the anandamide- or methanandamide-induced relaxation (data not shown). This suggests that anandamide/methanandamide produced vasorelaxation in the rabbit aortic rings through a gap junction-independent mechanism and therefore does not involve transfer of small molecules between cells. A recent study by White and colleagues (44) reported that anandamide-induced vasorelaxation in rat coronary arteries was not affected by the gap junction inhibitor 18alpha -glycerrhetinic acid.

Methanandamide produces vasorelaxation in a SR141716A-sensitive manner but not via the CB1 receptor. To address the issue of whether the anandamide-induced vasorelaxation in rabbit aortic rings could be due to stimulation of the CB1 receptor, we evaluated the vasoactivity of DALN and WIN55212-2, two potent and efficacious CB1 receptor agonists of the cannabinoid and aminoalkylindole chemical classes. Neither the classical cannabinoid agonist DALN nor the aminoalkylindole WIN55212-2 were able to produce a significant relaxation of the endothelium-intact aortic rings at any of the concentrations tested (100 nM-10 µM; Fig. 1A). Additionally, the CB1 agonists produced no response whether the endothelium was functionally intact or not (data not shown). Thus the anandamide and methanandamide response is not consistent with a CB1 pharmacological profile.

To further address the hypothesis that anandamide and methanandamide-induced relaxation is mediated by the CB1 receptor, studies were performed using the potent and selective CB1 antagonist SR141716A. Anandamide and methanandamide produced relaxation in endothelium-intact aortic rings at all concentrations tested with EC50 0.7 and 0.6 µM, respectively (Fig. 2, A and B). The anandamide response was reduced in the presence of 1 µM SR141716A (Fig. 2A). It was not possible to properly determine the EC50 because of concerns about lack of solubility at high concentrations of anandamide. The methanandamide log concentration-response curve also underwent a rightward parallel shift (EC50 = 4.2 µM) in the presence of 1 µM SR141716A without any alteration in the maximum response, consistent with competitive antagonism (Fig. 2B). When added to the incubation medium after methanandamide-induced relaxation, SR141716A partially reversed the response (data not shown). Higher concentrations of SR141716A were not used to induce a further antagonism because this drug has been reported to evoke miscellaneous cellular effects at a concentration >3 µM (31, 43). The CB2 cannabinoid receptor antagonist SR144528 did not block the anandamide- or methanandamide-induced vasorelaxation in the rabbit aortic ring preparation. This suggests that the anandamide- or methanandamide-induced response in rabbit aortic rings was not mediated through CB2 cannabinoid receptors.

SR141716A did not alter ACh-induced relaxation under conditions identical to those used for the anandamide and methanandamide responses (Fig. 2C), suggesting specificity of the antagonist for the anandamide-induced response. The endothelium-independent component of the methanandamide-induced relaxation, which was observed in denuded rings, was not attenuated in the presence of SR141716A (data not shown). Furthermore, SR141716A did not affect the vasorelaxation evoked by glyceryl trinitrate (data not shown), indicating that the antagonism is not at the level of the smooth muscle.

VR1 receptor and CGRP antagonists block methanandamide-induced endothelium-independent relaxation. The role of vascular innervation on the methanandamide-induced relaxation of rabbit aortic rings was addressed by examining the effects of VR1 receptor-dependent neuromediator release. Pretreatment (30 min at 37°C) of endothelium-intact rabbit aortic rings with either the VR1 antagonist capsazepine (3 µM) or the CGRP receptor antagonist CGRP-(8-37) (2 µM) partially blocked (15-20%) methanandamide-induced relaxation (Fig. 3A). In endothelium-denuded rings, pretreatment with both capsazepine and CGRP-(8-37) totally blocked the relaxation evoked by 20 µM methanandamide (Fig. 3B). These data suggest that the endothelium-independent component of the methanandamide-induced relaxation can be attributed to the activation of VR1 receptors known to be present on the primary sensory neurons embedded in the smooth muscle cell layer in the aortic vessel wall (46). Furthermore, these results suggest that the methanandamide-stimulated VR1 receptor evokes vasorelaxation primarily by the release of the vasodilator CGRP from the prearterial nerve endings.

The methanandamide-induced, endothelium-independent vasorelaxation is only a small fraction of the vasorelaxation that can be produced by VR1 receptors in the rabbit aortic ring preparation. Capsaicin and CGRP (10 µM) produced robust relaxation of endothelium-intact rabbit aortic rings [70% (Fig. 4A) and 60% (Fig. 4C), respectively]. This occurs in a concentration-dependent manner with EC50 of 1 and 0.3 µM, respectively (data not shown). Of importance, the capsaicin- and CGRP-mediated relaxation in endothelium-denuded rings was significantly less than that observed with endothelium-intact rings (25% and 15%, respectively; Fig. 4). This shows that capsaicin- and CGRP-evoked relaxation in the rabbit aortic ring preparation exhibits an endothelium-dependent component that is not related to the response that was produced by methanandamide.

L-NAME blocks both methanandamide- and capsaicin-induced relaxation. Pretreatment (30 min at 37°C) of endothelial intact rings with the NO synthase (NOS) inhibitor L-NAME (30 µM) almost completely blocked methanandamide- and capsaicin-induced relaxation in endothelium-intact (Fig. 4A) and -denuded (Fig. 4B) rabbit aortic rings. CGRP-mediated relaxation was also significantly blocked after L-NAME treatment in endothelium-intact and -denuded rings (Fig. 4C). These results indicate that methanandamide, capsaicin, and CGRP produced vasorelaxation via a NO-mediated mechanism.

Pertussis toxin treatment blocks methanandamide-induced relaxation in endothelium-intact rings. The CB1 and CB2 cannabinoid receptors are coupled to signal transduction pathways that are mediated via the pertussis toxin-sensitive Gi/o proteins (for a review, see Ref. 14). We performed a study to determine whether the methanandamide-induced vasorelaxation is mediated via Gi/o proteins. Treatment of rabbit aortic rings with pertussis toxin (250 ng/ml for 2 h at 37°C) significantly blocked the methanandamide-induced relaxation in endothelium-intact rings (Fig. 5A). However, in endothelial-denuded rings, pertussis toxin treatment had no effect on methanandamide-induced relaxation (Fig. 5B). Pertussis toxin treatment also blocked the anandamide-induced vasorelaxation in endothelium-intact rings (data not shown). The methanandamide-induced relaxation in pertussis toxin-treated endothelium-intact rings is equivalent in magnitude to that produced in endothelium-denuded rings (20%). This observation suggests that the endothelium-dependent component of the methanandamide-induced relaxation in the rabbit aorta is mediated by a pertussis toxin-sensitive G protein, whereas the endothelium-independent, VR1-mediated component of the relaxation is not.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study demonstrates that anandamide and methanandamide can activate at least two different pathways to produce vasorelaxation in the rabbit aortic ring model. When taken together, the data indicate that the endothelium-dependent, SR141716A-sensitive component of anandamide- or methanandamide-induced relaxation in the rabbit aorta is regulated by a pertussis toxin-sensitive G protein. The results of the present study have also provided evidence in support of the dual involvement of pertussis toxin-sensitive G proteins and NO in the vasoregulatory effects of anandamide and methanandamide. In contrast, the endothelium-independent, VR1-mediated component of the relaxation is Gi/o protein independent. This represents the first report of the involvement of pertussis toxin-sensitive G proteins with the endothelium-dependent component of anandamide/methanandamide relaxation and is important because it implicates a G protein-coupled receptor in the endothelium-dependent response.

The anandamide-induced relaxation was found to be endothelium dependent in bovine coronary arteries (31) but not in rat mesenteric (42), coronary (44), and hepatic (45) arteries. The finding of CB1 mRNA and protein in rat and human vascular endothelial cells (9, 21) allows for the possible involvement of CB1 receptors in vasoregulation at the level of endothelial cells. However, mediation by the CB1 receptor in the present studies is made untenable by the observation that very potent and efficacious cannabinoid receptor agonists DALN and WIN55212-2 failed to evoke relaxation of the rabbit aortic ring preparation. Antagonism of the anandamide- or methanandamide-evoked vasorelaxation response by SR141716A is in agreement with previous findings in rat coronary (33) and mesenteric (34) arteries. In the present studies, a greater concentration of SR141716A (1 µM) was necessary to shift the concentration-response curve eightfold for methanandamide in the aortic ring relaxation compared with a response believed to be mediated via the CB1 receptor. For example, for the inhibition by WIN55212-2 of the guinea pig myenteric plexus longitudinal muscle twitch response, a shift of one order of magnitude could be produced by SR141716A at a concentration of 100 nM (8). Similar to the observations made in this study, anandamide-induced relaxation in rat coronary arteries was attenuated by SR141716A at a relatively higher concentration (3 µM) (44). These findings suggest that a novel noncannabinoid receptor that nevertheless exhibits sensitivity to SR141716A exists in this preparation. Our studies support the existence of a SR141716A-sensitive "anandamide" receptor, which has been previously proposed by Kunos and colleagues (18, 40).

Anandamide has previously been proposed to be an endothelium-derived hyperpolarizing factor (EDHF) (32). Blockade of carbachol- and anandamide-mediated relaxation by SR141716A in isolated, buffer-perfused rat mesenteric and coronary vasculature as well as in mesenteric arterial segments implicated the CB1 receptor in these events (32-34). Unlike our experiments, in those studies, anandamide- or carbachol-induced relaxation was measured in the presence of L-NAME (300 µM) and indomethacin (10 µM) to detect a non-NO, EDHF response. However, these findings were not confirmed by other laboratories (5, 30). The "EDHF"-like response of anandamide measured in the presence of L-NAME was found to be pertussis toxin sensitive (42). Results of our study suggest that anandamide-induced vasorelaxation involves a pertussis toxin-sensitive G protein but that the final mediator is NO, obviating the notion of anandamide as an EDHF. Furthermore, our finding that SR141716A fails to alter the response to ACh but exhibits a competitive inhibition for methanandamide would eliminate the possibility that the ACh response occurred via the release of endogenously synthesized anandamide in the rabbit aorta.

Anandamide-derived arachidonic acid released by the action of fatty acid aminohydrolase and subsequently formed eicosatrienoic acid derivatives was held responsible for the vasodilatory action of anandamide in studies by Pratt et al. (31). The involvement of eicosanoid metabolites can be ruled out in the present study for the following reasons: 1) anandamide and its metabolically stable analog methanandamide produced an identical profile of relaxation; 2) arachidonic acid itself did not produce any response in the rabbit aortic ring preparation (data not shown); 3) the epoxyeicosatrienoic acid inhibitor 17-octadecanoic acid did not alter anandamide- or methanandamide-induced relaxation (data not shown); and 4) participation of cyclooxygenase products in methanandamide-induced vasorelaxation can be ruled out because indomethacin was used in all the experiments reported in the present study.

Zygmunt and colleagues (46) demonstrated that in isolated rat hepatic, rat mesenteric, and guinea pig basilar arterial preparations, anandamide-induced relaxation was almost completely blocked by the VR1 vanilloid receptor antagonist capsazepine but not by SR141716A. They suggested that the VR1-mediated release of neuromediators is responsible for the anandamide-evoked vasorelaxation (46). The response observed by Zygmunt et al. (46) appears to be comparable to that which we observed in the endothelium-denuded rings. This is consistent with a mechanism by which methanandamide activates the VR1 receptor to cause vasorelaxation via CGRP. In mesenteric arteries of rabbits (25), splenic, gastric, and hepatic arteries of rats (4), splenic arteries of pigs (29), cerebral arteries of cats (10, 35), and pulmonary arteries of guinea pigs (22) and humans (24), CGRP-induced vasorelaxation is endothelium independent. The relaxation that we observed in the rabbit aortic ring preparation was not pertussis toxin sensitive, which would be consistent with the actions of CGRP occurring via a non-Gi/o subtype of G proteins (6).

The significant attenuation by L-NAME of both components of the methanandamide-evoked vasorelaxation, as well as the capsaicin- and CGRP-evoked vasorelaxation, suggests the involvement of NO in both endothelium-dependent and -independent vasorelaxation. Anandamide has been reported to activate endothelial NOS in the human saphenous vein, and this effect was blocked by SR141716A (37). Although there is no consensus regarding the role of NO in capsaicin and CGRP-induced vasorelaxation, evidence suggests that capsaicin causes the release of CGRP, which in turn activates endothelial NOS activity to produce NO to evoke vasodilation (11, 12). In vascular smooth muscle cells (36) and in the rat thoracic aorta (12), CGRP increased NOS activity via a cAMP-dependent pathway. Results from other laboratories suggest that NO is involved in the capsaicin-stimulated release of CGRP (3).

In summary, the present findings indicate that anandamide and methanandamide induce vasorelaxation in the rabbit aorta by the activation of a non-CB1 "anandamide" receptor coupled to Gi/o protein(s) via an endothelium-dependent mechanism requiring NO synthesis. To the best of our knowledge, this is the first report of the involvement of a pertussis toxin-sensitive G protein in methanandamide-induced vasodilation. A less prominent endothelium-independent component of the vasorelaxation results from anandamide's stimulation of the VR1 receptor and subsequent release of the vasodilator CGRP.


    ACKNOWLEDGEMENTS

We thank Drs. Randy Sprague and Alan Stephenson for help in rabbit surgery and aortic ring preparation.


    FOOTNOTES

This work was supported by American Heart Association Grant 0060377Z (to S. Mukhopadhyay) and by National Institute on Drug Abuse Grants R01-DA-03690 and K05-DA-00182 (to A. C. Howlett).

Present address of A. C. Howlett: Program in Neuroscience/Drug Abuse Research, J. L. Chambers Biomedical/Biotechnology Research Institute, North Carolina Central Univ., Durham, NC 27707.

Address for reprint requests and present address of S. Mukhopadhyay: Program in Neuroscience/Drug Abuse Research, J. L. Chambers Biomedical/Biotechnology Research Institute, North Carolina Central Univ., Durham, NC 27707 (E-mail: smukhopadhyay{at}wpo.nccu.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published January 24, 2002;10.1152/ajpheart.00497.2001

Received 6 June 2001; accepted in final form 8 January 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Abadji, V, Lin S, Taha G, Griffin G, Stevenson LA, Pertwee RG, and Makriyannis A. (R)-methanandamide: a chiral novel anandamide possessing higher potency and metabolic stability. J Med Chem 37: 1899-1893, 1994[Web of Science][Medline].

2.   Benowitz, N, and Jones RT. Cardiovascular and metabolic considerations in prolonged cannabinoid administration in man. J Clin Pharmacol 21: 214S-223S, 1981[Abstract].

3.   Brain, SD, Hughes SR, Cambridge H, and O'Driscoll G. The contribution of CGRP to neurogenic vasodilator responses. Agents and Actions 38: C19-C21, 1993.

4.   Bratveit, M, Haugan A, and Helle KB. Effects of CGRP on regional haemodynamics and on selected hepato-splanchnic arteries from the rat: a comparison with VIP and atriopeptin II. Scand J Clin Lab Invest 51: 167-174, 1991[Web of Science][Medline].

5.   Chataigneau, T, Feletou M, Thollon C, Villeneuve N, Vilaine J-P, Duhault J, and Vanhoutte PM. Cannabinoid CB1 receptor and endothelium-dependent hyperpolarization in guinea pig carotid, rat mesenteric and porcine coronary arteries. Brit J Pharmacol 123: 968-974, 1998.

6.   Chatterjee, TK, and Fisher RA. Multiple affinity and guanine nucleotide sensitive forms of the calcitonin gene-related peptide (CGRP) receptor. Can J Physiol Pharmacol 73: 968-973, 1995[Web of Science][Medline].

7.   Chaytor, AT, Martin PM, Evans WH, Randall MD, and Griffith TM. The endothelial component of cannabinoid-induced relaxation in rabbit mesenteric artery depends on gap junctional communication. J Physiol 520: 539-550, 1999.

8.   Coutts, AA, and Pertwee RG. Inhibition by cannabinoid receptor agonists of acetylcholine release from the guinea-pig myenteric plexus. Brit J Pharmacol 121: 1557-1566, 1997.

9.   Deutsch, DG, Goligorsky MS, Schmid PC, Krebsbach RJ, Schmid HHO, Das SK, Dey SK, Arreaza G, Thorup C, Stefano G, and Moore LC. Production and physiological actions of anandamide in the vasculature of the rat kidney. J Clin Invest 100: 1538-1546, 1997[Web of Science][Medline].

10.   Edvinsson, L, Fredholm B, Hamel E, Jansen I, and Verrechia C. Perivascular peptides relax arteries concomitant with stimulation of cyclic AMP accumulation or release of an endothelium derived relaxing factor. Neurosci Lett 58: 213-220, 1985[Web of Science][Medline].

11.   Elhawary, AM, and Pang CCY Renal vascular and tubular actions of CGRP: effect of NG-nitro-L-arginine methyl ester. J Pharmacol Exp Therp 273: 56-63, 1995.

12.   Gray, DW, and Marshall I. NO synthesis inhibitors attenuate CGRP endothelium-dependent vasorelaxation in rat aorta. Eur J Pharmacol 212: 37-42, 1992[Web of Science][Medline].

13.   Hillard, CJ. Endocannabinoids and vascular function. J Pharmacol Exp Therp 294: 27-32, 2000.

14.   Howlett, AC. Pharmacology of cannabinoid receptors. Annu Rev Pharmacol Toxicol 35: 607-634, 1995.

15.   Howlett, AC, and Mukhopadhyay S. Cellular signal transduction by anandamide and 2-arachidonoylglycerol. Chem Phys Lipids 108: 53-70, 2000.

16.   Ishac, EJN, Jiang L, Lake KD, Varga K, Abood ME, and Kunos G. Inhibition of exocytotic noradrenaline release by presynaptic cannabinoid CB1 receptors on peripheral sympathetic nerves. Brit J Pharmacol 118: 2023-2028, 1996.

17.   Jarai, Z, Wagner JA, Goparaju SK, Wang L, Razdan RK, Sugiura T, Zimmer M, Bonner T, Zimmer A, and Kunos G. Cardiovascular effects of 2-arachidonoylglycerol in anesthetized mice. Hypertension 35: 679-690, 2000[Abstract/Free Full Text].

18.   Jarai, Z, Wagner JA, Varga K, Lake KD, Compton D, Martin BR, Zimmer AM, Bonner TI, Buckley NE, Mezey E, Razdan RK, Zimmer A, and Kunos G. Cannabinoid-induced mesenteric vasodilation through an endothelial site distinct from CB1 or CB2 receptors. Proc Natl Acad Sci USA 96: 14136-14141, 1999[Abstract/Free Full Text].

19.   Lake, KD, Compton DR, Varga K, Martin BR, and Kunos G. Cannabinoid-induced hypotension and bradycardia in rats is mediated by CB1-like cannabinoid receptors. J Pharmacol Expt Therp 281: 1030-1037, 1997.

20.   Lake, KD, Martin BR, Kunos G, and Varga K. Cardiovascular effects of anandamide in anesthetized and conscious normotensive and hypertensive rats. Hypertension 29: 1204-1210, 1997[Abstract/Free Full Text].

21.   Liu, J, Gao B, Mirshahi F, Sanyal AJ, Khanolkar AD, Makriyannis A, and Kunos G. Functional CB1 cannabinoid receptors in human vascular endothelial cells. Biochem J 346: 835-840, 2000.

22.   Maggi, CA, Patacchini R, Perretti F, Tramontana M, Manzini S, Geppetti P, and Santicioli P. Sensory nerves, vascular endothelium and neurogenic relaxation of guinea pig isolated pulmonary artery. Naunyn-Schiedeberg's Arch Pharmacol 342: 78-84, 1990.

23.   Malinowska, B, Godlewski G, Bucher B, and Schlicker E. Cannabinoid CB1 receptor-mediated inhibition of neurogenic vasorepressor response in the pithed rat. Naunyn Schmiedebergs Arch Pharmacol 356: 197-202, 1997[Web of Science][Medline].

24.   McCormack, DG, Mak JCW, Coupe MO, and Barnes PJ. CGRP vasodilation of human pulmonary vessels. J Appl Physiol 67: 1265-1270, 1989.

25.   Nelson, MT, Huang Y, Brayden JE, Hescheler J, and Standen NB. Arteriolar dilations in response to CGRP involve activation of K+ channels. Nature 344: 770-773, 1990[Medline].

26.   Niederhoffer, N, and Szabo B. Effect of the cannabinoid receptor agonist WIN55212-2 on sympathetic cardiovascular regulation. Br J Pharmacol 126: 457-466, 1999[Web of Science][Medline].

27.   Pawloski, JR, and Chapnick BM. LTD4 and bradykinin evoke endothelium-dependent relaxation of the renal vein: dissimilar mechanisms. Am J Physiol Heart Circ Physiol 261: H88-H95, 1991[Abstract/Free Full Text].

28.   Pawloski, JR, and Chapnick BM. Antagonism of LTD4-evoked relaxation in canine renal artery and vein. Am J Physiol Heart Circ Physiol 265: H980-H985, 1993[Abstract/Free Full Text].

29.   Pernow, J. Actions of constrictor (NPY and endothelin) and dilator (Substance P, CGRP and VIP) peptides on pig splenic and human skeletal muscle arteries: involvement of the endothelium. Brit J Pharmacol 97: 983-989, 1989.

30.   Plane, F, Holland M, Waldron GJ, Garland CJ, and Boyle JP. Evidence that anandamide and EDHF act via different mechanisms in rat isolated mesenteric arteries. Brit J Pharmacol 121: 1509-1511, 1997.

31.   Pratt, PF, Hillard CJ, Edgemond WS, and Campbell WB. N-arachidonylethanolamide relaxation of bovine coronary artery is not mediated by CB1 cannabinoid receptor. Am J Physiol Heart Circ Physiol 274: H375-H381, 1998[Abstract/Free Full Text].

32.   Randall, MD, Alexander SP, H, Bennet T, Boyd EA, Fry JR, Gardiner SH, Kemp PA, McCulloch AI, and Kendall DA. An endogenous cannabinoid as an endothelium-derived vasorelaxant. Biochem Biophys Res Comm 229: 114-120, 1996.

33.   Randall, MD, and Kendall DA. Involvement of a cannabinoid in endothelium-derived hyperpolarizing factor-mediated coronary vasorelaxation. Eur J Pharmacol 335: 205-209, 1997[Web of Science][Medline].

34.   Randall, MD, McCulloch AI, and Kendall DA. Comparative pharmacology of endothelium-derived hyperpolarizing factor and anandamide in rat isolated mesentery. Eur J Pharmacol 333: 191-197, 1997[Web of Science][Medline].

35.   Saito, A, Masaki T, Uchiyama Y, Lee TJF, and Goto K. CGRP and vasodilator nerves in large cerebral arteries of cats. J Pharmacol Expt Therp 248: 455-462, 1989.

36.   Schini-Kerth, VB, Fisslthaler B, and Busse R. CGRP enhances induction of NO synthase in vascular smooth muscle cells via a cyclic AMP-dependent mechanism. Am J Physiol Heart Circ Physiol 267: H2483-H2490, 1994[Abstract/Free Full Text].

37.   Stefano, GB, Salzet M, Magazine HI, and Bilfinger TV. Antagonism of LPS and IFN-gamma induction of iNOS in human saphenous vein endothelium by morphine and anandamide by nitric oxide inhibition of adenylate cyclase. J Cardiovasc Pharmacol 31: 813-820, 1998[Web of Science][Medline].

38.   Varga, K, Lake K, Martin BR, and Kunos G. Novel antagonist implicates the CB1 cannabinoid receptor in the hypotensive action of anandamide. Eur J Pharmacol 278: 279-283, 1995[Web of Science][Medline].

39.   Varga, K, Lake KD, Huangfu D, Guyenet PG, and Kunos G. Mechanism of the hypotensive action of anandamide in anesthetized rats. Hypertension 28: 682-686, 1996[Abstract/Free Full Text].

40.   Wagner, JA, Varga K, Jarai Z, and Kunos G. Mesenteric vasodilation mediated by endothelial anandamide receptors. Hypertension 33: 429-434, 1999[Abstract/Free Full Text].

41.   Wagner, JA, Varga K, and Kunos G. Cardiovascular actions of cannabinoids and their generation during shock. J Mol Med 76: 824-836, 1998[Web of Science][Medline].

42.   White, R, and Hiley CR. A comparison of EDHF-mediated and anandamide-induced relaxations in the rat isolated mesenteric artery. Brit J Pharmacol 121: 1573-1584, 1997.

43.   White, R, and Hiley CR. The actions of the cannabinoid receptor antagonist, SR141716A, in the rat isolated mesenteric artery. Brit J Pharmacol 125: 689-696, 1998.

44.   White, R, Venessa Ho W, Bottrill FE, Ford WR, and Hiley CR. Mechanism of anandamide-induced vasorelaxation in rat isolated coronary arteries. Brit J Pharmacol 134: 921-929, 2001.

45.   Zygmunt, PM, Edwards G, Weston AH, Larsson B, and Hogestatt ED. Involvement of voltage-dependent potassium channels in the EDHF-mediated relaxation of rat hepatic artery. Brit J Pharmacol 121: 141-149, 1997.

46.   Zygmunt, PM, Petersson J, Andersson DA, Chuang HH, Sorgard M, DiMarzo V, Julius D, and Hogestatt ED. Vanilloid receptors on sensory nerves mediate the vasodilator action of anandamide. Nature 400: 452-457, 1999[Medline].

Am J Physiol Heart Circ Physiol 282(6):H2046-H2054
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