Cannabinoid receptor interactions with the antagonists SR 141716A and SR 144528.

The G protein-coupled cannabinoid receptor subtypes CB1 and CB2 have been cloned from several species. The CB1 receptor is highly conserved across species, whereas the CB2 receptor shows considerable cross-species variations. The two human receptors share only 44% overall identity, ranging from 35% to 82% in the transmembrane regions. Despite this structural disparity, the most potent cannabinoid agonists currently available are largely undiscriminating and are therefore unsatisfactory tools for investigating the architecture of ligand binding sites. However, the availability of two highly specific antagonists, SR 141716A for the CB1 receptor and SR 144528 for the CB2 receptor, has allowed us to adopt a systematic approach to defining their respective binding sites through the use of chimeric CB1 receptor/CB2 receptor constructs, coupled with site-directed mutagenesis. We identified the region encompassed by the fourth and fifth transmembrane helices as being critical for antagonist specificity. Both the wild type human receptors overexpressed in heterologous systems are autoactivated; SR 141716A and SR 144528 exhibit classical inverse agonist properties with their respective target receptors. In addition, through its interaction with the CB1 receptor SR 141716A blocks the Gi protein-mediated activation of mitogen-activated protein kinase stimulated by insulin or insulin-like growth factor I. An in-depth analysis of this discovery has led to a modified three-state model for the CB1 receptor, one of which implicates the SR 141716A-mediated sequestration of Gi proteins, with the result that the growth factor-stimulated intracellular pathways are effectively impeded.     

Role of cannabinoid receptors in regulation of cardiac tolerance to ischemia and reperfusion

Coronary artery occlusion (45 min) and reperfusion (2 h) were modeled in vivo in anesthetized artificially ventilated Wistar rats. Total ischemia (45 min) and reperfusion (30 min) of the isolated rat heart were performed in vitro. The selective agonist of cannabinoid (CB) receptors HU-210 was injected intravenously at a dose of 0.1 mg/kg 15 min prior to the coronary artery ligation. The selective CB1 antagonist SR141716A and the selective CB2 antagonist SR144528 were injected intravenously 25 min prior to ischemia. In vitro, HU-210 and SR141716A were added to the perfusion solution at the final concentrations of 0.1 μM prior to total ischemia. Preliminary injection of HU-210 reduced the infarct size-to-area at risk (IS/AAR) ratio in vivo. This cardioprotective effect was completely abolished by SR141716A but remained after SR144528 injection. Both antagonists had no effect on the IS/AAR ratio. Preliminary injection of the K_ATP channel blocker glibenclamide did not abolish the cardioprotective effect of HU-210. The addition of HU-210 prior to ischemia reduced the creatine phosphokinase (CPK) level in the coronary effluent and decreased left ventricular developed pressure. SR141716A alone had no effect on cardiac contractility and CPK levels. These results suggest that cardiac CB1 receptor activation increases cardiac tolerance to ischemia-reperfusion and has a negative effect on the cardiac pump function. Endogenous cannabinoids are not involved in the regulation of cardiac contractility and tolerance to ischemia and reperfusion. ATP-sensitive k_ATP-channels are not involved in the mechanism of the cardioprotective effect of HU-210.     

Increase of the heart arrhythmogenic resistance and decrease of the myocardial necrosis zone during activation of cannabinoid receptors

We have found that intravenous administration of cannabinoid receptor (CB) agonist HU-210 (0.05 mg/kg), increases cardiac resistance against arrhythmogenic effect of epinephrine, aconitine, coronary artery occlusion and reperfusion in rats. Pretreatment with CB2-receptor antagonist, SR144528 (1 mg/kg), completely abolished the antiarrhythmic effect of HU-210. However this effect of HU-210 was not attenuated by pretreatment with CB1-receptor antagonist, SR141716A (3 mg/kg). We also found that HU-210 (0.05 mg/kg) decreased the relationship between infarction size and area of ischemia. It is concluded that CB2 receptor stimulation promotes an increase in the cardiac resistance against arrhythmogenic influences and probably increases myocardial tolerance of both ischemic and reperfusion damages in rats.     

Acute effects of the cannabinoid receptor agonist WIN55212-2 on dopamine release in rat: an in vivo electrochemical study

The aim of this study was to investigate the effects of the cannabinoid receptor agonist, WIN55212-2, and the cannabinoid receptor antagonist, SR141716A, on dopamine (DA) release evoked by KC1 (120 mM) microinjected into the striatum. The cannabinoid agonist WIN55212-2 (1 and 5 mg/kg, i.p.) dose-dependently attenuated DA release in the striatum, whereas the cannabinoid receptor antagonist SR141716A (3 mg/kg, i.p.) produced the opposite effect. SR141716A (3 mg/kg, i.p.) blocked the effects on DA release by WIN55212-2 (5 mg/kg, i.p.). Vehicle alone did not change DA release. These results suggest that cannabinoids modulate DA release in the striatum.     

Regulation of peripheral cannabinoid receptor CB2 phosphorylation by the inverse agonist SR 144528. Implications for receptor biological responses.

We recently demonstrated that the selective cannabinoid receptor antagonist SR 144528 acts as an inverse agonist that blocks constitutive mitogen-activated protein kinase activity coupled to the spontaneous autoactivated peripheral cannabinoid receptor (CB2) in the Chinese hamster ovary cell line stably transfected with human CB2. In the present report, we studied the effect of SR 144528 on CB2 phosphorylation. The CB2 phosphorylation status was monitored by immunodetection using an antibody specific to the COOH-terminal CB2 which can discriminate between phosphorylated and non-phosphorylated CB2 isoforms at serine 352. We first showed that CB2 is constitutively active, phosphorylated, and internalized at the basal level. By blocking autoactivated receptors, inverse agonist SR 144528 treatment completely inhibited this phosphorylation state, leading to an up-regulated CB2 receptor level at the cell surface, and enhanced cannabinoid agonist sensitivity for mitogen-activated protein kinase activation of Chinese hamster ovary-CB2 cells. After acute agonist treatment, serine 352 was extensively phosphorylated and maintained in this phosphorylated state for more than 8 h after agonist treatment. The cellular responses to CP-55,940 were concomitantly abolished. Surprisingly, CP-55,940-induced CB2 phosphorylation was reversed by SR 144528, paradoxically leading to a non-phosphorylated CB2 which could then be fully activated by CP-55,940. The process of CP-55,940-induced receptor phosphorylation followed by SR 144528-induced receptor dephosphorylation kept recurring many times on the same cells, indicating that the agonist switches the system off but the inverse agonist switches the system back on. Finally, we showed that autophosphorylation and CP-55, 940-induced serine 352 CB2 phosphorylation involve an acidotropic GRK kinase, which does not use Gibetagamma. In contrast, SR 144528-induced CB2 dephosphorylation was found to involve an okadaic acid and calyculin A-sensitive type 2A phosphatase.     

Influence of Cannabinoids on Electrically Evoked Dopamine Release and Cyclic AMP Generation in the Rat Striatum

Abstract: Using the endogenous cannabinoid receptor agonist anandamide, the synthetic agonist CP 55940 {[1α,2β( R )5α]-(−)-5-(1,1-dimethylheptyl)-2-[5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]phenol}, and the specific antagonist SR 141716 [ N -(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1 H -pyrazole-3-carboxamide hydrochloride], second messenger activation of the central cannabinoid receptor (CB1) was examined in rat striatal and cortical slices. The effects of these cannabinoid ligands on electrically evoked dopamine (DA) release from [³H]dopamine-prelabelled striatal slices were also investigated. CP 55940 (1 µ M ) and anandamide (10 µ M ) caused significant reductions in forskolin-stimulated cyclic AMP accumulation in rat striatal slices, which were reversed in the presence of SR 141716 (1 µ M ). CP 55940 (1 µ M ) had no effect on either KCl- or neurotransmitter-stimulated ³H-inositol phosphate accumulation in rat cortical slices. CP 55940 and anandamide caused significant reductions in the release of dopamine after electrical stimulation of [³H]dopamine-prelabelled striatal slices, which were antagonised by SR 141716. SR 141716 alone had no effect on electrically evoked dopamine release from rat striatal slices. These data indicate that the CB1 receptors in rat striatum are negatively linked to adenylyl cyclase and dopamine release. That the CB1 receptor may influence dopamine release in the striatum suggests that cannabinoids play a modulatory role in dopaminergic neuronal pathways.     

Anti-proliferative effect of a putative endocannabinoid, 2-arachidonylglyceryl ether in prostate carcinoma cells

Endocannabinoids (ECs), anandamide (AEA) and 2-arachidonoylglycerol (2-AG), inhibit proliferation of carcinoma cells. Several enzymes hydrolyze ECs to reduce endogenous EC concentrations and produce eicosanoids that promote cell growth. In this study, we determined the effects of EC hydrolysis inhibitors and a putative EC, 2-arachidonylglyceryl ether (noladin ether, NE) on proliferation of prostate carcinoma (PC-3, DU-145, and LNCaP) cells. PC-3 cells had the least specific hydrolysis activity for AEA and administration of AEA effectively inhibited cell proliferation. The proliferation inhibition was blocked by SR141716A (a selective CB1R antagonist) but not SR144528 (a selective CB2R antagonist), suggesting a CB1R-mediated inhibition mechanism. On the other hand, specific hydrolysis activity for 2-AG was high and 2-AG inhibited proliferation only in the presence of EC hydrolysis inhibitors. NE inhibited proliferation in a concentration-dependent manner; however, SR141716A, SR144528 and pertussis toxin did not block the NE-inhibited proliferation, suggesting a CBR-independent mechanism of NE. A peroxisome proliferator-activated receptor gamma (PPARγ) antagonist GW9662 did not block the NE-inhibited proliferation, suggesting that PPARγ was not involved. NE also induced cell cycle arrest in G(0)/G(1) phase in PC-3 cells. NE inhibited the nuclear translocation of nuclear factor-kappa B (NF-κB p65) and down-regulated the expression of cyclin D1 and cyclin E in PC-3 cells, suggesting the NF-κB/cyclin D and cyclin E pathways are involved in the arrest of G1 cell cycle and inhibition of cell growth. These results indicate therapeutic potentials of EC hydrolysis inhibitors and the enzymatically stable NE in prostate cancer.     

Inhibition of pain responses by activation of CB(2) cannabinoid receptors

Cannabinoid receptor agonists diminish responses to painful stimuli. Extensive evidence demonstrates that CB(1) cannabinoid receptor activation inhibits pain responses. Recently, the synthesis of CB(2) cannabinoid receptor-selective agonists has allowed testing whether CB(2) receptor activation inhibits pain. CB(2) receptor activation is sufficient to inhibit acute nociception, inflammatory hyperalgesia, and the allodynia and hyperalgesia produced in a neuropathic pain model. Studies using site-specific administration of agonist and antagonist have suggested that CB(2) receptor agonists inhibit pain responses by acting at peripheral sites. CB(2) receptor activation also inhibits edema and plasma extravasation produced by inflammation. CB(2) receptor-selective agonists do not produce central nervous system (CNS) effects typical of cannabinoids retaining agonist activity at the CB(1) receptor. Peripheral antinociception without CNS effects is consistent with the peripheral distribution of CB(2) receptors. CB(2) receptor agonists may have promise for the treatment of pain and inflammation without CNS side effects.     

CB(2) cannabinoid receptor antagonist SR144528 decreases mu-opioid receptor expression and activation in mouse brainstem: role of CB(2) receptor in pain

Formerly considered as an exclusively peripheral receptor, it is now accepted that CB(2) cannabinoid receptor is also present in limited amounts and distinct locations in the brain of several animal species, including mice. However, the possible roles of CB(2) receptors in the brain need to be clarified. The aim of our work was to study the mu-opioid receptor (MOR) mRNA expression level and functional activity after acute in vivo and in vitro treatments with the endocannabinoid noladin ether (NE) and with the CB(2) receptor antagonist SR144528 in brainstem of mice deficient in either CB(1) or CB(2) receptors. This study is based on our previous observations that noladin ether (NE) produces decrease in the activity of MOR in forebrain and this attenuation can be antagonized by the CB(2) cannabinoid antagonist SR144528, suggesting a CB(2) receptor mediated effect. We used quantitative real-time PCR to examine the changes of MOR mRNA levels, [(35)S]GTPgammaS binding assay to analyze the capability of mu-opioid agonist DAMGO to activate G-proteins and competition binding assays to directly measure the ligand binding to MOR in mice brainstem. After acute NE administration no significant changes were observed on MOR signaling. Nevertheless pretreatment of mice with SR144528 prior to the administration of NE significantly decreased MOR signaling suggesting the involvement of SR144528 in mediating the effect of MOR. mRNA expression of MORs significantly decreased both in CB(1) wild-type and CB(1) knockout mice after a single injection of SR144528 at 0.1mg/kg when compared to the vehicle treated controls. Consequently, MOR-mediated signaling was attenuated after acute in vivo treatment with SR144528 in both CB(1) wild-type and CB(1) knockout mice. In vitro addition of 1microM SR144528 caused a decrease in the maximal stimulation of DAMGO in [(35)S]GTPgammaS binding assays in CB(2) wild-type brainstem membranes whereas no significant changes were observed in CB(2) receptor knockouts. Radioligand binding competition studies showed that the noticed effect of SR144528 on MOR signaling is not mediated through MORs. Our data demonstrate that the SR144528 caused pronounced decrease in the activity of MOR is mediated via CB(2) cannabinoid receptors.     

Constitutive activity of the cannabinoid CB1 receptor regulates the function of co-expressed Mu opioid receptors

The human mu opioid receptor was expressed stably in Flp-In T-REx HEK293 cells. Occupancy by the agonist DAMGO (Tyr-d-Ala-Gly-N-methyl-Phe-Gly-ol) resulted in phosphorylation of the ERK1/2 MAP kinases, which was blocked by the opioid antagonist naloxone but not the cannabinoid CB1 receptor inverse agonist SR141716A. Expression of the human cannabinoid CB1 receptor in these cells from the inducible Flp-In T-REx locus did not alter expression levels of the mu opioid receptor. This allowed the cannabinoid CB1 agonist WIN55212-2 to stimulate ERK1/2 phosphorylation but resulted in a large reduction in the capacity of DAMGO to activate these kinases. Although lacking affinity for the mu opioid receptor, co-addition of SR141716A caused recovery of the effectiveness of DAMGO. In contrast co-addition of the CB1 receptor neutral antagonist O-2050 did not. Induction of the CB1 receptor also resulted in an increase of basal [(35)S]guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding and thereby a greatly reduced capacity of DAMGO to further stimulate [(35)S]GTPgammaS binding. CB1 inverse agonists attenuated basal [(35)S]GTPgammaS binding and restored the capacity of DAMGO to stimulate. Flp-In T-REx HEK293 cells were generated, which express the human mu opioid receptor constitutively and harbor a modified D163N cannabinoid CB1 receptor that lacks constitutive activity. Induction of expression of the modified cannabinoid CB1 receptor did not limit DAMGO-mediated ERK1/2 MAP kinase phosphorylation and did not allow SR141716A to enhance the function of DAMGO. These data indicate that it is the constitutive activity inherent in the cannabinoid CB1 receptor that reduces the capacity of co-expressed mu opioid receptor to function.     

Evidence that 2-arachidonoylglycerol but not N-palmitoylethanolamine or anandamide is the physiological ligand for the cannabinoid CB2 receptor. Comparison of the agonistic activities of various cannabinoid receptor ligands in HL-60 cells

We examined the effect of 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand, on the intracellular free Ca(2+) concentrations in HL-60 cells that express the cannabinoid CB2 receptor. We found that 2-arachidonoylglycerol induces a rapid transient increase in intracellular free Ca(2+) concentrations in HL-60 cells. The response was affected by neither cyclooxygenase inhibitors nor lipoxygenase inhibitors, suggesting that arachidonic acid metabolites are not involved. Consistent with this notion, free arachidonic acid was devoid of any agonistic activity. Importantly, the Ca(2+) transient induced by 2-arachidonoylglycerol was blocked by pretreatment of the cells with SR144528, a CB2 receptor-specific antagonist, but not with SR141716A, a CB1 receptor-specific antagonist, indicating the involvement of the CB2 receptor but not the CB1 receptor in this cellular response. G(i) or G(o) is also assumed to be involved, because pertussis toxin treatment of the cells abolished the response. We further examined the structure-activity relationship. We found that 2-arachidonoylglycerol is the most potent compound among a number of naturally occurring cannabimimetic molecules. Interestingly, anandamide and N-palmitoylethanolamine, other putative endogenous ligands, were found to be a weak partial agonist and an inactive ligand, respectively. These results strongly suggest that the CB2 receptor is originally a 2-arachidonoylglycerol receptor, and 2-arachidonoylglycerol is the intrinsic natural ligand for the CB2 receptor that is abundant in the immune system.     

Blockade of cannabinoid CB(1) receptor function protects against in vivo disseminating brain damage following NMDA-induced excitotoxicity

The ability of cannabinoid CB(1) receptors to influence glutamatergic excitatory neurotransmission has fueled interest in how these receptors and their endogenous ligands may interact in conditions of excitotoxic insults. The present study characterized the impact of stimulated and inhibited CB(1) receptor function on NMDA-induced excitotoxicity. Neonatal (6-day-old) rat pups received a systemic injection of a mixed CB(1) /CB(2) receptor agonist (WIN55,212-2) or their respective antagonists (SR141716A for CB(1) and SR144528 for CB(2) ) prior to an unilateral intrastriatal microinjection of NMDA. The NMDA-induced excitotoxic damage in the ipsilateral forebrain was not influenced by agonist-stimulated CB(1) receptor function. In contrast, blockade of CB(1), but not CB(2), receptor activity evoked a robust neuroprotective response by reducing the infarct area and the number of cortical degenerating neurons. These results suggest a critical involvement of CB(1) receptor tonus on neuronal survival following NMDA receptor-induced excitotoxicity in vivo.     

Endogenous cannabinoid anandamide inhibits nicotinic acetylcholine receptor function in mouse thalamic synaptosomes

The effects of the endogenous cannabinoid anandamide [arachidonylethanolamide (AEA)] on the function of nicotinic acetylcholine receptor (nAChR) were investigated using the ⁸⁶Rb⁺ efflux assay in thalamic synaptosomes. AEA reversibly inhibited ⁸⁶Rb⁺ efflux induced by 300 μM ACh with an IC₅₀ value of 0.9 ± 2 μM. Pre-treatment with the cannabinoid (CB₁) receptor antagonist SR141716A (1 μM), the CB₂ receptor antagonist SR144528 (1 μM), or pertussis toxin (0.2 mg/mL) did not alter the inhibitory effects of AEA, suggesting that known CB receptors are not involved in AEA inhibition of nAChRs. AEA inhibition of ⁸⁶Rb⁺ efflux was not reversed by increasing acetylcholine (ACh) concentrations. In radioligand binding studies, the specific binding of [³H]-nicotine was not altered in the presence of AEA, indicating that AEA inhibits the function of nAChR in a non-competitive manner. Neither the amidohydrolase inhibitor phenylmethylsulfonyl fluoride (0.2 mM) nor the cyclooxygenase inhibitor, indomethacin, (5 μM) affected AEA inhibition of nAChRs, suggesting that the effect of AEA is not mediated by its metabolic products. Importantly, the extent of AEA inhibition of ⁸⁶Rb⁺ efflux was significantly attenuated by the absence of 1% fatty acid free bovine serum albumin pre-treatment, supporting previous findings that fatty acid-like compounds modulate the activity of nAChRs. Collectively, the results indicate that AEA inhibits the function of nAChRs in thalamic synaptosomes via a CB-independent mechanism and that the background activity of these receptors is affected by fatty acids and AEA.     

Endocannabinoids effect on oxidative status of human platelets

Reactive oxygen species (ROS) are known to regulate platelet activation. Since endocannabinoids behave as platelet agonists, we investigated the effect of two endocannabinoids, 2-arachidonoylglycerol (2AG) and anandamide (AEA) on the oxidative status of human platelets. We have demonstrated that 2AG and AEA stimulate ROS production, superoxide anion formation and lipid peroxidation. The effect is dose and time dependent and mainly occurs through the involvement of cannabinoid receptor 1 (CB1) since all tested parameters are greatly reduced by SR141716, the CB1 specific inhibitor. The specific inhibitor of cannabinoid receptor 2 (CB2) SR144528 produces a very small inhibition. The involvement of syk/PI3K/AKT/mTor pathway in oxidative stress induced by endocannabinoids is shown. Nicotinamide adenine dinucleotide phosphate oxidase seems to be poorly involved in the endocannabinoids effect. Concerning the aerobic metabolism, it has been demonstrated that endocannabinoids reduce the oxygen consumption and adenosine triphosphate synthesis, both in the presence of pyruvate + malate or succinate. In addition, endocannabinoids inhibit the activity of respiratory complexes II, III and IV and increase the activity of respiratory complex I. The endocannabinoids effect on aerobic metabolism seems to be also a CB1 mediated mechanism. Thus, in human platelets oxidative stress induced by endocannabinoids, mainly generated in the respiratory chain through the activation of complex I and the inhibition of complex II, III and IV, may lead to thrombotic events, contributing to cardiovascular diseases.     

Inhibition of peristaltic activity by cannabinoids in the isolated distal colon of mouse.

The effects of the cannabinoid receptor agonist Win 55,212-2 and of the competitive cannabinoid receptor antagonist SR 141716A on the electrically-evoked peristalsis of isolated distal colon of mouse were studied. Intraluminal pressure, longitudinal displacement, ejected fluid volume and changes in morphology of external intestinal wall were simultaneously recorded in the pre-drug period and in presence of Win 55,212-2 alone or in combination with SR 141716A. In the pre-drug period (control), peristaltic activity was characterised by regular, monophasic waves and the intraluminal content propelled towards anterograde (oro-aboral) direction with a propulsion velocity of 1.25 +/- 0.1 mm x s(-1). Pressure and shortening waves showed a peak amplitude of 2.44 +/- 0.32 kPa and 1.8 +/- 0.72 mm, respectively. The mean amount of fluid volume ejected during each contraction was 80 +/- 12.6 microl. The addition of Win 55,212-2 [10(-7)-10(-4) M] to the organ bath determined a dose-related attenuation of peristaltic activity consequent to the decrease of circular and longitudinal muscle strength. The decrease of contractile activity was followed by dose-dependent decrease of the amount of fluid ejected during peristalsis. The effects of Win 55,212-2 [10(-7)-10(-5) M] were prevented by SR 141716A, indicating the presence of cannabinoid CB1 receptors in the mouse distal colon. SR 141716A alone enhanced both tonic and phasic motor activities in the colonic longitudinal smooth muscle, suggesting that CB1 receptor antagonists could act either through antagonising the effect of endogenous CB1 receptor agonist or by an agonist effect on these receptors. The present results further support the hypothesis that cannabinoids perform a neuromodulatory role in various tracts of gastrointestinal system and first demonstrate their action also in the distal colon of rodents.     

Cannabinoid receptor type 1 modulates excitatory and inhibitory neurotransmission in mouse colon

The effects of cannabinoid receptor agonists and antagonists on smooth muscle resting membrane potentials and on membrane potentials following electrical neuronal stimulation in a myenteric neuron/smooth muscle preparation of wild-type and cannabinoid receptor type 1 (CB1)-deficient mice were investigated in vitro. Double staining for CB1 and nitric oxide synthase (neuronal) was performed to identify the myenteric CB1-expressing neurons. Focal electrical stimulation of the myenteric plexus induced a fast (f) excitatory junction potential (EJP) followed by a fast and a slow (s) inhibitory junction potential (IJP). Treatment of wild-type mice with the endogenous CB1 receptor agonist anandamide reduced EJP while not affecting fIJP and sIJP. EJP was significantly higher in CB1-deficient mice than in wild-type littermate controls, and anandamide induced no effects in CB1-deficient mice. N-arachidonoyl ethanolamide (anandamide), R-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3,-de]- 1,4-benzoxazin-6-yl]-1-naphtalenylmethanone, a synthetic CB1 receptor agonist, nearly abolished EJP and significantly reduced the fIJP in wild-type mice. N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-caroxamide (SR141716A), a CB1-specific receptor antagonist, was able to reverse the agonist effects induced in wild-type mice. SR141716A, when given alone, significantly increased EJP in wild-type mice without affecting IJP in wild-type and EJP in CB1-deficient mice. Interestingly, SR141716A reduced fIJP in CB1-deficient mice. In the mouse colon, nitrergic myenteric neurons do not express CB1, implying that CB1 is expressed in cholinergic neurons, which is in line with the functional data. Finally, excitatory and inhibitory neurotransmission in the mouse colon is modulated by activation of CB1 receptors. The significant increase in EJP in CB1-deficient mice strongly suggests a physiological involvement of CB1 in excitatory cholinergic neurotransmission.     

Significance of cardiac cannabinoid receptors in regulation of cardiac rhythm,myocardial contractility,and electrophysiologic processes in heart

Intravenous administration of cannabinoid (CB) receptor agonists (HU-210, 0.1 mg/kg; ACPA, 0.125 mg/kg; methanandamide, 2.5 mg/kg; and anandamide, 2.5 mg/kg) induced bradycardia in chloralose-anesthetized rats irrespective of the solubilization method. Methanandamide, HU-210, and ACPA had no effect on the electrophysiological activity of the heart, while anandamide increased the duration of the QRS complex. The negative chronotropic effect of HU-210 was due to CB1 receptor activation since it was not observed after CB1 receptor blockade by SR141716A (1 mg/kg intravenously) but was present after pretreatment with CB2 receptor antagonist SR144528 (1 mg/kg intravenously). CB receptor antagonists SR141716A and SR144528 had no effect on cardiac rhythm or ECG indices. Hence, in the intact heart, endogenous CB receptor agonists are not involved in the regulation of cardiac rhythm and electrophysiological processes. The chronotropic effect of CBs was independent of the autonomic nervous system because it remained significant after autonomic ganglion blockade by hexamethonium (1 mg/kg intravenously). CB receptor activation by HU-210 (0.1 and 1 μM) in vitro decreased the rate and force of isolated heart contractions, the rates of contraction and relaxation, and end diastolic pressure. The negative chronotropic effect of HU-210 was less pronounced in vitro than in vivo. The maximum inotropic effect of HU-210 was reached at the concentration of 0.1 μM.     

Effects of Cannabinoids on Dopamine Release in the Corpus Striatum and the Nucleus Accumbens In Vitro

Cannabinoid receptors are widely distributed in the nuclei of the extrapyramidal motor and mesolimbic reward systems; their exact functions are, however, not known. The aim of the present study was to characterize the effects of cannabinoids on the electrically evoked release of endogenous dopamine in the corpus striatum and the nucleus accumbens. In rat brain slices dopamine release elicited by single electrical pulses was determined by fast cyclic voltammetry. Dopamine release was markedly inhibited by the OP2 opioid receptor agonist U-50488 and the D2/D3 dopamine receptor agonist quinpirole, indicating that our method is suitable for studying presynaptic modulation of dopamine release. In contrast, the CB1/CB2 cannabinoid receptor agonists WIN55212-2 (10(-6) M) and CP55940 (10(-6)-10(-5) M) and the CB1 cannabinoid receptor antagonist SR141716A (10(-6) M) had no effect on the electrically evoked dopamine release in the corpus striatum and the nucleus accumbens. The lack of a presynaptic effect on terminals of nigrostriatal and mesolimbic dopaminergic neurons is in accord with the anatomical distribution of cannabinoid receptors: The perikarya of these neurons in the substantia nigra and the ventral tegmental area do not synthesize mRNA, and hence protein, for CB1 and CB2 cannabinoid receptors. It is therefore unlikely that presynaptic modulation of dopamine release in the corpus striatum and the nucleus accumbens plays a role in the extrapyramidal motor and rewarding effects of cannabinoids.     

Effects of endogenous cannabinoid anandamide on excitation–contraction coupling in rat ventricular myocytes

A role for anandamide (N-arachidonoyl ethanolamide; AEA), a major endocannabinoid, in the cardiovascular system in various pathological conditions has been reported in earlier reports. In the present study, the effects of AEA on contractility, Ca²⁺ signaling, and action potential (AP) characteristics were investigated in rat ventricular myocytes. Video edge detection was used to measure myocyte shortening. Intracellular Ca²⁺ was measured in cells loaded with the fluorescent indicator fura-2 AM. AEA (1 μM) caused a significant decrease in the amplitudes of electrically evoked myocyte shortening and Ca²⁺ transients. However, the amplitudes of caffeine-evoked Ca²⁺ transients and the rate of recovery of electrically evoked Ca²⁺ transients following caffeine application were not altered. Biochemical studies in sarcoplasmic reticulum (SR) vesicles from rat ventricles indicated that AEA affected Ca²⁺-uptake and Ca²⁺-ATPase activity in a biphasic manner. [³H]-ryanodine binding and passive Ca²⁺ release from SR vesicles were not altered by 10 μM AEA. Whole-cell patch-clamp technique was employed to investigate the effect of AEA on the characteristics of APs. AEA (1 μM) significantly decreased the duration of AP. The effect of AEA on myocyte shortening and AP characteristics was not altered in the presence of pertussis toxin (PTX, 2 μg/ml for 4 h), AM251 and SR141716 (cannabinoid type 1 receptor antagonists; 0.3 μM) or AM630 and SR 144528 (cannabinoid type 2 receptor antagonists; 0.3 μM). The results suggest that AEA depresses ventricular myocyte contractility by decreasing the action potential duration (APD) in a manner independent of CB1 and CB2 receptors.