Activation of type II cannabinoid receptors improves myocardial tolerance to arrhythmogenic effects of coronary occlusion and reperfusion

Preliminary intravenous injection of cannabinoid receptor agonist HU-210 (0.05 mg/kg) reduced the incidence of ventricular arrhythmias during 10-min coronary occlusion and 10-min reperfusion in chloralose-anesthetized rats. Preliminary injection of type I cannabinoid receptor antagonist SR 141716A (3 mg/kg) had no effect on the antiarrhythmic effect of HU-210, while type II cannabinoid receptor antagonist SR 144528 (1 mg/kg) completely abolished the effect of HU-210. Preconditioning with glibenclamide (0.3 mg/kg), an inhibitor of ATP-dependent K⁺-channels, did not affect the antiarrhythmic activity of HU-210. These findings suggest that antiarrhythmic effect of HU-210 is mediated through activation of type II cannabinoid receptors rather than activation of K⁺-channels.     

A CB2 receptor agonist,A-836339, modulates wide dynamic range neuronal activity in neuropathic rats: Contributions of spinal and peripheral CB2 receptors

We investigated the systemic and site-specific actions of a selective CB₂ receptor agonist, A-836339 on mechanically evoked (10 g von Frey hair) and spontaneous firing of spinal wide dynamic range (WDR) neurons in neuropathic (L5 and L6 ligations) and sham rats. Systemic administration of A-836339 (0.3–3 μmol/kg, i.v.) reduced both evoked and spontaneous WDR neuronal activity in neuropathic, but not sham rats. The effects in neuropathic rats were blocked by pre-administration of a CB₂, but not a CB₁, receptor antagonist. Similar to systemic delivery, intra-spinal injection of A-836339 (0.3 and 1 nmol) also attenuated both von Frey–evoked and spontaneous firing of WDR neurons in neuropathic rats. Intra-spinal injections of A-836339 were ineffective in sham rats. Application of A-836339 (3–30 nmol) onto the ipsilateral L5 dorsal root ganglion (DRG) of neuropathic rats reduced the von Frey–evoked activity of WDR neurons, but spontaneous firing was unaltered. All effects of A-836339 on WDR neuronal activity following either intra-spinal or intra-DRG administration were blocked by pre-administration of a CB₂ receptor antagonist. Pre-administration of a CB₁ receptor antagonist did not alter the site-specific effects of A-836339. Injection of A-836339 (300 nmol) into the neuronal receptive field on the ipsilateral hind paw did not affect evoked or spontaneous firing of WDR neurons. Thus, the current data demonstrate that modulation of spinal neuronal activity by a CB₂ receptor agonist is enhanced following peripheral nerve injury, and further delineate the contribution of spinal and peripheral CB₂ receptors to this modulation.     

Activation of cannabinoid receptors decreases the area of ischemic myocardial necrosis

We studied the possibility of decreasing the area of ischemic necrosis during myocardial infarction with HU-210, a selective cannabinoid receptor agonist. Activation of cannabinoid receptors with HU-210 had practically no effect on collateral blood flow in the myocardium, but considerably decreased the area of necrosis. There results indicate that cannabinoid receptor agonist HU-210 possesses cardioprotective activity and delays the formation of necrotic zones during coronary occlusion and reperfusion.     

Involvement of central and peripheral cannabionoid receptors in the regulation of heart resistance to arrhythmogenic effects of epinephrine

Intravenous injection of the selective cannabinoid receptor agonist HU-210 in doses of 0.05 and 0.25 mg/kg increased heart resistance to arrhythmogenic effects of epinephrine, while intracerebroventricular infusion of this substance had no effect on the incidence of epinephrine-induced arrhythmia. The selective antagonist of type I cannabinoid receptors SR141716A in a dose of 3 mg/kg and ganglion blocker hexamethonium in a dose of 10 mg/kg did not modify the antiarrhythmic effect of HU-210. This effect of HU-210 is probably related to activation of type II peripheral cannabinoid receptors. Translated fromByulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 130, No. 11, pp. 552–554, November, 2000     

Involvement of central and peripheral cannabionoid receptors in the regulation of heart resistance to arrhythmogenic effects of epinephrine

Intravenous injection of the selective cannabinoid receptor agonist HU-210 in doses of 0.05 and 0.25 mg/kg increased heart resistance to arrhythmogenic effects of epinephrine, while intracerebroventricular infusion of this substance had no effect on the incidence of epinephrine-induced arrhythmia. The selective antagonist of type I cannabinoid receptors SR141716A in a dose of 3 mg/kg and ganglion blocker hexamethonium in a dose of 10 mg/kg did not modify the antiarrhythmic effect of HU-210. This effect of HU-210 is probably related to activation of type II peripheral cannabinoid receptors.     

Inhibitory effects of the cannabinoid agonist HU 210 on rat sexual behaviour

The present study investigates the effects induced by the cannabinoid agonist HU 210 (25-100 microg/kg, administered intraperitoneally [i.p.]) on the following parameters: (a) sexual behaviour of male rats, categorised on the basis of seven consecutive mating pretests as sexually active (SA) and sexually inactive (SI) and (b) sexual receptivity of ovariectomised female rats displaying hormonally induced heat. The data obtained show that HU 210, administered in acute or subchronic mode (once daily for 7 and 14 days), impaired the copulatory pattern of SA rats in a dose- and mode-dependent manner, decreasing their sexual drive, mainly as represented by an increase in mount and intromission latencies, and affecting ejaculation mechanisms (represented as a decrease in intromission frequency and increase in ejaculation latency). After subchronic treatment with the highest dose had been suspended for 2 weeks, SA males' performance was still impaired. In SI rats, acute injections of the drug (25 and 50 microg/kg, i.p.) at the higher dose increased contact latency and decreased genital exploration time towards the female. Acute HU 210 (25-100 microg/kg, i.p.) also inhibited female sexual behaviour, potently reducing lordosis quotient and lordosis intensity.     

The endocannabinoid transport inhibitor AM404 modulates nociception in cholestasis

Cholestasis is associated with increased activity of the endogenous opioid system that results in analgesia. Endocannabinoid system can reduce pain sensitivity. Considering the interaction that has been shown between the endogenous opioid and endocannabinoid systems in nociception processing, we studied the effect of AM404, an endocannabinoid transport inhibitor, on modulation of nociception in cholestasis, a model of elevated endogenous opioid tone. Cholestasis was induced by ligation of the main bile duct using two ligatures and transection of the duct at the midpoint between them. A significant increase (P < 0.01) in TF was observed in cholestatic rats compared to unoperated and sham rats. AM404 (10 mg/kg, i.p.) significantly increased TFL at 5, 30 min but not 60 min after injection in cholestatic animals compared to the vehicle treated cholestatic group (P < 0.05, P < 0.001, respectively). AM404 injection to unoperated and sham rats did not alter baseline TFL. The effect of AM404 in cholestatic rats was blocked by co-administration of a CB₁ receptor antagonist, AM251 (1 mg/kg, i.p.) but not by the CB2 receptor antagonist, SR144528 (1 mg/kg, i.p.). Naloxone injection blocked the antinociception induced by cholestasis in bile duct ligated group. Antinociception produced by injection of AM404 in cholestatics was also attenuated by co-administration of naloxone. These data show that AM404 potentiates antinociception induced by cholestasis and indicate that there are possible interactions between opioid and cannabinoid systems in this experimental model of elevated endogenous opioid tone. The inhibitory effects of AM404 in this model are mediated by cannabinoid CB₁ and not CB₂ receptors.     

The cannabinoid agonist WIN55212-2 decreases L-DOPA-induced PKA activation and dyskinetic behavior in 6-OHDA-treated rats

Chronic Levodopa (l-DOPA), the gold standard therapy for Parkinson's disease (PD), causes disabling motor complications (dyskinesias) that are associated with changes in the activity of striatal protein kinase A (PKA) and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32). In this study, we showed that systemic administration of the cannabinoid agonist WIN55212-2 ameliorated l-DOPA-induced abnormal involuntary movements (AIMs) in the 6-OHDA rat model of PD and reversed l-DOPA-induced PKA hyperactivity via a CB₁-mediated mechanism. This effect was accompanied by increased phosphorylation of DARPP-32 at threonine 34, which was partially blocked by CB₁ antagonism. Striatal PKA activity was positively correlated with the severity of l-DOPA-induced axial and limb dyskinesias, suggesting a role for the cAMP/PKA signaling pathway in the expression of these motor disturbances.Our results indicate that activation of CB₁ receptors, as well as reduction of striatal PKA hyperactivity, might be an effective strategy for the treatment of l-DOPA-induced dyskinesias.     

Effect of the cannabinoid ajulemic acid on rat models of neuropathic and inflammatory pain

There is increasing evidence that cannabinoid agonists alleviate the abnormal pain sensations associated with animal models of neuropathic and inflammatory pain. However, cannabinoids produce a number of motor and psychotropic side effects. In the present study we found that systemic administration of the cannabinoid acid derivative 1',1'-dimethylheptyl-delta-8-tetrahydrocannabinol-11-oic acid (ajulemic acid, IP-751) and the non-selective cannabinoid receptor agonist HU-210 reduced mechanical allodynia in a nerve-injury induced model of neuropathic pain and in the CFA-induced model of inflammatory pain. In contrast, HU-210, but not ajulemic acid reduced motor performance in the rotarod test. These findings suggest that ajulemic acid reduces abnormal pain sensations associated with chronic pain without producing the motor side effects associated with THC and other non-selective cannabinoid receptor agonists.     

Differential alterations of GABA(A) receptor (α1, β2, γ2 subunit) expression and increased seizure susceptibility in rat offspring from morphine-addicted mothers: beneficial effect of dextromethorphan

Recent study demonstrated a close relationship between cerebellum atrophy and symptom severity of pediatric maltreatment-related posttraumatic stress disorder (PTSD). It has also been known that females are more vulnerable than males in developing anxiety disorders after exposure to traumatic stress. The mechanisms are unknown. Because cannabinoid receptors (CB₁ and CB₂) are neuroprotective and highly expressed in the cerebellum, we investigated cerebellar CB expression in stressed rats. Young male and female Sprague-Dawley rats were given 40 unpredictable electric tail-shocks for 2 h daily on 3 consecutive days. CB₁ and CB₂ mRNA and protein levels in rat cerebellum and brain stem were determined using quantitative real-time PCR and Western blot, respectively. Two-way ANOVA revealed significant gender and stress effects on cerebellar CB₁ mRNA expression, with females and non-stressed rats exhibiting higher CB₁ mRNA levels than the males (3 fold, p < 0.01) and stressed rats (30%, p < 0.01), respectively. CB₁ and CB₂ mRNA levels in brain stem were also greater in female rats than males (p < 0.01, p < 0.05, respectively). Repeated stress increased the level of phosphorylated CB₁ receptors, the inactivated CB₁, in rat cerebellum (p < 0.01), particularly in female rats as revealed by the significant gender × stress interaction. Thus, repeated severe stress caused greater CB₁ mRNA suppression and CB₁ receptor phosphorylation in female cerebellum that could lead to increased susceptibility to stress-related anxiety disorders including PTSD.     

Duration-dependent growth of N1m for speech-modulated bone-conducted ultrasound

Cannabinoids classically act via CB₁ and CB₂ receptors to modulate nociception; however, recent findings suggest that some cannabinoids bind to atypical receptors. One such receptor is GPR55 which is activated by the abnormal cannabidiol analogue O-1602. This study investigated whether the synthetic GPR55 agonist O-1602 can alter joint nociception in a rat model of acute joint inflammation. Acute (24 h) inflammatory joint pain was induced in male Wistar rats by intra-articular injection of 2% kaolin and 2% carrageenan. Single unit extracellular recordings were made from arthritic joint afferents in response to mechanical rotation of the knee. Peripheral administration of O-1602 significantly reduced movement-evoked firing of nociceptive C fibres and this effect was blocked by the GPR55 receptor antagonist O-1918. Co-administration of the CB₁ and CB₂ antagonists (AM281 and AM630 respectively) had no effect on O-1602 responses. This study clearly shows that atypical cannabinoid receptors are involved in joint nociception and these novel targets may be advantageous for the treatment of inflammatory pain.     

CRIP1a switches cannabinoid receptor agonist/antagonist-mediated protection from glutamate excitotoxicity

A shared pathology among many neurological and neurodegenerative disorders is neuronal loss. Cannabinoids have been shown to be neuroprotective in multiple systems. However, both agonists and antagonists of the CB₁ cannabinoid receptor are neuroprotective, but the mechanisms responsible for these actions remain unclear. Recently a CB₁ receptor interacting protein, CRIP1a, was identified and found to alter CB₁ activity. Here we show that in an assay of glutamate neurotoxicity in primary neuronal cortical cultures CRIP1a disrupts agonist-induced neuroprotection and confers antagonist-induced neuroprotection.     

Anandamide enhances expression of heat shock protein 72 to protect against ischemia–reperfusion injury in rat heart

Anandamide (AEA), one of endocannabinoids, has been reported to exhibit a cardioprotective ability to limit the damage produced by ischemia–reperfusion injury. AEA reportedly enhanced heat shock protein 72 (HSP72) and HSP25 expression in lungs to protect against lung inflammation. This study tested the hypothesis that intravenously injected AEA would induce HSP72 in the heart and thus render cardioprotection against ischemia–reperfusion injury in rats. Cardiac expression of HSPs was quantitatively evaluated in rats by Western blot analysis. That intravenously injected AEA 1 mg/kg in vivo induced expression of HSP72, which peaked at 24 h after administration. The enhancement of HSP72 by AEA was blocked by cannabinoid 2 (CB₂) receptor antagonist AM630, but not cannabinoid 1 (CB₁) receptor antagonist AM251. Therefore, the rats were induced with a 30-min coronary occlusion followed by a 120-min reperfusion in vivo at 24 h after administration of drugs or vehicle, and then the infarct size was measured. AEA reduced myocardial infarct size compared to control group. Pretreatment with AM630 but not AM251 abolished the infarct size-limiting effect of AEA. Further study demonstrated pretreatment with phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin, Akt inhibitor MK-2206 and AM630 attenuated phosphorylation of Akt and AEA-induced HSP72 expression. The results suggest that AEA is cardioprotective against ischemia–reperfusion insult through its induction of HSP72, which might be mediated by the PI3K/Akt signaling pathway. These effects were mediated by CB₂ but not CB₁ receptors.     

Effects of the 5-HT4 receptor agonist RS67333 and paroxetine on hippocampal extracellular 5-HT levels

The 5-HT₄ receptor modulates activity of serotonergic neurons and is a new potential target for antidepressant treatment. This microdialysis study evaluated the effect of the 5-HT₄ receptor agonist, RS67333, on extracellular serotonin (5-hydroxytryptamine, 5-HT) and 5-HIAA levels in rat ventral hippocampus during chloral hydrate anaesthesia, and explored the ability of RS67333 to augment the effect of the selective serotonin reuptake inhibitor paroxetine. The effect of RS67333 was examined after acute and subchronic (3 days) administration. Acute RS67333 (1.5 mg/kg i.v.) had no effect on extracellular 5-HT or 5-HIAA levels, while acute paroxetine (0.5 mg/kg i.v.) increased 5-HT levels by 299 ± 16% and decreased 5-HIAA levels by 25 ± 4%. Administration of RS67333 80 min after paroxetine caused an additional transient increase in 5-HT levels (to 398 ± 52% of baseline). Subchronic RS67333 administration (1.5 mg/kg i.p.) increased basal 5-HT levels by 73 ± 15% and decreased 5-HIAA levels by 27 ± 13%. In conclusion, the 5-HT₄ receptor agonist RS67333 augmented the acute effect of paroxetine on extracellular 5-HT levels in the ventral hippocampus, and after 3 days increased basal hippocampal 5-HT levels.     

Activation of CB1 cannabinoid receptors impairs memory consolidation and hippocampal polysialylated neural cell adhesion molecule expression in contextual fear conditioning

We investigated the role of CB1 receptors in hippocampal-dependent memory consolidation mediated by polysialylated neural cell adhesion molecule (PSA-NCAM) during contextual fear conditioning (CFC). The CB1 receptor agonist 3-(1,1-dimethylheptyl)-(-)-11-hydroxy-Δ⁸-tetrahydrocannabinol (HU-210) (0.1 mg/kg) was given immediately after training during the memory consolidation phase, and freezing behavior was measured 24 h after conditioning. Administration of HU-210 attenuated freezing behavior measured in CFC. Western blot analysis showed that CFC induced a decrease in the expression of NCAM-180, but did not change the level of NCAM-140 and increased PSA-NCAM expression measured 24 h after training in the rat hippocampus. HU-210 (0.1 mg/kg) injection did not affect the reduction in NCAM-180 levels induced by CFC, but it blocked the increase in PSA-NCAM expression. Since the dentate gyrus (DG) of the hippocampus is known to be involved in memory consolidation and expresses a high level of PSA-NCAM protein, we measured the effects of CFC and HU-210 administration on PSA-NCAM-immunoreactive (IR) cells in the DG. CFC caused an increase in the number of PSA-NCAM-IR cells in the DG, but not K_i-67- or doublecortin (DCX)-IR cells. This increase in PSA-NCAM-IR cells was abolished by HU-210 injection. Administration of the CB1 receptor antagonist N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM-251) (3 mg/kg immediately before HU-210) inhibited the effects of HU-210 on freezing behavior and PSA-NCAM expression in the DG. These results indicate that activation of CB1 receptors disturbs consolidation of fear memory in CFC, likely by affecting PSA-NCAM expression in the DG, which plays an important role in synaptic rearrangement during the formation of memory traces.     

The CB1 receptor agonist, WIN 55,212-2, dose-dependently disrupts object recognition memory in adult rats

Drugs that act as agonists at the cannabinoid CB₁ receptor have been reported to interfere with a diverse range of cognitive functions, including object recognition memory. However, to date, most of the studies conducted on this aspect of memory have suggested that these effects occur mainly in pubertal or pre-pubertal, rather than adult, rats. In this study we revisited this issue and evaluated the effects of a single s.c. injection of the CB₁ receptor agonist, WIN 55,212-2 (‘WIN’), at 1, 3 or 5 mg/kg, on object recognition memory. We found that WIN significantly reduced the total exploration time for objects at the 5 mg/kg dose only (P < 0.05). This was presumably due to its sedative effects at this dose. However, the discrimination index, which controlled for the general effects of WIN on object exploration, was significantly reduced only for the 1 mg/kg WIN group (P < 0.05), suggesting that only at this low dose did WIN specifically interfere with object discrimination. These results suggest that WIN can interfere with object recognition memory even in adult rats following a single injection of a low dose.     

The effect of leptin receptor deficiency and fasting on cannabinoid receptor 1 mRNA expression in the rat hypothalamus, brainstem and nodose ganglion

Despite ample evidence for the involvement of the endocannabinoid system in the control of appetite, food intake and energy balance, relatively little is known about the regulation of cannabinoid receptor 1 (CB₁R) expression in respect to leptin signalling and fasting. In the present study, we examined CB₁R mRNA levels in lean (Fa/?) and obese (fa/fa) male Zucker rats under basal and food-restricted conditions. Using stereological sampling principles coupled with semi-quantitative radioactive in situ hybridization we provide semi-quantitative estimates of CB₁R mRNA expression in key appetite regulatory hypothalamic and brainstem areas, as well as in the nodose ganglia. Whereas no effect of fasting were determined on CB₁R mRNA levels in the paraventricular (PVN) and ventromedial hypothalamic (VMH) nucleus, in the brainstem dorsal vagal complex or nodose ganglion of lean Zucker rats, CB₁R mRNA levels were consistently elevated in obese Zucker rats pointing to a direct influence of disrupted leptin signalling on CB₁R mRNA regulation.     

Use of a Novel Mouse Genotype to Model Acute Benzodiazepine Withdrawal

Withdrawal from benzodiazepines in physically dependent rodents often requires that the drug be dislodged from its receptor with a competitive antagonist. Withdrawal Seizure-Prone (WSP) mice were selectively bred for their susceptibility to handling-induced withdrawal convulsions following chronic treatment with ethanol. Reflecting pleiotropic genetic influences, they also experience more severe withdrawal from other sedative-hypnotics including the benzodiazepine, diazepam. We used this susceptible genotype to test whether other benzodiazepine receptor (BZR) agonists also produce physical dependence following acute administration, comparing studies of spontaneous withdrawal with those where convulsions were precipitated by a BZR antagonist (flumazenil). Separate groups of mice were tested following a single injection of one of eight BZR agonists. Several doses of each drug were tested for spontaneous withdrawal, and a single dose of each drug was tested for precipitated withdrawal. Withdrawal convulsions were seen after all of the drugs by at least one method, suggesting that BZR agonists as a class elicit acute physical dependence in this susceptible genotype. Edited by Andrew Holmes     

Overexpressed protein disulfide isomerase in brains of patients with sporadic Creutzfeldt-Jakob disease

Cannabinoids produce antinociception via specific cannabinoid receptor activation, but there are also non-receptor mediated effects like for example the activation of the arachidonic acid cascade. Here we investigate the influence of cannabinoids (CB) on sleep duration after isoflurane anesthesia. We found that the CB receptor agonists R(-)-7-hydroxy-delta-6-tetra-hydrocannabinol-dimethylheptyl (HU-210) (0.1 mg/kg), 2-O-arachidonoylglycerylether (30 mg/kg) and arachidonyl-2-chloroethylamide (3 mg/kg) significantly prolong the duration of isoflurane induced sleep in mice (P<0.05). This effect was absent when co-injecting the selective CB(1) antagonist N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (1 mg/kg). Furthermore, HU-210 was ineffective in CB(1) receptor knockout mice (CB(1)-/-). Our behavioral tests (tail flick, rotarod) indicate that the sleep latency can be prolonged even at low drug dosages which do not influence thermal nociception. In the chosen dosages thimerosal (20 mg/kg), 2-AG (10 mg/kg), R(1)-methanandamide (R(1)-MAEA) (10 mg/kg) and flurbiprofen (27 mg/kg) were ineffective to increase sleep duration.