Hypercaloric low-carbohydrate high-fat diet protects against the development of nonalcoholic fatty liver disease in obese mice in contrast to isocaloric Western diet.

Objective: Obesity and metabolic complications, such as type 2 diabetes and nonalcoholic fatty liver disease (NAFLD), are one of the greatest public health challenges of the 21st century. The major role of high sugar and carbohydrate consumption rather than caloric intake in obesity and NAFLD pathophysiology remains a subject of debate. A low-carbohydrate but high-fat diet (LCHFD) has shown promising results in obesity management, but its effects in preventing NAFLD need to be detailed. This study aims to compare the effects of a LCHFD with a high-fat high-sugar obesogenic Western diet (WD) on the progression of obesity, type 2 diabetes, and nonalcoholic fatty liver disease. Methods: Male C57BL/6J mice were initially fed a WD for 10 weeks. Subsequently, they were either switched to a LCHFD or maintained on the WD for an additional 6 weeks. Hepatic effects of the diet were explored by histological staining and RT-qPCR. Results: After the initial 10 weeks WD feeding, LCHF diet demonstrated effectiveness in halting weight gain, maintaining a normal glucose tolerance and insulin levels, in comparison to the WD-fed mice, which developed obesity, glucose intolerance, increased insulin levels and induced NAFLD. In the liver, LCHFD mitigated the accumulation of hepatic triglycerides and the increase in Fasn relative gene expression compared to the WD mice. Beneficial effects of the LCHFD occurred despite a similar calorie intake compared to the WD mice. Conclusion: Our results emphasize the negative impact of a high sugar/carbohydrate and lipid association for obesity progression and NAFLD development. LCHFD has shown beneficial effects for NAFLD management, notably improving weight management, and maintaining a normal glucose tolerance and liver health.

Nischarin Is Not the Functional I1 Imidazoline Receptor Involved in Blood Pressure Regulation.

ABSTRACT: Imidazoline receptor antisera selected/Nischarin was proposed several years ago as the functional entity for the I1 medullary receptors (I1Rs) targeted, together with α2-adrenoceptors, by the centrally acting antihypertensive drugs, such as clonidine. The objective of this study was to test this assumption using a pyrroline analog of clonidine, LNP599, which, unlike clonidine and related compounds, displays high selectivity toward I1Rs. Cardiovascular effects of LNP599 (3 mg/kg intravenous) were evaluated in anesthetized, artificially ventilated nischarin mutant rats expressing a truncated form of nischarin lacking the putative imidazoline binding site. LNP599 induced a rapid and pronounced fall in arterial blood pressure in wild-type animals (-42.7% ± 11.0% after 15 minutes), associated with a ≈30% heart rate reduction. Similar effects were obtained in homozygous and heterozygous nischarin mutant rats. The observation that the hypotensive response to I1R activation is not affected by the absence of the putative imidazoline binding site on nischarin strongly suggests that nischarin cannot be regarded as the functional I1R. Carbohydrate regulation was improved in nischarin mutant rats, further supporting the conclusion that nischarin and I1R are 2 distinct molecular entities.

Protective effects of the imidazoline-like drug lnp599 in a marmoset model of obesity-induced metabolic disorders.

BACKGROUND/OBJECTIVES: Overweight and obesity are undoubtable risk factors for type 2 diabetes and cardiovascular diseases and significantly contribute to the global morbi-mortality. We previoulsy reported that LNP599, a pharmacological imidazoline-like activator of hepatic AMPK/adiponectin signaling, protects against the development of adiposity and obesity and the associated cardio-metabolic disorders, suggesting that it may be a suitable drug candidate for a therapeutic approach targeting the development of obesity at very early stages. The objective of the present study was to evaluate the metabolic effects of LNP599 in a model of diet-induced overweight and metabolic disorders in a nonhuman primate, the common marmoset (Callithrix jacchus), and more particularly to establish the impact of the compound on cholesterol homeostasis, i.e., HDL and LDL/VLDL lipoproteins. METHODS: Marmosets were fed normal (NC) or hypercaloric (HC) chow during 16 weeks. Diet-induced changes in body weight and metabolism were assessed. Effects of LNP599 were evaluated in a subset of HC animals (HC-LNP) receiving the compound at a daily dose of 10 mg/kg over the 16 weeks. RESULTS: HC-feeding induced significant overweight associated with a marked dyslipidemia (hypertriglyceridemia, hypercholesterolemia, and reduced HDL over LDL/VLDL cholesterol ratio). LNP599 blunted the diet-induced body weight gain and largely protected against the development of hypertriglyceridemia. Total cholesterol was unchanged but the ratio of HDL over LDL/VLDL cholesterol was more than doubled. CONCLUSIONS: The profile of metabolic troubles obtained upon enriched diet mimicked the disorders associated with spontaneous obesity in marmosets. HC marmosets represent an experimental model of high clinical relevance to study the pathophysiology of obesity and related dyslipidemia and to evaluate the effects of emerging therapies targeting these disorders. Our data confirm the preventing effects of LNP599 in a nonhuman primate model and demonstrate for the first time the high potency of this drug in promoting HDL-cholesterol.

Increased expression of blood muscarinic receptors in patients with reflex syncope.

AIMS: Pathophysiology of reflex syncope is not fully understood but a vagal overactivity might be involved in this syncope. Previously, overexpression of muscarinic M2 receptors and acetylcholinesterase was found in particular in the heart and in lymphocytes of rabbits with vagal overactivity as well as in hearts of Sudden Infant Death Syndromes. The aim of this present study was to look at M2 receptor expression in blood of patients with reflex syncope. The second objective was to measure acetylcholinesterase expression in these patients. METHODS AND RESULTS: 136 subjects were enrolled. This monocenter study pooled 45 adults exhibiting recurrent reflex syncope compared with 32 healthy adult volunteers (18-50 years) and 38 children exhibiting reflex syncope requiring hospitalization compared with 21 controls (1-17 years). One blood sample was taken from each subject and blood mRNA expression of M2 receptors was assessed by qRT-PCR. Taking into account the non-symmetric distributions of values in both groups, statistical interferences were assessed using bayesian techniques. A M2 receptor overexpression was observed in adult and pediatric patients compared to controls. The medians [q1;q3] were 0.9 [0.3;1.9] in patients versus 0.2 [0.1;1.0] in controls; the probability that M2 receptor expression was higher in patients than in controls (Pr[patients>controls]) was estimated at 0.99. Acetylcholinesterase expression was also increased 0.7 [0.4;1.6] in patients versus 0.4 [0.2;1.1] in controls; the probability that acetylcholinesterase expression was higher in patients than in controls (Pr[patients>controls]) was estimated at 0.97. Both in adults and children, the expression ratio of M2 receptors over acetylcholinesterase was greater in the patient group compared with the control group. CONCLUSION: M2 receptor overexpression has been detected in the blood of both, adults and children, exhibiting reflex syncope. As in our experimental model, i.e. rabbits with vagal overactivity, acetylcholinesterase overexpression was associated with M2 receptor overexpression. For the first time, biological abnormalities are identified in vagal syncope in which only clinical signs are, so far, taken into account for differential diagnosis and therapeutic management. Further work will be needed to validate potential biomarkers of risk or severity associated with the cholinergic system.

Effects of imidazoline-like drugs on liver and adipose tissues, and their role in preventing obesity and associated cardio-metabolic disorders.

BACKGROUND/OBJECTIVES: We previously observed that selective agonists of the sympatho-inhibitory I1 imidazoline receptors (LNP ligands) have favorable effects on several cardiovascular and metabolic disorders defining the metabolic syndrome, including body weight. The objectives of this study were to explore the effects of LNPs on adiposity and the mechanisms involved, and to evaluate their impact on metabolic homeostasis. METHODS: Young Zucker fa/fa rats were treated with LNP599 (10 mg/kg/day) for 12 weeks. Effects on body weight, adiposity (regional re-distribution, morphology, and function of adipose tissues), cardiovascular and metabolic homeostasis, and liver function were evaluated. Direct effects on insulin and AMP-activated protein kinase (AMPK) signaling were studied in human hepatoma HepG2 cells. RESULTS: LNP599 treatment limited the age-dependent remodeling and inflammation of subcutaneous, epididymal, and visceral adipose tissues, and prevented total fat deposits and the development of obesity. Body-weight stabilization was not related to reduced food intake but rather to enhanced energy expenditure and thermogenesis. Cardiovascular and metabolic parameters were also improved and were significantly correlated with body weight but not with plasma norepinephrine. Insulin and AMPK signaling were enhanced in hepatic tissues of treated animals, whereas blood markers of hepatic disease and pro-inflammatory cytokine levels were reduced. In cultured HepG2 cells, LNP ligands phosphorylated AMPK and the downstream acetyl-CoA carboxylase and prevented oleic acid-induced intracellular lipid accumulation. They also significantly potentiated insulin-mediated AKT activation and this was independent from AMPK. CONCLUSIONS: Selective I1 imidazoline receptor agonists protect against the development of adiposity and obesity, and the associated cardio-metabolic disorders. Activation of I1 receptors in the liver, leading to stimulation of the cellular energy sensor AMPK and insulin sensitization, and in adipose tissues, leading to improvement of morphology and function, are identified as peripheral mechanisms involved in the beneficial actions of these ligands.

Comparison of bioimpedance spectroscopy and X-Ray micro-computed tomography for total fat volume measurement in mice.

Obesity and the metabolic syndrome are two pathologies whose prevalence are in a constant increase. Evaluation of the total fat mass but also of the distribution between visceral and subcutaneous adipose tissue are important factors while assessing the pathophysiology of these two pathologies. Computed tomography (CT) and bioimpedance (BIS) are the translational methods the most frequently used in human beings as well as in rodent models in longitudinal studies on adiposity and obesity. Surprisingly, no direct comparison of micro-CT and BIS was reported yet in mice. Therefore, the present study was carried out to evaluate and compare the accuracy and the uncertainty of measurement of micro-CT and BIS in this species. The proportion of fat mass was measured with BIS, micro-CT and direct post-mortem tissue weight, and correlations between the data were established to evaluate the accuracy of the methods but also the uncertainty of BIS and micro-CT. There were significant correlations between weights of fat tissues on scale and proportion of total fat mass determined by BIS or micro-CT (r = 0.81 and 0.86 respectively) but both methods overestimated the total fat mass, especially in the smallest animals; overestimation of fat mass was amplified with BIS compared to micro-CT. In addition BIS and micro-CT were highly correlated (r = 0.94). Test-test reliability showed a greater variability of the BIS with respect to the micro-CT (coefficient of variation = 17.2 vs 5.6% respectively). Hence, as far as subtle differences between groups or changes within one group are awaited, micro-CT may appear as the most reliable method for determination of fat mass in mice. Micro-CT, unlike BIS, will also allow to qualitatively and quantitatively differentiate between subcutaneous and visceral adipose tissues, which is of major importance in studies on adiposity and its complications.

Imidazoline-like drugs improve insulin sensitivity through peripheral stimulation of adiponectin and AMPK pathways in a rat model of glucose intolerance.

Altered adiponectin signaling and chronic sympathetic hyperactivity have both been proposed as key factors in the pathogenesis of metabolic syndrome. We recently reported that activation of I1 imidazoline receptors (I1R) improves several symptoms of the metabolic syndrome through sympathoinhibition and increases adiponectin plasma levels in a rat model of metabolic syndrome (Fellmann L, Regnault V, Greney H, et al. J Pharmacol Exp Ther 346: 370-380, 2013). The present study was designed to explore the peripheral component of the beneficial actions of I1R ligands (i.e., sympathoinhibitory independent effects). Aged rats displaying insulin resistance and glucose intolerance were treated with LNP509, a peripherally acting I1R agonist. Glucose tolerance, insulin sensitivity, and adiponectin signaling were assessed at the end of the treatment. Direct actions of the ligand on hepatocyte and adipocyte signaling were also studied. LNP509 reduced the area under the curve of the intravenous glucose tolerance test and enhanced insulin hypoglycemic action and intracellular signaling (Akt phosphorylation), indicating improved glucose tolerance and insulin sensitivity. LNP509 stimulated adiponectin secretion acting at I1R on adipocytes, resulting in increased plasma levels of adiponectin; it also enhanced AMPK phosphorylation in hepatic tissues. Additionally, I1R activation on hepatocytes directly enhanced AMPK phosphorylation. To conclude, I1R ligands can improve insulin sensitivity acting peripherally, independently of sympathoinhibition; stimulation of adiponectin and AMPK pathways at insulin target tissues may account for this effect. This may open a promising new way for the treatment of the metabolic syndrome.

The Src homology and collagen A (ShcA) adaptor protein is required for the spatial organization of the costamere/Z-disk network during heart development.

Src homology and collagen A (ShcA) is an adaptor protein that binds to tyrosine kinase receptors. Its germ line deletion is embryonic lethal with abnormal cardiovascular system formation, and its role in cardiovascular development is unknown. To investigate its functional role in cardiovascular development in mice, ShcA was deleted in cardiomyocytes and vascular smooth muscle cells by crossing ShcA flox mice with SM22a-Cre transgenic mice. Conditional mutant mice developed signs of severe dilated cardiomyopathy, myocardial infarctions, and premature death. No evidence of a vascular contribution to the phenotype was observed. Histological analysis of the heart revealed aberrant sarcomeric Z-disk and M-band structures, and misalignments of T-tubules with Z-disks. We find that not only the ErbB3/Neuregulin signaling pathway but also the baroreceptor reflex response, which have been functionally associated, are altered in the mutant mice. We further demonstrate that ShcA interacts with Caveolin-1 and the costameric protein plasma membrane Ca(2+)/calmodulin-dependent ATPase (PMCA), and that its deletion leads to abnormal dystrophin signaling. Collectively, these results demonstrate that ShcA interacts with crucial proteins and pathways that link Z-disk and costamere.

A new pyrroline compound selective for I1-imidazoline receptors improves metabolic syndrome in rats.

Symptoms of the metabolic syndrome (MetS), such as insulin resistance, obesity, and hypertension, have been associated with sympathetic hyperactivity. In addition, the adiponectin pathway has interesting therapeutic potentials in MetS. Our purpose was to investigate how targeting both the sympathetic nervous system and the adipose tissue (adiponectin secretion) with a drug selective for nonadrenergic I1-imidazoline receptors (I1Rs) may represent a new concept in MetS pharmacotherapy. LNP599 [3-chloro-2-methyl-phenyl)-(4-methyl-4,5-dihydro-3H-pyrrol-2-yl)-amine hydrochloride], a new pyrroline derivative, displaced the specific [(125)I]para-iodoclonidine binding to I1R with nanomolar affinity and had no significant affinity for a large set of receptors, transporters, and enzymes. In addition, it can cross the blood-brain barrier and has good intestinal absorption, permitting oral as well as intravenous delivery. The presence of I1Rs was demonstrated in 3T3-L1 adipocytes; LNP599 had a specific stimulatory action on adiponectin secretion in adipocytes. Short-term administration of LNP599 (10 mg/kg i.v.) in anesthetized Sprague-Dawley rats markedly decreased sympathetic activity, causing hypotension and bradycardia. Long-term treatment of spontaneously hypertensive heart failure rats with LNP599 (20 mg/kg PO) had favorable effects on blood pressure, body weight, insulin resistance, glucose tolerance, and lipid profile, and it increased plasma adiponectin. The pyrroline derivative, which inhibits sympathetic activity and stimulates adiponectin secretion, has beneficial effects on all the MetS abnormalities. The use of one single drug with both actions may constitute an innovative strategy for the management of MetS.

Cardiac muscarinic receptor overexpression in sudden infant death syndrome.

BACKGROUND: Sudden infant death syndrome (SIDS) remains the leading cause of death among infants less than 1 year of age. Disturbed expression of some neurotransmitters and their receptors has been shown in the central nervous system of SIDS victims but no biological abnormality of the peripheral vago-cardiac system has been demonstrated to date. The present study aimed to seek vago-cardiac abnormalities in SIDS victims. The cardiac level of expression of muscarinic receptors, as well as acetylcholinesterase enzyme activity were investigated. METHODOLOGY/PRINCIPAL FINDINGS: Left ventricular samples and blood samples were obtained from autopsies of SIDS and children deceased from non cardiac causes. Binding experiments performed with [(3)H]NMS, a selective muscarinic ligand, in cardiac membrane preparations showed that the density of cardiac muscarinic receptors was increased as shown by a more than doubled B(max) value in SIDS (n = 9 SIDS versus 8 controls). On average, the erythrocyte acetylcholinesterase enzyme activity was also significantly increased (n = 9 SIDS versus 11 controls). CONCLUSIONS: In the present study, it has been shown for the first time that cardiac muscarinic receptor overexpression is associated with SIDS. The increase of acetylcholinesterase enzyme activity appears as a possible regulatory mechanism.

Constitutive overexpression of muscarinic receptors leads to vagal hyperreactivity.

BACKGROUND: Alterations in muscarinic receptor expression and acetylcholinesterase (AchE) activity have been observed in tissues from Sudden Infant Death Syndrome (SIDS). Vagal overactivity has been proposed as a possible cause of SIDS as well as of vasovagal syncopes. The aim of the present study was to seek whether muscarinic receptor overexpression may be the underlying mechanism of vagal hyperreactivity. Rabbits with marked vagal pauses following injection of phenylephrine were selected and crossed to obtain a vagal hyperreactive strain. The density of cardiac muscarinic receptors and acetylcholinesterase (AchE) gene expression were assessed. Blood markers of the observed cardiac abnormalities were also sought. METHODOLOGY/PRINCIPAL FINDINGS: Cardiac muscarinic M(2) and M(3) receptors were overexpressed in hyperreactive rabbits compared to control animals (2.3-fold and 2.5-fold, respectively) and the severity of the phenylephrine-induced bradycardia was correlated with their densities. A similar overexpression of M(2) receptors was observed in peripheral mononuclear white blood cells, suggesting that cardiac M(2) receptor expression can be inferred with high confidence from measurements in blood cells. Sequencing of the coding fragment of the M(2) receptor gene revealed a single nucleotide mutation in 83% of hyperreactive animals, possibly contributing for the transcript overexpression. Significant increases in AchE expression and activity were also assessed (AchE mRNA amplification ratio of 3.6 versus normal rabbits). This phenomenon might represent a compensatory consequence of muscarinic receptors overexpression. Alterations in M(2) receptor and AchE expression occurred between the 5th and the 7th week of age, a critical period also characterized by a higher mortality rate of hyperreactive rabbits (52% in H rabbits versus 13% in normal rabbits) and preceeded the appearance of functional disorders. CONCLUSIONS/SIGNIFICANCE: The results suggest that cardiac muscarinic receptor overexpression plays a critical role in the development of vagal hyperreactivity, whereas AchE hyperactivity appears as a compensatory consequence of it. Since similar vagal disorders were observed recently by us in SIDS, muscarinic receptor overexpression could become a marker of risk of vasovagal syncopes and SIDS.

Search for an endogenous cannabinoid-mediated effect in the sympathetic nervous system.

Activation of CB(1) cannabinoid receptors by exogenous agonists causes presynaptic inhibition of neurotransmitter release from axon terminals. In the central nervous system, presynaptic CB(1) receptors can also be activated by endogenous cannabinoids (endocannabinoids) released from postsynaptic neurons. Except in the vas deferens, there is no indication of endocannabinoid-mediated presynaptic inhibition in the sympathetic nervous system. The aim of the present study was to search for such inhibition in pithed rats. Artificial sympathetic tone was established by continuous electrical stimulation of preganglionic sympathetic axons. The CB(1) cannabinoid receptor antagonist rimonabant (0.5 and 2 mg kg(-1) i.v.) did not change blood pressure, heart rate or plasma noradrenaline concentration. Since activation of Galpha(q/11) protein-coupled receptors enhances endocannabinoid synthesis in the central nervous system, we attempted to stimulate endocannabinoid production by infusion of arginine vasopressin and phenylephrine (both activate Galpha(q/11) protein-coupled receptors). Rimonabant (2 mg kg(-1) i.v.) did not change blood pressure, heart rate or plasma noradrenaline concentration during infusion of phenylephrine or vasopressin. In the final series of experiments we verified that an exogenous cannabinoid agonist produces sympathoinhibition. The synthetic CB(1)/CB(2) receptor agonist WIN55212-2 (0.1 and 1 mg kg(-1) i.v.) markedly lowered blood pressure and plasma noradrenaline concentration in pithed rats with electrically stimulated sympathetic outflow. In contrast, in pithed rats with a pressor infusion of noradrenaline, WIN55212-2 did not change blood pressure or heart rate. The results verify that activation of peripheral presynaptic CB(1) receptors inhibits noradrenaline release from sympathetic nerve terminals. The lack of effect of the CB(1) receptor antagonist rimonabant indicates that, even under conditions favouring endocannabinoid synthesis, endocannabinoid-mediated presynaptic inhibition is not operating in the sympathetic nervous system of the pithed rat.

Central effects of the cannabinoid receptor agonist WIN55212-2 on respiratory and cardiovascular regulation in anaesthetised rats.

1 The primary aim was to study the central respiratory effects of cannabinoids (CB). To this end, the cannabinoid receptor agonist WIN55212-2 was injected into the cisterna magna of urethane-anaesthetised rats and changes in respiratory parameters were observed. The secondary aim was to observe the centrally elicited cardiovascular actions of WIN55212-2. Involvement of opioid mechanisms in the central effects of WIN55212-2 was also studied. 2 Intracisternal (i.c.) application of WIN55212-2 (1, 3, 10 and 30 microg kg(-1)) dose-dependently decreased the respiratory rate and minute volume. Tidal volume was slightly increased, whereas peak inspiratory flow remained unchanged. In addition, WIN55212-2 increased mean arterial pressure and the plasma noradrenaline concentration and decreased heart rate. 3 I.c. injection of WIN55212-3 (1, 3, 10 and 30 microg kg(-1)), an enantiomer of WIN55212-2 lacking affinity for cannabinoid receptors, elicited no effects. All effects of WIN55212-2 were prevented by the CB1 receptor antagonist SR141716 (2 mg kg(-1) i.v.). I.c. administration of the opioid receptor agonist DAMGO (0.1, 0.3, 1 and 3 microg kg(-1)) markedly lowered the respiratory rate, tidal volume, minute volume and peak inspiratory flow. These effects were attenuated by the opioid receptor antagonist naloxone (0.2 mg kg(-1) i.v.). In contrast, naloxone did not affect the respiratory and cardiovascular effects of i.c. administered WIN55212-2. 4 Our results show that activation of CB1 cannabinoid receptors in the brain stem depresses respiration and enhances sympathetic tone and cardiac vagal tone. Opioid mechanisms are not involved in these central cannabinoid effects.

Modulation of the baroreceptor reflex by alpha 2A-adrenoceptors: a study in alpha 2A knockout mice.

1. Our objective was to determine whether alpha(2A)-adrenoceptors modulate the baroreceptor reflex. The efficacy of the reflex was evaluated by measuring the spontaneous blood pressure and heart rate variability at rest and the heart rate responses to evoked changes in blood pressure. Experiments were carried out in conscious, unrestrained, and anaesthetized alpha(2A)-adrenoceptor-deficient (alpha(2A)-KO) mice and WT mice. 2. In conscious alpha(2A)-KO mice, the spontaneous blood pressure variability was greater, and the spontaneous heart rate variability was lower than in conscious WT mice. This was also observed in anaesthetized animals. 3. The reflex bradycardia after intravenous injection of phenylephrine was greatly attenuated in conscious alpha(2A)-KO compared to conscious WT mice; the baroreceptor reflex gain (ratio maximal change in heart rate/maximal change in mean arterial pressure) was decreased by 40%. 4. Similar results were obtained when reflex bradycardia was elicited by intra-arterial volume loading of conscious WT and alpha(2A)-KO mice. The baroreceptor reflex gain upon volume loading was also low in anaesthetized alpha(2A)-KO mice. 5. The reflex tachycardia evoked by intravenous sodium nitroprusside injection was also significantly less in alpha(2A)-KO mice as compared to WT, conscious as well as anaesthetized; the baroreceptor reflex gains were decreased by 50 and 65%, respectively. 6. Direct stimulation of cardiac beta-adrenoceptors by the agonist isoprenaline produced similar cardioacceleration in alpha(2A)-KO and WT animals. 7. Our results show that the baroreceptor reflex function is impaired in mice lacking alpha(2A)-adrenoceptors. We conclude that central alpha(2A)-adrenoceptors facilitate the reflex response to both loading and unloading of the arterial baroreceptors.

Analysis of the respiratory effects of cannabinoids in rats.

The objective of the present study was to evaluate the respiratory effects of cannabinoids and their influence on cardiovascular homeostasis.In spontaneously breathing urethane-anaesthetised rats, intravenous injection of the two synthetic cannabinoid receptor agonists WIN55212-2 and CP55940 strongly and dose-dependently lowered mean arterial pressure, heart rate and the plasma noradrenaline concentration. The cardiovascular depressive effects were associated with a large decrease in respiratory rate, hypoxia, hypercapnia and blood acidosis. All depressor effects of WIN55212-2 were abolished by the selective CB(1) cannabinoid receptor antagonist SR141716A. The bradycardia elicited by WIN55212-2 was inhibited by the muscarinic acetylcholine receptor antagonist methylatropine. The natural agonist Delta(9)-tetrahydrocannabinol also elicited cardiovascular and respiratory depression. In contrast, WIN55212-3, an enantiomer of WIN55212-2 lacking affinity for cannabinoid receptors, had no effect. The cannabinoid-evoked decreases in blood pressure and heart rate were much more pronounced in spontaneously breathing than in artificially ventilated urethane-anaesthetised rats. In contrast, the plasma noradrenaline concentration was lowered equally in both preparations. Our results show that activation of CB(1) cannabinoid receptors not only induces cardiovascular depression, but also markedly impairs ventilation. The second major finding of the present study is that the respiratory depression evoked by cannabinoids largely amplifies the cardiovascular depression.

The peripheral sympathetic nervous system is the major target of cannabinoids in eliciting cardiovascular depression.

Our objective was to identify the sites of interaction of cannabinoids with cardiovascular sympathetic regulation in the rat. Effects on sympathetic tone were first determined in anaesthetised animals following i.v. administration of the drugs. Central effects were evaluated in anaesthetised rats receiving microinjections of cannabinoids into brain stem nuclei. Peripheral effects were identified in pithed rats with electrically stimulated sympathetic outflow. In anaesthetised and artificially ventilated rats, i.v. injection of the cannabinoid agonists WIN55212-2 and CP55940 decreased mean arterial pressure, heart rate and the plasma noradrenaline concentration. These effects were antagonized by the CB(1) cannabinoid receptor antagonist SR141716A. The bradycardia was abolished by the muscarinic acetylcholine receptor antagonist methylatropine. The decreases in mean arterial pressure and heart rate caused by cannabinoids in ventilated rats were much less pronounced than in spontaneously breathing rats. Microinjection of WIN55212-2 into the nucleus tractus solitarii had no effect. Microinjected into the rostral ventrolateral medulla oblongata, WIN55212-2 lowered mean arterial pressure slightly without changing other parameters. In pithed rats, WIN55212-2 inhibited the increases in mean arterial pressure, heart rate and the plasma noradrenaline concentration evoked by electrical stimulation of the sympathetic outflow. Our results show that activation of CB(1) cannabinoid receptors induces sympathoinhibition and enhancement of cardiac vagal tone, leading to hypotension and bradycardia. Presynaptic inhibition of noradrenaline release from terminals of postganglionic sympathetic neurons is the major component of the sympathoinhibition, but an effect in the rostral ventrolateral medulla oblongata may also contribute. The cannabinoid-evoked cardiovascular depression depends strongly on the respiratory state of the animals.