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Am J Physiol Heart Circ Physiol 288: H2280-H2288, 2005. First published December 16, 2004; doi:10.1152/ajpheart.00851.2004
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Involvement of CB1-receptors and {beta}-adrenoceptors in the regional hemodynamic responses to lipopolysaccharide infusion in conscious rats

S. M. Gardiner, J. E. March, P. A. Kemp, and T. Bennett

Centre for Integrated Systems Biology and Medicine, School of Biomedical Sciences, Medical School, Queen's Medical Centre, Nottingham, United Kingdom

Submitted 8 August 2004 ; accepted in final form 24 December 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURE
 REFERENCES
 
A possible involvement of endocannabinoids in a chronic model of endotoxemia was assessed by measuring the regional (renal, mesenteric, hindquarters) hemodynamic responses to continuous 24-h LPS infusion (150 µg·kg–1·h–1) in conscious, male Sprague-Dawley rats, in the absence or presence of the cannabinoid (CB1) receptor antagonist AM-251 (3 mg/kg). AM-251 inhibited the tachycardic and hindquarters vasodilator effects of LPS, but did not influence the other hemodynamic changes. In subsequent experiments, it was shown that the tachycardic and hindquarters vasodilator effects of LPS were also inhibited by the nonselective {beta}-adrenoceptor antagonist propranolol. In addition, the late (at 24 h) hindquarters vasodilator effects of LPS were inhibited by the {beta}2-adrenoceptor antagonist ICI-118551. Against the background of our previous work showing {beta}-adrenoceptor involvement in the cardiovascular effects of exogenous cannabinoids, we conclude that AM-251 may have been inhibiting endocannabinoid-modulated, sympathoadrenal-mediated activation of vasodilator {beta}-adrenoceptors in LPS-infused rats rather than suppressing a direct vasodilator action of endocannabinoids.

AM-251; endotoxemia; vasodilatation

THE SYSTEMIC RESPONSE TO BACTERIAL infection, often referred to as sepsis, involves complex pathogenetic mechanisms and associated cardiovascular changes (26). Before the development of septic shock, there may be a hyperdynamic phase of sepsis with high cardiac output and peripheral vasodilatation (26). The underlying causes of the vasodilatation are multifactorial, involving the loss of vascular sensitivity to vasoconstrictors (25), some of which may be released through nonbaroreflex-mediated processes (39) and possibly contributed to by disorders of baroreflex control (29). In addition, there are numerous potential vasodilator mediators reported to be involved in the cardiovascular sequelae of sepsis (25).

Endotoxemia induced by the administration of the bacterial cell wall product, LPS, to experimental animals is often used to model some of the pathophysiological changes occurring in sepsis. In a model of endotoxemia, involving a large bolus dose of LPS in anesthetized male Sprague-Dawley rats, Varga et al. (33) have provided evidence indicating a possible involvement of endocannabinoids in the hypotension, because the cannabinoid-1 (CB1) receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR-141716A) prevented the fall in mean arterial blood pressure. In addition, LPS stimulated the production of endocannabinoids by platelets and macrophages, although increased circulating levels of these substances were not directly demonstrated in LPS-treated rats (33). The same group has also shown an involvement of endogenous cannabinoids in the hypotension associated with hemorrhagic shock (35) and after myocardial infarction (34). In vitro studies (28) have shown cannabinoids cause vasodilatation by a variety of mechanisms, including release of endothelium-derived hyperpolarizing factor, activation of potassium channels, inhibition of calcium channels, direct effects of cannabinoid receptors on vascular smooth muscle, release of mediators from sensory nerves, and presynaptic inhibition of norepinephrine release. Against this background, Varga et al. (33) have proposed that endocannabinoids may be a new class of neurohumoral vascular mediators in endotoxemia, although they did not demonstrate that regional vasodilatation was responsible for the hypotension in their experimental animals or that the effects of SR-141716A were due to an influence on peripheral vascular conductance. This is an important point, because the hypotension after bolus administration of LPS is primarily due to a fall in cardiac output rather than to vasodilatation (11). Furthermore, more recently, Godlewski et al. (19) indicated that in septic shock, endocannabinoids might exert an indirect action through prejunctional inhibition of sympathoexcitation.

An endotoxemic model we developed is characterized by marked peripheral vasodilatation offset by a substantial increase in cardiac output such that there is little change in mean arterial blood pressure (9). Thus this is not a model of endotoxic shock but represents the hyperdynamic circulatory status seen in the early stages of clinical sepsis (26). In a series of studies in this model of endotoxemia, which involves continuous infusion of LPS in conscious rats, we have assessed the involvement of opposing vasoconstrictor and vasodilator mechanisms. Of the former, endothelin, angiotensin II, and vasopressin play major roles (10), but, although there is some evidence for an involvement of nitric oxide, ATP-sensitive K+ (KATP) channels, and calcitonin gene-related peptide (9, 12, 14), the mediator(s) of a major component of the vasodilator response to LPS infusions is unidentified. From the findings of Varga et al. (33; see above), we hypothesized that endocannabinoids may mediate regional vasodilatation in our endotoxemic model. To test this hypothesis, we assessed the influence of pretreatment with the CB1 receptor antagonist N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM-251) (17, 18) on the regional hemodynamic responses to LPS infusion in conscious rats.

Our first experiment (see RESULTS) showed that pretreatment with AM-251 attenuated the LPS-induced tachycardia and hindquarters vasodilatation. In normal conscious rats, the combination of tachycardia and hindquarters vasodilatation is characteristic of {beta}-adrenoceptor activation (36), and we have recently shown an involvement of {beta}-adrenoceptors in the hindquarters vasodilator effect of synthetic and endogenous cannabinoids (15, 16). Therefore, we hypothesized that the LPS-induced cardiovascular changes that were inhibited by AM-251 were {beta}-adrenoceptor-mediated events, secondary to LPS-induced sympathoadrenal activation (30) that involved, in part, an action of endocannabinoids. This is consistent with recent evidence to suggest that, in rats, cannabinoids can act centrally to enhance sympathetic tone (27). To test this hypothesis, experiments were performed to assess the effects of {beta}-adrenoceptor antagonism in the absence and presence of AM-251.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 DISCLOSURE
 REFERENCES
 
Male Sprague-Dawley rats (Charles River Laboratories; Kent, UK) were housed by groups (4–5 rats/cage) with free access to food (cat. no. BK001E, standard rat diet; BeeKay Foods) and tap water for at least 2 wk before any procedures were carried out. After this period, rats (then weighing 300–350 g) were anesthetized by an intraperitoneal injection of a mixture of fentanyl and medetomidine (300 µg/kg of each, supplemented as required). Through a midline laparotomy, miniaturized pulsed-Doppler probes were implanted around the left renal and superior mesenteric arteries and the distal abdominal aorta (to monitor hindquarters flow). All procedures have been published in detail previously (e.g., Refs. 15 and 16). After surgery, anesthesia was reversed with atipamezole and analgesia provided by nalbuphine (1 mg/kg sc of each), and animals were housed individually. Seven to fourteen days after probe implantation, animals were anesthetized (as above) and had an intra-arterial catheter implanted (distal abdominal aorta via ventral caudal artery) to allow monitoring of arterial blood pressure and heart rate. Three intravenous catheters were implanted (right jugular vein) to allow separate administration of substances during the experimental days (15, 16). At this stage, the wires from the pulsed-Doppler probes were soldered into a microconnector (Microtech; Boothwyn, PA) clamped into a custom-designed harness fitted to the rat. The catheters, which emerged from the same site on the nape of the neck as the probe wires, ran through a counter-balanced flexible spring attached to the harness. After catheterization, animals were housed individually in the cages in which they remained for the duration of the experimental protocol, unrestrained, and with free access to food and water. During periods when recordings were not being made, fluid-filled, double-channel swivels (3) were used to permit infusion of heparinized (15 U/ml) saline through the arterial catheter (to ensure patency) and continuous intravenous substance administration. All procedures were approved by the local Ethical Review Committee and were performed under Home Office Licence authority.

One day after catheterization, recordings were begun, and the following experiments were performed,

Regional hemodynamic changes during LPS infusion in the presence of vehicle or AM-251. In other in vivo experiments with AM-251, we produced clear evidence that at a dose of 3 mg/kg (infused intravenously over 30 min at 2 ml/h), the antagonist significantly inhibited responses to exogenous CB1 receptor agonists (16). Therefore, in one group of rats (n = 8), we infused AM-251 (as above) 30 min before the onset of a 24-h infusion of LPS (150 µg·kg–1·h–1). From our previous experiments (16), we had evidence that the dose of AM-251 was effective for at least 6 h, but to ensure continued antagonism, AM-251 was readministered at 6 and 24 h after the onset of LPS infusion. In a separate group of rats (n = 10), the same protocol was followed but with the vehicle for AM-251 (isotonic saline containing 5% propylene glycol and 2% Tween 80) being administered together with LPS (as above).

This first experiment (see RESULTS) showed that only the LPS-induced tachycardia and hindquarter vasodilatation were attenuated by AM-251. Because {beta}-adrenoceptor activation causes tachycardia and hindquarter vasodilatation in rats (36) and {beta}-adrenoceptors mediate the hindquarter vasodilator effect of synthetic and endogenous cannabinoids (15, 16), we hypothesized that the LPS-induced cardiovascular changes inhibited by AM-251 (i.e., tachycardia and hindquarter vasodilatation) were {beta}-adrenoceptor-mediated events. We tested this hypothesis first by treating animals with propranolol, and then, to determine the extent of contribution of antagonism of {beta}2-adrenoceptors to the effects of propranolol, we carried out an additional experiment with the {beta}2-adrenoceptor-selective antagonist dl-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol (ICI-118551) (2).

Regional hemodynamic changes during LPS infusion in the presence of propranolol or ICI-118551. On day 1, a group of rats (n = 7) was given saline (0.1 ml bolus, 0.4 ml/h infusion) starting 15 min before the onset of saline infusion (0.4 ml/h), with saline then being continuously infused for 24 h. These animals on day 3 were given saline (as above) starting 15 min before LPS infusion (as above).

A second group of rats (n = 8) had propranolol (1 mg/kg bolus, 0.5 mg·kg–1·h–1 infusion) administered starting 15 min before saline infusion (as above) for 24 h starting on day 1. On day 3, these animals had propranolol (dose as above) administered before LPS (as above) with both substances coinfused for 24 h. During the period of recording, the LPS and propranolol were administered through separate catheters, but for overnight infusion via the swivel system, a mixture of propranolol and LPS was prepared. The dose of propranolol used has been shown to abolish hemodynamic responses to isoprenaline (87 ng/kg; Ref. 38).

On day 1, a third group of rats (n = 8) was given ICI-118551 (0.2 mg/kg bolus, 0.1 mg·kg–1·h–1 infusion) starting 15 min before saline infusion (0.4 ml/h), with both infusions being continued for 24 h thereafter. On day 3, these animals were given ICI-118551 (as above) together with LPS (150 µg·kg–1·h–1) with substance administrations through separate catheters during the recording period and in a mixture for overnight infusion. The dose of ICI-118551 used has been shown to markedly inhibit the hemodynamic responses to salbutamol (600 ng·kg–1·h–1 for 3 min; Ref. 16). A fourth group of rats (n = 7) was given propranolol (dose as above) 24 h after the onset of LPS infusion.

Regional hemodynamic changes during LPS infusion in the combined presence of propranolol and AM-251. Because there was some similarity between the effects of AM-251 and of propranolol on LPS-induced changes (see RESULTS), it was possible that the antagonists were acting through a common pathway. Thus, in the final experiment, rats were given LPS in the combined presence of AM-251 and propranolol (doses and timings as above; n = 8) or the vehicle for AM-251 plus saline (n = 6). On the first day, AM-251, propranolol, and LPS were administered through separate catheters. For overnight administration, a mixture of LPS and propranolol was prepared.

Data analysis. On the first experimental day, data were recorded continuously (starting at ~0700 h) for an initial control period of 45 min before any intervention and for 8 h after the onset of any infusion. On the second experimental day, data were collected for 1–2 h, beginning at around 0700 h (i.e., between 24 and 26 h after onset of any treatment). The data obtained during the control period on the first experimental day were averaged electronically, and all subsequent changes were calculated relative to this average. Measurements were made off-line, using software that interfaced with the data acquisition system (Instrumentation Laboratories, University of Maastricht, Maastricht, The Netherlands). The values represent electronic averages of 10–15 min epochs around the selected time points. These data were then extracted into a custom-designed statistical software package. Within-group data were analyzed by Friedman's test (32), and between-group analysis by Mann-Whitney U-tests or Kruskal-Wallis tests (as appropriate). Between-group differences were assessed at the time of peak hypotension (1.25 h) and at the end of the experiment (24 h and 24.25 h) after the onset of LPS infusion with the Holm-Bonferroni correction applied for multiple comparisons. Data are expressed as means ± SE; P ≤ 0.05 was taken as significant.

Drugs. Fentanyl citrate was from Janssen-Cilag (High Wycombe); medetomidine hydrochloride (Domitor) and atipamezole hydrochloride (Antisedan) were from Pfizer (Sandwich, UK); and nalbuphine hydrochloride (Nubain) was from Bristol-Myers Squibb (Hounslow, UK). AM-251, ICI-118551 hydrochloride, and propranolol hydrochloride were obtained from Tocris (Avonmouth, UK). LPS (Escherichia coli serotype 0127 B8) was purchased from Sigma (Dorset, UK) and dissolved in sterile isotonic saline. Solutions of AM-251 were prepared fresh daily in sterile saline containing 5% propylene glycol (Sigma) and 2% Tween 80 (BDH/VWR, Dorset, UK). ICI-118551 and propranolol were dissolved in sterile water. Bolus injections were given in a volume of 0.1 ml; AM-251 was infused at a rate of 2 ml/h for 30 min. All other infusions were given at a rate of 0.4 ml/h. There were no cardiovascular effects attributable to administration of vehicle at these volumes.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 DISCLOSURE
 REFERENCES
 
Cardiovascular changes during LPS infusion in the presence of vehicle or AM-251. As noted previously (15, 16), administration of AM-251 had no significant cardiovascular effects, although there were some behavioral actions, notably grooming, that influenced cardiovascular variables at the time they occurred but that had generally passed before administration of LPS was started. Thus resting hemodynamic variables before administration of LPS in the presence of vehicle or AM-251 were not significantly different [heart rate, 311 ± 14 and 333 ± 11 beats/min; mean arterial blood pressure, 99 ± 2 and 106 ± 2 mmHg; renal vascular conductance, 81 ± 8 and 87 ± 7 kHz/mmHg x 103; mesenteric vascular conductance, 116 ± 10 and 106 ± 11 kHz/mmHg x 103; hindquarters vascular conductance, 36 ± 3 and 37 ± 3 kHz/mmHg x 103, respectively]. In animals given vehicle/LPS, there was a marked and sustained tachycardia, a triphasic change in mean arterial blood pressure (rise, fall, rise), renal vasodilatation, variable mesenteric vasoconstriction, and an early marked but transient and a delayed more sustained hindquarter vasodilatation (Fig. 1). Animals receiving LPS after pretreatment with AM-251 showed a significantly less marked tachycardia and a reduction in the hindquarter vasodilatation, particularly the delayed phase (Figs. 1 and 2). Readministration of AM-251 at 6 and 24 h after the onset of LPS infusion had no discernible additional cardiovascular effects (Fig. 1).



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  		Fig. 1. Cardiovascular responses to continuous 24-h infusion of LPS (150 µg·kg–1·h–1) in conscious Sprague-Dawley rats pretreated with vehicle (closed circles, n = 10) or AM-251 (3 mg/kg; open circles, n = 8). Values are means, and vertical bars represent SE. *P ≤ 0.05 vs. the original baseline (Friedman's test). Arrows indicate additional administrations of AM-251.

 


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  		Fig. 2. Changes in cardiovascular variables at 1.25 h and 24 h after the onset of LPS infusion in rats treated with vehicle (open bars, n = 10) or AM-251 (filled bars, n = 8). Values are means, and vertical bars represent SE. *P ≤ 0.05 vs. vehicle-treated animals (Mann-Whitney U-test).

 
Cardiovascular changes during LPS infusion in the presence of propranolol or ICI-118551. Resting cardiovascular variables in the three groups of rats pretreated before administration of saline (day 1) and LPS (day 3) are shown in Table 1. Neither propranolol nor ICI-118551 had any significant effects on heart rate or hindquarter vascular conductance in these animals, possibly indicating a low level of sympathetic tone.


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  		Table 1. Resting cardiovascular variables

 
In animals treated with saline/saline, propranolol/saline, or ICI-118551/saline, there were no significant changes (Fig. 3). In animals treated with saline/LPS (Fig. 4), the general pattern of cardiovascular changes was similar to that described above for vehicle/LPS-treated rats, although, at the 24-h time point, mean arterial blood pressure was not above the baseline, and there was no significant mesenteric vasoconstriction (compare Figs. 1 and 4).



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  		Fig. 3. Cardiovascular responses to continuous 24-h infusion of saline (0.4 ml/h) in conscious Sprague-Dawley rats treated with saline (closed circles, n = 7), ICI-118551 (0.2 mg/kg, 0.1 mg·kg–1·h–1; open squares, n = 8). or propranolol (1 mg/kg, 0.5 mg·kg–1·h–1; open circles, n = 8). Values are means, and vertical bars represent SE.

 


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  		Fig. 4. Cardiovascular responses to continuous 24-h infusion of LPS (150 µg·kg–1·h–1) in conscious Sprague-Dawley rats treated with saline (closed circles, n = 7), ICI-118551 (0.2 mg/kg, 0.1 mg·kg–1·h–1; open squares, n = 8), or propranolol (1 mg/kg, 0.5 mg·kg–1·h–1; open circles, n = 8). Values are means, and vertical bars represent SE. *P ≤ 0.05 vs. the original baseline (Friedman's test).

 
Pretreatment with propranolol inhibited the early (at 1.25 h) tachycardia seen during LPS infusion, although heart rate still increased gradually (Figs. 4 and 5). However, by 24 h after the onset of LPS infusion in the presence of propranolol, the tachycardia was still inhibited (Figs. 4 and 5). Pretreatment with propranolol prevented the hypotensive effects of LPS (at 1.25 h, Figs. 4 and 5). In addition, propranolol prevented early (at 1.25 h) and late (at 24 h) hindquarter vasodilator effects of LPS infusion (Figs. 4 and 5).



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  		Fig. 5. Changes in cardiovascular variables at 1.25 h and 24 h after the onset of LPS infusion in rats treated with vehicle (open bars, n = 7), propranolol (filled bars, n = 8), or ICI-118551 (hatched bars, n = 8). Values are means, and vertical bars represent SE. *P ≤ 0.05 vs. saline-treated animals (Kruskal-Wallis test).

 
Pretreatment with ICI-118551 did not affect the early (at 1.25 h) increase in hindquarter vascular conductance. However, in the presence of ICI-118551, the delayed (at 24 h) hindquarter vasodilator response to LPS was significantly reduced (Figs. 4 and 5).

In contrast to the effects of pretreatment, administration of propranolol 24 h after the onset of LPS infusion reduced, but did not abolish, the tachycardia (83 ± 13 beats/min at 24 h down to 47 ± 10 beats/min at 25 h) and hindquarter vasodilatation (65 ± 17% at 24 h down to 35 ± 16% at 25 h); it had no effect on renal or mesenteric vascular beds (data not shown).

Cardiovascular changes during LPS infusion in the combined presence of propranolol and AM-251. In the presence of propranolol and AM-251, there was no tachycardia (–1 ± 14 beats/min) after infusion of LPS for 1.25 h (i.e., when the peak increase in heart rate occurred during infusion of LPS in the presence of vehicle; Fig. 6). This was in contrast to the residual tachycardia with LPS infusion in the presence of propranolol alone (37 ± 12 beats/min, Fig. 4) or AM-251 alone (29 ± 13 beats/min, Fig. 1). However, at this time, there was no significant change in hindquarter vascular conductance in the presence of propranolol and AM-251 (16 ± 16%, Fig. 6), as was the case in the presence of propranolol alone (15 ± 14%, Fig. 4).



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  		Fig. 6. Cardiovascular responses to continuous 24-h infusion of LPS (150 µg·kg–1·h–1) in conscious Sprague-Dawley rats treated with saline and AM-251 vehicle (closed circles, n = 6), or propranolol (1 mg/kg, 0.5 mg·kg–1·h–1) and AM-251 (3 mg/kg; open circles, n = 8). Values are means, and vertical bars represent SE. *P ≤ 0.05 vs. the original baseline (Friedman's test). Arrows indicate additional administrations of AM-251 or vehicle.

 
After infusion of LPS for 24 h in the presence of propranolol and AM-251, the changes in heart rate, mean blood pressure, and hindquarter vascular conductance (Fig. 6) were not different from the changes in the presence of propranolol alone (Fig. 4).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
DISCLOSURE
 REFERENCES
 
The major objective of the present work was to test the hypothesis that endocannabinoids are responsible for the regional vasodilatation in a model of endotoxemia that is characterized by marked peripheral vasodilatation offset by an increase in cardiac output with little or no change in blood pressure (9), i.e., a hyperdynamic profile similar to that seen at the onset of clinical sepsis, but before the progression to endotoxic shock (26). A secondary objective was to test the hypothesis that the cardiovascular events affected by the CB1 receptor antagonist AM-251 involved {beta}-adrenoceptors. The rationale behind this juxtaposition of objectives was our observation that a component of the hindquarter vasodilator response to cannabinoid agonists is mediated by {beta}2-adrenoceptors (15, 16), and others (33) have postulated an important vasodilator role for cannabinoids in endotoxic shock.

Hemodynamic responses to LPS: effects of AM-251. The hemodynamic changes that occurred during LPS infusion in the presence of either saline or vehicle were generally similar to those we (9, 10) described previously on several occasions. Thus an early onset and sustained renal vasodilatation, tendency for mesenteric vasoconstriction, and a biphasic hindquarter vasodilatation are characteristic of this model.

The most clear-cut effects of AM-251 on the responses to LPS infusion were inhibition of the tachycardia and of the delayed hindquarter vasodilatation. Three possible interpretations of this finding are that 1) activation of CB1 receptors by endocannabinoids was directly responsible for the AM-251-sensitive cardiovascular events; 2) pretreatment with AM-251 influenced the inflammatory response to LPS (6, 31) and, thereby, modulated the cardiovascular responses; or 3) the LPS-induced cardiovascular changes inhibited by AM-251 were {beta}-adrenoceptor-mediated events secondary to LPS-induced sympathoadrenal activation (30).

Because {beta}-adrenoceptor activation causes tachycardia and hindquarter vasodilatation (36), and we have recently shown an involvement of {beta}-adrenoceptors in the hindquarter vasodilator effect of synthetic and endogenous cannabinoids (15, 16), the third alternative formed the hypothesis we tested in experiments investigating the possible involvement of {beta}-adrenoceptors in the cardiovascular changes seen with LPS infusion.

Hemodynamic responses to LPS: effects of {beta}-adrenoceptor antagonism. We found that pretreatment with propranolol abolished both the LPS-induced hindquarter vasodilatation and the marked tachycardia at 24 h, consistent with these responses being due to increased sympathoadrenal activity. The latter has been demonstrated in conscious endotoxemic rats, albeit after bolus injection of LPS (30). Pretreatment with the {beta}2-adrenoceptor-selective antagonist ICI-118551 caused clear inhibition of the delayed (at 24 h) hindquarter vasodilatation seen during LPS infusion, indicating that the ability of propranolol to inhibit this phenomenon was likely due to its {beta}2-adrenoceptor antagonistic effects.

The results seen around 1.25 h after the onset of the LPS infusion are less straightforward to explain. Thus propranolol clearly inhibited the hindquarter vasodilatation and abolished the associated hypotension at that stage, whereas ICI-118551 did not affect the magnitude of the hindquarter vasodilatation. If {beta}-adrenoceptor activation was responsible for the hindquarter vasodilatation at that juncture, the difference between the effects of ICI-118551 and propranolol could theoretically be explained by more effective blockade of {beta}2-adrenoceptors by propranolol than by ICI-118551. But this is most unlikely because we have evidence that at the dose used here ICI-118551 causes almost complete inhibition of an increase of 134% in hindquarter conductance evoked by salbutamol (16). Conversely, it is feasible that the differing effects of ICI-118551 and propranolol were due to vascular {beta}1-adrenoceptors being involved in the early hindquarter vasodilatation after the onset of LPS infusion. Although most of the evidence suggests that adrenergic vasodilatation is mediated by {beta}2-adrenoceptors, there are some studies that show neurogenic vasodilatation, at least in some beds, may be mediated by vascular {beta}1-adrenoceptors (Ref. 24; for a review, see Ref. 20). However, experiments with the {beta}1-adrenoceptor-selective antagonist atenolol show no effect on the early hindquarter vasodilatation (S. M. Gardiner, J. E. March, P. A. Kemp, and T. Bennett, unpublished observations).

Alternatively, it is possible that propranolol was exerting effects that were not due to cardiovascular {beta}-adrenoceptor antagonism. For example, there is evidence that {beta}-adrenoceptors can influence LPS-induced production of a variety of pro- and anti-inflammatory cytokines (for review see Ref. 21) and inducible nitric oxide (NO) synthase-mediated NO production (4, 5). It is notable that treatment with propranolol 24 h after the onset of LPS infusion reduced, but did not abolish, the tachycardia and hindquarter vasodilatation. In contrast, when propranolol was given as a pretreatment, the tachycardia and hindquarter vasodilatation were inhibited to the extent that they did not differ from baseline. Hence, it might be speculated that the difference between the effects of propranolol given as a pretreatment versus posttreatment was due to the former inhibiting some aspect of the LPS-induced inflammatory process.

Hemodynamic responses to LPS: effects of combined CB1 and {beta}-adrenoceptor antagonism. Because there was some similarity between the effects of AM-251 and propranolol, inasmuch as both caused inhibition of the LPS-induced tachycardia and hindquarter vasodilatation at 24 h, experiments were performed with combined administration of the antagonists to determine whether or not the effects involved a common pathway.

Interestingly, the effect of combined administration of propranolol and AM-251 on the peak hindquarter vasodilator response to LPS infusion (at 1.25 h) was not different from that of propranolol, indicating that a common pathway involving {beta}-adrenoceptors was likely involved. In contrast, however, the combination of propranolol and AM-251 abolished the peak tachycardia at that time, whereas neither drug alone had such a marked effect. Thus it appears the increase in heart rate could have involved both propranolol- and AM-251-sensitive mechanisms.

Propranolol, AM-251, ICI-118551, and propranolol plus AM-251 all inhibited the delayed (at 24 h) hindquarter vasodilator response to LPS infusion, and AM-251, propranolol, and propranolol plus AM-251 inhibited the tachycardia. One interpretation of these findings is that endogenous cannabinoids were indirectly involved in {beta}-adrenoceptor activation at this time. Whereas another possible explanation of our findings is that, in vivo, AM-251 has antagonistic effects at {beta}-adrenoceptors, we found no influence of AM-251 on the hemodynamic response to salbutamol (S. M. Gardiner, J. E. March, P. A. Kemp, and T. Bennett, unpublished observations). In addition, there is no evidence that cannabinoids interact with {beta}-adrenoceptors (7).

A further interpretation of our results is that the propranolol- and AM-251-sensitive tachycardia and hindquarter vasodilatation seen after infusion of LPS for 24 h were due to modulation of the inflammatory process (see Hemodynamic responses to LPS: effects of {beta}-adrenoceptor antagonism). However, on balance, we consider the most likely explanation for our present findings is that enhanced {beta}-adrenoceptor activation is a major mechanism in the tachycardia and hindquarter vasodilatation seen in LPS-infused rats, and an involvement of endogenous cannabinoids in these effects, particularly the latter, could be via an influence on sympathoadrenal activity.

In conclusion, we have shown an involvement of {beta}-adrenoceptors in the cardiovascular consequences of LPS infusion and, on the basis of indirect evidence, suggest that modulation of these processes may account for the ability of the CB1 receptor antagonist AM-251 to suppress the positive chronotropic and hindquarter vasodilator responses to low-dose LPS infusion in conscious rats. Thus, although there is a wealth of in vitro evidence to suggest that endocannabinoids can cause vasodilatation via a number of different mechanisms (Ref. 28; see Introduction) and evidence that serum levels of endocannabinoids may be elevated in patients with endotoxic shock (37), our results indicate that it is unlikely that a direct vascular action of endogenous cannabinoids is involved in the vasodilatation associated with chronic LPS infusion. However, a limitation of this study is that we did not measure endocannabinoid levels under our experimental conditions.

Our results and conclusions clearly differ from those of Varga et al. (33), who showed that pretreatment of urethane-anaesthetized rats with a different CB1 receptor antagonist (SR-141716A) prevented the hypotensive response to a bolus dose of LPS (15 mg/kg) without affecting the tachycardia. Those authors also showed that LPS-activated macrophages contained anandamide and caused hypotension that was sensitive to SR-141716A and concluded that endocannabinoids "provided a novel paracrine mechanism for vasodilatation in endotoxic shock" (33). The two most obvious differences between their study and ours are the choice of antagonist and the endotoxemic model used.

In anesthetized rats, hypotensive responses to the endocannabinoid anandamide are blocked by SR-141716A, whereas the regional vascular effects of anandamide in conscious rats are not blocked by AM-251 (15). Recently, it has been proposed that there is a novel receptor involved in some of the cardiovascular effects of anandamide (8, 22) and that these effects are blocked by SR-141716A but not by AM-251 (8). Thus it is feasible that the reported effect of SR-141716A on LPS-induced hypotension (33) was not CB1 receptor mediated. Furthermore, because the hypotension associated with a bolus dose of LPS is more related to a fall in cardiac output than to vasodilatation (11), it is possible that the effects of SR-141716A were due to inhibition of a negative inotropic action of the endocannabinoids mediated by the recently identified novel receptor site (8). In this context, a very recent study from Kunos and colleagues (1) published after this work was completed has demonstrated that the ability of SR-141716A to block the hypotensive effect of bolus injection of LPS is not shared by AM-251. Moreover, the effect of SR-141716A was not due to inhibition of vasodilatation. These results fit well with our findings and interpretations (see above), but are inconsistent with the conclusion that endocannabinoids provide "a novel paracrine mechanism for vasodilatation in endotoxic shock" (33).


   DISCLOSURE
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURE
REFERENCES
 
Some of these results have been presented to the British Pharmacological Society (13).


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. M. Gardiner, Centre for Integrated Systems Biology and Medicine, School of Biomedical Sciences, Univ. of Nottingham, Nottingham NG7 2UH, UK (E-mail: sheila.gardiner{at}nottingham.ac.uk)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURE
 REFERENCES
 

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