Novel, not adenylyl cyclase-coupled cannabinoid binding site in cerebellum of mice

In this study we report data suggesting the presence of a non-CB1, non-CB2 cannabinoid site in the cerebellum of CB1-/- mice. We have carried out [(35)S]GTPgammaS binding experiments in striata, hippocampi, and cerebella of CB1-/- and CB1(+/+) mice with Delta(9)-THC, WIN55,212-2, HU-210, SR141716A, and SR144528. In CB1-/- mice Delta(9)-THC and HU-210 did not stimulate [(35)S]GTPgammaS binding. However, WIN55,212-2 was able to stimulate [(35)S]GTPgammaS binding in cerebella of CB1-/- mice. The maximal effect of this stimulation was 31% that of wild type animals. This effect was reversible neither by CB1 nor CB2 receptor antagonists. Similar results were obtained with the endogenous cannabinoid, anandamide. However, adenylyl cyclase was not inhibited by WIN55,212-2 or anandamide in the CB1(minus sign/minus sign) animals. In striata and hippocampi of CB1-/- mice no [(35)S]GTPgammaS stimulation curve could be obtained with WIN55,212. Our findings suggest that there is a non-CB1 non-CB2 receptor present in the cerebellum of CB1-/- mice.     

Delta9-tetrahydrocannabinol (Delta9-THC) prevents cerebral infarction via hypothalamic-independent hypothermia

Delta(9)-tetrahydrocannabinol (Delta(9)-THC), a primary psychoactive constituent of cannabis, has been reported to act as a neuroprotectant via the cannabinoid CB(1) receptor. In this study, Delta(9)-THC significantly decreased the infarct volume in a 4 h mouse middle cerebral artery occlusion mouse model. The neuroprotective effect of Delta(9)-THC was completely abolished by SR141716, cannabinoid CB(1) receptor antagonist, and by warming the animals to 31 degrees C. Delta(9)-THC significantly decreased the rectal temperature, and the hypothermic effect was also inhibited by SR141716 and by warming to 31 degrees C. At 24 h after cerebral ischemia, Delta(9)-THC significantly increased the expression level of CB(1) receptor in both the striatum and cortex, but not in the hypothalamus. Warming to 31 degrees C during 4 h cerebral ischemia did not increase the expression of CB(1) receptor at the striatum and cortex in MCA-occluded mice. These results show that the neuroprotective effect of Delta(9)-THC is mediated by a temperature-dependent mechanism via the CB(1) receptor. In addition, warming to 31 degrees C might attenuate both the neuroprotective and hypothermic effects of Delta(9)-THC through inhibiting the increase in CB(1) receptor in both the striatum and cortex but not in the hypothalamus, which may suggest a new thermoregulation mechanism of Delta(9)-THC.     

Contribution of endocannabinoids in the endothelial protection afforded by ischemic preconditioning in the isolated rat heart

The aim of the present study was to assess the contribution of endogenous cannabinoids in the protective effect of ischemic preconditioning on the endothelial function in coronary arteries of the rat. Isolated rat hearts were exposed to a 30-min low flow ischemia (1 ml/min) followed by 20-min reperfusion, after which the response to the endothelium-dependent vasodilator, serotonine (5-HT), was compared with that of the endothelium-independent vasodilator, sodium nitroprusside (SNP). In untreated hearts, ischemia-reperfusion diminished selectively 5-HT-induced vasodilatation, compared with time-matched sham hearts, the vasodilatation to SNP being unaffected. A 5-min zero-flow preconditioning ischemia in untreated hearts preserved the vasodilatation produced by 5-HT. Blockade of either CB(1)-receptors with SR141716A or CB(2)-receptors with SR144528 abolished the protective effect of preconditioning on the 5-HT vasodilatation. Perfusion with either palmitoylethanolamide or 2-arachidonoylglycerol 15 min before and throughout the ischemia mimicked preconditioning inasmuch as it protected the endothelium in a similar fashion. This protection was blocked by SR144528 in both cases, whereas SR141716A only blocked the effect of PEA. The presence of CB(1) and CB(2)-receptors in isolated rat hearts was confirmed by Western blots. In conclusion, the data suggest that endogenous cannabinoids contribute to the endothelial protective effect of ischemic preconditioning in rat coronary arteries.     

3-(1',1'-Dimethylbutyl)-1-deoxy-delta8-THC and related compounds: synthesis of selective ligands for the CB2 receptor

The synthesis and pharmacology of 15 1-deoxy-delta8-THC analogues, several of which have high affinity for the CB2 receptor, are described. The deoxy cannabinoids include 1-deoxy-11-hydroxy-delta8-THC (5), 1-deoxy-delta8-THC (6), 1-deoxy-3-butyl-delta8-THC (7), 1-deoxy-3-hexyl-delta8-THC (8) and a series of 3-(1',1'-dimethylalkyl)-1-deoxy-delta8-THC analogues (2, n = 0-4, 6, 7, where n = the number of carbon atoms in the side chain-2). Three derivatives (17-19) of deoxynabilone (16) were also prepared. The affinities of each compound for the CB1 and CB2 receptors were determined employing previously described procedures. Five of the 3-(1',1'-dimethylalkyl)-1-deoxy-delta8-THC analogues (2, n = 1-5) have high affinity (Ki = < 20 nM) for the CB2 receptor. Four of them (2, n = 1-4) also have little affinity for the CB1 receptor (Ki = > 295 nM). 3-(1',1'-Dimethylbutyl)-1-deoxy-delta8-THC (2, n = 2) has very high affinity for the CB2 receptor (Ki = 3.4 +/- 1.0 nM) and little affinity for the CB1 receptor (Ki = 677 +/- 132 nM).     

Inhibitory effect of anandamide on resiniferatoxin-induced sensory neuropeptide release in vivo and neuropathic hyperalgesia in the rat

Anandamide (AEA) is an endogenous cannabinoid ligand acting predominantly on the cannabinoid 1 (CB(1)) receptor, but it is also an agonist on the capsaicin VR(1)/TRPV(1) receptor. In the present study we examined the effects of AEA and the naturally occurring cannabinoid 2 (CB(2)) receptor agonist palmitylethanolamide (PEA) on basal and resiniferatoxin (RTX)-induced release of calcitonin gene-related peptide (CGRP) and somatostatin in vivo. Since these sensory neuropeptides play important role in the development of neuropathic hyperalgesia, the effect of AEA and PEA was also examined on mechanonociceptive threshold changes after partial ligation of the sciatic nerve. Neither AEA nor PEA affected basal plasma peptide concentrations, but both of them inhibited RTX-induced release. The inhibitory effect of AEA was prevented by the CB(1) receptor antagonist SR141716A. AEA abolished and PEA significantly decreased neuropathic mechanical hyperalgesia 7 days after unilateral sciatic nerve ligation, which was antagonized by SR141716A and the CB(2) receptor antagonist SR144528, respectively. Both SR141716A and SR144528 increased hyperalgesia, indicating that endogenous cannabinoids acting on CB(1) and peripheral CB(2)-like receptors play substantial role in neuropathic conditions to diminish hyperalgesia. AEA and PEA exert inhibitory effect on mechanonociceptive hyperalgesia and sensory neuropeptide release in vivo suggesting their potential therapeutical use to treat chronic neuropathic pain.     

Synthesis and testing of novel phenyl substituted side-chain analogues of classical cannabinoids

A series of novel phenyl substituted side-chain analogues of classical cannabinoids were synthesized and their CB1 and CB2 binding affinities were evaluated relative to Delta(8)-THC and compound 2. CB1 and CB2 binding assays indicate that the dimethyl and ketone analogues (3) and (6) display selectivity for the CB2 receptor in comparison to delta(8)-THC and compound 2. This study provides newer insights into the geometrical and functional group requirements of the ligand binding pockets of the CB1 and the CB2 receptors.     

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.     

Activation of phosphoinositide 3-kinase/PKB pathway by CB(1) and CB(2) cannabinoid receptors expressed in prostate PC-3 cells. Involvement in Raf-1 stimulation and NGF induction

Cannabinoids exert a variety of physiological and pharmacological responses in humans through interaction with specific cannabinoid receptors. Cannabinoid receptors described to date belong to the seven-transmembrane-domain receptor superfamily and are coupled through the inhibitory G(i) protein to adenylyl cyclase inhibition. However, downstream signal transduction mechanisms triggered by cannabinoids are poorly understood. We examined here the involvement of the phosphoinositide 3-kinase (PI3K)/PKB pathway in the mechanism of action of cannabinoids in human prostate epithelial PC-3 cells. Cannabinoid receptors CB(1) and CB(2) are expressed in these cells, as shown by RT-PCR, Western blot and immunofluorescence techniques. Treatment of PC-3 cells with either Delta(9)-tetrahydrocannabinol (THC), the major psychoactive ingredient of marijuana, or R-(+)-methanandamide (MET), an analogue of the endogenous cannabinoid anandamide, increased phosphorylation of PKB in Thr308 and Ser473. The stimulation of PKB induced by cannabinoids was blocked by the two cannabinoid receptor antagonists, SR 141716 and SR 144528, and by the PI3K inhibitor LY 294002. These results indicate that activation of cannabinoid receptors in PC-3 cells stimulate the PI3K/PKB pathway. We further investigated the involvement of Raf-1/Erk activation in the mechanism of action of cannabinoid receptors. THC and MET induced translocation of Raf-1 to the membrane and phosphorylation of p44/42 Erk kinase, which was reversed by cannabinoid receptor antagonists and PI3K inhibitor. These results point to a sequential connection between cannabinoid receptors/PI3K/PKB pathway and Raf-1/Erk in prostate PC-3 cells. We also show that this pathway is involved in the mechanism of NGF induction exerted by cannabinoids in PC-3 cells.     

Cytochrome P450 enzymes involved in the metabolism of tetrahydrocannabinols and cannabinol by human hepatic microsomes

In this study, tetrahydrocannabinols (THCs) were mainly oxidized at the 11-position and allylic sites at the 7alpha-position for Delta(8)-THC and the 8beta-position for Delta(9)-THC by human hepatic microsomes. Cannabinol (CBN) was also mainly metabolized to 11-hydroxy-CBN and 8-hydroxy-CBN by the microsomes. The 11-hydroxylation of three cannabinoids by the microsomes was markedly inhibited by sulfaphenazole, a selective inhibitor of CYP2C enzymes, while the hydroxylations at the 7alpha-(Delta(8)-THC), 8beta-(Delta(9)-THC) and 8-positions (CBN) of the corresponding cannabinoids were highly inhibited by ketoconazole, a selective inhibitor of CYP3A enzymes. Human CYP2C9-Arg expressed in the microsomes of human B lymphoblastoid cells efficiently catalyzed the 11-hydroxylation of Delta(8)-THC (7.60 nmol/min/nmol CYP), Delta(9)-THC (19.2 nmol/min/nmol CYP) and CBN (6.62 nmol/min/nmol CYP). Human CYP3A4 expressed in the cells catalyzed the 7alpha-(5.34 nmol/min/nmol CYP) and 7beta-hydroxylation (1.39 nmol/min/nmol CYP) of Delta(8)-THC, the 8beta-hydroxylation (6.10 nmol/min/nmol CYP) and 9alpha,10alpha-epoxidation (1.71 nmol/min/nmol CYP) of Delta(9)-THC, and the 8-hydroxylation of CBN (1.45 nmol/min/nmol CYP). These results indicate that CYP2C9 and CYP3A4 are major enzymes involved in the 11-hydroxylation and the 8-(or the 7-) hydroxylation, respectively, of the cannabinoids by human hepatic microsomes. In addition, CYP3A4 is a major enzyme responsible for the 7alpha- and 7beta-hydroxylation of Delta(8)-THC, and the 9alpha,10alpha-epoxidation of Delta(9)-THC.     

The CB2 cannabinoid receptor signals apoptosis via ceramide-dependent activation of the mitochondrial intrinsic pathway

Delta9-tetrahydrocannabinol and other cannabinoids exert pro-apoptotic actions in tumor cells via the CB2 cannabinoid receptor. However, the molecular mechanism involved in this effect has remained elusive. Here we used the human leukemia cell line Jurkat-that expresses CB2 as the unique CB receptor-to investigate this mechanism. Our results show that incubation with the selective CB2 antagonist SR144528 abrogated the pro-apoptotic effect of Delta9-tetrahydrocannabinol. Cannabinoid treatment led to a CB2 receptor-dependent stimulation of ceramide biosynthesis and inhibition of this pathway prevented Delta9-tetrahydrocannabinol-induced mitochondrial hypopolarization and cytochrome c release, indicating that ceramide acts at a pre-mitochondrial level. Inhibition of ceramide synthesis de novo also prevented caspase activation and apoptosis. Caspase 8 activation-an event typically related with the extrinsic apoptotic pathway-was also evident in this model. However, activation of this protease was post-mitochondrial since (i) a pan-caspase inhibitor as well as a selective caspase 8 inhibitor were unable to prevent Delta9-tetrahydrocannabinol-induced loss of mitochondrial-membrane transmembrane potential, and (ii) cannabinoid-induced caspase 8 activation was not observed in Bcl-xL over-expressing cells. In summary, results presented here show that CB2 receptor activation signals apoptosis via a ceramide-dependent stimulation of the mitochondrial intrinsic pathway.     

Activation of brain prostanoid EP3 receptors via arachidonic acid cascade during behavioral suppression induced by Delta8-tetrahydrocannabinol

We have previously shown that behavioral changes induced by cannabinoid were due to an elevation of prostaglandin E2 (PGE2) via the arachidonic acid cascade in the brain. In the present study, we investigated the participation of the prostanoid EP3 receptor, the target of PGE2 in the brain, in behavioral suppression induced by Delta8-tetrahydrocannabinol (Delta8-THC), an isomer of the naturally occurring Delta9-THC, using a one-lever operant task in rats. Intraperitoneal administration of Delta8-THC inhibited the lever-pressing behavior, which was significantly antagonized by both the selective cannabinoid CB1 receptor antagonist SR141716A and the cyclooxygenase inhibitor diclofenac. Furthermore, intracerebroventricular (i.c.v.) administration of PGE2 significantly inhibited the lever-pressing performance similar to Delta8-THC. Prostanoid EP3 receptor antisense-oligodeoxynucleotide (AS-ODN; twice a day for 3 days, i.c.v.) significantly decreased prostanoid EP3 receptor mRNA levels as determined by the RT-PCR analysis in the cerebral cortex, hippocampus and midbrain. AS-ODN also antagonized the PGE2-induced suppression of the lever pressing. In the same way, the suppression of lever-pressing behavior by Delta8-THC was significantly improved by AS-ODN. It is concluded that the suppression of lever-pressing behavior by cannabinoid is due to activation of the prostanoid EP3 receptor through an elevation of PGE2 in the brain.     

Cannabinoid CB1 receptor activation mediates the opposing effects of amphetamine on impulsive action and impulsive choice

It is well known that acute challenges with psychostimulants such as amphetamine affect impulsive behavior. We here studied the pharmacology underlying the effects of amphetamine in two rat models of impulsivity, the 5-choice serial reaction time task (5-CSRTT) and the delayed reward task (DRT), providing measures of inhibitory control, an aspect of impulsive action, and impulsive choice, respectively. We focused on the role of cannabinoid CB1 receptor activation in amphetamine-induced impulsivity as there is evidence that acute challenges with psychostimulants activate the endogenous cannabinoid system, and CB1 receptor activity modulates impulsivity in both rodents and humans. Results showed that pretreatment with either the CB1 receptor antagonist/inverse agonist SR141716A or the neutral CB1 receptor antagonist O-2050 dose-dependently improved baseline inhibitory control in the 5-CSRTT. Moreover, both compounds similarly attenuated amphetamine-induced inhibitory control deficits, suggesting that CB1 receptor activation by endogenously released cannabinoids mediates this aspect of impulsive action. Direct CB1 receptor activation by Δ9-Tetrahydrocannabinol (Δ9-THC) did, however, not affect inhibitory control. Although neither SR141716A nor O-2050 affected baseline impulsive choice in the DRT, both ligands completely prevented amphetamine-induced reductions in impulsive decision making, indicating that CB1 receptor activity may decrease this form of impulsivity. Indeed, acute Δ9-THC was found to reduce impulsive choice in a CB1 receptor-dependent way. Together, these results indicate an important, though complex role for cannabinoid CB1 receptor activity in the regulation of impulsive action and impulsive choice as well as the opposite effects amphetamine has on both forms of impulsive behavior.     

Cannabinoids, hippocampal function and memory.

Prior studies from this laboratory have shown that the psychoactive ingredient in marijuana, delta9-tetrahydrocannabinol (THC), interferes with short-term memory (1-3) in both delayed match and nonmatch to sample tasks (DMS/DNMS). Recent experiments have shown that other cannabinoids such as the potent CB1 receptor agonist, WIN 55,212-2 produces a delay-dependent deficit in the DNMS task at a dose range (0.10-0.50 mg/kg) well below that of delta9-THC which was blocked by the CB11 receptor antagonist SR141716A (Sanofi Inc). The effects of WIN 55,212-2 at low doses were similar to those of isolated lesions of the hippocampus, whereas high doses (0.50 mg/kg, i.p.) produced effects similar to lesions of both hippocampus and surrounding retrohippocampal areas. The low dose effect was delay-dependent while the high dose introduced an additional deficit at short delays that was sensitive to both SR141716A and the GABA(B) receptor antagonist, phaclofen. Comparison of lesion vs. cannabinoid effects on DNMS performance suggests that CB1 receptors on hippocampal neurons interfere with the processing of DNMS task-specific information within a trial. CB1 receptors on hippocampal GABAergic interneurons and in retrohippocampal areas appear to influence the ability to maintain segregation of information between trials in the task.     

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.     

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.     

Neuromodulatory role of the endocannabinoid signaling system in alcoholism: an overview

The current review evaluates the evidence that some of the pharmacological and behavioral effects of ethanol (EtOH), including EtOH-preferring behavior, may be mediated through the endocannabinoid signaling system. The recent advances in the understanding of the neurobiological basis of alcoholism suggest that the pharmacological and behavioral effects of EtOH are mediated through its action on neuronal signal transduction pathways and ligand-gated ion channels, receptor systems, and receptors that are coupled to G-proteins. The identification of a G-protein-coupled receptor, namely, the cannabinoid receptor (CB1 receptor) that was activated by Delta(9)-tetrahydrocannabinol (Delta(9)-THC), the major psychoactive component of marijuana, led to the discovery of endogenous cannabinoid agonists. To date, two fatty acid derivatives identified to be arachidonylethanolamide (AEA) and 2-arachidonylglycerol (2-AG) have been isolated from both nervous and peripheral tissues. Both these compounds have been shown to mimic the pharmacological and behavioral effects of Delta(9)-THC. The involvement of the endocannabinoid signaling system in the development of tolerance to the drugs of abuse including EtOH has not been known until recently. Recent studies from our laboratory have demonstrated for the first time the down-regulation of CB1 receptor function and its signal transduction by chronic EtOH. The observed down-regulation of CB1 receptor binding and its signal transduction results from the persistent stimulation of the receptors by the endogenous CB1 receptor agonists, AEA and 2-AG, the synthesis of which has been found to be increased by chronic EtOH treatment. This enhanced formation of endocannabinoids may subsequently influence the release of neurotransmitters. It was found that the DBA/2 mice, known to avoid EtOH intake, have significantly reduced brain-CB1-receptor function consistent with other studies, where the CB1 receptor antagonist SR141716A has been shown to block voluntary EtOH intake in rodents. Similarly, activation of the CB1 receptor system promoted alcohol craving, suggesting a role for the CB1 receptor gene in excessive EtOH drinking behavior and development of alcoholism. Ongoing investigations may lead to the development of potential therapeutic strategies for the treatment of alcoholism.     

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.     

Evaluation of the role of the arachidonic acid cascade in anandamide's in vivo effects in mice

The pharmacological profiles of the endocannabinoid anandamide and exogenous cannabinoids (e.g., Delta9-tetrahydrocannabinol) are similar, but not exactly the same. One notable difference is that anandamide's in vivo effects in mice are not blocked by the brain cannabinoid (CB1) receptor antagonist SR141716A. The degree to which the rapid metabolism of anandamide to arachidonic acid might be involved in this unexpected lack of effect was the focus of this study. Mice were tested in a tetrad of tests sensitive to cannabinoids, consisting of spontaneous locomotion, ring immobility, rectal temperature and tail flick nociception. Anandamide and arachidonic acid produced a similar profile of effects, but neither drug was blocked by SR141716A. When hydrolysis of anandamide was inhibited by an amidase inhibitor (phenylmethyl sulfonyl fluoride; PMSF), however, SR141716A significantly attenuated anandamide's effects but did not completely block them. Similarly, the effects of the metabolically stable anandamide analog O-1812 were attenuated by SR141716A. The role of oxidative metabolism in anandamide's effects in the tetrad was also investigated through pharmacological modulation of cyclooxygenase and lipoxygenase, two major classes of enzymes that degrade arachidonic acid. Whereas the non-selective cyclooxygenase inhibitor ibuprofen blocked the in vivo effects of arachidonic acid, it did not alter anandamide's effects. Other modulators of the cyclooxygenase and lipoxygenase pathways also failed to block anandamide's effects. Together, these results offer partial support for a pharmacokinetic explanation of the failure of SR141716A to antagonize the effects of anandamide; however, they also suggest that non-CB1, non-CB2 receptors may be involved in mediation of anandamide's in vivo actions, particularly at higher doses.     

Delayed treatment with cannabidiol has a cerebroprotective action via a cannabinoid receptor-independent myeloperoxidase-inhibiting mechanism

We examined the neuroprotective mechanism of cannabidiol, non-psychoactive component of marijuana, on the infarction in a 4 h mouse middle cerebral artery (MCA) occlusion model in comparison with Delta(9)-tetrahydrocannabinol (Delta(9)-THC). Release of glutamate in the cortex was measured at 2 h after MCA occlusion. Myeloperoxidase (MPO) and cerebral blood flow were measured at 1 h after reperfusion. In addition, infarct size and MPO were determined at 24 and 72 h after MCA occlusion. The neuroprotective effect of cannabidiol was not inhibited by either SR141716 or AM630. Both pre- and post-ischemic treatment with cannabidiol resulted in potent and long-lasting neuroprotection, whereas only pre-ischemic treatment with Delta(9)-THC reduced the infarction. Unlike Delta(9)-THC, cannabidiol did not affect the excess release of glutamate in the cortex after occlusion. Cannabidiol suppressed the decrease in cerebral blood flow by the failure of cerebral microcirculation after reperfusion and inhibited MPO activity in neutrophils. Furthermore, the number of MPO-immunopositive cells was reduced in the ipsilateral hemisphere in cannabidiol-treated group. Cannabidiol provides potent and long-lasting neuroprotection through an anti-inflammatory CB(1) receptor-independent mechanism, suggesting that cannabidiol will have a palliative action and open new therapeutic possibilities for treating cerebrovascular disorders.     

Cannabinoid pharmacology: implications for additional cannabinoid receptor subtypes

Delta(9)-Tetrahydrocannabinol (delta(9)-THC), the primary psychoactive constituent of marijuana (Cannabis sativa), is known to bind to two cannabinoid receptors: CB(1) receptors, located primarily in the brain, and CB(2) receptors, located primarily in the periphery. Recent research has suggested that other cannabinoids, including anandamide and WIN 55212-2, may also act at novel non-CB(1), non-CB(2) cannabinoid receptor(s). Anandamide produces a number of in vivo pharmacological effects in CB(1) knockout mice that are not produced by delta(9)-THC and cannot be explained by anandamide's rapid metabolism. In addition, in vitro anandamide and WIN 55212-2 stimulate [35S]GTPgammaS binding in both CB(1) knockout and wildtype mice while delta(9)-THC stimulates this binding only in wildtype mice. Although anandamide and vanilloid agonists share pharmacological effects, anandamide's actions in CB(1) knockout mice do not appear to be mediated by vanilloid VR(1) receptors. While not yet conclusive, these results suggest the possibility of additional cannabinoid receptors in the brain and periphery.