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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. REFERENCES

Rimonabant (RM) is a cannabinoid CB1 receptor antagonist useful in the treatment of obesity associated cardiovascular risk factors. Since cannabinoids are vasoactive compounds, the aim of this study is to evaluate the effect of chronic treatment with RM on systolic blood pressure (SBP), and endothelial and vascular reactivity. Obese Zucker rats (OZRs) and their lean counterparts were orally treated during 20 weeks with either RM (10 mg/kg/day). Endothelial and vascular function was assessed in aorta and small mesenteric arteries (SMAs) by concentration response curves to acetylcholine (ACh) and phenylephrine (Phe), respectively. Participation of nitric oxide (NO) was evaluated by incubation with the NO synthase (NOS) inhibitor NG-nitro-l-arginine methyl ester (L-NAME) and cyclooxygenase (COX)-derived products involvement was analyzed by incubation with indomethacin (INDO). Plasma lipid profile, insulin and adiponectin were also analyzed. Sympathetic activity was evaluated by urinary excretion of noradrenaline. As expected, RM decreased body weight gain and enhanced adiponectin concentration. Insulin resistance and sympathetic activity were also decreased. The increase in SBP observed in OZRs was reduced by treatment with RM. Aortae and SMAs from OZRs exhibited lower contractile response to Phe, being this effect prevented by RM administration. Although ACh-induced response and NO participation remained unaltered with obesity, enhanced COX-derived constrictor products were found in OZRs. RM treatment neither altered endothelium-dependent relaxation nor L-NAME-sensitive component of the response. Nevertheless, it was able to regulate COX-derived vasoactive products participation. Those effects may contribute to explain some of the cardiovascular protective actions elicited by this drug.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. REFERENCES

Obesity is a medical disorder characterized by the overweight but also by the association to comorbidities such as hypertension, dyslipidemia, and diabetes. The risk of hypertension is higher among obese people and up to two-thirds of cases of hypertension are linked to excess weight (1). Obesity is also associated to an increase in proatheromatous dense small-particle-sized low-density lipoprotein, a decrease of high-density lipoprotein (HDL) and a higher triglyceride concentration. The frequent simultaneous presence of these facts led to the definition of the metabolic syndrome (MS) as the presence of increased waist circumference and two other cardiovascular risk factors: hyperglycemia, hypertriglyceridemia, low HDL, and/or hypertension (2). Obesity has also been related to a great extent to an overactivation of the endocannabinoid system, what may explain the increase of food intake and the development of the cardiometabolic risk that accompanied the weight gain (3,4,5). Finally, it is well established that obesity and MS impair the vasodilating properties of the endothelium leading to a dysfunction, which in turn can be considered the first step in the progression of cardiovascular disease (6).

Rimonabant (RM) is an antagonist of the cannabinoid receptor CB1 that has demonstrated to be useful in the treatment of obesity associated to cardiovascular risk factors (7,8,9). The regulation of appetite and body weight exerted by cannabinoids is known to be CB1 receptor mediated. Thus, one of the most widely accepted explanation for the efficacy of CB1 receptors antagonists against obesity and related cardiometabolic disorders may be the reduction of the central and peripheral overactivity of endocannabinoid system (10,11,12). Endogenous cannabinoids such as anandamide, also contribute to the regulation of vascular tone evoking the release of prostanoids (13), NO (14) and endothelium-derived hyperpolarizing factor (EDHF) (15). However, the mechanism underlying those effects remains controversial and may involve activation of CB1 but also of TRPV-1 (16,17).

The aim of our study is to evaluate the effects of a chronic treatment with the CB1 antagonist RM on blood pressure and vascular and endothelial function in obese Zucker rats (OZRs), which is a common animal model of obesity and MS. These animals are characterized by the overweight, hyperlipidemia and hyperinsulinemia (18) and several studies have found that they develop mild hypertension at the 22–25 week of age (19,20,21,22).

Methods and Procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. REFERENCES

Animals

OZRs and their littermate controls, lean Zucker rats (LZRs) (11 weeks aged, Charles River Laboratories, Barcelona, Spain), were fed with standard diet and water ad libitum. OZRs and LZRs were divided into two groups and daily treated with either RM (10 mg/kg/day, RM-OZR-n = 10- and RM-LZRs-n = 5-, respectively) or its vehicle (carboximethylcelullose 0.5%, vehicle-OZR-n = 10- and vehicle-LZRs-n = 10-, respectively). Both treatments were administered during 20 weeks by intragastric gavage. The selected dose is reported to reduce OZR body weight gain and food intake (23). As pair-fed controls, a third group of OZRs (n = 5) and LZRs (n = 5) were dosed with vehicle and given a daily food allotment equal to that consumed by the RM-treated counterpart during the treatment period.

Body weight, food and water intake, and systolic blood pressure (SBP) were weekly evaluated. SBP and heart rate was measured by the pneumatic “tail-cuff” method with pressure meter (Niprem 645, Cibertec, Madrid, Spain). Urine was collected for 24 h in a metabolic cage. Rats were anesthetized and killed with chloral hydrate 12% intraperitoneally. Blood samples were collected by intracardiac puncture for biochemical assays. Visceral adipose tissue was removed and weighted and thoracic aorta and mesenteric vascular bed were isolated. The protocol for animal handling and experimentation agreed with the European Union European Community guidelines for the ethical treatment of animals (EEC Directive of 1986; 86/609/EEC) and was approved by the Ethical Committee for Animal Research of the University of Seville.

Blood and urine biochemical assays

Serum samples were obtained from blood by centrifugation. Triglycerides, total cholesterol, HDL-cholesterol were assessed by UV/visible spectrophotometry (Roche/Hitachi Modular P, Roche Diagnostics, Mannheim, Germany). Plasma adiponectin and insulin were measured by commercial enzyme-linked immunosorbent assay (ELISA) kits (Linco Research, St. Charles, MO and Spi-Bio, Montigny le Bretonneux, France, respectively). The homeostasis model assessment of insulin resistance was calculated as described (24). Urinary noradrenalin concentrations were measured using high-performance liquid chromatography as reported by Zydron et al. (25).

Assessment of vascular reactivity

Arterial preparation and mounting. Aortas and small mesenteric arteries (SMAs) were dissected and placed in modified Krebs–Henseleit bicarbonate solution, composition in mmol/l: NaCl 118, KCl 4.75, NaHCO3 25, MgSO4 1.2, CaCl2 1.8, KH2PO4 1.18, and glucose 11. Aortic rings and SMAs were mounted on myographs and mechanical activity was measured as previously described (26). Contractile capacity of the vessels was assessed with either phenylephrine (Phe) 1 µmol/l or KCl 80 mmol/l prior to relaxation or contraction experiments, respectively. The presence of functional endothelium was assessed by the ability of acetylcholine (Ach) 1 µmol/l to induce more than 50% relaxation of precontracted vessels.

Contraction and relaxation experiments. Arteries were exposed to cumulative concentrations of Phe (0.01–30 µmol/l for aortas and 1 nmol/l–10 µmol/l for SMAs) to obtain concentration–response curves. Endothelium-dependent relaxation was studied by cumulatively adding ACh (0.01–30 µmol/l) to precontracted arteries with Phe. Concentration–response curves to ACh were constructed in the absence or in the presence of the indicated inhibitors: the NO synthase (NOS) inhibitor NG-nitro-l-arginine methyl ester (L-NAME, 300 µmol/l), the nonselective cyclooxygenase (COX) inhibitor, indomethacin (INDO 10 µmol/l). Besides, the putative “EDHF-mediated,” non-NO and nonprostacyclin endothelium-dependent relaxations of SMAs were assessed using the combination L-NAME plus INDO. KCl 25 mmol/l was added to the Krebs–Henseleit bicarbonate solution to completely abolish ACh-induced relaxation.

Expression of results and statistical analysis

Data represented are means ± s.e.m. of n = 5 or 10 rats depending on the group. SBP was expressed in mm Hg. Vascular contraction was expressed in g/mg of dry tissue for the aorta and in mN/mm for the SMAs. Relaxation was expressed as a percentage of the precontraction. Two-way ANOVA and unpaired Student's t-test were used to compare data. Differences were considered significant when P < 0.05. A StatView Software 5.0 (SAS Institute Inc., Cary, NY) was used for statistical analysis.

Drugs and chemicals

RM was gently provided by Sanofi-Aventis, Madrid, Spain. All the chemicals were purchased from Sigma Chemical Co (St. Louis, MO). All drugs were prepared in distilled water, except for INDO that was dissolved in dimethylsulfoxide. The final concentration of dimethylsulfoxide was <0.01%, which was shown to have no effect on the vascular reactivity.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. REFERENCES

Effect of chronic treatment with RM on body weight and biochemical parameters

OZRs showed higher values of body weight and visceral adipose tissue than LZRs (Figure 1, P < 0.001 and Table 1, P < 0.001). RM significantly reduced body weight gain over the 20-week study in OZRs (P < 0.01), but not in LZRs. Pair-fed obese controls exhibited a similar drop in body weight to that showed by their RM-treated obese counterparts (Figure 1). RM treatment did not alter visceral fat content from neither OZRs nor LZRs. This value was neither affected in the pair-fed control group (Table 1).

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Figure 1. Weight evolution of obese and lean Zucker rats (OZRs and LZRs) during the 20 weeks of treatment with either vehicle or rimonabant (RM). Data are mean ± s.e.m. of n = 5 or 10 depending on the group. *P < 0.05; **P < 0.01 vs. vehicle-treated group. ###P < 0.001 LZRs vs. OZRs.

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Table 1.  In vivo measures to OZR and LZR treated with either RM or vehicle
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Daily food intake was significantly higher in OZRs compared to LZRs (Table 1, P < 0.001). RM decreased food consumption in OZRs but not in LZRs throughout the treatment toward values similar to that observed in control LZRs. Both vehicle- and RM-treated OZRs drunk less water than LZRs (Table 1, P < 0.01).

OZRs presented eight and twofold higher level in serum of triglycerides and cholesterol, respectively, than the lean (Table 2, P < 0.001). Treatment of OZRs with RM led to a decrease in the triglyceride concentration (Table 2, P < 0.05). On the other hand, both groups receiving RM exhibited higher values of total cholesterol. Finally, HDL cholesterol was higher in RM-treated OZRs than in both vehicle-treated groups (Table 2, P < 0.05). Total cholesterol/HDL cholesterol ratio was higher in OZRs, treated or not with RM, than in LZRs (Table 2, P < 0.001). After RM treatment, an appreciable reduction of cholesterol/HDL cholesterol ratio was found in OZRs, even though a statistically significant difference was not achieved (Table 2). OZRs exhibited higher concentrations of insulin than the lean (Table 2, P < 0.05). Although RM reduced insulin concentration in OZRs, the differences among values were not statistically significant. RM did not altered the fasting hyperglycemia (Table 2, P < 0.05), but it was able to reduce the increased homeostasis model assessment of insulin resistance displayed by the OZRs (Table 2, P < 0.001 vs. LZRs and P < 0.05 vs. RM-OZRs). OZRs showed lower adiponectin in serum (Table 2, P < 0.01), being this level enhanced by RM treatment (P < 0.05).

Table 2.  Blood and urine measures made to LZR and OZR treated with either RM or vehicle
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Effect of RM SBP, heart rate, and sympathetic activity

Initial SBP values were higher in LZRs (141.79 ± 2.13 mm Hg) than in OZRs (121.11 ± 1.22 mm Hg, P < 0.001), what resulted inversed at the end of the study (140.87 ± 1.47 mm Hg in vehicle-LZRs and 164.74 ± 3.79 mm Hg in vehicle-OZRs, P < 0.001). Consequently, the increase in SBP found in OZRs from the beginning to the end of the treatment was higher than that displayed by LZRs (Table 1, P < 0.01). Although RM treatment did not affect the final SBP values (151.79 ± 4.28 mm Hg and 159.36 ± 2.86 mm Hg in RM-LZRs and RM-OZRs, respectively), the increase in SBP was partially prevented in both groups (Table 1, P < 0.05 vs. LZRs and P < 0.01 vs. OZRs, respectively). A similar decrease in the SBP to that displayed by RM-treated rats was observed in the pair-fed control groups (Table 1). Nevertheless, any statistical difference was found among those results. During the whole treatment, RM-treated OZRs exhibited lower heart rate than LZRs (Table 1, P < 0.001). RM increased OZRs heart rate toward values observed in LZRs (Table 1, P < 0.001 vs. OZRs). Such effect seems to be weight loss–independent because it was not observed in the pair-fed fatty control group (Table 1, P < 0.001 vs. LZRs).

There was not any difference in the sympathetic activity evaluated by urinary excretion of noradrenalin among lean and obese animals (Table 2). RM treatment reduced urinary noradrenalin in LZRs and OZRs (Table 2, P < 0.05).

Effect of RM chronic treatment on contraction response to phenylephrine

Phe induced concentration-dependent contraction of both types of arteries (Figure 2a,b). Aorta and SMAs from OZRs showed a lower response to Phe than LZRs (Figure 2a and b, P < 0.001). Arteries from OZRs receiving the CB1 antagonist exhibited a greater response to Phe than those from vehicle-treated (Figure 2a,b, P < 0.05 and P < 0.01, respectively).

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Figure 2. Concentration–response curves to phenylephrine (Phe) (a and b) and ACh (c and d) of aorta (a and c) and SMAs (b and d) from obese (OZRs, open squares and closed squares) and lean Zucker rats (LZRs, open circles and closed circles) treated with either vehicle (open circles and open squares) or rimonabant (RM, closed circles and closed squares). Data are mean ± s.e.m. of n = 5 or 10 depending on the group. *P < 0.05; **P < 0.01 vehicle vs. RM-treated. #P < 0.05; ###P < 0.001 OZRs vs. LZRs.

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Effect of RM on endothelium-dependent relaxation and characterization of endothelial factors involved

ACh induced relaxation of Phe-contracted aortas and SMAs with endothelium, but failed to induce relaxation in endothelium-denuded arteries (data not shown). OZRs showed a diminished response induced by ACh in the aortas (Figure 2c, P < 0.05), whereas not differences between LZRs and OZRs were observed in the effect of the muscaric agonist in the SMAs (Figure 2d). RM treatment did not alter ACh-induced relaxation or aorta or SMAs (Figure 2c,d).

To investigate the role of NO in concentration–response curves to ACh, those were constructed in the presence or in the absence of the NOS inhibitor, L-NAME. Relaxation to ACh was abolished in aorta after L-NAME incubation (Figure 3, P < 0.001). However, SMAs showed a residual relaxation resistant to NOS inhibition (Figure 4, P < 0.01 and P < 0.001). These effects were neither affected by obesity nor by RM treatment.

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Figure 3. Endothelium-dependent response to acetylcholine (ACh) of aortic rings from lean (LZRs, a and b) and obese Zucker rats (OZRs, c and d) treated with either vehicle (a and c) or rimonabant (RM, b and d). Curves were done in the absence (open circles) or in the presence of indomethacin (INDO, closed circles) or L-NAME (closed squares). Curves in the absence of inhibitors were taken as control. Data are mean ± s.e.m. of n = 5 or 10 depending on the group. *P < 0.05; ***P < 0.001 vs. control.

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image

Figure 4. Endothelium-dependent response to acetylcholine (ACh) of small mesenteric arteries (SMAs) from lean (LZRs, a and b) and obese Zucker rats (OZRs, c and d) treated with either vehicle (a and c) or rimonabant (RM, b and d). Curves were done in the absence (open circles) or in the presence of indomethacin (INDO, inverted triangles), L-NAME (closed circles), indomethacin plus L-NAME (closed triangles) or indomethacin plus L-NAME plus KCl 25 mmol/l (closed squares). Curves in the absence of inhibitors were taken as control. Data are mean ± s.e.m. of n = 5 or 10 depending in the group. *P < 0.05; **P < 0.01; ***P < 0.001 vs. control.

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After COX inhibition by INDO, vasodilatation to ACh was partially inhibited in aortas from control LZRs (Figure 3a, P < 0.05), but not from control OZRs (Figure 3c), suggesting the involvement of COX-derived relaxant products. After RM treatment, INDO did not exert any effect on ACh-induced response of aortas from either LZRs or OZRs (Figure 3b,d, respectively). In SMAs, INDO increased the relaxant response to ACh in control LZRs (Figure 4a) and OZRs (Figure 4c, P < 0.05), but decreased (Figure 4b, P < 0.05) or remained unaltered (Figure 4d) such response in RM-treated LZRs and OZRs, respectively.

The combination INDO plus l-name inhibited the relaxation induced by ACh in SMAs from all the groups (Figure 4, P < 0.01 and P < 0.001). However, it was not able to completely abolish the L-NAME-resistant residual relaxation described above. These data indicate that the EDHF could participate in the response to ACh of these arteries. To test the involvement of the hyperpolarization in the persisting relaxation, concentration–response curves to ACh in the presence and the absence of the combination L-NAME+INDO+KCl 25 mmol/l (Figure 4). As expected, this combination led to a total inhibition of the ACh-induced effect, which was observed in all groups (Figure 4, P < 0.001).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. REFERENCES

Obesity is characterized by increased body weight, excessive fat tissue and deregulation of the endocrine function of adipose tissue and is currently associated to insulin resistance, hypertriglyceridemia, and inflammation. When central obesity, insulin resistance, dyslipidemia, and hypertension appear as a cluster in the same patient, the existence of a generalized metabolic disorder could be considered, being this disorder known to a great extent as MS (2). RM is a CB1 receptor antagonist, useful in the treatment of obesity when it is accompanied by diabetes mellitus type 2 and/or dyslipemia since clinical trials indicate that RM not only reduces body weight but also improves cardiovascular risk factors (7,8,9). Indeed, treatment with RM has been described to decrease systolic and diastolic blood pressure, being this effect greater among patients with hypertension (27). In contrast, treatment of spontaneously hypertensive rats with RM resulted in an increase of blood pressure (28). Moreover, endocannabinoids have been traditionally considered to play a favorable role in the cardiovascular system because their vasodilatatory effect (13,14,15) and their capacity to decrease blood pressure in anesthetized animals (29). Nevertheless, there is no general consensus regarding its molecular target(s), being some of the vascular actions of cannabinoids explained through TRPV-1 activation (16,17).

The aim of this work was to clarify the hemodynamic and vascular effects of the oral and chronic treatment with RM in an animal model of MS, as well as to highlight the possible mechanisms that lead to such effects. Our results show that the CB1 antagonist prevented the obesity-induced increase of SBP observed in OZRs. An improvement of the insulin resistance, lipid profile or an increase of adiponectin plasma levels could be involved in such prevention. RM also prevented the decrease of heart rate observed in OZRs, decreased sympathetic activity, and increased vascular contraction induced by α-adrenergic agonists. However, further studies are needed to better understand the later effects.

As expected, long-term oral treatment with RM led to a decrease of the body weight, probably due to the parallel decrease in food intake in OZRs (30). Although the lower lipid intake within diet may contribute to explain the decrease in triglyceride plasma concentrations, additional reasons are required to completely explain that result, being the decrease on insulin resistance found after treatment with RM probably involved in that finding. Insulin resistance is defined as the condition in which normal amounts of insulin are inadequate to produce a normal insulin response from fat, muscle and liver cells. As a result, hyperglycemia, hypertriglyceridemia, high free fatty acids levels and hyperinsulinemia are achieved. High homeostasis model assessment of insulin resistance values (24), as well as low adiponectin plasma concentrations (31) are being used as predictors of such situation. Accordingly, our results showed both an insulin resistance state in OZRs, but not in LZRs and a substantial improvement of the sensitivity to insulin after chronic treatment with RM. An enhanced capability of fatty storage on the part of adipocytes may well be responsible for such decrease of the triglyceride plasma concentration. The increase of blood adiponectin levels agrees with that hypothesis and can also be involved in the improvement of the insulin resistance state. Another remarkable effect of treatment with RM was the increase in HDL cholesterol in OZRs. In agreement with that result, most of the RIO studies showed that the increase in HDL cholesterol levels elicited by RM was greater than the average increase induced by fibrates and similar to that associated with nicotinic acid administration (32). In this study, such result is accompanied by a likely consequent increase of total cholesterol. Nevertheless, these higher levels of cholesterol were not enough to increase the total cholesterol/HDL cholesterol ratio, value that was even slightly lower in OZRs receiving chronic treatment with RM.

As obesity is closely related to the development of endothelial dysfunction and cannabinoids can act on endothelial cells, one of the objectives of this study was to evaluate the effects on endothelial and vascular function after selectively antagonize the CB1 receptor as antiobesity treatment during 20 weeks. There are several mechanisms by which obesity negatively influences endothelial physiology (for review see ref. 5). In this study, endothelial function was evaluated by the responses to the vasodilator agonist ACh. Some authors found that either aortic rings (22) or mesenteric arteries (21) from OZRs exhibited lower response to ACh. On the contrary, an enhanced vasorelaxant response induced by the same agonist has been previously described in OZRs compared to LZRs (33). In our study, a lower response to ACh was observed in the aorta of OZRs, but not in the SMAs. Although global response to ACh was not affected by obesity in resistance arteries, the presence of the COX inhibitor was able to enhance the vasodilatation from OZRs but not in LZRs, revealing the presence of contracting factors derived from COX in that arteries. This result agrees with the previous observation of a higher urinary excretion of TXB2, stable metabolite of the vasoconstrictor COX product, TXA2 (19).

Treatment with RM did not elicit any changes in the endothelium-dependent response to ACh in aortic rings or SMAs. Further, the inhibition of this response observed after blocking NOS remained unaltered after administration of RM. On the contrary, the presence of INDO revealed the absence of contracting COX-derived products in SMAs from OZRs treated with RM. Incubation with this inhibitor of resistance arteries from LZRs treated with RM, was even able to reduce the relaxant response to ACh, suggesting the involvement of the vasodilatatory PGI2. The fact that the ratio of PGI2 to TXA2 metabolite excretion was reduced in 30-week-old obese rats (19), can help to explain the greater effect of RM in LZRs than in OZRs. Several facts contribute to explain the effect of RM on COX-derived vasoactive action. Although accurate mechanisms explaining endocannabionids effects on endothelium have not been elucidated, several studies report that a COX-2 related pathway may be involved. As a mechanism of vasodilatation, it has been suggested that anandamide may be converted to arachidonic acid by fatty acid amidohydrolase and this arachidonic acid converted to prostaglandins by COX. More exactly, a CB1 receptor-mediated upregulation of COX-2 expression and a consequent production of vasodilatadory factors have been involved in the effect of anandamide in the cerebral microcirculation (34). Accordingly, RM decreased COX-2 expression in human peripheral blood mononuclear cells (35) and amnion (36). Thus, the CB1antagonism may well regulate COX-2-derived production of prostanoids. Supporting this hypothesis an increase of COX-2 derivate vasoconstrictors factors and a consequent increase in pulmonary arterial pressure has been reported in isolated rabbits lungs after perfusion with endocannabinoids (37). In contrast, other studies pointed out a CB1 receptor-independent anandamide effect on COX-2 expression regulation (17). Further studies are needed to clarify the effects of long-term CB1 blockade on vasoactive COX-derived products and on COX expression and/or activity in arterial vessels.

The enhanced sensitivity to insulin resulting of RM treatment can contribute to explain that effects of RM on COX-derived vasoactive products, because insulin resistance reduces arterial prostacyclin synthase activity by increasing endothelial fatty acid oxidation (38). The existence of a residual ACh-induced relaxation after the incubation of both OZR and LZR arteries with INDO plus L-NAME revealed the likely participation of the EDHF in the endothelial response. After the CB1 antagonist chronic administration, the EDHF vasorelaxant component seemed to be higher in LZRs and lower in OZRs compared to their vehicle-treated counterparts. A decrease in the EDFH participation in the ACh-induced vasorelaxation in the mesenteric artery could be a compensatory effect, secondary to the enhancement of PGI2 production, which may well be greater in OZRs.

Obesity has been closely related to an overactivation of the endogenous cannabinoid system (3,4,5), what may lead, among other cardiometabolic effects, to a reduction of blood pressure (5) as the initial SBP values confirmed. In contrast, obesity is a well established risk factor for the development of hypertension, being most of the cases of hypertension associated to overweight (1). In fact, the increase of SBP developed by fatty animals was higher than that found in LZRs. Due to these observations, concerns may be established regarding the net pro- or antihypertensive effect of long-term CB1 blockade. Our results show that this CB1 antagonist did not exert any effects in the final SBP values of obese animals, but it was able to prevent the obesity-induced increase of SBP. It has been previously suggested that antihypertensive effects of RM in patients with high blood pressure might be explained by weight loss (27). Indeed, in our study, a similar effect was observed in the pair-fed control fatty group. However, the caloric restriction was not enough to obtain a statistically significant effect, so an improvement of the insulin resistance and lipid profile, an increase of adiponectin plasma levels or a direct effect on the endothelium could also be involved such prevention. Obesity-related hypertension is associated to both a decrease in adiponectin concentration and the development of insulin resistance, which are in turn related (31). The reasons for the association of insulin resistance and essential hypertension can be sought in at least four general types of mechanisms: Na+ retention, sympathetic nervous system overactivity, disturbed membrane ion transport, and proliferation of vascular smooth muscle cells. Regarding to a possible greater sympathetic activity in OZRs secondary to insulin resistance, although we did not observed higher noradrenalin urine levels in obese animals compared to LZRs, RM was able to reduce sympathetic activity. Restoration of fatty acid oxidation and reduction of fatty acid synthesis by RM could potentially be responsible for the decrease of circulating catecholamine levels (30).

Finally, OZRs displayed lower heart rates than LZRs and, after RM chronic administration, the OZRs heart rate turned into values similar to those observed in LZRs. The previously described overactivation of the endogenous cannabinoid system in obesity that lead to bradychardia (39), may contribute to explain such effect. Then, CB1 receptor antagonism might evoke an increase in the heart rate in OZRs. Other studies found that CB1 blockade with RM led to an increase in the heart rate in isolated hearts and anesthetized rats, after bradychardia was previously induced by endocannabionids injection (40,41,42). Nevertheless, the increase in the cardiac rhythm in RM-treated OZRs is not completely consistent with the lower sympathetic activity also described in these animals. In anesthetized rats, endocannabinoids induced bradychardia through a CB1-dependent peripheral sympathoinhibition and enhancement of cardiac vagal tone (41). Nevertheless when conscious animals were used, the bradychardic effect of endocannabinoids was sensitive to COX inhibition and ascribed to the production of arachidonic acid metabolites (43). Moreover, cannabinoids caused CB1-mediated central sympathoexcitation together with bradychardia in conscious rabbits (44). Our results may be therefore in agreement with a sympathetic activity–independent effect of CB1 blockade on the heart rate. In fact, the decrease in the sympathetic activity was observed in both RM-treated LZRs and OZRs, whereas the increase in heart rate was only observed in fatty animals treated with RM.

In conclusion, we have found that RM prevents development of hypertension in a rat model of obesity. In addition, decrease of either sympathetic activity or insulin resistance was found. Although RM treatment did not alter endothelium-dependent relaxations, it was able to regulate COX-derived vasoactive products participation in endothelial function. Those effects may contribute to explain some of the cardiovascular protective actions elicited by this drug.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. REFERENCES

This research was supported by funds obtained from Andalucia Regional Government. Mingorance has a fellow of the Spanish Government. We would also like to extend our appreciation to Sanofi-Aventis for supplying the drug for this study.

REFERENCES

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. REFERENCES