Unresponsiveness to cannabinoids and reduced addictive effects of opiates in CB1 receptor knockout mice.

The function of the central cannabinoid receptor (CB1) was investigated by invalidating its gene. Mutant mice did not respond to cannabinoid drugs, demonstrating the exclusive role of the CB1 receptor in mediating analgesia, reinforcement, hypothermia, hypolocomotion, and hypotension. The acute effects of opiates were unaffected, but the reinforcing properties of morphine and the severity of the withdrawal syndrome were strongly reduced. These observations suggest that the CB1 receptor is involved in the motivational properties of opiates and in the development of physical dependence and extend the concept of an interconnected role of CB1 and opiate receptors in the brain areas mediating addictive behavior.

Behavioral profile of CCK2 receptor-deficient mice.

CCK2 receptor-deficient mice were used to investigate in vivo the role of this receptor in behavior. Mutant mice showed a neuromuscular impairment in the traction and rotarod tests but not in the chimney test. Brain cholecystokinin has been shown to participate in stress-related behaviors. However, CCK2 receptor-deficient mice did not show behavioral modifications compared to wild-type mice in the elevated plus maze and in the motility conditioned suppression test, indicating that compensatory mechanisms very likely occur following CCK2 receptor invalidation. On the other hand, a hyperlocomotor activity was observed in actimeter which can be related to an impairment in environmental habituation. Finally, CCK2 receptor-deficient mice showed an impairment of performance in the spontaneous alternation behavior as expected from the opposite effects evoked by CCK2 agonists, supporting the physiological role of CCK2 receptors in attention and/or memory processes. This result is reinforced by the defects observed in these functions after the administration of CCK2 antagonists.

Presynaptic beta-adrenoceptors in rat atria: evidence for the presence of stereoselective beta 1-adrenoceptors.

1. Presynaptic beta-adrenoceptor activity was studied in rat isolated atria, previously loaded with [3H]-noradrenaline. The stimulation-induced release of 3H transmitter was measured in the presence of cocaine, and adrenaline was used as a facilitatory beta-adrenoceptor agonist. 2. Adrenaline (0.1 and 2 nM) increased, by about 50%, the evoked efflux of tritium. With phenoxybenzamine present, the same activity was shown with 10 nM adrenaline. 3. The beta 2-selective adrenoceptor blocking drugs: IPS 339 and ICI 118 551 caused a concentration-dependent decrease in the activity of adrenaline. Cardioselective beta-blocking drugs: acebutolol, beta-xolol, nebivolol and its isomers (R 67 138 and R 67 145) also reduced dose-dependently the agonistic action of adrenaline. The order of potency for nebivolol and its isomers was R 67 138 greater than nebivolol greater than R 67 145. The activity of pindolol was not concentration-dependent. The inhibitory effect of acebutolol was also observed in the presence of blockade of alpha-adrenoceptors. 4. The postsynaptic beta-adrenoceptor blocking activity of nebivolol and its isomers was studied in pithed rats. They reduced isoprenaline-induced tachycardia without altering hypotensive responses. The order of potency was: R 67 138 greater than nebivolol greater than R 67 145. 5. It is concluded that in rat isolated atria, presynaptic beta 2- and beta 1-adrenoceptors coexist and that facilitatory beta 1-adrenoceptors are stereospecific.

Effect of endopeptidase-24.11 inhibitors and C-ANP receptor ligand on responses evoked in arterioles of rat cremaster muscle by atrial natriuretic peptide.

1. The present study examined the effect of exogenous atrial natriuretric peptide (ANP), alone or in presence of inhibitors of the two major mechanisms for clearing ANP, metabolism by neutral endopeptidase-24.11 (NEP) and internalization by C-ANP receptors, on arteriolar responses using intravital microscopy on the rat cremaster muscle after intravenous or topical administration of the peptide. 2. Topical application of ANP (3 x 10(-10) to 3 x 10(-8) M) produced a gradual increase in arteriolar diameter. NEP inhibitors, thiorphan (30 mg kg-1, i.v.), kelatorphan (10 mg kg-1, i.v.) and retrothiorphan (25 mg kg-1, i.v.) alone, did not significantly affect vascular tone but caused significant potentiation of the arteriolar responses to topically applied ANP. 3. When given as an i.v. bolus, ANP dilates skeletal arterioles at a high dose (20 micrograms kg-1). At a lower dose (10 micrograms kg-2), ANP alone or with retrothiorphan or the C-ANP receptor ligand C-ANP (4-23) did not produce any arteriolar responses, while after the combined administration of the two inhibitors, an increase in arteriolar diameter was induced. 4. These results indicate that low doses of topically applied ANP dilate rat cremaster arterioles and that the vasodilator responses can be potentiated by NEP inhibition. When given as an i.v. bolus, a high dose of ANP can also dilate skeletal arterioles. However at a lower dose the rapid metabolism of the peptide prevents it from producing its action.

Mutant mice lacking the cholecystokinin2 receptor show a dopamine-dependent hyperactivity and a behavioral sensitization to morphine.

Cholecystokinin2 (CCK2) receptor-deficient mice were used to analyze the in vivo function of CCK2 receptor and especially the incidence of this gene invalidation on enkephalinergic and dopaminergic systems. Hyperlocomotor activity of CCK2 receptor-deficient mice was suppressed by a selective D2 antagonist but not by a D1 antagonist. Injection of amphetamine induced a hyperlocomotor activity in both groups of mice while mutant mice were less sensitive to cocaine. Administration of 6 mg/kg of morphine once every 2 days for 5 days significantly (P<0.05) enhanced motor activity the last day compared to the first day, only in CCK2 receptor-deficient mice. These results emphasize the role of CCK2 receptors in counteracting the effects of dopaminergic systems and suggest that CCK2 receptor invalidation could lead to a slight behavioral sensitization.

Chronic subcutaneous treatment with acebutolol: haemodynamic effects and metabolism in spontaneously hypertensive rats.

Chronic administration of acebutolol (15 mg kg-1 s.c. three times a week for five weeks, then 30 mg kg-1 for three weeks) did not lower blood pressure in 17 and 33 weeks-old spontaneously hypertensive rats (SHR). At the end of this treatment, the plasma concentrations of acebutolol and diacetolol were measured by HPLC. After 24 h, acebutolol was absent from plasma while diacetolol was lower after chronic treatment than after acute administration. Twenty-four hours after the last injection of acebutolol, both isoprenaline-induced tachycardia and vasodilatation were reduced. The vasomotor agents, noradrenaline, bradykinin and angiotensin, exhibited the same activity in control and treated SHR. These findings suggest that the lack of antihypertensive effect of acebutolol in SHR may be the result of a decrease in diacetolol formation together with blockade of beta 2 vascular receptors.

Cardiovascular effects of intracerebroventricular injections of (+/-)-nebivolol and its enantiomers--a comparison with those of metoprolol in the rat.

The cardiovascular effects of (+/-)-nebivolol, a potent beta 1-adrenoceptor antagonist, and its enantiomers, (+)-nebivolol (SRRR) and (-)-nebivolol (RSSS) in normotensive anaesthetized rats, have been investigated using metoprolol as a reference substance. The drugs decreased blood pressure and heart rate immediately after i.c.v. injection. These effects paralleled the beta-blocking potencies ((+)- greater than (+/-)-greater than (-)-nebivolol). Metoprolol induced a weaker hypotension than (+/-)-nebivolol, and a long-lasting reduction in stroke volume. As reported after i.v. administration, (+/-)-nebivolol and isomers by the i.c.v. route decreased peripheral vascular resistance following i.c.v. administration while metoprolol increased it. These effects are centrally mediated since cardiovascular responses to isoprenaline i.v. remained unchanged.

Tissue distribution of acebutolol in mature normotensive and stroke-prone spontaneously hypertensive rats.

Tissue distribution of acebutolol was studied in 33-week-old normotensive (WKY) and Okamoto stroke-prone (SHR-SP) rats, 30 min after an i.v. administration, by using 14C-acebutolol. Plasma level of acebutolol was higher in WKY than in SHR-SP. Aorta, kidney, liver and muscle radioactivity/plasma radioactivity ratios were higher in SHR-SP than in WKY. The brain/plasma radioactivity ratio was very low and similar in the two groups. The drug distribution was the same in the two groups except in medulla + corpus trapezoides where drug concentration was greater in SHR-SP. These results, compared with previous ones, show an age-related evolution in pathological state in SHR-SP. They point out a specific concentration of the beta-blocking drug in a defined part of the brain, namely medulla + corpus trapezoides.

Study on the mechanism of 5-HT-induced tachycardia in the pithed rat.

1. Intravenous infusion of serotonin (5-HT) (2.5, 5, 10 and 20 micrograms/kg/min) in pithed rats induced a dose-dependent sustained tachycardia. 2. Pretreatment by phentolamine or diltiazem did not modify the chronotropic response to 5-HT. In contrast, atenolol antagonized this tachycardia and the 5-HT antagonists methysergide, ketanserin and MDL 72222 reduced it. 3. The 5-HT-induced tachycardia was abolished by desipramine and was not affected by fluvoxamine, a specific 5-HT uptake inhibitor. Surrenalectomy did not change the response to 5-HT but catecholamine depletion by reserpine markedly inhibited it. 4. Infusion of 5-HT increased the ratio of noradrenaline (NA) in the heart to NA in plasma, from 1.70 in control group to 2.76 in treated group (P less than 0.05). Desipramine inhibited this effect. 5. It was concluded that the tachycardia induced by an infusion of 5-HT in pithed rat results from a complex mechanism involving mainly the release of NA from the cardiac sympathetic nerves and a less important direct 5-HT2 mechanism.

Uptake of 5-hydroxytryptamine in rat isolated atria.

1. The mode of 5-hydroxytryptamine (5-HT) uptake by the rat isolated atria was studied and compared to noradrenaline (NA) uptake. 2. Rat isolated atria were incubated with 14C-5-HT (46 microM) or 3H-NA 0.4 microM). After washing, the radioactivity fixed in atria was counted and the total NA, 5-HT and 5-hydroxyindol-3-acetic acid (5-HIAA) atria contents were measured by HPLC. 3. 14C-5-HT uptake was reduced in atria from 6-hydroxydopamine (100 mg/kg, i.p., 48 hr before experiments) or reserpine (2.5 mg/kg, i.p., 24 and 48 hr before experiments) pretreated rats. 4. The incubation of atria with 5-HT (50 microM) at the same time as 3H-NA reduced the 3H-NA value fixed. 5. Addition of desipramine (1 microM) or hydrocortisone (150 microM) before the incubation of atria with 14C-5-HT was without any effect on 14C-5-HT uptake. In contrast, fluvoxamine (1 microM) or indalpine (5 microM) caused a slight inhibition. 6. These data indicate that 5-HT is taken into the NA storage vesicles within the atria sympathetic nerves. This uptake does not use the NA carrier and involves partly the 5-HT carrier. An extraneuronal accumulation was noticed and a part of it is intracellular.

[Autonomic cardiovascular regulation and permanent catheterization in normotensive conscious (WKY) and spontaneously hypertensive (SHR) rats]. / Régulation cardiovasculaire autonome et cathétérisme permanent chez le rat éveillé normotendu (WKY) et spontanément hypertendu (SHR).

In cardiovascular research, methods of indwelling catheterism have been frequently described. In the present work, we used normotensive and SHR rats to compare carotid catheterism to left ventricular catheterism, this last method being proposed by some investigators for cardiac index measurement. Our results of plasma catecholamines and autonomous nervous system activity show that ventricular catheterism results in an important disturb in cardiovascular regulation and question its validity to study cardiac function.

Peripheral and central cardiovascular effects of ketanserin in conscious normotensive rats.

The aim of the present study was to evaluate the effects of low doses of ketanserin, close to therapeutic ones, in conscious normotensive rats and to compare the different thresholds of 5-HT2- and alpha-antagonist properties and central sympatho-inhibitory activity. Male Sprague-Dawley rats (250-300 g) were used. Fourty-eight hr before experimentation, indwelling venous and arterial catheters were placed in each rat under light anaesthesia, and exteriorized through the interscapular skin. Drugs injected by i.v. route were ketanserin (50, 100, 200 micrograms/kg), serotonin (50 micrograms/kg), noradrenaline (1 microgram/kg), angiotensin (0.5 microgram/kg), nitroglycerin (200 micrograms/kg) and histamine (100 micrograms/kg). Ketanserin induced a short-lasting reduction in blood pressure and did not modify heart rate. At the dose of 50 micrograms/kg, this compound fully inhibited the pressor effect of serotonin, without modifying its hypotensive responses; after 200 micrograms/kg, ketanserin reduced by 50% the pressor effect of noradrenaline. Comparison of antagonistic activities on phenylephrine and noradrenaline pressor responses showed a selective blockade of alpha 1-adrenoceptors by 100 micrograms/kg ketanserin. In contrast, the angiotensin hypertensive response was unchanged. Reflex bradycardia after noradrenaline and angiotensin was reduced by 100 micrograms/kg ketanserin, whereas nitroglycerin and histamine effects were unaffected. In conclusion, administration of increasing low doses of ketanserin showed that blockade of 5-HT2-receptors appears in first, then a central sympatho-inhibitory effect and a selective alpha 1-adrenolytic activity occur. In addition, baroreceptor sensitivity seems not to be affected.

Reduction of stress-induced analgesia but not of exogenous opioid effects in mice lacking CB1 receptors.

CB1 cannabinoid receptors are widely distributed in the central nervous system where they mediate most of the cannabinoid-induced responses. Here we have evaluated the interactions between the CB1 cannabinoid receptors and the endogenous opioid system by assaying a number of well-characterized opioid responses, e.g. antinociception and stress-mediated effects, on mutant mice in which the CB1 receptor gene was invalidated. The spontaneous responses to various nociceptive stimuli (thermal, mechanical and visceral pain) were not changed in mutant CB1 mice. Furthermore, the absence of the CB1 cannabinoid receptor did not modify the antinociceptive effects induced by different opioid agonists: morphine (preferential mu opioid agonist), D-Pen2-D-Pen5-enkephalin (DPDPE) and deltorphin II (selective delta opioid agonists), and U-50,488H (selective kappa opioid agonist) in the hot-plate and tail-immersion tests. In contrast, the stress-induced opioid mediated responses were modified in CB1 mutants. Indeed, these mutants did not exhibit antinociception following a forced swim in water at 34 degrees C and presented a decrease in the immobility induced by the previous exposure to electric footshock. However, the antinociception induced by a forced swim in water at 10 degrees C was preserved in CB1 mutants. These results indicate that CB1 receptors are not involved in the antinociceptive responses to exogenous opioids, but that a physiological interaction between the opioid and cannabinoid systems is necessary to allow the development of opioid-mediated responses to stress.

Cardiovascular effects of drugs acting on the autonomous nervous system, in awake, normotensive and hypertensive rats.

A study has been carried out in awake normotensive (WKY), spontaneously hypertensive (SHR) and renal hypertensive (RHR) rats in order to compare the reactivity of the autonomous nervous system using different indwelling catheterizations. In all cases, a jugular catheter was implanted for i.v. injections. The arterial cannula was implanted in the femoral artery (group 1) or in the right carotid artery (group 2), where the catheter was routed over 20 mm towards the heart, reaching the aortic arch. The plasma catecholamines levels were lower in the RHR than in the WKY or SHR, and whatever the strain, lower in group 2 than in group 1. In the WKY and the RHR, the mean blood pressure was higher in group 2 than in group 1. The tachycardia induced by isoprenaline and the pressor response to noradrenaline were higher in the SHR than in the WKY and in the RHR. By contrast, the reflex bradycardia was lower. Furthermore, the procedure of catheterization could modify the cardiovascular responses induced by adrenergic and cholinergic agents. These results demonstrate that indwelling catheterizations cannot be performed safely in the rat when studying effects on cardiac performance or vascular reactivity.

Modulation of noradrenergic transmission in the rat isolated portal vein: role of prejunctional alpha 2-adrenoceptors and beta-adrenoceptors.

1. The effect of several adrenoceptor agonists and antagonists on the spontaneous and stimulus-evoked release of [3H]noradrenaline was studied in rat isolated portal vein. 2. Yohimbine (10(-6)M) increased the stimulus-evoked [3H]noradrenaline efflux. Adrenaline alone (3 x 10(-6)M) was without effect, while it increased the resting efflux when added together with yohimbine. 3. Propranolol alone was without effect on the release of [3H]noradrenaline. When added (2 x 10(-6)M) at the same time as yohimbine, it reduced the stimulation-induced 3H efflux. When added before adrenaline and yohimbine, propranolol (10(-5)M) reduced both spontaneous and stimulus-evoked release of [3H]noradrenaline. 4. The effect of several beta-blocking drugs was measured on the enhancing effect of yohimbine on the stimulation-evoked 3H efflux. The beta 1-adrenoceptor blocking drugs: atenolol (5 x 10(-6) and 10(-5) M), metoprolol (5 x 10(-6) and 10(-5) M), like the more selective bisoprolol (2 x 10(-6) and 4 x 10(-6) M) inhibited yohimbine activity. Likewise, propranolol (2 x 10(-6) and 5 x 10(-6)M) and the beta 2-adrenoceptor blocker ICI 118551 exhibited an antagonistic effect. 5. These results indicate the possibility for noradrenaline to activate presynaptic beta-adrenoceptors in rat portal vein. They show an interaction between the presynpatic alpha 2- and beta-adrenoceptor mediated systems in the release of noradrenaline. They suggest the presence and the activity of facilitatory beta 1-adrenoceptors.

Inhibition of both angiotensin-converting enzyme and neutral endopeptidase by S21402 (RB105) in rats with experimental myocardial infarction.

The vasoconstrictor angiotensin II and atrial natriuretic peptide (ANP) are oppositely involved in the development of heart failure, as modeled by myocardial infarction (MI) in rats. MI is a model also characterized by sodium retention despite the elevated plasma ANP levels, showing a desensitization of responses to ANP. S21402 (RB105) {N-[2S,3R-(2-mercaptom-ethyl-1-oxo-3-phenylbutyl) L-alanine]} is a dual inhibitor that inhibits both neutral endopeptidase (Ki = 1.7 +/- 0.3 nM) and angiotensin-converting enzyme (Ki = 4.2 +/- 0.5 nM). Inhibition of neutral endopeptidase protects endogenous ANP, and inhibition of angiotensin-converting enzyme blocks angiotensin II production, whereas inhibition of both peptidases is required to protect endogenous bradykinin (BK). Induction of MI in rats, by ligation of the left coronary artery, increased the base-line plasma ANP, cyclic GMP (cGMP) and renin concentrations, which were related to the degree of MI (moderate and severe MI rats). Urinary excretion of ANP, cGMP and BK was also increased in MI rats and was linked to the infarction size. S21402 (RB105) (25 mg/kg bolus plus 25 mg/kg/hr i.v.) decreased the mean blood pressure and increased natriuresis in MI rats whatever the degree of MI. S21402 (RB105) induced an increase in plasma renin in MI rats despite the elevated base-line levels. S21402 (RB105) did not alter the plasma in ANP in MI rats. However, plasma cGMP was increased by the dual inhibitor, as a function of the infarction severity. Urinary excretion of ANP, cGMP and BK was also increased by S21402 (RB105), proportionally to the infarction size. Whatever the degree of MI, S21402 (RB105) was able to induce natriuresis, characterized by a desensitization of ANP-induced renal responses. Inhibition of both angiotensin-converting enzyme and neutral endopeptidase by potentiating endogenous ANP and BK and blocking angiotensin II production could be an interesting therapeutic approach in heart failure.

Delta9-tetrahydrocannabinol releases and facilitates the effects of endogenous enkephalins: reduction in morphine withdrawal syndrome without change in rewarding effect.

Recent studies have suggested that cannabinoids might initiate the consumption of other highly addictive substances, such as opiates. In this work, we show that acute administration of Delta9-tetrahydrocannabinol in mice facilitates the antinociceptive and antidepressant-like responses elicited by the endogenous enkephalins protected from their degradation by RB 101, a complete inhibitor of enkephalin catabolism. This emphasizes the existence of a physiological interaction between endogenous opioid and cannabinoid systems. Accordingly, Delta9-tetrahydrocannabinol increased the release of Met-enkephalin-like material in the nucleus accumbens of awake and freely moving rats measured by microdialysis. In addition, this cannabinoid agonist displaced the in vivo [3H]diprenorphine binding to opioid receptors in total mouse brain. The repetitive pretreatment during 3 weeks of Delta9-tetrahydrocannabinol in mice treated chronically with morphine significantly reduces the naloxone-induced withdrawal syndrome. However, this repetitive administration of Delta9-tetrahydrocannabinol did not modify or even decrease the rewarding responses produced by morphine in the place preference paradigm. Taken together, these behavioural and biochemical results demonstrate the existence of a direct link between endogenous opioid and cannabinoid systems. However, chronic use of high doses of cannabinoids does not seem to potentiate the psychic dependence to opioids.

Acute haemodynamic effects and tissue distribution of acebutolol in normotensive and spontaneously hypertensive rats.

Acute i.v. administration of 15 mg/kg acebutolol in normotensive (WKY), Okamoto (SHR) and Okamoto stroke-prone (SHR-SP) awake rats resulted in weak effects on blood pressure and in bradycardia more marked in SHR-SP. Thirty minutes after i.v. administration, lung and renal uptake of [14C]acebutolol was reduced in SHR compared to other rats. Muscle uptake was higher in SHR and blood concentration was higher in SHR-SP. Brain levels were low and similar in all rats. Plasma protein binding was identical in all rats. These results are discussed according to the characteristics of the three strains studied.

In vivo properties of thiol inhibitors of the three vasopeptidases NEP, ACE and ECE are improved by introduction of a 7-azatryptophan in P2' position.

Three zinc metallopeptidases are implicated in the regulation of fluid homeostasis and vascular tone and represent interesting targets for the treatment of chronic heart failure. We have previously reported the synthesis of a triple inhibitor able to simultaneously inhibit neprilysin (NEP, EC 3.4.24.11), angiotensin-converting enzyme (ACE, EC 3.4.15.1) and endothelin-converting enzyme (ECE-1, EC 3.4.24.71) with nanomolar potency towards NEP and ACE and a lesser affinity for ECE. Here, we report the optimization and biological activities of analogs derived from lead compound 1 (2S)-2-[(2R)-2-((1S)-5-bromo-indan-1-yl)-3-mercapto-propionylamino]-3- (1H-indol-3-yl)-propionic acid by a structural approach. Among several inhibitors, compound 21, (2S)-2-[(2R)-2-((1S)-5-bromo-indan-1-yl)-3-mercapto-propionylamino]-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)-propionic acid was selected by taking into account its good molecular adaptation with the recently published structures of the three vasopeptidases. This optimization procedure led to an improved pharmacologic activity when compared with 1.

Cannabinoid withdrawal is dependent upon PKA activation in the cerebellum.

Region-specific up-regulation of the cyclic AMP pathway is considered an important molecular mechanism in the origin of the somatic manifestations of the withdrawal syndrome to known drugs of abuse. Nevertheless, the existence of a withdrawal syndrome after prolonged cannabinoid administration has long been a controversial issue. Recent studies, in different species, have shown that withdrawal to prolonged cannabinoid exposure precipitated by the cannabinoid antagonist SR141716A is characterized by physical signs underlying impairment of motor coordination. Interestingly, cannabinoid withdrawal is accompanied by an increase of adenylyl cyclase activity in the cerebellum. Here, we investigate the functional role of the cyclic AMP pathway in the cerebellum in the establishment of cannabinoid withdrawal. We show that after SR141716A precipitation of cannabinoid withdrawal, basal and calcium-calmodulin-stimulated adenylyl cyclase activities as well as active PKA in the cerebellum increase in a transient manner with a temporal profile which matches that of the somatic expression of abstinence. Selectively blocking the up-regulation of the cyclic AMP pathway in the cerebellum, by microinfusing the cyclic AMP blocker Rp-8Br-cAMPS in this region, markedly reduced both PKA activation and the somatic expression of cannabinoid withdrawal. Our results (i) directly link the behavioural manifestations of cannabinoid withdrawal with the up-regulation of the cyclic AMP pathway in the cerebellum, pointing towards common molecular adaptive mechanisms for dependence and withdrawal to most drugs of abuse; (ii) suggest a particular role for the cerebellum as a major neurobiological substrate for cannabinoid withdrawal.