Nathalie Niederhoffer

Aortic Calcification Produced by Vitamin D3 plus Nicotine

Calcification of the elastic arteries of the young rat by treatment with vitamin D and nicotine (VDN) has been proposed as an animal model of arterial calcification associated with age and age-related vascular pathology in man. The calcium-binding protein, S-100, which is found in human atherosclerotic lesions was associated with medial calcification of the aorta in VDN rats, especially in cases of severe calcification. Calcification (total calcium content: 366 +/- 87, n = 12 in VDN vs. 24 +/- 2 micromol g(-1) aortic dry weight in controls, n = 13) involved elastocalcinosis leading to elastolysis as revealed by a fall in the amount of desmosine and isodesmosine in the aortic wall (266 +/- 17 and 254 +/- 15 in VDN vs. 655 +/- 56 and 588 +/- 30 microg g(-1) aortic dry weight in controls). The decrease in elastin was associated with an increase in the stiffness of the aortic wall (elastic modulus: 15.1 +/- 1.8 in VDN vs. 6.7 +/- 0.5 10(6) dyn cm(-2) in controls), an increase in end-systolic stress (central systolic aortic pressure: 152 +/- 6 in VDN vs. 136 +/- 2 mm Hg in controls) (at a normotensive mean pressure level) and left ventricular hypertrophy (heart weight/body weight 2.51 +/- 0.10 in VDN vs. 2.24 +/- 0.07 g kg(-1) in controls). In conclusion, the mechanisms and consequences of aortic calcification in VDN show several similarities with calcification occurring in human athero- and arteriosclerosis.

Effect of the cannabinoid receptor agonist WIN55212-2 on sympathetic cardiovascular regulation

The aim of the present study was to analyse the cardiovascular actions of the synthetic CB1/CB2 cannabinoid receptor agonist WIN55212‐2, and specifically to determine its sites of action on sympathetic cardiovascular regulation. Pithed rabbits in which the sympathetic outflow was continuously stimulated electrically or which received a pressor infusion of noradrenaline were used to study peripheral prejunctional and direct vascular effects, respectively. For studying effects on brain stem cardiovascular regulatory centres, drugs were administered into the cisterna cerebellomedullaris in conscious rabbits. Overall cardiovascular effects of the cannabinoid were studied in conscious rabbits with intravenous drug administration. In pithed rabbits in which the sympathetic outflow was continuously electrically stimulated, intravenous injection of WIN55212‐2 (5, 50 and 500 μg kg−1) markedly reduced blood pressure, the spillover of noradrenaline into plasma and the plasma noradrenaline concentration, and these effects were antagonized by the CB1 cannabinoid receptor‐selective antagonist SR141716A. The hypotensive and the sympathoinhibitory effect of WIN55212‐2 was shared by CP55940, another mixed CB1/CB2 cannabinoid receptor agonist, but not by WIN55212‐3, the enantiomer of WIN55212‐2, which lacks affinity for cannabinoid binding sites. WIN55212‐2 had no effect on vascular tone established by infusion of noradrenaline in pithed rabbits. Intracisternal application of WIN55212‐2 (0.1, 1 and 10 μg kg−1) in conscious rabbits increased blood pressure and the plasma noradrenaline concentration and elicited bradycardia; this latter effect was antagonized by atropine. In conscious animals, intravenous injection of WIN55212‐2 (5 and 50 μg kg−1) caused bradycardia, slight hypotension, no change in the plasma noradrenaline concentration, and an increase in renal sympathetic nerve firing. The highest dose of WIN55212‐2 (500 μg kg−1) elicited hypotension and tachycardia, and sympathetic nerve activity and the plasma noradrenaline concentration declined. The results obtained in pithed rabbits indicate that activation of CB1 cannabinoid receptors leads to marked peripheral prejunctional inhibition of noradrenaline release from postganglionic sympathetic axons. Intracisternal application of WIN55212‐2 uncovered two effects on brain stem cardiovascular centres: sympathoexcitation and activation of cardiac vagal fibres. The highest dose of systemically administered WIN55212‐2 produced central sympathoinhibition; the primary site of this action is not known.

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 CB1 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 CB1 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.

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

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. Intracisternal (i.c.) application of WIN55212‐2 (1, 3, 10 and 30 μg 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. I.c. injection of WIN55212‐3 (1, 3, 10 and 30 μg 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 μg 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. 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.

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 CB1 cannabinoid receptor antagonist SR141716A. The bradycardia elicited by WIN55212-2 was inhibited by the muscarinic acetylcholine receptor antagonist methylatropine. The natural agonist Δ9-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 CB1 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.

Effects of cannabinoids on adrenaline release from adrenal medullary cells

The objective of the present study was to analyse the peripheral effects of cannabinoids on adrenaline release from adrenal chromaffin cells. In pithed rabbits with electrically stimulated sympathetic outflow, intravenous injection of the cannabinoid receptor agonists WIN55212‐2 and CP55940 (5, 50 and 500 μg kg−1) markedly lowered the plasma adrenaline concentration. The effect of WIN55212‐2 was attenuated by the selective CB1 cannabinoid receptor antagonist SR141716A (500 μg kg−1). WIN55212‐3 (same doses as WIN55212‐2), the enantiomer of WIN55212‐2 lacking affinity for cannabinoid receptors, had no effect on the plasma adrenaline concentration. In rabbit isolated adrenal glands, the release of adrenaline elicited by electrical stimulation was measured by fast cyclic voltammetry. Electrically‐evoked adrenaline release was inhibited by WIN55212‐2 (0.3, 1, 3 and 10 μM) and this effect was antagonized by SR141716A (1 μM). The non‐cholinergic component of adrenaline release observed after blockade of nicotinic (by hexamethonium 100 μM) and muscarinic (by atropine 0.5 μM) acetylcholine receptors was not depressed by WIN55212‐2. WIN55212‐3 (10 μM) had no effect on adrenaline release. No detectable specific CB1 receptor binding and mRNA expression were found in rabbit adrenal glands with autoradiography and in situ hybridization. The results show that cannabinoids inhibit adrenaline secretion in rabbit isolated adrenal glands; the likely mechanism is a presynaptic CB1 receptor‐mediated inhibition of acetylcholine release from preganglionic sympathetic neurons. The inhibition of adrenaline secretion in adrenal glands most probably accounts for the decrease in the plasma adrenaline concentration observed after cannabinoid administration in pithed rabbits.

Analysis of the receptor involved in the central hypotensive effect of rilmenidine and moxonidine

Abstract The aim of this study was to determine whether α2-adrenoceptors or imidazoline I1-receptors are responsible for the central sympathoinhibition produced by rilmenidine and moxonidine, two clonidine-like antihypertensive drugs. Rilmenidine and moxonidine were compared with the indirectly acting α2-adrenoceptor agonist α-methyldopa. Three antagonists were used. Yohimbine and SK & F86466 were used as selective α2-adrenoceptor antagonists. They were compared with efaroxan which is also an α2-adrenoceptor antagonist, but, in addition, possesses affinity for imidazoline I1-receptors. According to some but not all studies, the affinity of efaroxan for I1-receptors is much higher than its affinity for α2-adrenoceptors.Drugs were administered into the cisterna cerebellomedullaris of conscious rabbits by a catheter implanted previously under halothane anaesthesia. Rilmenidine (10 μg kg–1), moxonidine (0.3 μg kg–1) and α-methyldopa (0.4 mg kg–1) lowered blood pressure and the plasma noradrenaline concentration; the degree of sympathoinhibition produced by the three agonists was very similar. When injected after the agonists, efaroxan (0.1–14 μg kg–1; cumulative doses), yohimbine (0.4–14 μg kg–1) and SK & F86466 (0.4–44 μg kg–1) counteracted the effects of the agonists on blood pressure and the plasma noradrenaline concentration. Efaroxan was about tenfold more potent than yohimbine and SK & F86466 at antagonizing the hypotensive effects of α-methyldopa. Similarly, efaroxan was two- to tenfold more potent than yohimbine and SK & F86466 against rilmenidine and moxonidine. Finally, efaroxan was about as potent against α-methyldopa as against rilmenidine and moxonidine.The results confirm previous observations that selective α2-adrenoceptor antagonists are capable of completely antagonizing effects of rilmenidine and moxonidine. The effects of the α2-adrenoceptor antagonist with an additional high affinity for imidazoline I1-receptors, efaroxan, can also be explained by blockade of α2-adrenoceptors. Efaroxan was more potent against rilmenidine and moxonidine than the selective α2-adrenoceptor antagonists. This was probably due to the fact that the affinity of efaroxan for α2-adrenoceptors is higher than the affinity of yohimbine and SK & F86466, since efaroxan was also the most potent of the three antagonists against the indirectly acting α2-adrenoceptor agonist α-methyldopa. The observation that efaroxan was equally potent against rilmenidine and moxonidine and against α-methyldopa suggests that the same receptors were involved in the effects of the three agonists, α2-adrenoceptors; this observation is not compatible with the high I1/α2 selectivity of efaroxan and the hypothesis that rilmenidine and moxonidine activate I1-receptors, whereas α-methyldopa activates α2-adrenoceptors. Thus, the data do not indicate involvement of I1 imidazoline receptors in the central sympathoinhibition elicited by ril-menidine and moxonidine in rabbits. It is likely that ril-menidine and moxonidine produce sympathoinhibition by activating the same receptors which are activated by the indirectly acting catecholamine α-methyldopa, namely α2-adrenoceptors.

Mechanism of the Sympathoinhibition Produced by the Clonidine‐Like Drugs Rilmenidine and Moxonidine

The mechanism of the sympathoinhibition produced by two new derivatives of clonidine, rilmenidine and moxonidine, was studied. One aim was to determine the receptor responsible for the central sympathoinhibition by these drugs. Rilmenidine and moxonidine were injected into the cisterna cerebellomedullaris. They decreased blood pressure and the plasma noradrenaline concentration. After rilmenidine and moxonidine, two selective alpha 2-adrenoceptor antagonists (devoid of affinity for I1 binding sites), yohimbine and SK&F86466, were given intracisternally. They completely counteracted the hypotensive effects of rilmenidine and moxonidine, indicating that alpha 2-adrenoceptors are involved in the central sympathoinhibition produced by these drugs. The other aim was to determine if peripheral presynaptic inhibition of noradrenaline release from postganglionic sympathetic neurons contributes to the overall reduction of sympathetic tone. Rilmenidine and moxonidine were injected i.v. in pithed rabbits with electrically stimulated sympathetic outflow. They dose-dependently lowered blood pressure and the plasma noradrenaline concentration and inhibited stimulation-evoked cardioacceleration. Moreover, the doses necessary for these peripheral effects were identical to the doses that reduce the sympathetic nerve firing rate and blood pressure in conscious rabbits. These observations indicate that peripheral presynaptic inhibition of noradrenaline release from postganglionic sympathetic neurons contributes to the overall reduction of sympathetic tone produced by rilmenidine and moxonidine in intact animals.

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

Activation of CB1 cannabinoid receptors by exogenous agonists causes presynaptic inhibition of neurotransmitter release from axon terminals. In the central nervous system, presynaptic CB1 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 CB1 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 Gαq/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 Gαq/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 CB1/CB2 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 CB1 receptors inhibits noradrenaline release from sympathetic nerve terminals. The lack of effect of the CB1 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.

Involvement of CB1 cannabinoid receptors in the EDHF‐dependent vasorelaxation in rabbits

It was recently suggested that an endogenous cannabinoid could represent an endothelium‐derived hyperpolarizing factor (EDHF). The aim of the present study was to clarify whether CB1 cannabinoid receptors are involved in the nitric oxide (NO)‐ and prostanoid‐independent vasodilation produced by acetylcholine in rabbits. Pithed rabbits received indomethacin. Noradrenaline was infused to raise blood pressure, and vasodilation was elicited by bolus injections of acetylcholine. The NO‐synthase inhibitor Nω‐nitro‐L‐arginine methylester inhibited the acetylcholine‐evoked vasodilation by about 40%. The remaining vasodilation was unaffected by the CB1 cannabinoid receptor antagonist SR141716A, but was inhibited by the potassium channel blocker tetraethylammonium. In addition, the mixed CB1/CB2 cannabinoid receptor agonist WIN55212‐2 did not elicit vasodilation. No CB1 cannabinoid receptors were involved in the prostanoid‐ and NO‐independent vasodilation produced by acetylcholine. An exogenous cannabinoid also did not cause vasodilation. Therefore, it is unlikely that an endogenous cannabinoid serves as an EDHF acting at smooth muscle CB1 cannabinoid receptors in the rabbit.