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CARDIOVASCULAR
School of Biomedical Sciences, University of Nottingham Medical School, Queen's Medical Centre, Nottingham, United Kingdom
Received March 10, 2004; accepted June 15, 2004.
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Abstract |
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9-tetrahydrocannabinol (THC) (1 µM) attenuated sensory neurogenic relaxations, as did the CB2 agonist JWH-015 [(2-methyl-1-propyl-1H-indol-3-yl)-1-naphthalenylmethanone]. The inhibitory actions of both THC and JWH-015 were still evident in the presence of SR141716A (1 µM) and SR144528 (1 µM). None of the cannabinoid agonists investigated had an effect on vasorelaxation elicited by exogenous CGRP, indicating a prejunctional mechanism. These data demonstrate that different classes of cannabinoid agonists attenuate sensory neurotransmission via a prejunctional site and provide evidence for mediation by a CB1 and/or a non-CB1/CB2 receptor.
). There is evidence, however, that the cannabinoid receptor classification is incomplete. There are data that suggest the presence of multiple CB1 subtypes in the spinal cord (Welch et al., 1998), and putative CB2-like receptors have been implicated in antinociceptive and hypotensive effects of cannabinoid agonists (Hanu
et al., 1999; Calignano et al., 2001). Experiments using CB receptor knockout mice have uncovered a putative novel cannabinoid receptor in the mouse brain (Di Marzo et al., 2000; Breivogel et al., 2001; Hájos et al., 2001; Monory et al., 2002). In addition, studies of the rat mesentery have described a novel anandamide-sensitive, SR141716A-sensitive endothelial receptor (Járai et al., 1999). Recently, Zygmunt et al. (2002) reported that 9-tetrahydrocannabinol (THC) and cannabidiol induce a CB1/CB2-independent release of calcitonin gene-related peptide (CGRP) from capsaicin-sensitive nerves in isolated rat and mouse mesenteric arterial rings that could be blocked by the vanilloid receptor (TRPV1) blocker ruthenium red but that was still present in TRPV1 knockout animals.
Capsaicin-sensitive sensory nerves are widely distributed in the cardiovascular system and have a dual function; an afferent function whereby they participate in reflex activation of motor nerves, and an efferent function, whereby neurotransmitter is released from the nerve terminal being stimulated (Maggi and Meli, 1988). Studies on dorsal root ganglia (DRG) and F-11 cells (DRG x neuroblastoma hybridomas) indicate that both CB1 and CB2 receptor proteins exist in these cells (Ross et al., 2001). However, only CB1 receptors have been demonstrated to be transported from DRG cell bodies to the periphery of the sensory nerves (Hohmann and Herkenham, 1999).
Recently, functional evidence has been provided which suggests that non-CB1/CB2 cannabinoid receptors are located on capsaicin-sensitive sensory nerves in the rat mesenteric bed (Ralevic and Kendall, 2001). The potent cannabinoid CB1/CB2 receptor agonist HU-210 [(-)-11-hydroxy-dimethylheptyl-8-tetrahydrocannabinol] was found to attenuate sensory neurogenic relaxation evoked by electrical field stimulation (EFS) in preconstricted mesenteric beds. This inhibitory action was resistant to the CB1-selective antagonists SR141716A and LY320135 and the CB2-selective antagonist SR144528. HU-210 had no effect on the vasorelaxant response to exogenous CGRP, suggesting prejunctional modulation of CGRP release (Ralevic and Kendall, 2001).
The present study investigated the effects of different classes of cannabinoid compounds represented by the amino-alkylindoles WIN55,212 and JWH-015, the bicyclic CP55,940, and the classical cannabinoid agonist THC on sensory neurogenic vasorelaxation to EFS in the rat isolated mesenteric arterial bed. The effects of CB1-(SR141716A and LY320135) and CB2 (SR144528)-selective antagonists on agonist responses were determined. Electrical stimulation of sensory nerve terminals releases CGRP, the principal sensory vasorelaxant in the rat isolated mesenteric arterial bed, which can be reversed by the CGRP antagonist CGRP(8-37) (Kawasaki et al., 1988; Han et al., 1990). The effects of cannabinoids on responses to exogenous CGRP were, therefore, investigated to determine whether the actions were mediated at a pre- or postjunctional site. Because there has been reported cross-talk between cannabinoid and vanilloid receptors, the actions of capsaicin in the presence of cannabinoids were investigated. Some of this work has been published in abstract form.
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Materials and Methods |
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), consistent with it being mediated by capsaicin-sensitive sensory nerves (Kawasaki et al., 1988). Moreover, the relaxation is mimicked by CGRP, whereas substance P and neurokinin A are inactive and vasoactive intestinal polypeptide causes relaxation only at relatively high concentrations, and there are numerous CGRP-like immunoreactive fibers but few substance P and vasoactive intestinal polypeptide-like immunoreactive fibers present in the mesenteric arteries (Kawasaki et al., 1988). In addition, the neurogenic relaxation to electrical field stimulation is abolished by the CGRP receptor antagonist CGRP(8-37) (Han et al., 1990). Responses were measured as changes in perfusion pressure (mm Hg) with a pressure transducer (model P23XL; Viggo-Spectramed, Oxnard, CA), situated on a sidearm proximal to the preparation, and recorded using A to D converter (PowerLab/400; ADInstruments, Pty Ltd., Castle Hill, Australia). Preparations were allowed to equilibrate for 30 min before experimentation. Experimental Protocol. Three consecutive relaxant response curves to EFS at 1 to 12 Hz, EFS control, EFS I, and EFS II, were generated in each preconstricted mesenteric arterial bed. The tone of the preparation was allowed to return to preconstricted tone before each stimulus was applied. The first response curve acted as a control, the compound under investigation was then added to the perfusate and after 15 min, response curves EFS I and EFS II were generated. Antagonists were added at the start of the equilibration period. Only a single concentration of agonist was used per preparation. In a separate series of experiments, a dose-response curve was constructed to CGRP (0.05 pmol–0.5 nmol) and capsaicin (0.05 pmol–5 nmol) by applying 50-µl bolus injections via norprene tubing proximal to the preparation.
Drugs. SR141716A [N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichloro phenyl)-4-methyl-3-pyrazole-carboxamide] and SR144528 [N-[1S)-endo-1,3,3,-trimetyl bicyclo [2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide] were gifts from SANOFI Research Center (Montpellier, France). LY320135 [6-methoxy-2-(4-methoxyphenyl)benzo[b]-thien-3-yl][4-cyanophenyl] methanone] was a gift from Eli Lilly (Hampshire, UK). CP55,940 [(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl) cyclohexanol] and JWH-015 [(2-methyl-1-propyl-1H-indol-3-yl)-1-naphthalenylmethanone] were from Tocris Cookson Inc. (Bristol, UK). (+)-WIN55,212-2 [R-(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)-methyl]pyrrolo [1,2,3-de]-1,4-benzoazinyl]-(1-naphthalenyl)methanone mesylate] and (-)-WIN55,212-3 [S-(-)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoazinyl]-(1-naphthalenyl)methanone mesylate] were also purchased from Tocris Cookson Inc. Calcitonin gene-related peptide was from Sigma Chemical (Poole, Dorset, UK). Guanethidine (Ismelin) was from Alliance Pharmaceuticals (Wiltshire, UK). All cannabinoids were dissolved in ethanol at a concentration of 10-2 M. CGRP was dissolved in distilled water.
Data Analysis. Vasorelaxant responses (mm Hg) of the mesenteric beds were expressed as a percentage of the methoxamine-induced increase in tone above baseline. Data were compared by Student's t test and one-way/two-way analysis of variance with Tukey's post hoc test. A value of P < 0.05 was taken to indicate a statistically significant difference. The software package Prism GraphPad (version 3.0; GraphPad Software Inc., San Diego, CA) was used to perform the analyses. RMAX represents maximal relaxation.
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Results |
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Effect of SR141716A (1 µM) on Vasorelaxant Responses to EFS. SR141716A has previously been reported to augment sensory neurogenic relaxations to EFS in the rat isolated arterial mesenteric bed (Ralevic and Kendall, 2001). In this study, SR141716A (1 µM; n = 9) was found to have no significant effect on the vasorelaxant response (Fig. 1B). At a submaximal frequency of 8 Hz, EFS control was 54.7 ± 5.4%; EFS I, 48.6 ± 5.8%; and EFS II, 45.3 ± 6.1% (P > 0.05). The RMAX was unaffected; EFS control from 56.0 ± 4.8% to EFS II 48.5 ± 5.9%. SR141716A had no significant effect on the tone of the preparations.
Effect of LY320135 (1 µM) on Vasorelaxant Responses to EFS. The experiments were carried out in the presence of LY320135 (1 µM; n = 3) to determine whether the antagonist had any effect on the vasorelaxant response to EFS. At a submaximal frequency of 8 Hz, LY320135 had no effect on the vasorelaxant response (Fig. 1C) (EFS control, 50.3 ± 2.1%; EFS I; 51.7 ± 6.0%; and EFS II, 41.0 ± 8.0%; P > 0.05). The RMAX was unaffected; EFS control from 66.4 ± 5.8% to EFS II 56.7 ± 4.2%. LY320135 had no significant effect on the tone of the preparations.
Effect of SR144528 (1 µM) on Vasorelaxant Responses to EFS. To rule out any involvement of CB2 receptors, the experiments were repeated in the presence of the CB2 antagonist SR144528 (1 µM; n = 3, Fig. 1D). At a submaximal frequency of 8 Hz, SR144528 had no effect on the vasorelaxant response (EFS control, 68.8 ± 7.2%; EFS I, 64.2 ± 4.2%; and EFS II, 57.5 ± 2.5%; P > 0.05). The RMAX was reduced slightly but significantly; EFS control from 80.3 ± 2.4%, EFS I 71.5 ± 0.8%, and EFS II 66.3 ± 2.4% (P < 0.05). SR144528 had no significant effect on the tone of the preparations.
Effect of WIN55,212 on Vasorelaxant Responses to EFS. WIN55,212, a high-potency cannabinoid agonist at both CB1 and CB2 receptors, attenuated EFS I and EFS II in a concentration- and time-dependent manner, illustrated in the representative trace in Figs. 2 and 3. At the lowest concentration, (0.01 µM; n = 9), there was a small difference at 4 Hz but no significant difference in the maximal relaxation (value obtained at 12 Hz; RMAX) (Fig. 3). At concentrations of 0.1 and 1 µM WIN55,212, the relaxation response was significantly reduced; at a submaximal frequency of 8 Hz, 0.1 µM reduced EFS control 50.5 ± 4.4% to EFS II 26.2 ± 2.8% (P < 0.05; n = 6) and 1 µM reduced EFS control 53.4 ± 5.5% to EFS II 24.3 ± 2.3% (P < 0.05; n = 6). The RMAX was significantly reduced at both concentrations; at 1 µM, WIN55,212 reduced EFS control from 58.8 ± 4.9% to EFS II 33.0 ± 5.1%. WIN55,212 had no significant effect on the tone of the preparations.
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Effect of SR141716A and SR144528 on the Inhibitory Actions of WIN55,212 on the Vasorelaxant Response to EFS. To determine whether the inhibitory actions exerted by WIN55,212 were mediated by either CB1 or CB2 receptors, the experiments were repeated in the presence of 1 µM CB1-selective antagonist SR141716A or the CB2-selective antagonist SR144528 (Fig. 4). A submaximal concentration, 0.1 µM WIN55,212, was used, and 1 µM SR141716A was used to minimize nonselective effects, which have been reported at higher concentrations. In the presence of SR141716A, the inhibitory actions of WIN55,212 (0.1 µM) on sensory neurogenic vasorelaxation were largely absent. At a submaximal frequency of 8 Hz, there was no significant difference between the responses (EFS control, 46.7 ± 3.6%; EFS I, 39.3 ± 3.9%; and EFS II, 36.1 ± 3.8%; n = 8; P > 0.05). WIN55,212 was still able to cause inhibition of sensory neurogenic vasorelaxation in the presence of SR144528 (1 µM). At 8 Hz, there was a significant inhibition of neurogenic relaxations by 0.1 µM WIN55,212 (EFS control 44.2 ± 3.9% to EFS II 17.5 ± 3.0%; n = 5; P < 0.05).
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Effect of the WIN55,212 Stereoisomer (-)-WIN55,212 on Vasorelaxant Responses to EFS. The stereoisomer (-)-WIN55,212 was used to investigate whether a stereospecificity for this compound exists; this is a useful tool to determine whether the actions are receptor-mediated (Fig. 5). (-)-WIN55,212 (1 µM) had no significant effect on the sensory neurogenic vasorelaxation evoked by EFS, at 8 Hz EFS (control 63.0 ± 7.8% and EFS II 51.2 ± 9.0%; n = 6; P > 0.05).
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Effect of CP55,940 on Vasorelaxant Responses to EFS. CP55,940, an agonist that has equivalent high potency at both CB1 and CB2 receptor subtypes, attenuated sensory neurogenic relaxations in a concentration-dependent manner (Fig. 6). At a concentration of 0.01 µM, CP55,940 blocked relaxation only at 8 Hz but had no effect on the RMAX. At concentrations of 0.1 and 1 µM CP55,940, the relaxation response was significantly reduced. At a submaximal frequency of 8 Hz, 0.1 µM CP55,940 reduced EFS control 38.0 ± 3.9% to EFS II 17.7 ± 3.3% (n = 6; P < 0.05); 1 µM CP55,940 reduced EFS control 38.6 ± 4.6% to EFS II 18.0 ± 2.9% (n = 8; P < 0.05). There was a significant reduction in RMAX at both concentrations; at 1 µM CP55,940, the EFS control 46.4 ± 3.9% was reduced to 21.9 ± 3.0% (P < 0.05). CP55,940 had no significant effect on the tone of the preparations at all concentrations.
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Effect of LY320135 and SR144528 on Inhibition by CP55,940 of the Vasorelaxant Response to EFS. Because CP55,940 is equally effective at both cannabinoid receptor subtypes, to characterize the receptor mediating the inhibitory effects of CP55,940, the experiment was repeated in the presence of LY320135, a CB1-selective antagonist, and SR144528, a CB2-selective antagonist (Fig. 7). CP55,940 (0.1 µM) had no inhibitory actions on sensory neurogenic vasorelaxation in the presence of LY320135 (1 µM; n = 6). At a submaximal frequency of 8 Hz, there was no significant difference between the curves. CP55,940 (0.1 µM) was still able to cause inhibition in the presence of SR144528 (1 µM). At a submaximal frequency of 8 Hz, EFS control 54.0 ± 7.8% was reduced to EFS II 23.8 ± 5.8% (n = 6; P < 0.05).
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Effect of JWH-015 on Vasorelaxant Responses to EFS. To further characterize the receptor involved in the inhibition of the neurogenic response, the CB2-selective agonist JWH-015 was investigated (Fig. 8, A and B). At a concentration of 0.1 µM JWH-015, there was no significant difference between the frequency-response curves or the RMAX values. In the presence of 1 µM JWH-015, the relaxation response was significantly reduced; at a submaximal frequency of 8 Hz, EFS control 51.7 ± 2.7% to EFS II 33.5 ± 2.8% (n = 6; P < 0.05). The RMAX was significantly reduced; at 1 µM EFS control 56.7 ± 1.93% was reduced to EFS II 36.0 ± 3.3% (P < 0.05). JWH-015 had no significant effect on the tone of the preparations.
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Effect of SR141716A and SR144528 on Inhibition by JWH-015 of the Vasorelaxant Response to EFS. To determine whether the inhibitory actions of JWH-015 were mediated by the CB1 or CB2 receptor, we challenged with selective antagonists for CB1 and CB2 (Fig. 8, C and D). JWH-015 (1 µM) was still able to cause inhibition of sensory neurogenic vasorelaxation in the presence of the CB2-selective antagonist SR144528 (1 µM). At a submaximal frequency of 8 Hz, EFS control 59.4 ± 6.6% was reduced to EFS II 33.4 ± 5.1% (n = 9; P < 0.05). In the presence of the CB1-selective antagonist SR141716A (1 µM), JWH-015 (1 µM) was still able to inhibit sensory neurogenic vasorelaxation, although this effect seemed to be attenuated compared with control responses in the absence of SR141716A. At a submaximal frequency of 8 Hz, there was a significant difference between the curves (EFS control, 74.8 ± 5.1%; EFS I, 57.4 ± 7.3%; and EFS II, 40.7 ± 8.9%; n = 5; P < 0.05).
Effect of THC on Vasorelaxant Responses to EFS. The classical plant-derived cannabinoid THC, which acts as a partial agonist at both CB1 and CB2 receptors, attenuated sensory neurogenic relaxation (Fig. 9, A and B). In the presence of 0.1 µM THC, at a submaximal frequency of 8 Hz, the response was reduced from EFS control 60.6 ± 7.6% to EFS II 31.1 ± 3.9% (n = 6; P < 0.05). THC (1 µM) reduced 8 Hz EFS control 53.1 ± 5.0% to EFS II 11.1 ± 1.6%. The maximal response was also significantly reduced, from 59.3 ± 7.5% to 17.3 ± 2.0% (n = 4; P < 0.05). THC initially produced a transient pressor effect (30.14 ± 2.6 mm Hg) followed by a reduction in the tone of the preparation. The secondary vasorelaxation required methoxamine to maintain tone.
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Effect of SR141716A and SR144528 on Inhibition by THC of the Vasorelaxant Response to EFS. THC (1 µM) was challenged with antagonists for both the CB1 and CB2 receptors to determine its site of action (Fig. 9, C and D). THC was still able to cause inhibition of sensory neurogenic vasorelaxation in the presence of the CB1-selective antagonist SR141716A (1 µM). At a submaximal frequency of 8 Hz, there was a significant difference between the curves (EFS control, 71.78 ± 6.9%; EFS I, 33.2 ± 3.2%; and EFS II, 27.9 ± 5.7%; n = 5; P < 0.05). THC was still able to cause inhibition of sensory neurogenic vasorelaxation in the presence of the CB2-selective antagonist SR144528 (1 µM). At a submaximal frequency of 8 Hz, EFS control 52.1 ± 4.7% was reduced to EFS II 24.0 ± 7.2% (n = 8; P < 0.05).
Effects of WIN55,212, CP55,940, JWH-015, and THC on the Vasorelaxant Response to Exogenous CGRP. To determine whether the inhibitory actions on sensory neuro-transmission by the cannabinoid agonists are via a pre- or postjunctional site, dose-response curves to CGRP, the principal motor neuropeptide involved in sensory neurogenic relaxation, were constructed in the presence of 1 µM cannabinoid agonists. The pD2 value was unaltered in the presence of all cannabinoid agonists investigated (Table 1).
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Effects of WIN55,212, CP55,940, JWH-015, and THC on the Vasorelaxant Response to Capsaicin. To investigate the possibility of cross-talk between actions of the cannabinoid agonists and TRPV1 receptors, dose-response curves to capsaicin, which elicits CGRP release via TRPV1 activation, were constructed in the presence of 1 µM cannabinoid agonists. The pD2 value for capsaicin was unaltered in the presence of CP55,940, THC, and JWH-015; however, the capsaicin response was enhanced in the presence of WIN55,212 (Table 2).
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Discussion |
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), in which CP55,940 and anandamide were demonstrated to inhibit capsaicin-evoked CGRP release in rat paw skin, which is richly innervated with sensory nerves, in a SR141716A-sensitive manner. Furthermore, studies using [3H]CP55,940 in radioligand binding assays have uncovered a saturatable binding site using the trigeminal ganglia as a model for sensory nerves (Richardson et al., 1998a). This would indicate that a CP55,940-sensitive receptor is present on sensory nerves, in agreement with the functional pharmacological studies. CB1 mRNA has been located in DRG cells, and CB1 receptors are trafficked to peripheral sensory nerve terminals (Hohmann and Herkenham, 1999) and so cannabinoids would be expected to exert their inhibitory effects by prejunctional modulation of neurotransmitter release from the peripheral nerve terminal. This is in agreement with our findings that none of the cannabinoids investigated exerted an inhibitory effect on vasorelaxation in response to exogenous CGRP.
The concentrations of cannabinoid agonist used in this study are in the micromolar range, much higher than concentrations used in some other bioassay preparations such as mouse vas deferens and CB receptor-transfected cell lines (Pertwee, 1999; Howlett et al., 2002). However, the concentrations used are in agreement with previous studies on isolated blood vessels (White and Hiley, 1998). Because cannabinoids are highly lipophilic compounds, the true concentration at the site of action could be much lower than the micromolar range. Indeed, the time dependence of the inhibitory action (EFS II is more sensitive to the inhibitory actions than EFS I) could be, in part, due to the slow diffusion of the compounds through the blood vessel wall.
The inhibitory actions of both WIN55,212 and CP55,940 in the rat mesenteric bed, which were selectively antagonized by CB1 but not CB2 antagonists, indicate the likely involvement of CB1- or CB1-like receptors. Neither of the two compounds are agonists at TRPV1 receptors (Zygmunt et al., 1999). Moreover, the inactivity of the stereoisomer (-)-WIN55,212 indicates a clear structure-activity relationship. Although we cannot rule out the possibility that at least part of the action of SR141716A was due to functional antagonism (Ralevic and Kendall, 2001), this is unlikely to account for all its inhibitory actions because another CB1 antagonist, LY320135, was similarly effective. However, there is growing evidence indicating the existence of novel cannabinoid receptors. Indeed, WIN55,212 is reported to be active at a putative novel cannabinoid receptor identified in the brain. This receptor, described by Breivogel et al. (2001), was SR141716A-sensitive, whereas the novel WIN55,212-sensitive receptors reported by Hájos et al. (2001) and Monory et al. (2002) were SR141716A-resistant. Our data describing the effects of the other cannabinoid agonists suggest that CB1/CB1-like receptors are not the only subtype expressed on the perivascular sensory nerves.
JWH-015, a CB2-selective agonist, inhibited sensory neurogenic vasorelaxation, which was unexpected because there is little evidence for the expression of CB2 receptors in sensory nerves. However, this action was not blocked by a selective CB2 antagonist SR144528. Griffin et al. (1997) reported that JWH-015 inhibited electrically evoked contractions in the mouse vas deferens and the guinea pig myenteric plexus and that this effect could be reversed by SR141716A in the myenteric plexus, suggesting that the inhibitory actions are CB1-mediated (Griffin et al., 1997). This is in agreement with our study in that the inhibitory actions of JWH-015 seemed to be attenuated in the presence of the CB1 antagonist SR141716A. Recently, Ross et al. (2001) described CB2 receptor protein in F-11 (DRG x neuroblastoma hybridoma) cells using antibodies raised against the N terminus. However, in nerve ligation studies using antisense cRNA probes, mRNA for CB2 receptors could not be detected in the DRG, and CB2 receptors were not found to be transported to peripheral nerve endings (Hohmann and Herkenham, 1999). Thus, the pharmacological and molecular studies do not indicate expression of CB2 receptors in sensory nerves. There is evidence for cardiovascular actions of CB2 receptors because a bicyclic CB2-specific agonist has been shown to significantly reduce blood pressure in vivo, and this action can be blocked by the CB2 antagonist SR144528 (Hanu et al., 1999). Therefore, a novel JWH-015-sensitive, SR144528-resistant cannabinoid receptor could be involved in the regulation of perivascular sensory nerves. However, an indirect action cannot be ruled out; JWH-015 could be activating cannabinoid receptors and releasing vasoactive mediators from cells associated with sensory nerves such as mast cells, which have been reported to express CB2 receptors, although these inhibitory effects were SR144528-sensitive (Facci et al., 1995).
THC, a plant-derived cannabinoid that acts equally well at both CB receptor subtypes, also inhibited vasorelaxation to EFS. This effect could not be blocked by either SR141716A or SR144528. A postjunctional effect was ruled out, because THC had no effect on CGRP vasorelaxation. It is, therefore, possible that THC is mediating its inhibitory actions via a novel prejunctional cannabinoid receptor. In contrast to the other cannabinoids investigated, THC produced a vasoconstriction when added to the perfusate; this is not unexpected because THC has been reported to produce a pressor response followed by a prolonged hypotension in vivo (Wagner et al., 1998). The hypotensive effect can be abolished by surgical/pharmacological removal of sympathetic tone, and this effect is also sensitive to SR141716A, leading to the possibility of sympatho-inhibition by presynaptic CB1 receptors. The pressor response could not be abolished by the elimination of sympathetic tone and so is thought to be due to a peripheral vasoconstriction; it is therefore possible that the constriction we observed in the mesenteric bed contributes to the pressor effect observed in vivo. Vasoconstriction is unlikely, however, to have caused a functional antagonism of the neurogenic relaxation responses, because it was not sustained (the relaxation-response curves were generated after reversal of the constriction) and because relaxation responses to CGRP were unaffected.
THC and cannabidiol have recently been described as acting via a novel receptor subtype to release CGRP in capsaicin-sensitive sensory nerves (Zygmunt et al., 2002). The ability of THC to release CGRP was blocked by the vanilloid blocker ruthenium red but was present in TRPV1 knockout mice, which raises the possibility of THC activating a transient receptor potential ion channel to release CGRP. It is possible that the constriction we observed to THC could mask the effect of the coincident CGRP release, and indeed, a relaxation followed. Thus, vasorelaxation to EFS could be attenuated because the sensory nerves were depleted of CGRP, or the CGRP receptors were desensitized; this would explain why the inhibitory action could not be blocked by SR141716A. However, ruthenium red (1 µM) does not block the inhibitory actions of THC on sensory neurotransmission in the rat isolated mesenteric arterial bed (Duncan et al., 2003), so a vasorelaxant mechanism dependent on CGRP depletion is unlikely. Interestingly, Zygmunt et al. (2002) did not report a constrictor effect of THC, but their study was carried out in arterial segments, whereas the present experiments were in whole vascular beds, so more complex mechanisms could be involved such as the release of endothelial vasoconstrictors.
From the data presented, THC seems to mediate the inhibition of sensory neurotransmission via a non-CB1CB2 site. The apparently differential sensitivities of JWH-015 and THC to the actions of SR141716A would suggest they are not acting at a common site. We recently reported that the inhibitory actions of the putative endocannabinoid noladin ether on sensory neurotransmission could not be blocked by CB1 and CB2 antagonists (Duncan et al., 2004) or ruthenium red (Duncan et al., 2003), but they could be abolished by the pretreatment of animals with pertussis toxin. The inhibitory actions of THC are also abolished by pertussis toxin pretreatment (our unpublished observations). Thus, there does seem to be a Gi/o protein-coupled, cannabinoid-sensitive receptor on the capsaicin-sensitive perivascular sensory nerves.
In this study, the antagonists alone had no effect on mesenteric arterial contraction, indicating a potential lack of effect of endogenous cannabinoid tone as a modulator in the vascular beds. This is in contrast with a previous report from this laboratory showing that SR141716A augmented sensory neurotransmission in the same model (Ralevic and Kendall, 2001). The reason for this is not clear because the sex, strain, and ages of the rats and the experimental conditions were similar.
In the investigation of possible cross talk between the cannabinoid and vanilloid systems, we found no modulation of the capsaicin response by CP55,940, THC, and JWH-015. WIN55,212 enhanced the capsaicin response for reasons that are not clear (but that are unrelated to its inhibitory effects on electrically evoked sensory neurotransmitter release), indicating possible complex effects of this compound on sensory nerves.
In conclusion, it is not possible to fully characterize the receptors involved in cannabinoid-mediated attenuation of sensory nerve-mediated vasorelaxation using the antagonists presently available, and the inhibitory prejunctional effects cannot be readily characterized as CB1- or CB2-mediated. However, we propose that a heterogeneous population of cannabinoid receptors exist on capsaicin-sensitive nerves in the rat mesenteric bed. Our findings suggest at least two types of receptor: one sensitive to SR141716A and LY320135, possibly CB1 or CB1-like, and the other resistant to SR141716A but distinct from SR144528-sensitive CB2 receptors.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: CB, cannabinoid; THC, 9-tetrahydrocannabinol; CGRP, calcitonin gene-related peptide; TRPV1, vanilloid receptor; DRG, dorsal root ganglia; EFS, electrical field stimulation.
Address correspondence to: Dr. V. Ralevic, School of Biomedical Sciences, University of Nottingham Medical School, Queen's Medical Centre, Nottingham NG7 2UH, UK. E-mail: vera.ralevic{at}nottingham.ac.uk
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References |
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Breivogel CS, Griffin G, Di Marzo V, and Martin BR (2001) Evidence for a new G protein coupled cannabinoid receptor in the mouse brain. Mol Pharmacol 60: 155-163.
Calignano A, La Rana G, and Piomelli D (2001) Antinociceptive activity of the endogenous fatty acid amide, palmitoylethanolamide. Eur J Pharmacol 419: 191-198.[CrossRef][Medline]
Di Marzo V, Breivogel CS, Tao Q, Bridgen DT, Razdan RK, Zimmer AM, Zimmer A, and Martin BR (2000) Levels, metabolism and pharmacological activity of anandamide in CB1 cannabinoid receptor knockout mice: evidence for non CB1, non CB2 receptor-mediated actions of anandamide in the mouse brain. J Neurochem 75: 2434-2444.[CrossRef][Medline]
Duncan M, Kendall DA, and Ralevic V (2003) The effects of ruthenium red on the inhibitory actions of noladin ether, THC and HU210 on sensory neurotransmission in the rat isolated mesenteric arterial bed. 1st European Workshop on Cannabinoid Research; 2003 Apr 4–5; Madrid, Spain. http://www.ucm.es/info/seic-web/abstract-book.pdf
Duncan M, Kendall DA, and Ralevic V (2004) Noladin ether, a putative endocannabinoid, attenuates sensory neurotransmission in the rat isolated mesenteric arterial bed via a non-CB1/CB2 Gi/o linked receptor. Br J Pharmacol 142: 509-518.[CrossRef][Medline]
Ellington HC, Cotter MA, Cameron NE, and Ross RA (2002) The effect of cannabinoids on capsaicin-evoked calcitonin gene-related peptide (CGRP) release from the isolated paw skin of diabetic and non-diabetic rats. Neuropharmacology 42: 966-975.[CrossRef][Medline]
Facci L, Dal Toso R, Romanello S, Buriani A, Skaper SD, and Leon A (1995) Mast cells express a peripheral cannabinoid receptor with differential sensitivity to anandamide and palmitoylethanolamide. Proc Natl Acad Sci USA 92: 3376-3380.
Griffin G, Fernando SR, Ross RA, McKay NG, Ashford MLJ, Shire D, Huffman JW, Yu S, Lainton JAH, and Pertwee RG (1997) Evidence for the presence of CB2-like cannabinoid receptors on peripheral nerve terminals. Eur J Pharmacol 339: 53-61.[CrossRef][Medline]
Hájos N, Ledent C, and Freund TF (2001) Novel cannabinoid-sensitive receptor mediates inhibition of glutamateric synaptic transmission in the hippocampus. Neuroscience 106: 1-4.[CrossRef][Medline]
Han SP, Naes L, and Westfall TC (1990) Inhibition of periarterial nerve stimulation-induced vasodilation of the mesenteric arterial bed by CGRP (8–37) and CGRP receptor desensitization. Biochem Biophys Res Commun 168: 786-791.[CrossRef][Medline]
Hanu L, Breuer A, Tchilibon S, Shiloah S, Goldenberg D, Horowitz M, Pertwee RG, Ross RA, Mechoulam R, and Fride E (1999) HU-308: a specific agonist for CB2, a peripheral cannabinoid receptor. Proc Natl Acad Sci USA 96: 14228-14233.
Hohmann AG and Herkenham M (1999) Cannabinoid receptors undergo axonal flow in sensory nerves. Neuroscience 92: 1171-1175.[CrossRef][Medline]
Howlett AC, Barth F, Bonner TI, Cabral G, Casellass P, Devane WA, Felder CC, Herkenham M, Mackie K, Martin BR, et al. (2002) International Union of Pharmacology. XXVII. Classification of cannabinoid receptors. Pharmacol Rev 54: 161-202.
Járai Z, Wagner JA, Varga K, Lake KD, Compton DR, Martin BR, Zimmer AM, Bonner TI, Buckley NE, Mezey E, et al. (1999) Cannabinoid-induced mesenteric vasodilation through an endothelial site distinct from CB1 or CB2 receptors. Proc Natl Acad Sci USA 96: 14136-14141.
Kawasaki H, Takasaki K, Saito A, and Goto K (1988) Calcitonin gene-related peptide acts as a novel vasodilator neurotransmitter in mesenteric resistance vessels of the rat. Nature (Lond) 335: 164-167.[CrossRef][Medline]
Maggi CA and Meli A (1988) The sensory-efferent function of capsaicin-sensitive neurons. Gen Pharmacol 19: 1-43.[Medline]
Monory K, Tzavara EH, Lexime J, Ledent C, Parmentier M, Borsodi A, and Hanoune J (2002) Novel, non-adenylyl cyclase-coupled cannabinoid binding site in cerebellum of mice. Biochem Biophys Res Commun 292: 231-235.[CrossRef][Medline]
Pertwee RG (1999) Pharmacology of cannabinoid receptor ligands Curr Med Chem 6: 635-664.[Medline]
Ralevic V, Aberdeen JA, and Burnstock G (1991) Acrylamide-induced autonomic neuropathy of rat mesenteric vessels: histological and pharmacological studies. J Auton Nerv Syst 34: 77-88.[Medline]
Ralevic V and Kendall DA (2001) Cannabinoid inhibition of capsaicin-sensitive sensory neurotransmitter in the rat isolated mesenteric arterial bed. Eur J Pharmacol 418: 117-125.[CrossRef][Medline]
Richardson JD, Aanonsen L, and Hargreaves KM (1998a) Antihyperalgersic effects of spinal cannabinoids. Eur J Pharmacol 345: 145-153.[CrossRef][Medline]
Richardson JD, Kilo S, and Hargreaves KM (1998b) Cannabinoids reduce hyperalgesia and inflammation via interaction with peripheral CB1 receptors. Pain 75: 111-119.[CrossRef][Medline]
Ross RA, Coutts AA, McFarlane SM, Anavi-Goffer S, Irving AJ, Pertwee RG, MacEwan DJ, and Scott RH (2001) Actions of cannabinoid receptor ligands on rat cultured sensory neurones: implications for antinociception. Neuropharmacology 40: 221-232.[CrossRef][Medline]
Wagner JA, Varga K, and Kunos G (1998) Cardiovascular actions of cannabinoids and their generation during shock. J Mol Med 76: 824-836.[CrossRef][Medline]
Welch SP, Huffman JW, and Lowe J (1998) Differential blockade of the antinociceptive effects of centrally administered cannabinoids by SR141716A. J Pharmacol Exp Ther 286: 1301-1308.
White R and Hiley CR (1998) The actions of come cannabinoid receptor ligands in the rat isolated mesenteric artery. Br J Pharmacol 125: 533-541.[Medline]
Zygmunt PM, Andersson DA, and Högestätt ED (2002) 9-Tetrahydrocannabidiol and Cannabidiol activate capsaicin-sensitive sensory nerves via a CB1 and CB2 cannabinoid receptor-independent mechanism. J Neurosci 22: 4720-4727.
Zygmunt PM, Petersson J, Andersson DA, Chuang H, Sørgard M, Di Marzo V, Julius D, and Högestätt ED (1999) Vanilloid receptors on sensory nerves mediate the vasodilator action of anandamide. Nature (Lond) 400: 452-456.[CrossRef][Medline]
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