Jim Finnel
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Influence of the CB1 receptor antagonist, AM251, on the regional haemodynamic effects of WIN-55212-2 or HU210 in conscious rats
S M Gardiner,1* J E March,1 P A Kemp,1 and T Bennett1
11School of Biomedical Sciences, University of Nottingham Medical School, Queen's Medical Centre, Nottingham NG7 2UH
*Author for correspondence: Email: sheila.gardiner@nottingham.ac.uk
Abstract
In conscious, freely-moving, male, Sprague-Dawley rats, the regional haemodynamic responses to the synthetic cannabinoids, WIN-55212-2 and HU210, were compared. The possible involvement of cannabinoid, CB1-receptors, or β2-adrenoceptors in the responses to WIN-55212-2 and HU210 were investigated using the CB1-receptor antagonist, AM251, or the β2-adrenoceptor antagonist, ICI118551, respectively.
Both WIN-55212-2 (150μgkg−1) and HU210 (100μgkg−1) had pressor, renal, and mesenteric vasoconstrictor and hindquarters vasodilator actions, although the effects of HU210 were much more sustained than those of WIN-55212-2. Lower doses of the cannabinoids (WIN-55212-2, 50μgkg−1, HU210, 10μgkg−1) had less consistent actions.
All the significant cardiovascular effects of WIN-55212-2 and HU210 were antagonized by pretreatment with AM251 (3mgkg−1). Furthermore, pretreatment with the β2-adrenoceptor antagonist, ICI118551, inhibited the hindquarters vasodilator effects of WIN-55212-2 and of HU210.
On the basis of the present findings, and our earlier work, it is suggested that, in conscious rats, the pressor and vasoconstrictor effects of HU210 and WIN-55212-2 involve cannabinoid-receptor-mediated increases in sympathetic activity. The accompanying hindquarters vasodilator actions of these agonists are cannabinoid receptor-mediated and appear to involve β2-adrenoceptors.
Keywords: Cannabinoids, HU210, WIN-55212-2, haemodynamics
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Introduction
Previously (Gardiner et al., 2001), we have drawn attention to the complexity of the cardiovascular effects of cannabinoids as described in the literature, and have suggested that this might reflect variations in experimental conditions.
Considering the in vivo studies, it appears that the state of anaesthesia influences the cardiovascular response to synthetic cannabinoids, since we have recently reported that WIN-55212-2, which has been shown to have marked hypotensive effects in anaesthetized rats (Lake et al., 1997), has dose-dependent pressor and renal and mesenteric vasoconstrictor effects in conscious rats, with hindquarters vasodilator actions being seen only with the higher doses of the cannabinoid (Gardiner et al., 2001).
In our previous study, we made no formal assessment of the involvement of cannabinoid receptors in the vasoconstrictor or vasodilator responses to WIN-55212-2. Thus, the first aim of the present study was to determine the effects of the cannabinoid, CB1-receptor antagonist, AM251 (Gatley et al., 1996, 1997) on the cardiovascular responses to WIN-55212-2 in conscious rats. We considered this an important experiment since, unlike SR141716A, AM251 is commercially available and yet there are no published in vivo data on the cardiovascular effects of AM251, either on baseline haemodynamic status, or on responses to cannabinoids. In addition, since we (Gardiner et al., 2001) have hypothesized that the hindquarters vasodilator action of WIN-55212-2 could be mediated by β2-adrenoceptors, our second aim was to assess the effects of the β2-adrenoceptor antagonist, ICI118551 (Bilski et al., 1983), on responses to WIN-55212-2.
In anaesthetized rats, other cannabinoid agonists, such as HU210, which differ structurally from WIN-55212-2 (for review see Pertwee, 1997), have also been reported to cause CB1-receptor-mediated hypotension, and this has been taken as a sign of vasodilatation (e.g., Lake et al., 1997). However, it is clear from our studies with WIN-55212-2, that it is only by monitoring regional haemodynamics that one can get a proper picture of the processes underlying any effects substances, such as cannabinoids, have on systemic arterial blood pressure. Very recently, some work has been published which indicates a complex regional haemodynamic profile associated with administration of HU210 in urethane-anaesthetized rats (Wagner et al., 2001). However, because of the technique they used, Wagner et al. (2001) were restricted to making measurements at a single point in time following HU210 administration. We know of only one report of the cardiovascular effects of HU210 in conscious rats (Vidrio et al., 1996), and in that study, the cannabinoid was administered i.p., and measurements were restricted to blood pressure and heart rate, monitored indirectly. Therefore, the final aim of the present study was to make continuous measurements of the regional haemodynamic effects of HU210 in conscious rats, to ascertain whether or not they resembled those of WIN-55212-2, and to determine the effects of cannabinoid receptor antagonism (with AM251), or β2-adrenoceptor antagonism (with ICI118551), thereupon.
Some of the results have been presented to the British Pharmacological Society (Gardiner et al., 2002a,2002b).
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Methods
All experiments were carried out on male, Sprague-Dawley rats (Charles River, U.K.), weighing between 420 and 470g at the time of study. Animals were housed in a temperature-controlled environment (20–22°C), with a 12h light-dark cycle (lights on at 06:00h), with free access to food and water throughout. The procedures were approved by the University of Nottingham Ethical Review Committee, and were performed under Home Office Project Licence authority.
Surgical preparation
Surgery was performed in two stages, under general anaesthesia (fentanyl and meditomidine, 300μgkg−1 of each, i.p., reversed with nalbuphine and atipamezole 1mgkg−1 of each, s.c.). The first stage involved implantation of miniaturized pulsed Doppler flow probes around the left renal artery, the superior mesenteric artery and the distal abdominal aorta (below the level of the ileocaecal artery, to monitor flow to the hindquarters). The second stage involved placement of catheters in the distal abdominal aorta (via the caudal artery) to monitor arterial blood pressure and heart rate, and in the right jugular vein for drug administrations. The two surgical stages were separated by at least 10 days and, prior to the second stage, the fitness of all animals was certified by the named veterinary surgeon.
Experiments began 24h after catheterization, when the animals were fully conscious, freely moving, and with access to food and water ad libitum.
Cardiovascular recordings
Continuous recordings of cardiovascular variables were made using a customized, computer-based system (Haemodynamics Data Acquisition System (HDAS), University of Limburg, Maastricht, Netherlands) connected to the transducer amplifier (Gould model 13-4615-50) and the Doppler flowmeter (Crystal Biotech VF-1 mainframe (pulse repetition frequency 125kHz) fitted with high velocity (HVPD-20) modules). Raw data were sampled by HDAS every 2ms, averaged every cardiac cycle and stored to disc at 5s intervals. Data were analysed offline using software (Datview, University of Limburg, Maastricht, Netherlands) which interfaced with HDAS.
Cardiovascular responses to WIN-55212-2 in the absence or presence of AM251.
Animals (n=8) were given i.v. bolus doses of WIN-55212-2 (50 and 150μgkg−1) at 45min intervals, starting 30min after the end of administration of the cannabinoid antagonist AM251 (3mgkg−1 infused i.v. over 30min at 2mlh−1), or the vehicle for AM251 (saline containing 5% propylene glycol and 2% Tween 80). The low dose of WIN-55212-2 was always given first. Animals were randomized to receive vehicle or AM251 on the first day and the other treatment on the third day, with the intervening day being allowed to ensure adequate washout of any active treatment.
Cardiovascular responses to HU210 in the absence or presence of AM251
The cardiovascular effects of HU210 develop slowly and are long-lasting (see Results). Therefore, in one group of animals (n=8), we administered HU210 (10μgkg−1) on the first experimental day, vehicle (saline containing 5% propylene glycol and 2% Tween 80) on the second day, and HU210 (100μgkg−1) on the third day. Cardiovascular variables were recorded continuously for 5h on each day.
In separate groups of animals, responses to HU210 (100μgkg−1) were assessed 45min after administration of AM251 (3mgkg−1 as above, n=7) or vehicle (as above, n=6), and monitored for 5h. As a time control, a separate group of animals (n=7) was given AM251 followed by the vehicle for HU210 (doses and timings as above).
The experiments involving administration of WIN-55212-2 showed clear-cut antagonism of its cardiovascular effects by AM251 (see below). Therefore, at the end of the 5h recording period following HU210 administration, all animals were given a single dose of WIN-55212-2 (150μgkg−1) to check for adequacy of blockade by AM 251.
Cardiovascular responses to WIN-55212-2 in the absence or presence of ICI118551
In preliminary experiments (n=2) we confirmed the reproducibility of the responses to WIN-55212-2 (150μgkg−1) in the absence of ICI118551 (see Results).
In the main experiment, rats (n=7) were given WIN-55212-2 (150μgkg−1) before and 90min after administration of ICI118551 (0.2mgkg−1 bolus, 0.1mgkg−1h−1 infusion) (Gardiner & Bennett, 1988; Gardiner et al., 1992). To check the effectiveness of this dose of ICI118551 in the present conditions, four animals were given a 3min infusion of salbutamol (600ngkg−1min−1) before and 90min after the onset of administration of ICI118551. Before ICI118551, 3min after the onset of infusion of salbutamol, there was an increase in heart rate (+86±18beatsmin−1) and hindquarters vascular conductance (+134±24%), a fall in blood pressure (−9±4mmHg) and no change in renal or mesenteric vascular conductances. In the presence of ICI118551, salbutamol caused no change in heart rate or blood pressure, and the increase in hindquarters vascular conductance (+22±3%) was markedly inhibited.
Cardiovascular responses to HU210 in the presence of ICI118551
Rats (n=8) were given HU210 (100μgkg−1) 90min after the start of administration of ICI118551 (0.2mgkg−1 bolus, 0.1mgkg−1h−1 infusion).
Data analysis
Within-group analyses were by non-parametric analysis of variance (Friedman's test; Theodorsson-Norheim, 1987).
All between-group comparisons were confined to the integrated responses to the cannabinoids, measured as the areas under or over the time curves (0–5min for WIN-55212-2, 0–300min for HU210). Between-group analyses were by the Wilcoxon or Mann–Whitney U-test (two groups), or the Kruskal–Wallis test with Dunn's post-test (more than two groups). A P value 0.05 was taken as significant. Statistical tests were performed using GraphPad Prism version 3.00 for Windows (GraphPad Software, San Diego, CA, U.S.A.; licensed to School of Biomedical Sciences, University of Nottingham, U.K.).
Drugs
Fentanyl citrate was obtained from Martindale; meditomidine hydrochloride (Domitor) and atipamezole hydrochloride (Antisedan) were obtained from Pfizer; nalbuphine hydrochloride (Nubain) was obtained from Du Pont: WIN-55212-2, ((R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl) pyrrolo [1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate); AM251, (N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide); HU210 ((6aR)-trans-3-(1,1-Dimethylheptyl)-6a,7,10,10a-tetrahydro-1-hydroxy-6,6-dimethyl-6H-dibenzo[b,d]pyran-9-methanol) and ICI118551 hydrochloride were obtained from Tocris (U.K.). Solutions of AM251, HU210 and WIN-55212-2 were prepared fresh daily in sterile saline containing 5% propylene glycol (Sigma) and 2% Tween 80 (B.D.H.). ICI118551 was dissolved in sterile water. Bolus injections were given in a volume of 0.1ml; AM251 was infused at a rate of 2mlh−1 for 30min. There were no cardiovascular effects attributable to administration of vehicle at these volumes.
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Results
Cardiovascular responses to WIN-55212-2 in the absence or presence of AM251
Administration of AM251 had no significant cardiovascular effects, although there were some behavioural actions, notably grooming, which influenced cardiovascular variables at the time they occurred, but which had generally passed before administration of WIN-55212-2 was started. Thus, resting haemodynamic variables in this group of animals (n=8) prior to administration of the first dose of WIN-55212-2 in the presence of vehicle or AM251 were not significantly different (heart rate, 346±15 and 336±13beatsmin−1; mean arterial blood pressure, 102±3 and 104±4mmHg; renal vascular conductance, 86±3 and 86±4 (kHzmmHg−1)103; mesenteric vascular conductance, 105±8 and 109±10 (kHzmmHg−1)103; hindquarters vascular conductance, 35±2 and 39±5 (kHzmmHg−1)103), respectively). As noted previously (Gardiner et al., 2001), in the presence of vehicle, WIN-55212-2 had variable effects on heart rate, but caused a dose-dependent pressor response associated with renal and mesenteric vasoconstriction and hindquarters vasodilatation (Figure 1). All
the significant cardiovascular effects of WIN-55212-2 were markedly inhibited by AM251 (Figure 1). Figure 1
Cardiovascular responses to WIN-55212-2 (50 or 150μgkg−1) after vehicle or AM251 (3mgkg−1) on different days in the same, conscious, Sprague-Dawley rats (n=8). Values are mean (more ...)
Cardiovascular responses to HU210 in the absence or presence of AM251
In the first experiment, one group of animals was given HU210 (10μgkg−1) on Day 1, vehicle on Day 2 and HU210 (100μgkg−1) on Day 3. There were no cardiovascular effects of HU210 (10μgkg−1) which differed from the vehicle (data not shown). The responses to HU210 (100μgkg−1) were generally similar to those obtained when HU210 (100μgkg−1) was given to naïve rats (see below), but there were some differences which may have been due to de-sensitization, following exposure to the lower dose of the drug on Day 1 (data not shown).
Administration of AM251 had no significant cardiovascular actions and, hence, cardiovascular variables immediately prior to administration of HU210 (100μgkg−1) in the presence (n=7) or absence (n=6) of AM251 were similar, and not different to those in the animals (n=7) given the vehicle for HU210 in the presence of AM251 (heart rate, 320±10, 331±12 and 326±11 beatsmin−1; mean arterial blood pressure, 102±2, 101±3 and 107±3mmHg; renal vascular conductance, 84±6, 87±10 and 103±7 (kHzmmHg−1)103; mesenteric vascular conductance, 99±6, 100±14 and 95±6 (kHzmmHg−1)103; hindquarters vascular conductance, 44±4, 39±3 and 44±5 (kHzmmHg−1)103), respectively.
Following administration of vehicle for AM251, HU210 (100μgkg−1) caused bradycardia, a rise followed by a fall in blood pressure, short-lived renal vasoconstriction, longer-lasting mesenteric vasoconstriction and marked, prolonged hindquarters vasodilatation (Figure 2). Following administration
of AM251, there was substantial inhibition of all the cardiovascular responses to HU210 (Figure 2). Thus, over the
5h recording period, there were no differences between the integrated changes in heart rate, blood pressure, or renal and mesenteric vascular conductances in the animals given AM251 followed by vehicle for HU210 and those given AM251 followed by HU210 (Figure 2). Moreover, the residual
increase in hindquarters vascular conductance in response to HU210 in the presence of AM251 was significantly (P0.001) less than in the absence of AM251 (Figure 2). Figure 2
Cardiovascular responses to vehicle for HU210 after AM251 (3mgkg−1; n=7), or HU210 (100μgkg−1) after AM251 (n=7) or HU210 (100μgkg (more ...)
The cardiovascular effects of WIN-55212-2 (150μgkg−1), administered at the end of the 5h recording period, were markedly inhibited in the animals treated with AM251 (e.g. integrated (0–5min) increases in hindquarters vascular conductances were +172±75, +30±18 and +33±13%min in animals given vehicle for AM251 followed by HU210, AM251 followed by HU210 and AM251 followed by vehicle for HU210 respectively).
Cardiovascular responses to WIN-55212-2 in the absence or presence of ICI118551
In pilot experiments (n=2) it was shown that the cardiovascular responses to administration of WIN-55212-2 (150μgkg−1) before and after a 90min infusion of saline were consistent (changes in two animals at 3min following WIN-55212-2 administration: blood pressure, before saline: +22 , +17mmHg; after saline: +27, +22mmHg; renal vascular conductance, before saline: −37, −28%; after saline: −43, −31%; mesenteric vascular conductance, before saline: −43, −39%; after saline: −42, −45%; hindquarters vascular conductance, before saline: +50, +63%; after saline: +61, +56%).
Resting cardiovascular variables in animals (n=7), prior to administration of WIN-55212-2 in the absence and presence of ICI118551 were as follows:- heart rate, 337±17 and 322±12beatsmin−1; mean arterial blood pressure, 108±7 and 110±7mmHg; renal vascular conductance, 79±8 and 76±7 (kHzmmHg−1)103; mesenteric vascular conductance, 79±6 and 74±5 (kHzmmHg−1)103; hindquarters vascular conductance, 46±4 and 38±3 (kHzmmHg−1)103), respectively. Hindquarters vascular conductance was significantly (P0.01) lower in the presence of ICI118551 than in its absence, but the other cardiovascular variables were not different.
Responses to WIN-55212-2 in the absence and presence of ICI118551 are shown in Figure 3. In the absence of ICI
118551, the cardiovascular responses to WIN-55212-2 (150μgkg−1) were as described above. In the presence of ICI118551, the integrated pressor response to WIN-55212-2 and the associated falls in renal and mesenteric vascular conductances were not significantly affected, but the rise in hindquarters vascular conductance (+34±12%min) was significantly attenuated (P0.02). In the presence of ICI118551, the variable heart rate response to WIN-55212-2 was not significantly affected (Figure 3). Figure 3
Effects of ICI118551 (0.2mgkg−1, 0.1mgkg−1h−1) on the cardiovascular responses to WIN-55212-2 (150μgkg−1) and HU210 (100μgkg (more ...)
Cardiovascular responses to HU210 in the presence of ICI118551
Resting cardiovascular variables in rats (n=8) receiving ICI118551, prior to the administration of HU210 (100μgkg−1) were as follows: heart rate, 320±17beats min−1, mean arterial blood pressure, 105±5mmHg, renal vascular conductance, 99±10 (kHzmmHg−1)103, mesenteric vascular conductance, 105±12 (kHzmmHg−1)103, hindquarters vascular conductance, 43±5 (kHzmmHg−1)103. Responses to HU210 in the absence and presence of ICI118551 are shown in Figure 3. In the presence of ICI11551, the
integrated (0–5h) changes in heart rate, mean arterial blood and renal and mesenteric vascular conductances in response to HU210 (100μgkg−1) were similar to those seen in the animals given HU210 in the presence of the vehicle for AM251 However, the increase in hindquarters vascular conductance in response to HU210 in the presence of ICI118551 was significantly (P0.001) less than in its absence.
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Discussion
The aims of this study were to compare the regional haemodynamic effects of the structurally different cannabinoid agonists, HU210 and WIN-55212-2, in conscious rats, and to examine the extent of involvement of cannabinoid receptors in the cardiovascular responses observed. In addition, we explored the possible contribution of β2 adrenoceptors to cannabinoid-induced hindquarters vasodilatation.
The most notable findings are that: (i) HU210 and WIN-55212-2 caused similar patterns of cardiovascular change (pressor, renal and mesenteric vasoconstrictor, hindquarters vasodilator), but with markedly different time courses; (ii) the cardiovascular effects of HU210 and WIN-55212-2 were substantially antagonized by AM251, although the latter had no significant effects on resting haemodynamics; (iii) the hindquarters vasodilator responses to WIN-55212-2 and HU210, which appeared to involve CB1-receptors, were susceptible to β2-adrenoceptor antagonism.
Previously (Gardiner et al., 2001), we have drawn attention to the fact that, in conscious rats, the haemodynamic effects of WIN-55212-2 are quite different from those reported in anaesthetized rats (Lake et al., 1997), but resemble, in some regards, the effects reported in conscious rabbits (Niederhoffer & Szabo, 1999, 2000). Here we show, again, dose-dependent pressor, renal and mesenteric vasoconstrictor and hindquarters vasodilator effects of WIN-55212-2 and, in addition, now demonstrate marked inhibition of those effects by the CB1-receptor antagonist, AM251 (Gatley et al., 1996; 1997).
To our knowledge, this is the first study to describe antagonism of cannabinoid-mediated cardiovascular effects by AM251 in vivo. Most studies involving CB1-receptor antagonists in vivo have used SR141716A, which is not commercially available. AM251 and AM281 are analogues of SR141716A, which bind with high affinity to cannabinoid CB1-receptors following in vivo administration (Gatley et al., 1996, 1997). We chose to give a dose of 3mgkg−1 AM251, since that was the dose of SR141716A used by Lake et al. (1997) which blocked the cardiovascular effects of cannabinoid agonists in vivo, and because Izzo et al. (2000) showed equivalent antagonism by equimolar doses of SR141716A and AM281 in vitro. It is clear from our results that we had achieved effective cannabinoid receptor antagonism, but it is notable that AM251 itself had no significant cardiovascular effects. This suggests that cannabinoid receptor-mediated events are unlikely to be involved in normal cardiovascular regulation. Interestingly, we have recently shown that AM251, at the dose used here, has no effect on the cardiovascular actions of exogenous anandamide (Gardiner et al., 2002a), suggesting that the latter may not involve cannabinoid receptors.
We reported earlier that the pressor and vasoconstrictor effects of WIN-55212-2 were susceptible to ganglion blockade and were, therefore, likely to be due to sympathoexcitation (Gardiner et al., 2001). The present results indicate that these effects are CB1-receptor-mediated, but do not allow us to localize the cannabinoid receptors involved although, on the basis of the work of others, we would expect this to be a centrally-mediated action (Niederhoffer & Szabo, 1999, 2000). Our earlier work showed that the hindquarters vasodilator effect of WIN-55212-2 was not susceptible to ganglion blockade (indeed, it was enhanced under those conditions), and we speculated about a possible involvement of β2-adrenoceptors.
We have previously shown (Gardiner et al., 1992), and confirmed here, that administration of a β2-adrenoceptor agonist to conscious rats causes selective vasodilatation in the hindquarters, and that the dose of ICI118551 used presently ablates that response. Our present findings, which show that both AM251 and ICI118551, independently, antagonize the hindquarters vasodilator response to WIN-55212-2, are therefore entirely consistent with an involvement of β2-adrenoceptors in the CB1-receptor-mediated hindquarters vasodilator response. Although one theoretical possibility is that CB1-receptors stimulate adrenal medullary adrenaline release, this is not consistent with the only published paper that we are aware of on the topic (Niederhoffer et al., 2001), which shows that, in pithed rabbits, cannabinoids inhibit adrenaline release.
Although the time course of the effect of HU210 was far longer than that of WIN-55212-2, which is consistent with the higher affinity of binding of the former to CB1-receptors (Pertwee, 1997), the patterns of response to the cannabinoids were remarkably similar, inasmuch as both had pressor and renal and mesenteric vasoconstrictor effects, accompanied by hindquarters vasodilatation, and all these effects were antagonized by AM251. Furthermore, the hindquarters vasodilator effect of HU210, like that of WIN-55212-2 was reduced substantially by ICI118551.
It is worth noting that, whilst an increase in locomotor activity would result in hindquarters vasodilatation, that cannot be the explanation for our results since, particularly following administration of HU210, locomotor activity was substantially suppressed.
Our results with HU210 differ in some respects from those in the literature, where prolonged hypotension and bradycardia following administration of HU210 has been described (Vidrio et al., 1996; Lake et al., 1997). For example, in anaesthetized rats, HU210 (100μgkg−1) has been reported to cause a fall in blood pressure of ~50mmHg which was sustained for the duration of the 90min recording period (Vidrio et al., 1996). In the recent report of Wagner et al. (2001) it was suggested that the hypotension caused by HU210 was entirely due to a fall in cardiac output. Furthermore, it was proposed that the underlying mechanism was a cannabinoid receptor-mediated prejunctional inhibition of cardiac sympathetic tone, since the fall in cardiac output was not evident in ganglion-blocked animals (Wagner et al., 2001). However, in the ganglion-blocked state, there was still a substantial fall in blood pressure in response to HU210 (−57mmHg) which was not accounted for either by a fall in cardiac output, or by peripheral vasodilatation. Wagner et al. (2001) did not explain these findings, and we cannot see an obvious explanation for their discordant results. However, considering our results against the background of published findings it seems that responses to HU210, like those of WIN-55212-2 (see Introduction), may be influenced substantially by anaesthesia. Thus, it is unfortunate that the unratified dogma is that cannabinoids are hypotensive and vasodilator, considering this view derives from studies in anaesthetised animals, and is being used as a basis for proposing that cannabinoids might be useful in treating hypertension (e.g., Porter & Felder, 2001).
Acknowledgments
This work was supported by the British Heart Foundation (Project Grant 99/063)
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Influence of the CB1 receptor antagonist, AM251, on the regional haemodynamic effects of WIN-55212-2 or HU210 in conscious rats
S M Gardiner,1* J E March,1 P A Kemp,1 and T Bennett1
11School of Biomedical Sciences, University of Nottingham Medical School, Queen's Medical Centre, Nottingham NG7 2UH
*Author for correspondence: Email: sheila.gardiner@nottingham.ac.uk
Abstract
In conscious, freely-moving, male, Sprague-Dawley rats, the regional haemodynamic responses to the synthetic cannabinoids, WIN-55212-2 and HU210, were compared. The possible involvement of cannabinoid, CB1-receptors, or β2-adrenoceptors in the responses to WIN-55212-2 and HU210 were investigated using the CB1-receptor antagonist, AM251, or the β2-adrenoceptor antagonist, ICI118551, respectively.
Both WIN-55212-2 (150μgkg−1) and HU210 (100μgkg−1) had pressor, renal, and mesenteric vasoconstrictor and hindquarters vasodilator actions, although the effects of HU210 were much more sustained than those of WIN-55212-2. Lower doses of the cannabinoids (WIN-55212-2, 50μgkg−1, HU210, 10μgkg−1) had less consistent actions.
All the significant cardiovascular effects of WIN-55212-2 and HU210 were antagonized by pretreatment with AM251 (3mgkg−1). Furthermore, pretreatment with the β2-adrenoceptor antagonist, ICI118551, inhibited the hindquarters vasodilator effects of WIN-55212-2 and of HU210.
On the basis of the present findings, and our earlier work, it is suggested that, in conscious rats, the pressor and vasoconstrictor effects of HU210 and WIN-55212-2 involve cannabinoid-receptor-mediated increases in sympathetic activity. The accompanying hindquarters vasodilator actions of these agonists are cannabinoid receptor-mediated and appear to involve β2-adrenoceptors.
Keywords: Cannabinoids, HU210, WIN-55212-2, haemodynamics
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Introduction
Previously (Gardiner et al., 2001), we have drawn attention to the complexity of the cardiovascular effects of cannabinoids as described in the literature, and have suggested that this might reflect variations in experimental conditions.
Considering the in vivo studies, it appears that the state of anaesthesia influences the cardiovascular response to synthetic cannabinoids, since we have recently reported that WIN-55212-2, which has been shown to have marked hypotensive effects in anaesthetized rats (Lake et al., 1997), has dose-dependent pressor and renal and mesenteric vasoconstrictor effects in conscious rats, with hindquarters vasodilator actions being seen only with the higher doses of the cannabinoid (Gardiner et al., 2001).
In our previous study, we made no formal assessment of the involvement of cannabinoid receptors in the vasoconstrictor or vasodilator responses to WIN-55212-2. Thus, the first aim of the present study was to determine the effects of the cannabinoid, CB1-receptor antagonist, AM251 (Gatley et al., 1996, 1997) on the cardiovascular responses to WIN-55212-2 in conscious rats. We considered this an important experiment since, unlike SR141716A, AM251 is commercially available and yet there are no published in vivo data on the cardiovascular effects of AM251, either on baseline haemodynamic status, or on responses to cannabinoids. In addition, since we (Gardiner et al., 2001) have hypothesized that the hindquarters vasodilator action of WIN-55212-2 could be mediated by β2-adrenoceptors, our second aim was to assess the effects of the β2-adrenoceptor antagonist, ICI118551 (Bilski et al., 1983), on responses to WIN-55212-2.
In anaesthetized rats, other cannabinoid agonists, such as HU210, which differ structurally from WIN-55212-2 (for review see Pertwee, 1997), have also been reported to cause CB1-receptor-mediated hypotension, and this has been taken as a sign of vasodilatation (e.g., Lake et al., 1997). However, it is clear from our studies with WIN-55212-2, that it is only by monitoring regional haemodynamics that one can get a proper picture of the processes underlying any effects substances, such as cannabinoids, have on systemic arterial blood pressure. Very recently, some work has been published which indicates a complex regional haemodynamic profile associated with administration of HU210 in urethane-anaesthetized rats (Wagner et al., 2001). However, because of the technique they used, Wagner et al. (2001) were restricted to making measurements at a single point in time following HU210 administration. We know of only one report of the cardiovascular effects of HU210 in conscious rats (Vidrio et al., 1996), and in that study, the cannabinoid was administered i.p., and measurements were restricted to blood pressure and heart rate, monitored indirectly. Therefore, the final aim of the present study was to make continuous measurements of the regional haemodynamic effects of HU210 in conscious rats, to ascertain whether or not they resembled those of WIN-55212-2, and to determine the effects of cannabinoid receptor antagonism (with AM251), or β2-adrenoceptor antagonism (with ICI118551), thereupon.
Some of the results have been presented to the British Pharmacological Society (Gardiner et al., 2002a,2002b).
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Methods
All experiments were carried out on male, Sprague-Dawley rats (Charles River, U.K.), weighing between 420 and 470g at the time of study. Animals were housed in a temperature-controlled environment (20–22°C), with a 12h light-dark cycle (lights on at 06:00h), with free access to food and water throughout. The procedures were approved by the University of Nottingham Ethical Review Committee, and were performed under Home Office Project Licence authority.
Surgical preparation
Surgery was performed in two stages, under general anaesthesia (fentanyl and meditomidine, 300μgkg−1 of each, i.p., reversed with nalbuphine and atipamezole 1mgkg−1 of each, s.c.). The first stage involved implantation of miniaturized pulsed Doppler flow probes around the left renal artery, the superior mesenteric artery and the distal abdominal aorta (below the level of the ileocaecal artery, to monitor flow to the hindquarters). The second stage involved placement of catheters in the distal abdominal aorta (via the caudal artery) to monitor arterial blood pressure and heart rate, and in the right jugular vein for drug administrations. The two surgical stages were separated by at least 10 days and, prior to the second stage, the fitness of all animals was certified by the named veterinary surgeon.
Experiments began 24h after catheterization, when the animals were fully conscious, freely moving, and with access to food and water ad libitum.
Cardiovascular recordings
Continuous recordings of cardiovascular variables were made using a customized, computer-based system (Haemodynamics Data Acquisition System (HDAS), University of Limburg, Maastricht, Netherlands) connected to the transducer amplifier (Gould model 13-4615-50) and the Doppler flowmeter (Crystal Biotech VF-1 mainframe (pulse repetition frequency 125kHz) fitted with high velocity (HVPD-20) modules). Raw data were sampled by HDAS every 2ms, averaged every cardiac cycle and stored to disc at 5s intervals. Data were analysed offline using software (Datview, University of Limburg, Maastricht, Netherlands) which interfaced with HDAS.
Cardiovascular responses to WIN-55212-2 in the absence or presence of AM251.
Animals (n=8) were given i.v. bolus doses of WIN-55212-2 (50 and 150μgkg−1) at 45min intervals, starting 30min after the end of administration of the cannabinoid antagonist AM251 (3mgkg−1 infused i.v. over 30min at 2mlh−1), or the vehicle for AM251 (saline containing 5% propylene glycol and 2% Tween 80). The low dose of WIN-55212-2 was always given first. Animals were randomized to receive vehicle or AM251 on the first day and the other treatment on the third day, with the intervening day being allowed to ensure adequate washout of any active treatment.
Cardiovascular responses to HU210 in the absence or presence of AM251
The cardiovascular effects of HU210 develop slowly and are long-lasting (see Results). Therefore, in one group of animals (n=8), we administered HU210 (10μgkg−1) on the first experimental day, vehicle (saline containing 5% propylene glycol and 2% Tween 80) on the second day, and HU210 (100μgkg−1) on the third day. Cardiovascular variables were recorded continuously for 5h on each day.
In separate groups of animals, responses to HU210 (100μgkg−1) were assessed 45min after administration of AM251 (3mgkg−1 as above, n=7) or vehicle (as above, n=6), and monitored for 5h. As a time control, a separate group of animals (n=7) was given AM251 followed by the vehicle for HU210 (doses and timings as above).
The experiments involving administration of WIN-55212-2 showed clear-cut antagonism of its cardiovascular effects by AM251 (see below). Therefore, at the end of the 5h recording period following HU210 administration, all animals were given a single dose of WIN-55212-2 (150μgkg−1) to check for adequacy of blockade by AM 251.
Cardiovascular responses to WIN-55212-2 in the absence or presence of ICI118551
In preliminary experiments (n=2) we confirmed the reproducibility of the responses to WIN-55212-2 (150μgkg−1) in the absence of ICI118551 (see Results).
In the main experiment, rats (n=7) were given WIN-55212-2 (150μgkg−1) before and 90min after administration of ICI118551 (0.2mgkg−1 bolus, 0.1mgkg−1h−1 infusion) (Gardiner & Bennett, 1988; Gardiner et al., 1992). To check the effectiveness of this dose of ICI118551 in the present conditions, four animals were given a 3min infusion of salbutamol (600ngkg−1min−1) before and 90min after the onset of administration of ICI118551. Before ICI118551, 3min after the onset of infusion of salbutamol, there was an increase in heart rate (+86±18beatsmin−1) and hindquarters vascular conductance (+134±24%), a fall in blood pressure (−9±4mmHg) and no change in renal or mesenteric vascular conductances. In the presence of ICI118551, salbutamol caused no change in heart rate or blood pressure, and the increase in hindquarters vascular conductance (+22±3%) was markedly inhibited.
Cardiovascular responses to HU210 in the presence of ICI118551
Rats (n=8) were given HU210 (100μgkg−1) 90min after the start of administration of ICI118551 (0.2mgkg−1 bolus, 0.1mgkg−1h−1 infusion).
Data analysis
Within-group analyses were by non-parametric analysis of variance (Friedman's test; Theodorsson-Norheim, 1987).
All between-group comparisons were confined to the integrated responses to the cannabinoids, measured as the areas under or over the time curves (0–5min for WIN-55212-2, 0–300min for HU210). Between-group analyses were by the Wilcoxon or Mann–Whitney U-test (two groups), or the Kruskal–Wallis test with Dunn's post-test (more than two groups). A P value 0.05 was taken as significant. Statistical tests were performed using GraphPad Prism version 3.00 for Windows (GraphPad Software, San Diego, CA, U.S.A.; licensed to School of Biomedical Sciences, University of Nottingham, U.K.).
Drugs
Fentanyl citrate was obtained from Martindale; meditomidine hydrochloride (Domitor) and atipamezole hydrochloride (Antisedan) were obtained from Pfizer; nalbuphine hydrochloride (Nubain) was obtained from Du Pont: WIN-55212-2, ((R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl) pyrrolo [1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate); AM251, (N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide); HU210 ((6aR)-trans-3-(1,1-Dimethylheptyl)-6a,7,10,10a-tetrahydro-1-hydroxy-6,6-dimethyl-6H-dibenzo[b,d]pyran-9-methanol) and ICI118551 hydrochloride were obtained from Tocris (U.K.). Solutions of AM251, HU210 and WIN-55212-2 were prepared fresh daily in sterile saline containing 5% propylene glycol (Sigma) and 2% Tween 80 (B.D.H.). ICI118551 was dissolved in sterile water. Bolus injections were given in a volume of 0.1ml; AM251 was infused at a rate of 2mlh−1 for 30min. There were no cardiovascular effects attributable to administration of vehicle at these volumes.
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Results
Cardiovascular responses to WIN-55212-2 in the absence or presence of AM251
Administration of AM251 had no significant cardiovascular effects, although there were some behavioural actions, notably grooming, which influenced cardiovascular variables at the time they occurred, but which had generally passed before administration of WIN-55212-2 was started. Thus, resting haemodynamic variables in this group of animals (n=8) prior to administration of the first dose of WIN-55212-2 in the presence of vehicle or AM251 were not significantly different (heart rate, 346±15 and 336±13beatsmin−1; mean arterial blood pressure, 102±3 and 104±4mmHg; renal vascular conductance, 86±3 and 86±4 (kHzmmHg−1)103; mesenteric vascular conductance, 105±8 and 109±10 (kHzmmHg−1)103; hindquarters vascular conductance, 35±2 and 39±5 (kHzmmHg−1)103), respectively). As noted previously (Gardiner et al., 2001), in the presence of vehicle, WIN-55212-2 had variable effects on heart rate, but caused a dose-dependent pressor response associated with renal and mesenteric vasoconstriction and hindquarters vasodilatation (Figure 1). All
the significant cardiovascular effects of WIN-55212-2 were markedly inhibited by AM251 (Figure 1). Figure 1
Cardiovascular responses to WIN-55212-2 (50 or 150μgkg−1) after vehicle or AM251 (3mgkg−1) on different days in the same, conscious, Sprague-Dawley rats (n=8). Values are mean (more ...)
Cardiovascular responses to HU210 in the absence or presence of AM251
In the first experiment, one group of animals was given HU210 (10μgkg−1) on Day 1, vehicle on Day 2 and HU210 (100μgkg−1) on Day 3. There were no cardiovascular effects of HU210 (10μgkg−1) which differed from the vehicle (data not shown). The responses to HU210 (100μgkg−1) were generally similar to those obtained when HU210 (100μgkg−1) was given to naïve rats (see below), but there were some differences which may have been due to de-sensitization, following exposure to the lower dose of the drug on Day 1 (data not shown).
Administration of AM251 had no significant cardiovascular actions and, hence, cardiovascular variables immediately prior to administration of HU210 (100μgkg−1) in the presence (n=7) or absence (n=6) of AM251 were similar, and not different to those in the animals (n=7) given the vehicle for HU210 in the presence of AM251 (heart rate, 320±10, 331±12 and 326±11 beatsmin−1; mean arterial blood pressure, 102±2, 101±3 and 107±3mmHg; renal vascular conductance, 84±6, 87±10 and 103±7 (kHzmmHg−1)103; mesenteric vascular conductance, 99±6, 100±14 and 95±6 (kHzmmHg−1)103; hindquarters vascular conductance, 44±4, 39±3 and 44±5 (kHzmmHg−1)103), respectively.
Following administration of vehicle for AM251, HU210 (100μgkg−1) caused bradycardia, a rise followed by a fall in blood pressure, short-lived renal vasoconstriction, longer-lasting mesenteric vasoconstriction and marked, prolonged hindquarters vasodilatation (Figure 2). Following administration
of AM251, there was substantial inhibition of all the cardiovascular responses to HU210 (Figure 2). Thus, over the
5h recording period, there were no differences between the integrated changes in heart rate, blood pressure, or renal and mesenteric vascular conductances in the animals given AM251 followed by vehicle for HU210 and those given AM251 followed by HU210 (Figure 2). Moreover, the residual
increase in hindquarters vascular conductance in response to HU210 in the presence of AM251 was significantly (P0.001) less than in the absence of AM251 (Figure 2). Figure 2
Cardiovascular responses to vehicle for HU210 after AM251 (3mgkg−1; n=7), or HU210 (100μgkg−1) after AM251 (n=7) or HU210 (100μgkg (more ...)
The cardiovascular effects of WIN-55212-2 (150μgkg−1), administered at the end of the 5h recording period, were markedly inhibited in the animals treated with AM251 (e.g. integrated (0–5min) increases in hindquarters vascular conductances were +172±75, +30±18 and +33±13%min in animals given vehicle for AM251 followed by HU210, AM251 followed by HU210 and AM251 followed by vehicle for HU210 respectively).
Cardiovascular responses to WIN-55212-2 in the absence or presence of ICI118551
In pilot experiments (n=2) it was shown that the cardiovascular responses to administration of WIN-55212-2 (150μgkg−1) before and after a 90min infusion of saline were consistent (changes in two animals at 3min following WIN-55212-2 administration: blood pressure, before saline: +22 , +17mmHg; after saline: +27, +22mmHg; renal vascular conductance, before saline: −37, −28%; after saline: −43, −31%; mesenteric vascular conductance, before saline: −43, −39%; after saline: −42, −45%; hindquarters vascular conductance, before saline: +50, +63%; after saline: +61, +56%).
Resting cardiovascular variables in animals (n=7), prior to administration of WIN-55212-2 in the absence and presence of ICI118551 were as follows:- heart rate, 337±17 and 322±12beatsmin−1; mean arterial blood pressure, 108±7 and 110±7mmHg; renal vascular conductance, 79±8 and 76±7 (kHzmmHg−1)103; mesenteric vascular conductance, 79±6 and 74±5 (kHzmmHg−1)103; hindquarters vascular conductance, 46±4 and 38±3 (kHzmmHg−1)103), respectively. Hindquarters vascular conductance was significantly (P0.01) lower in the presence of ICI118551 than in its absence, but the other cardiovascular variables were not different.
Responses to WIN-55212-2 in the absence and presence of ICI118551 are shown in Figure 3. In the absence of ICI
118551, the cardiovascular responses to WIN-55212-2 (150μgkg−1) were as described above. In the presence of ICI118551, the integrated pressor response to WIN-55212-2 and the associated falls in renal and mesenteric vascular conductances were not significantly affected, but the rise in hindquarters vascular conductance (+34±12%min) was significantly attenuated (P0.02). In the presence of ICI118551, the variable heart rate response to WIN-55212-2 was not significantly affected (Figure 3). Figure 3
Effects of ICI118551 (0.2mgkg−1, 0.1mgkg−1h−1) on the cardiovascular responses to WIN-55212-2 (150μgkg−1) and HU210 (100μgkg (more ...)
Cardiovascular responses to HU210 in the presence of ICI118551
Resting cardiovascular variables in rats (n=8) receiving ICI118551, prior to the administration of HU210 (100μgkg−1) were as follows: heart rate, 320±17beats min−1, mean arterial blood pressure, 105±5mmHg, renal vascular conductance, 99±10 (kHzmmHg−1)103, mesenteric vascular conductance, 105±12 (kHzmmHg−1)103, hindquarters vascular conductance, 43±5 (kHzmmHg−1)103. Responses to HU210 in the absence and presence of ICI118551 are shown in Figure 3. In the presence of ICI11551, the
integrated (0–5h) changes in heart rate, mean arterial blood and renal and mesenteric vascular conductances in response to HU210 (100μgkg−1) were similar to those seen in the animals given HU210 in the presence of the vehicle for AM251 However, the increase in hindquarters vascular conductance in response to HU210 in the presence of ICI118551 was significantly (P0.001) less than in its absence.
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Discussion
The aims of this study were to compare the regional haemodynamic effects of the structurally different cannabinoid agonists, HU210 and WIN-55212-2, in conscious rats, and to examine the extent of involvement of cannabinoid receptors in the cardiovascular responses observed. In addition, we explored the possible contribution of β2 adrenoceptors to cannabinoid-induced hindquarters vasodilatation.
The most notable findings are that: (i) HU210 and WIN-55212-2 caused similar patterns of cardiovascular change (pressor, renal and mesenteric vasoconstrictor, hindquarters vasodilator), but with markedly different time courses; (ii) the cardiovascular effects of HU210 and WIN-55212-2 were substantially antagonized by AM251, although the latter had no significant effects on resting haemodynamics; (iii) the hindquarters vasodilator responses to WIN-55212-2 and HU210, which appeared to involve CB1-receptors, were susceptible to β2-adrenoceptor antagonism.
Previously (Gardiner et al., 2001), we have drawn attention to the fact that, in conscious rats, the haemodynamic effects of WIN-55212-2 are quite different from those reported in anaesthetized rats (Lake et al., 1997), but resemble, in some regards, the effects reported in conscious rabbits (Niederhoffer & Szabo, 1999, 2000). Here we show, again, dose-dependent pressor, renal and mesenteric vasoconstrictor and hindquarters vasodilator effects of WIN-55212-2 and, in addition, now demonstrate marked inhibition of those effects by the CB1-receptor antagonist, AM251 (Gatley et al., 1996; 1997).
To our knowledge, this is the first study to describe antagonism of cannabinoid-mediated cardiovascular effects by AM251 in vivo. Most studies involving CB1-receptor antagonists in vivo have used SR141716A, which is not commercially available. AM251 and AM281 are analogues of SR141716A, which bind with high affinity to cannabinoid CB1-receptors following in vivo administration (Gatley et al., 1996, 1997). We chose to give a dose of 3mgkg−1 AM251, since that was the dose of SR141716A used by Lake et al. (1997) which blocked the cardiovascular effects of cannabinoid agonists in vivo, and because Izzo et al. (2000) showed equivalent antagonism by equimolar doses of SR141716A and AM281 in vitro. It is clear from our results that we had achieved effective cannabinoid receptor antagonism, but it is notable that AM251 itself had no significant cardiovascular effects. This suggests that cannabinoid receptor-mediated events are unlikely to be involved in normal cardiovascular regulation. Interestingly, we have recently shown that AM251, at the dose used here, has no effect on the cardiovascular actions of exogenous anandamide (Gardiner et al., 2002a), suggesting that the latter may not involve cannabinoid receptors.
We reported earlier that the pressor and vasoconstrictor effects of WIN-55212-2 were susceptible to ganglion blockade and were, therefore, likely to be due to sympathoexcitation (Gardiner et al., 2001). The present results indicate that these effects are CB1-receptor-mediated, but do not allow us to localize the cannabinoid receptors involved although, on the basis of the work of others, we would expect this to be a centrally-mediated action (Niederhoffer & Szabo, 1999, 2000). Our earlier work showed that the hindquarters vasodilator effect of WIN-55212-2 was not susceptible to ganglion blockade (indeed, it was enhanced under those conditions), and we speculated about a possible involvement of β2-adrenoceptors.
We have previously shown (Gardiner et al., 1992), and confirmed here, that administration of a β2-adrenoceptor agonist to conscious rats causes selective vasodilatation in the hindquarters, and that the dose of ICI118551 used presently ablates that response. Our present findings, which show that both AM251 and ICI118551, independently, antagonize the hindquarters vasodilator response to WIN-55212-2, are therefore entirely consistent with an involvement of β2-adrenoceptors in the CB1-receptor-mediated hindquarters vasodilator response. Although one theoretical possibility is that CB1-receptors stimulate adrenal medullary adrenaline release, this is not consistent with the only published paper that we are aware of on the topic (Niederhoffer et al., 2001), which shows that, in pithed rabbits, cannabinoids inhibit adrenaline release.
Although the time course of the effect of HU210 was far longer than that of WIN-55212-2, which is consistent with the higher affinity of binding of the former to CB1-receptors (Pertwee, 1997), the patterns of response to the cannabinoids were remarkably similar, inasmuch as both had pressor and renal and mesenteric vasoconstrictor effects, accompanied by hindquarters vasodilatation, and all these effects were antagonized by AM251. Furthermore, the hindquarters vasodilator effect of HU210, like that of WIN-55212-2 was reduced substantially by ICI118551.
It is worth noting that, whilst an increase in locomotor activity would result in hindquarters vasodilatation, that cannot be the explanation for our results since, particularly following administration of HU210, locomotor activity was substantially suppressed.
Our results with HU210 differ in some respects from those in the literature, where prolonged hypotension and bradycardia following administration of HU210 has been described (Vidrio et al., 1996; Lake et al., 1997). For example, in anaesthetized rats, HU210 (100μgkg−1) has been reported to cause a fall in blood pressure of ~50mmHg which was sustained for the duration of the 90min recording period (Vidrio et al., 1996). In the recent report of Wagner et al. (2001) it was suggested that the hypotension caused by HU210 was entirely due to a fall in cardiac output. Furthermore, it was proposed that the underlying mechanism was a cannabinoid receptor-mediated prejunctional inhibition of cardiac sympathetic tone, since the fall in cardiac output was not evident in ganglion-blocked animals (Wagner et al., 2001). However, in the ganglion-blocked state, there was still a substantial fall in blood pressure in response to HU210 (−57mmHg) which was not accounted for either by a fall in cardiac output, or by peripheral vasodilatation. Wagner et al. (2001) did not explain these findings, and we cannot see an obvious explanation for their discordant results. However, considering our results against the background of published findings it seems that responses to HU210, like those of WIN-55212-2 (see Introduction), may be influenced substantially by anaesthesia. Thus, it is unfortunate that the unratified dogma is that cannabinoids are hypotensive and vasodilator, considering this view derives from studies in anaesthetised animals, and is being used as a basis for proposing that cannabinoids might be useful in treating hypertension (e.g., Porter & Felder, 2001).
Acknowledgments
This work was supported by the British Heart Foundation (Project Grant 99/063)
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References
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Influence of the CB1 receptor antagonist, AM251, on the regional haemodynamic effects of WIN-55212-2 or HU210 in conscious rats
