piperidines and 3-(2-hydroxy-4-(1-1-dimethylheptyl)phenyl)-4-(3-hydroxypropyl)cyclohexanol

In October 2019, the first X-ray crystal structure of a ternary cannabinoid receptor 1 (CB1) complex (PDB ID: 6KQI) was published, including the well-known orthosteric agonist, CP55940, and the well-studied negative allosteric modulator, ORG27569. Prior to the release of 6KQI, we applied binding pocket analysis and molecular docking to carefully prepared computational models of the ternary CB1 complex, in order to predict the binding site for ORG27569 with the CP55940-bound CB1 receptor. We carefully studied the binding pose of agonist ligands in the CB1 orthosteric pocket, including CP55940. Our computational studies identified the most favorable binding site for ORG27569, in the CP55940-CB1 complex, to be at the intracellular end of the receptor. However, in the 6KQI structure, ORG27569 was found at an extrahelical, intramembrane site on the complex, a site that partially overlaps with the site predicted in our calculations to be second-best. We performed molecular dynamics simulations of the CP55940-bound CB1 complex with ORG27569 at different binding sites. Our analysis of the simulations indicated that ORG27569 bound favorably and stably in each simulation, but, as in the earlier calculations, bound best at the intracellular site, which is different than that found in the crystal structure. These results suggest that the intracellular site might serve as an alternative binding site in CB1. Our studies show that the computational techniques we used are valuable in identifying ligand-binding pockets in proteins, and could be useful for the study of the interaction mode of other allosteric modulators.

Topics: Allosteric Regulation; Allosteric Site; Cyclohexanols; Indoles; Molecular Docking Simulation; Piperidines; Protein Binding; Receptors, Cannabinoid

The endocannabinoid, anandamide (AEA), stimulates cannabinoid receptors (CBRs) and is enriched in the kidney, especially the renal medulla. AEA infused into the renal outer medulla of mice stimulates urine flow rate and salt excretion. Here we show that these effects are blocked by the CBR type 1 (CB1) inverse agonist, rimonabant. Immunohistochemical analysis demonstrated the presence of CB1 in thick ascending limb (TAL) tubules. Western immunoblotting demonstrated the presence of CB1 (52 kDa) in the cortex and outer medulla of mouse kidney. The effect of direct [CP55940 (CP) or AEA] or indirect [fatty acyl amide hydrolase (FAAH) inhibitor, PF3845 (PF)] cannabinoidimetics on Na

Topics: Amidohydrolases; Animals; Arachidonic Acids; Cannabinoid Receptor Agonists; Cannabinoid Receptor Antagonists; Cyclohexanols; Diuresis; Endocannabinoids; Loop of Henle; Male; Mice; Mice, Inbred C57BL; Natriuresis; Ouabain; Piperidines; Polyunsaturated Alkamides; Pyridines; Rimonabant; Sodium; Sodium-Potassium-Exchanging ATPase

To determine if diminished orthosteric agonist binding due to mutations in extracellular loops 1 or 2 of the cannabinoid receptor 1 (CB. Binding assays were performed using a range of concentrations of orthosteric compound, in the presence or absence of a set concentration of the allosteric modulator PSNCBAM-1 to determine the EC. Single mutations in extracellular loop 1 or 2 of CB. Based on our findings, we provide evidence of a therapeutic application for allosteric modulators in situations where a mutation in the receptor may hinder its function. By utilizing allosteric modulators, restoration of orthosteric binding may be possible.

Topics: Binding Sites; Cannabinoid Receptor Agonists; Cyclohexanols; HEK293 Cells; Humans; Indoles; Ligands; Mutation; Phenylurea Compounds; Piperidines; Protein Binding; Protein Conformation; Pyridines; Receptor, Cannabinoid, CB1; Rimonabant; Structure-Activity Relationship

Animal models suggest that the endocannabinoid system (eCS) helps regulate various aspects of social behavior, including play behavior and social reward, during adolescence. Properly tuned endocannabinoid signaling may be a critical developmental component in the emergence of normal adult sociability. In the current experiment, we attempted to pharmacologically disrupt endocannabinoid tone during early adolescence, and then measure the behavioral effects at two subsequent time points. 36 male and 36 female Long Evans rats received daily injections of one of three treatments between post-natal day (PND) 25-39: 1) vehicle treatment, 2) 0.4 mg/kg CP55,940 (a potent CB1/CB2 receptor agonist), or 3) 0.5 mg/kg AM251 (a CB1 receptor antagonist/inverse agonist). Both soon after treatment (PND 40-44) and several weeks later (PND 66-70), subjects were tested in an elevated plus maze (EPM) for anxiety and in a three-chambered apparatus for sociability. For the latter test, the number of entries into each chamber and the amount of time spent investigating each target were measured. Analyses revealed significant main effects of both sex and age on sociability: males expressed greater sociability compared to females, and sociability was higher in adolescence than adulthood. Most importantly, drug treatment (both CP55,940 and AM251) attenuated sociability in adolescence without having a significant effect on anxiety in the EPM. However, this effect did not persist into adulthood. These results indicate that pharmacological disruption of endocannabinoid tone - through either chronic agonism or antagonism of cannabinoid receptors - during early adolescence has a detrimental effect on sociability. This effect may be caused by transient, compensatory alterations in the eCS.

Topics: Age Factors; Analgesics; Animals; Conditioning, Operant; Cyclohexanols; Endocannabinoids; Female; Male; Maze Learning; Piperidines; Pyrazoles; Rats; Rats, Long-Evans; Receptor, Cannabinoid, CB1; Social Interaction

The CB1 cannabinoid receptors have been found in the rodent suprachiasmatic nucleus, and their activation suppresses the light-induced phase shift in locomotor rhythmicity of mice and hamsters. Here, we show that the CB1 receptor agonist CP55940 significantly attenuates the light-induced phase delay in rats as well. Furthermore, it blocks the light induction of c-Fos and light-induced downregulation of pERK1/2 in the SCN, and the CB1 antagonist AM251 prevents the photic induction of pERK1/2 and reduces pGSK3β after photic stimulation. Our data suggest that the modulation of the cannabinoid receptor activity may affect the photic entrainment via the setting of the SCN sensitivity to light.

Topics: Animals; Cannabinoid Receptor Agonists; Cannabinoid Receptor Antagonists; Cyclohexanols; Light; Male; Motor Activity; Piperidines; Pyrazoles; Rats, Wistar; Suprachiasmatic Nucleus

Increasing the available repertoire of effective treatments for mood and anxiety disorders represents a critical unmet need. Pharmacological augmentation of endogenous cannabinoid (eCB) signaling has been suggested to represent a novel approach to the treatment of anxiety disorders; however, the functional interactions between two canonical eCB pathways mediated via anandamide (N-arachidonylethanolamine [AEA]) and 2-arachidonoylglycerol (2-AG) in the regulation of anxiety are not well understood.. We utilized pharmacological augmentation and depletion combined with behavioral and electrophysiological approaches to probe the role of 2-AG signaling in the modulation of stress-induced anxiety and the functional redundancy between AEA and 2-AG signaling in the modulation of anxiety-like behaviors in mice.. Selective 2-AG augmentation reduced anxiety in the light/dark box assay and prevented stress-induced increases in anxiety associated with limbic AEA deficiency. In contrast, acute 2-AG depletion increased anxiety-like behaviors, which was normalized by selective pharmacological augmentation of AEA signaling and via direct cannabinoid receptor 1 stimulation with Δ. Although AEA and 2-AG likely subserve distinct physiological roles, a pharmacological and functional redundancy between these canonical eCB signaling pathways exists in the modulation of anxiety-like behaviors. These data support development of eCB-based treatment approaches for mood and anxiety disorders and suggest a potentially wider therapeutic overlap between AEA and 2-AG augmentation approaches than was previously appreciated.

Topics: Adaptation, Ocular; Animals; Anti-Anxiety Agents; Anxiety; Arachidonic Acids; Benzodioxoles; Brain; Cannabinoid Receptor Agonists; Cyclohexanols; Disease Models, Animal; Dronabinol; Endocannabinoids; Excitatory Postsynaptic Potentials; Glycerides; Heterocyclic Compounds, 1-Ring; Locomotion; Male; Mice; Mice, Inbred ICR; Piperidines; Polyunsaturated Alkamides; Pyridines; Signal Transduction

While allosteric modulators of the cannabinoid type-1 receptor (CB

Topics: Allosteric Regulation; Animals; Cannabinoid Receptor Modulators; Cerebellum; Cyclohexanols; Discrimination, Psychological; Dose-Response Relationship, Drug; Dronabinol; Drug Interactions; Guanosine 5'-O-(3-Thiotriphosphate); Male; Mice, Inbred C57BL; Mice, Inbred ICR; Motor Activity; Phenylurea Compounds; Piperidines; Pregnenolone; Pyrazoles; Pyridines; Receptor, Cannabinoid, CB1; Rimonabant

Undesirable side effects associated with orthosteric agonists/antagonists of cannabinoid 1 receptor (CB1R), a tractable target for treating several pathologies affecting humans, have greatly limited their translational potential. Recent discovery of CB1R negative allosteric modulators (NAMs) has renewed interest in CB1R by offering a potentially safer therapeutic avenue. To elucidate the CB1R allosteric binding motif and thereby facilitate rational drug discovery, we report the synthesis and biochemical characterization of first covalent ligands designed to bind irreversibly to the CB1R allosteric site. Either an electrophilic or a photoactivatable group was introduced at key positions of two classical CB1R NAMs: Org27569 (1) and PSNCBAM-1 (2). Among these, 20 (GAT100) emerged as the most potent NAM in functional assays, did not exhibit inverse agonism, and behaved as a robust positive allosteric modulator of binding of orthosteric agonist CP55,940. This novel covalent probe can serve as a useful tool for characterizing CB1R allosteric ligand-binding motifs.

Topics: Affinity Labels; Allosteric Site; Animals; Arrestins; Binding Sites; CHO Cells; Cricetinae; Cricetulus; Cyclic AMP; Cyclohexanols; Drug Discovery; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Indoles; Ligands; Models, Molecular; Phenylurea Compounds; Piperidines; Pyridines; Radioligand Assay; Rats; Receptor, Cannabinoid, CB1; Structure-Activity Relationship

Previous studies have shown that social withdrawal in the phencyclidine (PCP) rat model of schizophrenia results from deficient endocannabinoid-induced activation of CB1 receptors. To understand the underlying cognitive mechanisms of the social deficit in PCP-treated rats, we examined the impact of pharmacological manipulation of the endocannabinoid system on sociability (i.e. social approach) and social novelty preference (which relies on social recognition). Control rats showed a clear preference for a "social" cage (i.e. unfamiliar stimulus rat placed under a wire mesh cage) versus an "empty" cage, and spent more time exploring a "novel" cage (i.e. new stimulus rat) versus a "familiar" cage. In contrast, rats receiving PCP (5 mg/kg, b.i.d. for 7 days, followed by a 7 day-washout period) showed intact sociability, but lacked social novelty preference. This PCP-induced deficit was due to increased activity at CB1 receptors as it was reversed by systemic administration of the CB1 antagonist AM251 (1 mg/kg). In agreement with this hypothesis, the cannabinoid agonist CP55,940 (0.003-0.03 mg/kg) dose-dependently suppressed social novelty preference in control animals without affecting sociability. Taken together, these data suggest that PCP-treated rats have a deficit in social cognition, possibly induced by increased stimulation of CB1 receptors. This deficit, however, is distinct from the social withdrawal previously observed in these animals, as the latter is due to deficient, rather than increased, CB1 stimulation.

Topics: Analysis of Variance; Animals; Cannabinoid Receptor Modulators; Cyclohexanols; Discrimination, Psychological; Disease Models, Animal; Endocannabinoids; Excitatory Amino Acid Antagonists; Exploratory Behavior; Male; Odorants; Phencyclidine; Piperidines; Pyrazoles; Rats; Rats, Wistar; Schizophrenia; Schizophrenic Psychology; Social Behavior Disorders; Social Perception

This study was designed to evaluate the effects of CP55,940 on normal bladder function in vivo and examine whether it suppresses urinary frequency induced by nociceptive stimuli in the bladder. Cannabinoid receptor (CBR) activity may be involved in the regulation of bladder function. However, the role of CBR subtypes in micturition has yet to be established. CP55,940 is a synthetic analogue of tetrahydrocannabidiol, which is a psychoactive ingredient of the Cannabis plant.. Cystometry under urethane anaesthesia was performed to evaluate the effect of intravesical delivery of CP55,940 with or without administration of CB1 antagonist AM251 or CB2 antagonist AM630 on bladder function in female rats. The effects of CP55,940 were also examined in rats with urinary irritation induced by intravesical infusion of acetic acid.. Infusion of CP55,940 significantly (p < 0.05) increased micturition interval (MI) and bladder capacity (BC) by 52 % and decreased maximal voiding pressure (MP) by 25 %. Pretreatment with AM251 or AM630 before CP55,940 administration prevented CP55,940-induced increases in MI, BC and reduced MP. Acetic acid induced urinary frequency as evidenced by a reduction in MI and was suppressed by CP55,940.. CP55,940 decreases bladder activity and urinary frequency induced by nociceptive stimuli, probably by suppression of bladder afferent activity. Effects of CP55,940 were abolished by both CBR antagonists. This data implicates a role for the endocannabinoid system in bladder mechanoafferent function in rats. In addition, our results show that CP55,940 reverses urinary frequency exemplified in an overactive bladder model, suggesting it could be an effective treatment for patients with lower urinary tract symptoms.

Topics: Acetic Acid; Administration, Intravesical; Animals; Cannabinoid Receptor Agonists; Cyclohexanols; Disease Models, Animal; Female; Indoles; Piperidines; Pyrazoles; Rats; Rats, Sprague-Dawley; Treatment Outcome; Urinary Bladder; Urinary Bladder, Overactive; Urination; Urodynamics

Cannabinoid receptor interacting protein 1a (CRIP1a) is a CB

Topics: Animals; Benzoxazines; Carrier Proteins; Cell Line; Cell Membrane; Cyclohexanols; Endocannabinoids; Gene Dosage; Gene Knockdown Techniques; Mice; Morpholines; Naphthalenes; Piperidines; Protein Transport; Pyrazoles; Receptor, Cannabinoid, CB1; Receptors, Cell Surface; Rimonabant; RNA, Small Interfering; Signal Transduction

Current concepts suggest that exposure to THC during adolescence may act as a risk factor for the development of psychiatric disorders later in life. However, the molecular underpinnings of this vulnerability are still poorly understood. To analyze this, we investigated whether and how THC exposure in female rats interferes with different maturational events occurring in the prefrontal cortex during adolescence through biochemical, pharmacological and electrophysiological means. We found that the endocannabinoid system undergoes maturational processes during adolescence and that THC exposure disrupts them, leading to impairment of both endocannabinoid signaling and endocannabinoid-mediated LTD in the adult prefrontal cortex. THC also altered the maturational fluctuations of NMDA subunits, leading to larger amounts of gluN2B at adulthood. Adult animals exposed to THC during adolescence also showed increased AMPA gluA1 with no changes in gluA2 subunits. Finally, adolescent THC exposure altered cognition at adulthood. All these effects seem to be triggered by the disruption of the physiological role played by the endocannabinoid system during adolescence. Indeed, blockade of CB1 receptors from early to late adolescence seems to prevent the occurrence of pruning at glutamatergic synapses. These results suggest that vulnerability of adolescent female rats to long-lasting THC adverse effects might partly reside in disruption of the pivotal role played by the endocannabinoid system in the prefrontal cortex maturation.

Topics: Age Factors; Animals; Cannabinoid Receptor Agonists; Cyclohexanols; Developmental Disabilities; Dizocilpine Maleate; Dronabinol; Estradiol; Estrous Cycle; Excitatory Amino Acid Antagonists; Female; In Vitro Techniques; Neurites; Piperidines; Prefrontal Cortex; Pyrazoles; Radionuclide Imaging; Rats; Rats, Sprague-Dawley; Receptors, Glutamate; Synaptic Potentials; Tritium

The brain endocannabinoid system is a potential target for the treatment of psychiatric and metabolic conditions. Here, a novel CB1 receptor antagonist (ABD459) was synthesized and assayed for pharmacological efficacy in vitro and for modulation of food consumption, vigilance staging and cortical electroencephalography in the mouse. ABD459 completely displaced the CB1 agonist CP99540 at a Ki of 8.6 nmol/l, and did not affect basal, but antagonized CP55940-induced GTPγS binding with a KB of 7.7 nmol/l. Acute ABD459 (3-20 mg/kg) reliably inhibited food consumption in nonfasted mice, without affecting motor activity. Active food seeking was reduced for 5-6 h postdrug, with no rebound after washout. Epidural recording of electroencephalogram confirmed that ABD459 (3 mg/kg) robustly reduced rapid eye movement (REM) sleep, with no alterations of wakefulness or non-REM sleep. Effects were strongest during 3 h postdrug, followed by a progressive washout period. The CB1 antagonist AM251 (3 mg/kg) and agonist WIN-55,212-2 (WIN-2: 3 mg/kg) also reduced REM, but variously affected other vigilance stages. WIN-2 caused a global suppression of normalized spectral power. AM251 and ABD459 lowered delta power and increased power in the theta band in the hippocampus, but not the prefrontal cortex. The neutral antagonist ABD459 thus showed a specific role of endocannabinoid release in attention and arousal, possibly through modulation of cholinergic activity.

Topics: Animals; Benzoxazines; Brain; Cannabinoid Receptor Antagonists; Cyclohexanols; Electroencephalography; Feeding Behavior; Male; Mice; Mice, Inbred C57BL; Morpholines; Motor Activity; Naphthalenes; Piperidines; Pyrazoles; Receptor, Cannabinoid, CB1; Sleep; Sleep, REM; Wakefulness

CB1 cannabinoid receptors (CB1R) are one of the most abundantly expressed G protein coupled receptors (GPCR) in the CNS and regulate diverse neuronal functions. The identification of GPCR interacting proteins has provided additional insight into the fine-tuning and regulation of numerous GPCRs. The cannabinoid receptor interacting protein 1a (CRIP1a) binds to the distal carboxy terminus of CB1R, and has been shown to alter CB1R-mediated neuronal function [1]. The mechanisms by which CRIP1a regulates CB1R activity have not yet been identified; therefore the focus of this investigation is to examine the cellular effects of CRIP1a on CB1R signaling using neuronal N18TG2 cells stably transfected with CRIP1a over-expressing and CRIP1a knockdown constructs. Modulation of endogenous CRIP1a expression did not alter total levels of CB1R, ERK, or forskolin-activated adenylyl cyclase activity. When compared to WT cells, CRIP1a over-expression reduced basal phosphoERK levels, whereas depletion of CRIP1a augmented basal phosphoERK levels. Stimulation of phosphoERK by the CB1R agonists WIN55212-2, CP55940 or methanandamide was unaltered in CRIP1a over-expressing clones compared with WT. However, CRIP1a knockdown clones exhibited enhanced ERK phosphorylation efficacy in response to CP55940. In addition, CRIP1a knockdown clones displayed a leftward shift in CP55940-mediated inhibition of forskolin-stimulated cAMP accumulation. CB1R-mediated Gi3 and Go activation by CP99540 was attenuated by CRIP1a over-expression, but robustly enhanced in cells depleted of CRIP1a. Conversely, CP55940-mediated Gi1 and Gi2 activation was significant enhanced in cells over-expressing CRIP1a, but not in cells deficient of CRIP1a. These studies suggest a mechanism by which endogenous levels of CRIP1a modulate CB1R-mediated signal transduction by facilitating a Gi/o protein subtype preference for Gi1 and Gi2, accompanied by an overall suppression of G-protein-mediated signaling in neuronal cells.

Topics: Animals; Carrier Proteins; Cell Line, Tumor; Cyclic AMP; Cyclohexanols; GTP-Binding Protein alpha Subunits, Gi-Go; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Phosphorylation; Piperidines; Pyrazoles; Receptor, Cannabinoid, CB1; Rimonabant; RNA Interference; RNA, Messenger; RNA, Small Interfering; Signal Transduction

Synthetic cannabinoids have emerged as a significant public health concern. To increase the knowledge of how these molecules interact on brain reward processes, we investigated the effects of CP55,940, a high efficacy synthetic CB1 receptor agonist, in a frequency-rate intracranial self-stimulation (ICSS) procedure.. The impact of acute and repeated administration (seven days) of CP55,940 on operant responding for electrical brain stimulation of the medial forebrain bundle was investigated in C57BL/6J mice.. CP55,940 attenuated ICSS in a dose-related fashion (ED50 (95% C.L.)=0.15 (0.12-0.18)mg/kg). This effect was blocked by the CB1 receptor antagonist rimonabant. Tolerance developed quickly, though not completely, to the rate-decreasing effects of CP55,940 (0.3mg/kg). Abrupt discontinuation of drug did not alter baseline responding for up to seven days. Moreover, rimonabant (10mg/kg) challenge did not alter ICSS responding in mice treated repeatedly with CP55,940.. The finding that CP55,940 reduced ICSS in mice with no evidence of facilitation at any dose is consistent with synthetic cannabinoid effects on ICSS in rats. CP55,940-induced ICSS depression was mediated through a CB1 receptor mechanism. Additionally, tolerance and dependence following repeated CP55,940 administration were dissociable. Thus, CP55,940 does not produce reward-like effects in ICSS under these conditions.

Topics: Animals; Brain; Catalepsy; Conditioning, Operant; Cyclohexanols; Dose-Response Relationship, Drug; Drug Tolerance; Male; Medial Forebrain Bundle; Mice; Piperidines; Pyrazoles; Receptor, Cannabinoid, CB1; Reward; Rimonabant; Self Stimulation

Previous studies have indicated a role for beta-arrestin2 in the regulation of brain cannabinoid effects and cannabinoid CB1 receptors, but whether beta-arrestin1 has a role has not been investigated.. To determine the role of beta-arrestin1 in cannabinoid activity.. Beta-arrestin1 -/- mice and their wild-type (+/+) counterparts were assayed for antinociceptive and temperature-decreasing effects of two ligands, Δ(9)-tetrahydrocannabinol (THC) and CP55940, after both single and repeated administration. In vitro assays examined the effects of deletion on CB1 receptor density, agonist-binding and G-protein activation.. Deletion of beta-arrestin1 diminished the effects of CP55940 in both antinociception (latency to tail withdrawal) and temperature-depression assays in mice. However, deleting beta-arrestin1 had no effect on the actions of THC in either assay. Antagonist radioligand ([(3)H]SR141716A) saturation binding indicated no difference between beta-arrestin1 +/+ and -/- mice in the density or affinity for cannabinoid CB1 receptors in brain membranes. CP55940 agonist binding in brain membranes from beta-arrestin1 +/+ mice exhibited high- and intermediate-affinity sites, but beta-arrestin1 -/- membranes exhibited an additional site with low affinity. CP55940 produced greater stimulation of [(35)S]GTPγS binding to membranes from whole brain of beta-arrestin1 -/- than +/+ mice. The rates of the development of tolerance to chronic THC or CP55940 administration did not appear to be affected by genotype.. Beta-arrestin1 appeared to mediate the actions of CP55940, but did not affect the activity of THC.. Beta-arrestin1 regulates cannabinoid CB1 receptor sensitivity in an agonist-selective manner, but may not be the primary mediator of tolerance to cannabinoid agonists.

Topics: Animals; Arrestins; beta-Arrestins; Brain; Cannabinoid Receptor Agonists; Cannabinoids; Cyclohexanols; Dronabinol; Mice; Mice, Knockout; Piperidines; Pyrazoles; Receptor, Cannabinoid, CB1; Rimonabant; Sequence Deletion

Recent studies showed that the pharmacological inhibition of endocannabinoid degrading enzymes such as fatty acid amide hydrolase (FAAH) and monoacyl glycerol lipase (MAGL) elicit promising analgesic effects in a variety of nociceptive models without serious side effects. However, the full spectrum of activities is not observed upon inhibition of either FAAH or MAGL enzymes alone and thus dual FAAH and MAGL inhibitors have been described. Visceral pain is strongly associated with inflammation and distension of the gut. Thus, we explored the comparable effects of FAAH, MAGL, and dual FAAH/MAGL inhibitors on inflammatory and mechanically evoked visceral pain models.. Visceral inflammatory and distension-induced pain were assessed with the 0.6% acetic acid writhing test in mice and colorectal distension (CRD) test in rats, respectively. The selective FAAH inhibitor PF 3845, MAGL inhibitor JZL 184, dual inhibitor JZL 195, and the cannabis analog CP 55,940 were given systemically 30 min prior to nociceptive testing.. PF 3845 (5, 10, and 20 mg/kg), JZL 184 (5, 10, and 20 mg/kg), and JZL 195 (5, 10, and 20 mg/kg) elicit dose-dependent antinociceptive in the acetic acid writhing test. In the CRD model, while JZL 195 (5, 10, or 20 mg/kg) and PF3845 (10, 20, and 40 mg/kg) produced dose-dependent antinociceptive effects comparable to those of CP 55,940 (0.1, 0.3, or 1 mg/kg), JZL 184 (10, 20, and 40 mg/kg) alone did not alter the visceromotor response (VMR).. The selective FAAH inhibitor and dual FAAH/MAGL inhibitors were effective in both inflammatory and mechanically evoked visceral pain, while the MAGL inhibitor elicited an analgesic effect in inflammatory, but not in distension-induced, visceral pain.

Topics: Amidohydrolases; Animals; Benzodioxoles; Carbamates; Colon; Cyclohexanols; Inflammation; Male; Mice; Mice, Inbred BALB C; Monoacylglycerol Lipases; Pain Measurement; Piperazines; Piperidines; Pyridines; Rats; Rats, Sprague-Dawley; Visceral Pain

CB1 cannabinoid receptors (CB1Rs) are attractive therapeutic targets for numerous central nervous system disorders. However, clinical application of cannabinoid ligands has been hampered owing to their adverse on-target effects. Ligand-biased signaling from, and allosteric modulation of, CB1Rs offer pharmacological approaches that may enable the development of improved CB1R drugs, through modulation of only therapeutically desirable CB1R signaling pathways. There is growing evidence that CB1Rs are subject to ligand-biased signaling and allosterism. Therefore, in the present study, we quantified ligand-biased signaling and allosteric modulation at CB1Rs. Cannabinoid agonists displayed distinct biased signaling profiles at CB1Rs. For instance, whereas 2-arachidonylglycerol and WIN55,212-2 [(R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-napthalenylmethanone] showed little preference for inhibition of cAMP and phosphorylation of extracellular signal-regulated kinase 1/2 (pERK1/2), N-arachidonoylethanolamine (anandamide), methanandamide, CP55940 [2-[(1R,2R,5R)-5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]-5-(2-methyloctan-2-yl)phenol], and HU-210 [11-hydroxy-Δ(8)-THC-dimethylheptyl] were biased toward cAMP inhibition. The small-molecule allosteric modulator Org27569 [5-chloro-3-ethyl-1H-indole-2-carboxylic acid [2-(4-piperidin-1-yl-phenyl)ethyl]amide] displayed biased allosteric effects by blocking cAMP inhibition mediated by all cannabinoid ligands tested, at the same time having little or no effect on ERK1/2 phosphorylation mediated by a subset of these ligands. Org27569 also displayed negative binding cooperativity with [(3)H]SR141716A [5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide]; however, it had minimal effects on binding of cannabinoid agonists. Furthermore, we highlight the need to validate the reported allosteric effects of the endogenous ligands lipoxin A4 and pregnenolone at CB1Rs. Pregnenolone but not lipoxin A4 displaced [(3)H]SR141716A, but there was no functional interaction between either of these ligands and cannabinoid agonists. This study demonstrates an approach to validating and quantifying ligand-biased signaling and allosteric modulation at CB1Rs, revealing ligand-biased "fingerprints" that may ultimately allow the development of improved CB1R-targeted therapies.

Topics: Allosteric Regulation; Animals; Benzoxazines; Cannabinoid Receptor Agonists; CHO Cells; Cricetulus; Cyclohexanols; Dronabinol; Humans; Indoles; MAP Kinase Signaling System; Morpholines; Naphthalenes; Phosphorylation; Piperidines; Pyrazoles; Receptor, Cannabinoid, CB1; Rimonabant; Signal Transduction

The activation of cannabinoid CB1 receptors decreases and increases blood pressure (BP) in anaesthetized and conscious rats, respectively. The aim of our study was to check the possible involvement of CB1 receptors in the paraventricular nucleus of the hypothalamus (PVN) in the cardiovascular effects of cannabinoids in rats. Methanandamide (metabolically stable analogue of the endocannabinoid anandamide) and the synthetic cannabinoid receptor agonist CP55940 were microinjected into the PVN of urethane-anaesthetized rats twice (S1 and S2, 20 min apart). Receptor antagonists were administered intravenously (i.v.) 5 min before S1. Methanandamide and CP55940 decreased blood pressure by 15 - 20%. The CB1 receptor antagonist AM251 reversed the depressor effect into a pressor response of 20 - 30%. The pressor effect of CP55940 observed in the presence of AM251 i.v. was reduced by AM251 given additionally into the PVN but not by the i.v. injection of the CB2 antagonist SR144528 or the vanilloid TRPV1 antagonist ruthenium red. In the presence of the peripherally restricted CB1 receptor antagonist AM6545, CP55940 given into the PVN increased BP by 40%. AM6545 reversed the decrease in BP induced by CP55940 i.v. into a marked increase. Bilateral chemical lesion of the PVN by kainic acid abolished all cardiovascular effects of CP55940 i.v. In conclusion, the cannabinoid CP55940 administered to the PVN of urethane-anaesthetized rats can induce depressor and pressor effects. The direction of the response probably depends on the sympathetic tone. The centrally induced hypertensive response of CP55940 can, in addition, be masked by peripheral CB1 receptors.

Topics: Amidohydrolases; Anesthesia; Animals; Arachidonic Acids; Blood Pressure; Cannabinoids; Cerebral Cortex; Cyclohexanols; Endocannabinoids; Heart Rate; Male; Microinjections; Morpholines; Paraventricular Hypothalamic Nucleus; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats, Wistar; Receptor, Cannabinoid, CB1

G protein-coupled receptors (GPCRs) are surprisingly flexible molecules that can do much more than simply turn on G proteins. Some even exhibit biased signaling, wherein the same receptor preferentially activates different G-protein or arrestin signaling pathways depending on the type of ligand bound. Why this behavior occurs is still unclear, but it can happen with both traditional ligands and ligands that bind allosterically outside the orthosteric receptor binding pocket. Here, we looked for structural mechanisms underlying these phenomena in the marijuana receptor CB1. Our work focused on the allosteric ligand Org 27569, which has an unusual effect on CB1-it simultaneously increases agonist binding, decreases G--protein activation, and induces biased signaling. Using classical pharmacological binding studies, we find that Org 27569 binds to a unique allosteric site on CB1 and show that it can act alone (without need for agonist cobinding). Through mutagenesis studies, we find that the ability of Org 27569 to bind is related to how much receptor is in an active conformation that can couple with G protein. Using these data, we estimated the energy differences between the inactive and active states. Finally, site-directed fluorescence labeling studies show the CB1 structure stabilized by Org 27569 is different and unique from that stabilized by antagonist or agonist. Specifically, transmembrane helix 6 (TM6) movements associated with G-protein activation are blocked, but at the same time, helix 8/TM7 movements are enhanced, suggesting a possible mechanism for the ability of Org 27569 to induce biased signaling.

Topics: Algorithms; Animals; Binding, Competitive; Cannabinoid Receptor Agonists; Cannabinoid Receptor Antagonists; Chlorocebus aethiops; COS Cells; Cyclohexanols; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Indoles; Kinetics; Models, Biological; Models, Molecular; Mutation; Piperidines; Protein Conformation; Protein Structure, Secondary; Protein Structure, Tertiary; Pyrazoles; Radioligand Assay; Receptor, Cannabinoid, CB1; Rimonabant

Despite the fact that the cannabinoid receptor type 1 (CB1R) plays a pivotal role in emotional memory processing in different regions of the brain, its function in the retrosplenial cortex (RSC) remains unknown. Here, using contextual fear conditioning in rats, we showed that a post-training intra-RSC infusion of the CB1R antagonist AM251 impaired, and the agonist CP55940 improved, long-term memory consolidation. Additionally, a post-reactivation infusion of AM251 enhanced memory reconsolidation, while CP55940 had the opposite effect. Finally, AM251 blocked extinction, whereas CP55940 facilitated it and maintained memory extinguished over time. Altogether, our data strongly suggest that the cannabinoid system of the RSC modulates emotional memory.

Topics: Animals; Cannabinoid Receptor Agonists; Cannabinoid Receptor Antagonists; Catheters, Indwelling; Cerebral Cortex; Conditioning, Psychological; Cyclohexanols; Extinction, Psychological; Fear; Male; Memory Consolidation; Piperidines; Pyrazoles; Rats, Wistar; Receptor, Cannabinoid, CB1

The "subjective high" from marijuana ingestion is likely due to Δ(9)-tetrahydrocannabinol (THC) activating the central cannabinoid receptor type 1 (CB1R) of the endocannabinoid signaling system. THC is a weak partial agonist according to in vitro assays, yet THC mimics the behavioral effects induced by more efficacious cannabinergics. This distinction may be important for understanding similarities and differences in the dose-effect spectra produced by marijuana/THC and designer cannabimimetics ("synthetic marijuana").. We evaluated if drug discrimination is able to functionally detect/differentiate between a full, high-efficacy CB1R agonist [(±)AM5983] and the low-efficacy agonist THC in vivo.. Rats were trained to discriminate between four different doses of AM5983 (0.10 to 0.56 mg/kg), and vehicle and dose generalization curves were determined for both ligands at all four training doses of AM5983. The high-efficacy WIN55,212-2 and the lower-efficacy (R)-(+)-methanandamide were examined at some AM5983 training conditions. Antagonism tests involved rimonabant and WIN55,212-2 and AM5983. The separate (S)- and (R)-isomers of (±)AM5983 were tested at one AM5983 training dose (0.30 mg/kg). The in vitro cyclic adenosine monophosphate (cAMP) assay examined AM5983 and the known CB1R agonist CP55,940.. Dose generalization ed50 values increased as a function of the training dose of AM5983, but more so for the partial agonists. The order of potency was (R)-isomer > (±)AM5983 > (S)-isomer and AM5983 > WIN55,212-2 ≥ THC > (R)-(+)-methanandamide. Surmountable antagonism of AM5983 and WIN55,212-2 occurred with rimonabant. The cAMP assay confirmed the cannabinergic nature of AM5983 and CP55,940.. Drug discrimination using different training doses of a high-efficacy, full CB1R agonist differentiated between low- and high-efficacy CB1R agonists.

Topics: Animals; Arachidonic Acids; Benzoxazines; Cyclic AMP; Cyclohexanols; Discrimination, Psychological; Dose-Response Relationship, Drug; Dronabinol; Indoles; Isomerism; Male; Molecular Structure; Morpholines; Naphthalenes; Piperidines; Pyrazoles; Random Allocation; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Rimonabant

Benzophenanthridine alkaloids (chelerythrine and sanguinarine) inhibited binding of [(3)H]SR141716A to mouse brain membranes (IC50s: <1µM). Piperonyl butoxide and (S)-methoprene were less potent (IC50s: 21 and 63µM respectively). Benzophenanthridines and piperonyl butoxide were more selective towards brain CB1 receptors versus spleen CB2 receptors. All compounds reduced Bmax of [(3)H]SR141716A binding to CB1 receptors, but only methoprene and piperonyl butoxide increased Kd (3-5-fold). Benzophenanthridines increased the Kd of [(3)H]CP55940 binding (6-fold), but did not alter Bmax. (S)-methoprene increased the Kd of [(3)H]CP55940 binding (by almost 4-fold) and reduced Bmax by 60%. Piperonyl butoxide lowered the Bmax of [(3)H]CP55940 binding by 50%, but did not influence Kd. All compounds reduced [(3)H]SR141716A and [(3)H]CP55940 association with CB1 receptors. Combined with a saturating concentration of SR141716A, only piperonyl butoxide and (S)-methoprene increased dissociation of [(3)H]SR141716A above that of SR141716A alone. Only piperonyl butoxide increased dissociation of [(3)H]CP55940 to a level greater than CP55940 alone. Binding results indicate predominantly allosteric components to the study compounds action. 4-Aminopyridine-(4-AP-) evoked release of l-glutamate from synaptosomes was partially inhibited by WIN55212-2, an effect completely neutralized by AM251, (S)-methoprene and piperonyl butoxide. With WIN55212-2 present, benzophenanthridines enhanced 4-AP-evoked l-glutamate release above 4-AP alone. Modulatory patterns of l-glutamate release (with WIN-55212-2 present) align with previous antagonist/inverse agonist profiling based on [(35)S]GTPγS binding. Although these compounds exhibit lower potencies compared to many classical CB1 receptor inhibitors, they may have potential to modify CB1-receptor-dependent behavioral/physiological outcomes in the whole animal.

Topics: Animals; Benzophenanthridines; Benzoxazines; Binding Sites; Brain; Cyclohexanols; Glutamic Acid; Isoquinolines; Male; Methoprene; Mice; Morpholines; Naphthalenes; Piperidines; Piperonyl Butoxide; Pyrazoles; Radioligand Assay; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Rimonabant; Spleen; Synaptosomes

Synthetic cannabinoid abuse and case reports of adverse effects have raised concerns about the pharmacologic mechanisms underlying in vivo effects. Here, a synthetic cannabinoid identified in abused products (HU-210) was compared to the effects of Δ(9)-THC and two other synthetic cannabinoid agonists used extensively in pre-clinical studies (CP 55,940 and WIN 55,212-2). One group of monkeys discriminated ∆(9)-THC (0.1mg/kg i.v.); a separate group received chronic ∆(9)-THC (1mg/kg/12h s.c.) and discriminated rimonabant (1mg/kg i.v.). CP 55,940, HU-210, ∆(9)-THC, and WIN 55,212-2 produced ∆(9)-THC lever responding. HU-210 had a long duration (i.e., 1-2 days), whereas that of the other cannabinoids was 5h or less. Rimonabant (1mg/kg) produced surmountable antagonism; single dose-apparent affinity estimates determined in the presence of ∆(9)-THC, CP 55,940, and WIN 55,212-2 did not differ from each other. In contrast, rimonabant (1mg/kg) produced a smaller rightward shift in the HU-210 dose-effect function. In ∆(9)-THC treated monkeys, the relative potency of CP 55,940, ∆(9)-THC, and WIN 55,212-2 to attenuate the discriminative stimulus effects of rimonabant was the same as that evidenced in the ∆(9)-THC discrimination, whereas HU-210 was unexpectedly more potent in attenuating the effects of rimonabant. In conclusion, the same receptor subtype mediates the discriminative stimulus effects of ∆(9)-THC, CP 55,940 and WIN 55,212-2. The limited effectiveness of rimonabant to either prevent or reverse the effects of HU-210 appears to be due to very slow dissociation or pseudo-irreversible binding of HU-210 at cannabinoid receptors.

Topics: Animals; Behavior, Animal; Benzoxazines; Cannabinoid Receptor Agonists; Cannabinoid Receptor Antagonists; Cyclohexanols; Discrimination, Psychological; Dose-Response Relationship, Drug; Dronabinol; Female; Macaca mulatta; Male; Morpholines; Naphthalenes; Piperidines; Pyrazoles; Receptors, Cannabinoid; Rimonabant; Time Factors

Several allosteric modulators (AMs) of the CB1 receptor have been characterized in vitro, including Org27569, which enhances CB1-specific binding of [H]CP55,940, but behaves as an insurmountable CB1-receptor antagonist in several biochemical assays. Although a growing body of research has investigated the molecular actions of this unusual AM, it is unknown whether these actions translate to the whole animal. The purpose of the present study was to determine whether Org27569 would produce effects in well-established mouse behavioral assays sensitive to CB1 orthosteric agonists and antagonists. Similar to the orthosteric CB1 antagonist/inverse agonist rimonabant, Org27569 reduced food intake; however, this anorectic effect occurred independently of the CB1 receptor. Org27569 did not elicit CB1-mediated effects alone and lacked efficacy in altering antinociceptive, cataleptic, and hypothermic actions of the orthosteric agonists anandamide, CP55,940, and Δ-tetrahydrocannabinol. Moreover, it did not alter the discriminative stimulus effects of anandamide in FAAH-deficient mice or Δ-tetrahydrocannabinol in wild-type mice in the drug discrimination paradigm. These findings question the utility of Org27569 as a 'gold standard' CB1 AM and underscore the need for the development of CB1 AMs with pharmacology that translates from the molecular level to the whole animal.

Topics: Allosteric Regulation; Amidohydrolases; Animals; Arachidonic Acids; Cannabinoid Receptor Agonists; Cannabinoid Receptor Antagonists; Cannabinoid Receptor Modulators; Catalepsy; Cyclohexanols; Dronabinol; Drug Evaluation; Eating; Endocannabinoids; Female; Hypothermia; Indoles; Male; Mice; Mice, Inbred C57BL; Mice, Inbred ICR; Mice, Knockout; Nociception; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB1; Rimonabant

Several effects of the abused synthetic cannabinoid JWH-018 were compared to those of Δ9-tetrahydrocannabinol (Δ9-THC) in rhesus monkeys. JWH-018 (0.1 mg/kg i.v.) was established as a discriminative stimulus and rimonabant was used to examine mechanisms responsible for discrimination as well as operant response rate-decreasing and hypothermic effects. JWH-018 dose-dependently increased drug-lever responding (ED50=0.01 mg/kg) and decreased response rate (ED50=0.064 mg/kg). Among various cannabinoids, the relative potency for producing discriminative stimulus and rate-decreasing effects was the same: CP-55940=JWH-018>Δ9-THC=WIN-55212-2=JWH-073. The benzodiazepine agonist midazolam and the NMDA antagonist ketamine did not exert JWH-018 like discriminative stimulus effects up to doses that disrupted responding. JWH-018 and Δ9-THC decreased rectal temperature by 2.2 and 2.8°C, respectively; the doses decreasing temperature by 2°C were 0.21 and 1.14 mg/kg, respectively. Antagonism did not differ between JWH-018 and Δ9-THC, but did differ among effects. The apparent affinities of rimonabant calculated in the presence of JWH-018 and Δ9-THC were not different from each other for antagonism of discriminative stimulus effects (6.58 and 6.59, respectively) or hypothermic effects (7.08 and 7.19, respectively). Apparent affinity estimates are consistent with the same receptors mediating the discriminative stimulus and hypothermic effects of both JWH-018 and Δ9-THC. However, there was more limited and less orderly antagonism of rate-decreasing effects, suggesting that an additional receptor mechanism is involved in mediating the effects of cannabinoids on response rate. Overall, these results strongly suggest that JWH-018 and Δ9-THC act at the same receptors to produce several of their shared psychopharmacological effects.

Topics: Animals; Benzoxazines; Body Temperature; Cannabinoid Receptor Agonists; Cannabinoid Receptor Antagonists; Cannabinoids; Conditioning, Operant; Cyclohexanols; Discrimination Learning; Dronabinol; Hypothermia; Indoles; Macaca mulatta; Male; Morpholines; Naphthalenes; Piperidines; Pyrazoles; Receptor, Cannabinoid, CB1; Rimonabant

Paediatric traumatic brain injury (TBI) is a leading cause of death and disability. Previous studies showed neuroprotection after TBI by (endo)cannabinoid mechanisms, suggesting involvement of cannabinoid receptors (CBR). We therefore determined CBR densities and expression of the translocator protein 18 kDA (TSPO) in newborn piglets after experimental TBI. Newborn female piglets were subjected to sham operation (n=6) or fluid-percussion (FP) injury (n=7) under controlled physiological conditions. After six hours, brains were frozen, sagittally cut and incubated with radioligands for CBR ([3HCP-55,940, [3H]SR141716A) and TSPO ([3H]PK11195), an indicator of gliosis/brain injury. Early after injury, FP-TBI elicited a significant ICP increase at a temporary reduced cerebral perfusion pressure; however, CBF and CMRO2 remained within physiological range. At 6 hours post injury, we found a statistically significant increase in binding of the non-selective agonist [3H]CP-55,940 in 15 of the 24 investigated brain regions of injured animals. By contrast, no significant changes in binding of the CB1R-selective antagonist [3H]SR141716A were observed. A non-significant trend towards increased binding of [3H]PK11195 was observed, suggesting an incipient microglial activation. We therefore conclude that in this model and time span after injury, the increase in [3H]CP-55,940 binding reflects changes in CB2R density, while CB1R density is not affected. The results may provide explanation for the neuroprotective properties of cannabinoid ligands and future therapeutic strategies of TBI.

Topics: Analysis of Variance; Animals; Animals, Newborn; Autoradiography; Brain; Brain Injuries; Cannabinoid Receptor Modulators; Cyclohexanols; Disease Models, Animal; Female; Isoquinolines; Piperidines; Protein Binding; Pyrazoles; Radionuclide Imaging; Receptors, Cannabinoid; Receptors, GABA; Rimonabant; Swine; Time Factors; Tritium

It has been reported that direct activation of the cannabinoid CB1 receptor in epidermal growth factor (EGR)-stimulated PC-3 prostate cancer cells results in an anti-proliferative effect accompanied by a down-regulation of EGF receptors (EGFR). In the present study, we investigated whether similar effects are seen following inhibition of the endocannabinoid hydrolytic enzyme monoacylglycerol lipase (MGL).. CB1 receptor expression levels were found to differ greatly between two experimental series conducted using PC-3 cells. The monoacylglycerol lipase inhibitor JZL184 increased levels of 2-arachidonoylglycerol in the PC-3 cells without producing changes in the levels of anandamide and related N-acylethanolamines. In the first series of experiments, JZL184 produced a small mitogenic effect for cells that had not been treated with EGF, whereas an anti-proliferative effect was seen for EGF-treated cells. An anti-proliferative effect for the EGF-treated cells was also seen with the CB receptor agonist CP55,940. In the second batch of cells, there was an interaction between JZL184 and CB1 receptor expression densities in linear regression analyses with EGFR expression as the dependent variable.. Inhibition of MGL by JZL184 can affect EGFR expression. However, the use in our hands of PC-3 cells as a model to investigate the therapeutic potential of MGL inhibitors and related compounds is compromised by their variability of CB1 receptor expression.

Topics: Arachidonic Acids; Benzodioxoles; Cannabinoids; Cell Line, Tumor; Cell Proliferation; Cyclohexanols; Endocannabinoids; Enzyme Inhibitors; ErbB Receptors; Ethanolamines; Gene Expression Regulation, Neoplastic; Glycerides; Humans; Male; Monoacylglycerol Lipases; Piperidines; Polyunsaturated Alkamides; Prostate; Receptor, Cannabinoid, CB1; Signal Transduction

The monoacylglycerol lipase (MAGL) inhibitor 4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184) produces antinociceptive and anti-inflammatory effects. However, repeated administration of high-dose JZL184 (40 mg/kg) causes dependence, antinociceptive tolerance, cross-tolerance to the pharmacological effects of cannabinoid receptor agonists, and cannabinoid receptor type 1 (CB1) downregulation and desensitization. This functional CB1 receptor tolerance poses a hurdle in the development of MAGL inhibitors for therapeutic use. Consequently, the present study tested whether repeated administration of low-dose JZL184 maintains its antinociceptive actions in the chronic constriction injury of the sciatic nerve neuropathic pain model and protective effects in a model of nonsteroidal anti-inflammatory drug-induced gastric hemorrhages. Mice given daily injections of high-dose JZL184 (≥16 mg/kg) for 6 days displayed decreased CB1 receptor density and function in the brain, as assessed in [(3)H]SR141716A binding and CP55,940 [(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl) cyclohexanol]-stimulated guanosine 5'-O-(3-[(35)S]thio)triphosphate binding assays, respectively. In contrast, normal CB1 receptor expression and function were maintained following repeated administration of low-dose JZL184 (≤8 mg/kg). Likewise, the antinociceptive and gastroprotective effects of high-dose JZL184 underwent tolerance following repeated administration, but these effects were maintained following repeated low-dose JZL184 treatment. Consistent with these observations, repeated high-dose JZL184, but not repeated low-dose JZL184, elicited cross-tolerance to the common pharmacological effects of Δ(9)-tetrahydrocannabinol. This same pattern of effects was found in a rimonabant [(5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide)]-precipitated withdrawal model of cannabinoid dependence. Taken together, these results indicate that prolonged, partial MAGL inhibition maintains potentially beneficial antinociceptive and anti-inflammatory effects, without producing functional CB1 receptor tachyphylaxis/tolerance or cannabinoid dependence.

Topics: Analgesics; Animals; Anti-Inflammatory Agents, Non-Steroidal; Anti-Ulcer Agents; Arachidonic Acids; Benzodioxoles; Brain Chemistry; Cyclohexanols; Diclofenac; Dose-Response Relationship, Drug; Dronabinol; Drug Tolerance; Endocannabinoids; Glycerides; Guanosine 5'-O-(3-Thiotriphosphate); Male; Mice; Mice, Inbred C57BL; Monoacylglycerol Lipases; Pain Measurement; Piperidines; Pyrazoles; Receptor, Cannabinoid, CB1; Rimonabant; Stomach Ulcer; Substance Withdrawal Syndrome; Substance-Related Disorders

Activation of the cannabinoid CB1 receptor (CB1) is modulated by aspartate residue D2.63(176) in transmembrane helix (TMH) 2. Interestingly, D2.63 does not affect the affinity for ligand binding at the CB1 receptor. Studies in class A G protein-coupled receptors have suggested an ionic interaction between residues of TMH2 and 7. In this report, modeling studies identified residue K373 in the extracellular-3 (EC-3) loop in charged interactions with D2.63. We investigated this possibility by performing reciprocal mutations and biochemical studies. D2.63(176)A, K373A, D2.63(176)A-K373A, and the reciprocal mutant with the interacting residues juxtaposed D2.63(176)K-K373D were characterized using radioligand binding and guanosine 5'-3-O-(thio)triphosphate functional assays. None of the mutations resulted in a significant change in the binding affinity of N-(piperidiny-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR141716A) or (-)-3cis -[2-hydroxyl-4-(1,1-dimethyl-heptyl)phenyl]-trans-4-[3-hydroxyl-propyl] cyclohexan-1-ol (CP55,940). Modeling studies indicated that binding-site interactions and energies of interaction for CP55,940 were similar between wild-type and mutant receptors. However, the signaling of CP55,940, and (R)-(+)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]-pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthalenyl)-methanone mesylate (WIN55,212-2) was impaired at the D2.63(176)A-K373A and the single-alanine mutants. In contrast, the reciprocal D2.63(176)K-K373D mutant regained function for both CP55,940 and WIN55,212-2. Computational results indicate that the D2.63(176)-K373 ionic interaction strongly influences the conformation(s) of the EC-3 loop, providing a structure-based rationale for the importance of the EC-3 loop to signal transduction in CB1. The putative ionic interaction results in the EC-3 loop pulling over the top (extracellular side) of the receptor; this EC-3 loop conformation may serve protective and mechanistic roles. These results suggest that the ionic interaction between D2.63(176) and K373 is important for CB1 signal transduction.

Topics: Amino Acid Sequence; Benzoxazines; Binding, Competitive; Cell Line; Cyclohexanols; Energy Metabolism; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Immunosuppressive Agents; Models, Chemical; Molecular Sequence Data; Morpholines; Mutagenesis, Site-Directed; Naphthalenes; Piperidines; Protein Conformation; Protein Structure, Secondary; Pyrazoles; Radioligand Assay; Receptor, Cannabinoid, CB1; Rimonabant; Signal Transduction

Compelling evidence has documented the anxiolytic and mood-enhancing properties of cannabis. In susceptible users, however, consumption of this drug is conducive to panic, paranoia and dysphoria. We hypothesized that the up-regulation of CB₁ receptors (CB₁Rs) in select brain regions may enhance the vulnerability to cannabinoid-induced anxiety. To test this possibility, we assessed the behavioral impact of a potent cannabinoid agonist (CP55,940; 0.05-0.1 mg/kg, IP) on C57BL/6 male mice, respectively subjected to a prolonged pre-treatment of either the selective CB₁R antagonist/inverse agonist AM251 (1 mg/kg/day IP, for 21 days, followed by a 3-day clearance period before testing) or its vehicle (VEH1). Anxiety-like responses were studied in the novel open field, elevated plus maze (EPM) and social interaction assays. While CP55,940 induced anxiolytic-like effects in the EPM in VEH1-exposed animals, it elicited opposite actions in AM251-exposed mice. In this last group, CP55,940 also reduced rearing and social interaction in comparison to its vehicle (VEH2). The divergent effects of CP55,940 in AM251- and VEH1-pretreated animals were confirmed in 129SvEv mice. Immunoblotting analyses on brain samples of C57BL/6 mice revealed that AM251 pre-treatment caused a significant up-regulation of CB₁R expression in the prefrontal cortex and striatum, but also a down-regulation of these receptors in the hippocampus and midbrain. Notably, CB₁R levels in the prefrontal cortex were negatively correlated with anxiolysis-related indices in the EPM; furthermore, midbrain CB₁R expression was positively correlated with the total duration of social interaction. These results suggest that regional variations in brain CB₁R expression may differentially condition the behavioral effects of cannabinoids with respect to anxiety-related responses.

Topics: Animals; Anxiety; Behavior, Animal; Brain; Cannabinoid Receptor Agonists; Cannabinoid Receptor Antagonists; Cyclohexanols; Dose-Response Relationship, Drug; Down-Regulation; Drug Interactions; Male; Mice; Piperidines; Pyrazoles; Receptor, Cannabinoid, CB1; Up-Regulation

The neuronal mechanisms underlying social withdrawal, one of the core negative symptoms of schizophrenia, are not well understood. Recent studies suggest an involvement of the endocannabinoid system in the pathophysiology of schizophrenia and, in particular, of negative symptoms. We used biochemical, pharmacological, and behavioral approaches to investigate the role played by the endocannabinoid system in social withdrawal induced by sub-chronic administration of phencyclidine (PCP). Pharmacological enhancement of endocannabinoid levels via systemic administration of URB597, an inhibitor of endocannabinoid degradation, reversed social withdrawal in PCP-treated rats via stimulation of CB1 receptors, but reduced social interaction in control animals through activation of a cannabinoid/vanilloid-sensitive receptor. In addition, the potent CB agonist CP55,940 reversed PCP-induced social withdrawal in a CB₁-dependent manner, whereas pharmacological blockade of CB₁ receptors by either AM251 or SR141716 reduced the time spent in social interaction in control animals. PCP-induced social withdrawal was accompanied by a decrease of anandamide (AEA) levels in the amygdala and prefrontal cortex, and these deficits were reversed by URB597. As CB₁ receptors are predominantly expressed on GABAergic interneurons containing the anxiogenic peptide cholecystokinin (CCK), we also examined whether the PCP-induced social withdrawal resulted from deficient CB₁-mediated modulation of CCK transmission. The selective CCK2 antagonist LY225910 blocked both PCP- and AM251-induced social withdrawal, but not URB597 effect in control rats. Taken together, these findings indicate that AEA-mediated activation of CB₁ receptors is crucial for social interaction, and that PCP-induced social withdrawal results from deficient endocannabinoid transmission.

Topics: Amidohydrolases; Amygdala; Animals; Arachidonic Acids; Benzamides; Cannabinoid Receptor Agonists; Cannabinoid Receptor Antagonists; Carbamates; Cyclohexanols; Dose-Response Relationship, Drug; Endocannabinoids; Male; Phencyclidine; Piperidines; Polyunsaturated Alkamides; Prefrontal Cortex; Pyrazoles; Quinazolinones; Rats; Receptor, Cannabinoid, CB1; Receptor, Cholecystokinin B; Rimonabant; Schizophrenia; Social Behavior

Cannabinoids are natural or synthetic compounds related chemically to (-)-(6aR,10aR)-6,6,9-trimethyl-3-pentyl-6a,7,8,10a-tetrahydro-6H-benzo[c]chromen-1-ol (Δ(9)-THC), the principle psychotropic constituent of the hemp plant, Cannabis sativa L. Here we examine the effects of the cannabinoids Δ(9)-THC, (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo [1,2,3-de]-1,4-benzoxazin-6-yl]-1-napthalenylmethanone and 2-[(1R,2R,5R)-5-hydroxy-2-(3-hydroxypropyl) cyclohexyl]-5-(2-methyloctan-2-yl)phenol, and the cannabinoid antagonist (AM 251). Exposures were either acute (1-12-h exposure at 108 hours of postfertilization [hpf]) or chronic (96-h exposure starting at 24 hpf). Geometric range finding was used to determine the experimental concentrations. The concentration of the chemical that kills 50% of the test animals in a given time (LC50) was determined based on cumulative mortality at 5 days of postfertilization. At day 5, behavioral analysis (visual motor response test) was carried out in which movement of individual larvae was analysed using automated video-tracking. With acute exposure, embryos showed a biphasic response to the dark challenge with all three cannabinoids tested. This response consisted of stimulation of the locomotor activity at low concentrations, suppression at high doses. With chronic exposure, embryos habituated to the effects of all three cannabinoids when assayed with the dark challenge phase. Further, the excitation was ameliorated when the antagonist AM 251 was coadministered with the cannabinoid. When AM 251 was administered on its own (chronically or acutely), the locomotor activity was suppressed at high concentrations. We examined the embryos for a range of malformations after chronic exposure to cannabinoid. Only Δ(9)-THC was associated with a significant increase in malformations at 5d (yolk sac and pericardial edema, bent tail/body axis). We conclude that cannabinoids have behavioral effects in zebrafish that are comparable to some of those reported in the literature for mammals. In particular, the acute exposure response resembles behavioral effects reported for adult rodents. Our data are consistent with these behavioral effects being mediated, at least in part, by the CB1 receptor.

Topics: Animals; Behavior, Animal; Benzoxazines; Cannabinoid Receptor Agonists; Cyclohexanols; Dronabinol; Embryo, Nonmammalian; Female; Larva; Lethal Dose 50; Locomotion; Male; Morpholines; Naphthalenes; Piperidines; Pyrazoles; Toxicity Tests, Acute; Toxicity Tests, Chronic; Zebrafish

The cannabinoid receptor type 1 (CB1 ) has an allosteric binding site. The drugs ORG27569 {5-chloro-3-ethyl-N-[2-[4-(1-piperidinyl)phenyl]ethyl]-1H-indole-2-carboxamide} and PSNCBAM-1 {1-(4-chlorophenyl)-3-[3-(6-pyrrolidin-1-ylpyridin-2-yl)phenyl]urea} have been extensively characterized with regard to their effects on signalling of the orthosteric ligand CP55,940 {(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol}, and studies have suggested that these allosteric modulators increase binding affinity but act as non-competitive antagonists in functional assays. To gain a deeper understanding of allosteric modulation of CB1 , we examined real-time signalling and trafficking responses of the receptor in the presence of allosteric modulators.. Studies of CB1 signalling were carried out in HEK 293 and AtT20 cells expressing haemagglutinin-tagged human and rat CB1 . We measured real-time accumulation of cAMP, activation and desensitization of potassium channel-mediated cellular hyperpolarization and CB1 internalization.. ORG27569 and PSNCBAM-1 produce a complex, concentration and time-dependent modulation of agonist-mediated regulation of cAMP levels, as well as an increased rate of desensitization of CB1 -mediated cellular hyperpolarization and a decrease in agonist-induced receptor internalization.. Contrary to previous studies characterizing allosteric modulators at CB1, this study suggests that the mechanism of action is not non-competitive antagonism of signalling, but rather that enhanced binding results in an increased rate of receptor desensitization and reduced internalization, which results in time-dependent modulation of cAMP signalling. The observed effect of the allosteric modulators is therefore dependent on the time frame over which the signalling response occurs. This finding may have important consequences for the potential therapeutic application of these compounds.

Topics: Animals; Cannabinoid Receptor Agonists; Cannabinoid Receptor Antagonists; Cell Line, Tumor; Cyclic AMP; Cyclohexanols; Dose-Response Relationship, Drug; HEK293 Cells; Humans; Indoles; Kinetics; Ligands; Membrane Potentials; Mice; Phenylurea Compounds; Piperidines; Potassium Channels, Inwardly Rectifying; Protein Transport; Pyridines; Rats; Receptor, Cannabinoid, CB1; Signal Transduction; Transfection

The human cannabinoid receptor, CB1, a G protein-coupled receptor (GPCR), contains a relatively long (∼110 a.a.) amino terminus, whose function is still not defined. Here we explore a potential role for the CB1 N-terminus in modulating ligand binding to the receptor. Although most of the CB1 N-terminus is not necessary for ligand binding, previous studies have found that mutations introduced into its conserved membrane proximal region (MPR) do impair the receptors ability to bind ligand. Moreover, within the highly conserved MPR (∼ residues 90-110) lie two cysteine residues that are invariant in all CB1 receptors. We find these two cysteines (C98 and C107) form a disulfide in heterologously expressed human CB1, and this C98-C107 disulfide is much more accessible to reducing agents than the previously known disulfide in extracellular loop 2 (EL2). Interestingly, the presence of the C98-C107 disulfide modulates ligand binding to the receptor in a way that can be quantitatively analyzed by an allosteric model. The C98-C107 disulfide also alters the effects of allosteric ligands for CB1, Org 27569 and PSNCBAM-1. Together, these results provide new insights into how the N-terminal MPR and EL2 act together to influence the high-affinity orthosteric ligand binding site in CB1 and suggest that the CB1 N-terminal MPR may be an area through which allosteric modulators can act.

Topics: Allosteric Regulation; Amino Acid Sequence; Animals; Binding Sites; Chlorocebus aethiops; COS Cells; Cyclohexanols; Cysteine; Disulfides; Dithiothreitol; Humans; Indoles; Ligands; Phenylurea Compounds; Piperidines; Pyridines; Rats; Receptor, Cannabinoid, CB1

Chronic cannabinoid exposure results in tolerance due to region-specific desensitization and down-regulation of CB1 cannabinoid receptors (CB1Rs). For most G-protein-coupled receptors, internalization closely follows rapid desensitization as an important component of long-term down-regulation. However, in vivo patterns of CB1R internalization are not known. Here we investigate the subcellular redistribution of CB1Rs in the rat forebrain following activation by agonist CP55 940 or inhibition by antagonist/inverse agonist AM251. At steady state, CB1Rs are mainly localized to the cell membrane of preterminal axon shafts and, to a lesser degree, to synaptic terminals. A high proportion of CB1Rs is also localized to somatodendritic endosomes. Inhibition of basal activation by acute AM251 administration decreases the number of cell bodies containing CB1R-immunoreactive endosomes, suggesting that CB1Rs are permanently activated and internalized at steady state. On the contrary, acute agonist treatment induces rapid and important increase of endosomal CB1R immunolabeling, likely due to internalization and retrograde transport of axonal CB1Rs. Repeated agonist treatment is necessary to significantly reduce initially high levels of axonal CB1R labeling, in addition to increasing somatodendritic endosomal CB1R labeling in cholecystokinin-positive interneurons. This redistribution displays important region-specific differences; it is most pronounced in the neocortex and hippocampus and absent in basal ganglia.

Topics: Animals; Cyclohexanols; Endosomes; Intracellular Space; Male; Neurons; Piperidines; Prosencephalon; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1

Evidence indicates cannabinoid receptor agonists impair performance in procedures to assess memory that may also be confounded by motivational or motor effects, both of which occur with cannabinoids. Thus, convergence of evidence from a variety of procedures that differ in motivation, attention, arousal and response requirements, but share a common reliance on memory, is required. There are no current reports on cannabinoid effects on mice tested in the radial arm maze.. The objective was to determine the effects of the cannabinoid agonist CP 55940 and the dependence of any such effects on the CB1 receptor using the CB1 receptor antagonist SR 141716A on two strains of mice in the eight-arm radial maze procedure.. Male C57BL/6J (N = 36) and C3H/HEJ (N = 12) mice were trained to a criterion and then were treated (IP) with vehicle + vehicle, SR 141716A + vehicle, vehicle + CP 55940 and SR 141716A + CP 55940 in a fully balanced mixed design prior to further tests in the maze. Reference (long-term) and working (short-term) memory were assessed.. CP 55940 impaired performance of the reference memory task in the C57BL/6J strain but not the C3H/HEJ strain; SR 141716A reversed the effect of CP 55940 on these measures. CP 55940 also increased working memory errors in the C57BL/6J mice only, which was not affected by SR 141716A.. The present study provides evidence for a strain-specific effect of a dose of CP 55940 on reference memory. While the cannabinoid agonist also impaired working memory in one strain, this effect was apparently not mediated by CB1 receptors.

Topics: Animals; Cannabinoid Receptor Agonists; Cannabinoid Receptor Antagonists; Cannabinoids; Cyclohexanols; Male; Maze Learning; Memory; Memory, Short-Term; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Piperidines; Pyrazoles; Receptor, Cannabinoid, CB1; Rimonabant; Species Specificity

We have previously identified allosteric modulators of the cannabinoid CB(1) receptor (Org 27569, PSNCBAM-1) that display a contradictory pharmacological profile: increasing the specific binding of the CB(1) receptor agonist [(3)H]CP55940 but producing a decrease in CB(1) receptor agonist efficacy. Here we investigated the effect one or both compounds in a broad range of signaling endpoints linked to CB(1) receptor activation. We assessed the effect of these compounds on CB(1) receptor agonist-induced [(35)S]GTPγS binding, inhibition, and stimulation of forskolin-stimulated cAMP production, phosphorylation of extracellular signal-regulated kinases (ERK), and β-arrestin recruitment. We also investigated the effect of these allosteric modulators on CB(1) agonist binding kinetics. Both compounds display ligand dependence, being significantly more potent as modulators of CP55940 signaling as compared with WIN55212 and having little effect on [(3)H]WIN55212 binding. Org 27569 displays biased antagonism whereby it inhibits: agonist-induced guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPγS) binding, simulation (Gα(s)-mediated), and inhibition (Gα(i)-mediated) of cAMP production and β-arrestin recruitment. In contrast, it acts as an enhancer of agonist-induced ERK phosphorylation. Alone, the compound can act also as an allosteric agonist, increasing cAMP production and ERK phosphorylation. We find that in both saturation and kinetic-binding experiments, the Org 27569 and PSNCBAM-1 appeared to influence only orthosteric ligand maximum occupancy rather than affinity. The data indicate that the allosteric modulators share a common mechanism whereby they increase available high-affinity CB(1) agonist binding sites. The receptor conformation stabilized by the allosterics appears to induce signaling and also selectively traffics orthosteric agonist signaling via the ERK phosphorylation pathway.

Topics: Allosteric Regulation; Animals; Arrestins; Benzoxazines; beta-Arrestins; Brain; Cell Line; Cell Membrane; CHO Cells; Colforsin; Cricetinae; Cyclic AMP; Cyclohexanols; Guanosine 5'-O-(3-Thiotriphosphate); HEK293 Cells; Humans; Indoles; Kinetics; Ligands; Male; MAP Kinase Signaling System; Mice; Morpholines; Naphthalenes; Phosphorylation; Piperidines; Protein Binding; Receptor, Cannabinoid, CB1; Signal Transduction

Activation of type 1 cannabinoid receptors (CB1R) decreases GABA and glutamate release in cortical and subcortical regions, with complex outcomes on cortical network activity. To date there have been few attempts to disentangle the region- and cell-specific mechanisms underlying the effects of cannabinoids on cortical network activity in vivo. Here we addressed this issue by combining in vivo electrophysiological recordings with local and systemic pharmacological manipulations in conditional mutant mice lacking CB1R expression in different neuronal populations. First we report that cannabinoids induce hypersynchronous thalamocortical oscillations while decreasing the amplitude of faster cortical oscillations. Then we demonstrate that CB1R at striatonigral synapses (basal ganglia direct pathway) mediate the thalamocortical hypersynchrony, whereas activation of CB1R expressed in cortical glutamatergic neurons decreases cortical synchrony. Finally we show that activation of CB1 expressed in cortical glutamatergic neurons limits the cannabinoid-induced thalamocortical hypersynchrony. By reporting that CB1R activations in cortical and subcortical regions have contrasting effects on cortical synchrony, our study bridges the gap between cellular and in vivo network effects of cannabinoids. Incidentally, the thalamocortical hypersynchrony we report suggests a potential mechanism to explain the sensory "high" experienced during recreational consumption of marijuana.

Topics: Animals; Cannabinoids; Cerebral Cortex; Corpus Striatum; Cortical Synchronization; Cyclohexanols; Electromyography; GABAergic Neurons; Glutamic Acid; Mice; Mice, Mutant Strains; Nerve Net; Piperidines; Pyrazoles; Receptor, Cannabinoid, CB1; Statistics, Nonparametric; Substantia Nigra; Thalamus

This study examined the role of endocannabinoid signaling in stress-induced reinstatement of cocaine seeking and explored the interaction between noradrenergic and endocannabinergic systems in the process. A well-validated preclinical model for human relapse, the rodent conditioned place preference assay, was used. Cocaine-induced place preference was established in C57BL/6 mice using injections of 15 mg/kg cocaine. Following extinction of preference for the cocaine-paired environment, reinstatement of place preference was determined following 6 min of swim stress or cocaine injection (15 mg/kg, i.p.). The role of endocannabinoid signaling was studied using the cannabinoid antagonist AM-251 (3 mg/kg, i.p.). Another cohort of mice was tested for reinstatement following administration of the cannabinoid agonist CP 55,940 (10, 20, or 40 μg/kg, i.p.). The alpha-2 adrenergic antagonist BRL-44408 (5 mg/kg, i.p.) with or without CP 55,940 (20 μg/kg) was administered to a third group of mice. We found that: (1) AM-251 blocked forced swim-induced, but not cocaine-induced, reinstatement of cocaine-seeking behavior; (2) the cannabinoid agonist CP 55,940 did not reinstate cocaine-seeking behavior when administered alone but did synergize with a non-reinstating dose of the alpha-2 adrenergic antagonist BRL-44408 to cause reinstatement. These results are consistent with the hypothesis that stress exposure triggers the endogenous activation of CB1 receptors and that activation of the endocannabinoid system is required for the stress-induced relapse of the mice to cocaine seeking. Further, the data suggest that the endocannabinoid system interacts with noradrenergic mechanisms to influence stress-induced reinstatement of cocaine-seeking behavior.

Topics: Adrenergic alpha-Antagonists; Animals; Behavior, Addictive; Cannabinoids; Cocaine; Conditioning, Psychological; Cyclohexanols; Dopamine Uptake Inhibitors; Drug-Seeking Behavior; Extinction, Psychological; Imidazoles; Isoindoles; Male; Mice; Piperidines; Pyrazoles; Receptor, Cannabinoid, CB1; Self Administration; Stress, Psychological

The prototypic cannabinoid type 1 (CB₁) receptor antagonist/inverse agonist, rimonabant, is comprised of a pyrazole core surrounded by a carboxyamide with terminal piperidine group (3-substituent), a 2,4-dichlorophenyl group (1-substituent), a 4-chlorophenyl group (5-substituent), and a methyl group (4-substituent). Previous structure-activity relationship (SAR) analysis has suggested that the 3-position may be involved in receptor recognition and agonist activity. The goal of the present study was to develop CB₁-selective compounds and explore further the SAR of 3-substitution on the rimonabant template. 3-Substituted analogs with benzyl and alkyl amino, dihydrooxazole, and oxazole moieties were synthesized and evaluated in vitro and in vivo. Several notable patterns emerged. First, most of the analogs exhibited CB₁ selectivity, with many lacking affinity for the CB₂ receptor. Affinity tended to be better when [³H]5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide (SR141716), rather than [³H](-)-cis-3-[2-hydroxy-4(1,1-dimethyl-heptyl)phenyl]-trans-4-(3-hydroxy-propyl)cyclohexanol (CP55,940), was used as the binding radioligand. Second, many of the analogs produced an agonist-like profile of effects in mice (i.e., suppression of activity, antinociception, hypothermia, and immobility); however, their potencies were not well correlated with their CB₁ binding affinities. Further assessment of selected analogs showed that none were effective antagonists of the effects of Δ⁹-tetrahydrocannabinol in mice, their agonist-like effects were not blocked by rimonabant, they were active in vivo in CB₁⁻/⁻ mice, and they failed to stimulate guanosine-5'-O-(3-[³⁵S]thio)-triphosphate binding. Several analogs were inverse agonists in the latter assay. Together, these results suggest that this series of 3-substituted pyrazole analogs represent a novel class of CB₁-selective cannabinoids that produce agonist-like effects in mice through a non-CB₁, non-CB₂ mechanism.

Topics: Animals; Binding, Competitive; Body Temperature; Camphanes; Cell Membrane; CHO Cells; Cricetinae; Cyclohexanols; Dronabinol; Drug Inverse Agonism; Female; Freezing Reaction, Cataleptic; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Male; Mice; Mice, Inbred C57BL; Mice, Inbred ICR; Mice, Knockout; Motor Activity; Pain Threshold; Piperidines; Pyrazoles; Radioligand Assay; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Rimonabant; Structure-Activity Relationship; Transfection

The purpose of this study was to determine whether sex differences in cannabinoid (CB)-induced antinociception and motoric effects can be attributed to differential activation of CB(1) or CB(2) receptors. Rats were injected intraperitoneally with vehicle, rimonabant [5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide (SR141716A), a putative CB(1) receptor-selective antagonist; 0.1-10 mg/kg] or 5-(4-chloro-3-methylphenyl)-1-[(4-methylphenyl)methyl]-N-[(1S,2S,4R)-1,3,3-trimethylbicyclo[2.2.1]hept-2-yl]-1H-pyrazole-3-carboxamide (SR144528) (a putative CB(2) receptor-selective antagonist; 1.0-10 mg/kg). Thirty minutes later, Δ(9)-tetrahydrocannabinol (THC; 1.25-40 mg/kg) or 5-(1,1-dimethylheptyl)-2-[5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]phenol (CP55,940) (0.05-1.6 mg/kg) was injected. Paw pressure and tail withdrawal antinociception, locomotor activity, and catalepsy were measured. Rimonabant dose-dependently antagonized THC and CP55,940 in each test, but was up to 10 times more potent in female than male rats on the nociceptive tests; estimates of rimonabant affinity (apparent pK(B)) for the CB(1) receptor were approximately 0.5 to 1 mol/kg higher in female than male rats. SR144528 partially antagonized THC-induced tail withdrawal antinociception and locomotor activity in females, but this antagonism was not dose-dependent or consistent; no SR144528 antagonism was observed in either sex tested with CP55,940. Neither the time course of rimonabant antagonism nor the plasma levels of rimonabant differed between the sexes. Rimonabant and SR144528 did not antagonize morphine-induced antinociception, and naloxone did not antagonize THC-induced antinociception in either sex. These results suggest that THC produces acute antinociceptive and motoric effects via activation of CB(1), and perhaps under some conditions, CB(2) receptors, in female rats, whereas THC acts primarily at CB(1) receptors in male rats. Higher apparent pK(B) for rimonabant in female rats suggests that cannabinoid drugs bind with greater affinity to CB(1) receptors in female than male rats, probably contributing to greater antinociceptive effects observed in female compared with male rats.

Topics: Analgesics; Animals; Camphanes; Cyclohexanols; Dose-Response Relationship, Drug; Dronabinol; Female; Male; Morphine; Motor Activity; Naloxone; Piperidines; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Rimonabant; Sex Characteristics

Endocannabinoids (eCBs) regulate neuronal activity in the dorso-lateral striatum (DLS), a brain region that is involved in habitual behaviors. How synaptic eCB signaling contributes to habitual behaviors under physiological and pathological conditions remains unclear. Using a mouse model of cannabinoid tolerance, we found that persistent activation of the eCB pathway impaired eCB-mediated long-term depression (LTD) and synaptic depotentiation in the DLS. The loss of eCB LTD, occurring preferentially at cortical connections to striatopallidal neurons, was associated with a shift in behavioral control from goal-directed action to habitual responding. eCB LTD and behavioral alterations were rescued by in vivo modulation of small-conductance calcium activated potassium channel (SK channel) activity in the DLS, which potentiates eCB signaling. Our results reveal a direct relationship between drug tolerance and changes in control of instrumental performance by establishing a central role for eCB LTD in habit expression. In addition, SK channels emerge as molecular targets to fine tune the eCB pathway under pathological conditions.

Topics: Animals; Apamin; Benzamides; Biophysics; Cannabinoids; Carbamates; Conditioning, Operant; Corpus Striatum; Cyclohexanols; Dose-Response Relationship, Drug; Dronabinol; Drug Tolerance; Electric Stimulation; Enzyme Inhibitors; Excitatory Postsynaptic Potentials; Guanosine 5'-O-(3-Thiotriphosphate); Habits; Long-Term Synaptic Depression; Male; Mice; Mice, Inbred C57BL; Motor Activity; Okadaic Acid; Patch-Clamp Techniques; Piperidines; Protein Binding; Pyrazoles; Rimonabant; Small-Conductance Calcium-Activated Potassium Channels; Sodium Channel Blockers; Tritium

The cannabinoid receptor 1 (CB1), a member of the class A G protein-coupled receptor family, is expressed in brain tissue where agonist stimulation primarily activates the pertussis toxin-sensitive inhibitory G protein (G(i)). Ligands such as CP55940 ((1R,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3- hydroxypropyl)cyclohexan-1-ol) and Δ(9)-tetrahydrocannabinol are orthosteric agonists for the receptor, bind the conventional binding pocket, and trigger G(i)-mediated effects including inhibition of adenylate cyclase. ORG27569 (5-chloro-3-ethyl-1H-indole-2-carboxylic acid [2-(4-piperidin-1-yl-phenyl)ethyl]amide) has been identified as an allosteric modulator that displays positive cooperativity for CP55940 binding to CB1 yet acts as an antagonist of G protein coupling. To examine this apparent conundrum, we used the wild-type CB1 and two mutants, T210A and T210I (D'Antona, A. M., Ahn, K. H., and Kendall, D. A. (2006) Biochemistry 45, 5606-5617), which collectively cover a spectrum of receptor states from inactive to partially active to more fully constitutively active. Using these receptors, we demonstrated that ORG27569 induces a CB1 receptor state that is characterized by enhanced agonist affinity and decreased inverse agonist affinity consistent with an active conformation. Also consistent with this conformation, the impact of ORG27569 binding was most dramatic on the inactive T210A receptor and less pronounced on the already active T210I receptor. Although ORG27569 antagonized CP55940-induced guanosine 5'-3-O-(thio)triphosphate binding, which is indicative of G protein coupling inhibition in a concentration-dependent manner, the ORG27569-induced conformational change of the CB1 receptor led to cellular internalization and downstream activation of ERK signaling, providing the first case of allosteric ligand-biased signaling via CB1. ORG27569-induced ERK phosphorylation persisted even after pertussis toxin treatment to abrogate G(i) and occurs in HEK293 and neuronal cells.

Topics: Allosteric Regulation; Animals; Cyclohexanols; Drug Synergism; Endocytosis; Enzyme Activation; GTP-Binding Protein alpha Subunits, Gi-Go; Guanosine 5'-O-(3-Thiotriphosphate); HEK293 Cells; Hippocampus; Humans; Indoles; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; Neurons; Pertussis Toxin; Phosphorylation; Piperidines; Protein Binding; Protein Structure, Secondary; Pyrazoles; Rats; Receptor, Cannabinoid, CB1; Rimonabant

The extent to which behavioural effects vary as a function of CB₁ receptor agonist efficacy is not clear. These studies tested the hypothesis that cannabinoid tolerance and cross-tolerance depend upon the CB₁ agonist efficacy of drugs to which tolerance/cross-tolerance develops.. Sensitivity to cannabinoids, including the cannabinoid antagonist rimonabant, low efficacy agonist Δ⁹-tetrahydrocannabinol (Δ⁹-THC), and high efficacy agonists CP 55940 and WIN 55212-2, was determined before and after chronic Δ⁹-THC treatment in rhesus monkeys. Two measures of behavioural effect were assessed: effects of drugs to decrease fixed ratio responding for food presentation and stimulus-shock termination and discriminative stimulus effects in monkeys discriminating Δ⁹-THC (0.1 mg·kg⁻¹, i.v.).. Δ⁹-THC decreased responding for both food presentation and stimulus-shock termination; these effects were antagonized by the CB₁ antagonist rimonabant. Chronic Δ⁹-THC (1 mg·kg⁻¹ per 12 h, s.c.) resulted in tolerance to the rate-decreasing effects of Δ⁹-THC and cross-tolerance to CP 55940 and WIN 55212-2; however, cross-tolerance was less than tolerance. Chronic Δ⁹-THC increased sensitivity to rimonabant without changing sensitivity to the non-cannabinoids midazolam and ketamine. In monkeys discriminating Δ⁹-THC (0.1 mg·kg⁻¹, i.v.), both CP 55940 and WIN 55212-2 produced high levels of drug-lever responding. Chronic Δ⁹-THC (1 mg·kg⁻¹ per day, s.c.) decreased sensitivity to Δ⁹-THC without producing cross-tolerance to CP 55940 or WIN 55212-2.. In Δ⁹-THC-treated monkeys, the magnitude of tolerance and cross-tolerance to other CB₁ receptor agonists varied inversely with agonist efficacy, suggesting that CB₁ agonist efficacy is an important determinant of behavioural effects.

Topics: Animals; Behavior, Animal; Benzoxazines; Cannabinoids; Cyclohexanols; Discrimination Learning; Dose-Response Relationship, Drug; Dronabinol; Drug Tolerance; Female; Macaca mulatta; Male; Morpholines; Naphthalenes; Piperidines; Pyrazoles; Receptor, Cannabinoid, CB1; Rimonabant

Ischaemic insult results in short-term changes in cannabinoid-1 (CB(1)) receptor expression in the brain, but it is not known whether long-term changes occur, which could potentially mean a change in the intrinsic ability of the brain to withstand new ischaemic episodes. In this study, we have investigated the expression and functionality of CB(1) receptors in coronal brain slices obtained from ovariectomised female rats 46days after middle cerebral artery occlusion (MCAO). The animals were treated with either 17ß-oestradiol or placebo pellets 6h after MCAO and thereafter housed either in isolated or enriched environments. [(3)H]CP55,940 autoradiography indicated no significant effect of 17ß-oestradiol treatment or housing environment upon CB(1) receptor densities. There was, however, a modest but significant decrease in the CB(1) receptor density on the ipsilateral side relative to the contralateral side in the frontal cortex, parietal cortex, CA1-CA3 regions of the hippocampus, thalamus and hypothalamus. CB(1) receptor functionality was assessed by measurement of basal and CP55,940-stimulated [(35)S]GTPγS autoradiography. In the frontal cortex, parietal cortex, CA1-CA3 regions of the hippocampus and dentate gyrus, a robust stimulation, blocked by the CB(1) receptor inverse agonist AM251, was seen. There were no significant changes in the response to CP55,940 with respect either to the 17ß-oestradiol treatment, housing environment or MCAO. Our results reveal that although there are modest long-term decreases in ipsilateral CB(1) receptor densities following MCAO in female rats, these decreases do not result in a functional CB(1) receptor deficit.

Topics: Animals; Autoradiography; Brain; Brain Ischemia; Cyclohexanols; Disease Models, Animal; Estradiol; Estrogens; Female; Functional Laterality; Gene Expression Regulation; Guanosine 5'-O-(3-Thiotriphosphate); Infarction, Middle Cerebral Artery; Piperidines; Protein Binding; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Sulfur Isotopes; Time Factors; Tritium

Many G protein-coupled receptors internalize following agonist binding. The studies were designed to identify novel means to effectively quantify this process using the orexin OX(1) receptor and the cannabinoid CB(1) receptor as exemplars.. The human OX(1) and CB(1) receptors were modified to incorporate both epitope tags and variants (SNAP and CLIP) of the enzyme O(6)-alkylguanine-DNA-alkyltransferase within their extracellular, N-terminal domain. Cells able to regulate expression of differing amounts of these constructs upon addition of an antibiotic were developed and analysed.. Cell surface forms of each receptor construct were detected by both antibody recognition of the epitope tags and covalent binding of fluorophores to the O(6)-alkylguanine-DNA-alkyltransferase variants. Receptor internalization in response to agonists but not antagonists could be monitored by each approach but sensitivity was up to six- to 10-fold greater than other approaches when employing a novel, time-resolved fluorescence probe for the SNAP tag. Sensitivity was not enhanced, however, for the CLIP tag, possibly due to higher levels of nonspecific binding.. These studies demonstrate that highly sensitive and quantitative assays that monitor cell surface CB(1) and OX(1) receptors and their internalization by agonists can be developed based on introduction of variants of O(6)-alkylguanine-DNA-alkyltransferase into the N-terminal domain of the receptor. This should be equally suitable for other G protein-coupled receptors.

Topics: Alkyl and Aryl Transferases; Benzoxazoles; Cell Line; Cell Membrane; Cloning, Molecular; Cyclohexanols; Extracellular Signal-Regulated MAP Kinases; Humans; Intracellular Signaling Peptides and Proteins; Ligands; Naphthyridines; Neuropeptides; Orexin Receptors; Orexins; Phenylurea Compounds; Phosphorylation; Piperidines; Plasmids; Pyrazoles; Pyrrolidines; Receptor, Cannabinoid, CB1; Receptors, G-Protein-Coupled; Receptors, Neuropeptide; Rimonabant; Thiazoles; Urea

The inflammatory response plays an important role in the pathogenesis of many diseases in the central nervous system. Cannabinoids exhibit diverse pharmacological actions including anti-inflammatory activity. In this study, we tried to elucidate possible effects of cannabinoids on lipopolysaccharide (LPS)-induced expression of inflammatory cytokine mRNAs in rat cerebellar granule cells.. Inhibitory effects of cannabinoids on cytokine induction in cerebellar granule cells were determined by RT-PCR method.. In these cells, both mRNA and protein of cannabinoid receptor 1 (CB(1) ), but not CB(2) , were expressed. LPS (1 µg/ml) produced a marked increase in the induction of inflammatory cytokines, including interleukin-1β, interleukin-6 and tumour necrosis factor-α. CP55940, a synthetic cannabinoid analogue, concentration-dependently inhibited inflammatory cytokine expression induced by LPS. On the other hand, the endocannabinoids 2-arachidonoylglycerol and anandamide were not able to inhibit this inflammatory response. Notably, a CB(1) /CB(2) antagonist NESS0327 (3 µm) did not reverse the inhibition of cytokine mRNA expression induced by CP55940. GPR55, a putative novel cannabinoid receptor, mRNA was also expressed in cerebellar granule cells. Although it has been suggested that G(q) associates with GPR55, cannabinoids including CP55940 did not promote phosphoinositide hydrolysis and consequent elevation of intracellular Ca([2+]) concentration. Furthermore, a putative GPR55 antagonist, cannabidiol, also showed a similar inhibitory effect to that of CP55940.. These results suggest that the synthetic cannabinoid CP55940 negatively modulates cytokine mRNA expression in cerebellar granule cells by a CB and GPR55 receptor-independent mechanism.

Topics: Animals; Anti-Inflammatory Agents; Arachidonic Acids; Calcium; Cannabidiol; Cannabinoid Receptor Antagonists; Cannabinoid Receptor Modulators; Cannabinoids; Cerebellum; Cyclohexanols; Cytokines; Dose-Response Relationship, Drug; Endocannabinoids; Glycerides; Inflammation; Lipopolysaccharides; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Receptors, Cannabinoid; Receptors, G-Protein-Coupled; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Δ(9)-Tetrahydrocannabinol (THC) discrimination in rodents is a behavioral assay that has been used to probe differences among classes of cannabinoids in rats. The purpose of this study was to determine whether traditional and anandamide-like cannabinoids were distinguishable in cannabinoid discrimination procedures in mice. Male mice were trained to discriminate 30 mg/kg THC or 70 mg/kg methanandamide from vehicle in a two-lever milk-reinforced drug discrimination procedure. After acquisition, agonist tests with THC, methanandamide, CP 55940, and anandamide were conducted, as were antagonism tests with rimonabant. Substitution (agonism) and antagonism tests were also carried out in female mice trained to discriminate THC. THC and CP 55940 fully substituted in THC-trained mice of both sexes. Further, THC substitution was rimonabant reversible. In contrast, mice injected with methanandamide or anandamide failed to respond substantially on the THC lever, even up to doses that decreased overall responding. In methanandamide-trained mice, methanandamide fully generalized to the methanandamide training dose. Rimonabant did not reverse this generalization. Although THC, CP 55940, and anandamide also increased responding on the methanandamide lever, the magnitude of substitution was less than for methanandamide. These results suggest incomplete overlap in the underlying mechanisms mediating endocannabinoid pharmacology and marijuana intoxication. Further, they suggest that methanandamide discrimination may involve a non-CB(1) receptor mechanism that is particularly prominent at higher doses.

Topics: Animals; Arachidonic Acids; Cyclohexanols; Discrimination Learning; Dose-Response Relationship, Drug; Dronabinol; Female; Generalization, Stimulus; Male; Mice; Mice, Inbred C57BL; Piperidines; Psychotropic Drugs; Pyrazoles; Receptor, Cannabinoid, CB1; Rimonabant; Sex Factors

This research examines the in vitro interaction of phthalate diesters and monoesters with the G protein-coupled cannabinoid 1 (CB(1)) receptor, a presynaptic complex involved in the regulation of synaptic activity in mammalian brain. The diesters, n-butylbenzylphthalate (nBBP), di-n-hexylphthalate (DnHP), di-n-butylphthalate (DnBP), di-2-ethylhexylphthalate (DEHP), di-isooctylphthalate (DiOP) and di-n-octylphthalate (DnOP) inhibited the specific binding of the CB(1) receptor agonist [(3)H]CP-55940 to mouse whole brain membranes at micromolar concentrations (IC(50)s: nBBP 27.4 μM; DnHP 33.9 μM; DnBP 45.9 μM; DEHP 47.4 μM; DiOP 55.4 μM; DnOP 75.2 μM). DnHP, DnBP and nBBP achieved full (or close to full) blockade of [(3)H]CP-55940 binding, whereas DEHP, DiOP and DnOP produced partial (55-70%) inhibition. Binding experiments with phenylmethane-sulfonylfluoride (PMSF) indicated that the ester linkages of nBBP and DnBP remain intact during assay. The monoesters mono-2-ethylhexylphthalate (M2EHP) and mono-isohexylphthalate (MiHP) failed to reach IC(50) at 150 μM and mono-n-butylphthalate (MnBP) was inactive. Inhibitory potencies in the [(3)H]CP-55940 binding assay were positively correlated with inhibition of CB(1) receptor agonist-stimulated binding of [(35)S]GTPγS to the G protein, demonstrating that phthalates cause functional impairment of this complex. DnBP, nBBP and DEHP also inhibited binding of [(3)H]SR141716A, whereas inhibition with MiHP was comparatively weak and MnBP had no effect. Equilibrium binding experiments with [(3)H]SR141716A showed that phthalates reduce the B(max) of radioligand without changing its K(d). DnBP and nBBP also rapidly enhanced the dissociation of [(3)H]SR141716A. Our data are consistent with an allosteric mechanism for inhibition, with phthalates acting as relatively low affinity antagonists of CB(1) receptors and cannabinoid agonist-dependent activation of the G-protein. Further studies are warranted, since some phthalate esters may have potential to modify CB(1) receptor-dependent behavioral and physiological outcomes in the whole animal.

Topics: Animals; Benzoxazines; Cyclohexanols; Guanosine 5'-O-(3-Thiotriphosphate); In Vitro Techniques; Kinetics; Male; Mice; Morpholines; Naphthalenes; Phthalic Acids; Piperidines; Protein Binding; Pyrazoles; Receptor, Cannabinoid, CB1; Receptors, G-Protein-Coupled; Rimonabant

The 'cannabinoid hypothesis' of schizophrenia tulates that over-activity of the endocannabinoid system might contribute to the aetiology of schizophrenia. In keeping with this hypothesis, increased expression of CB1 receptors, elevation of the endocannabinoid anandamide (AEA) and cannabinoid-induced cognitive changes have been reported in animal models of schizophrenia and psychotic patients. In this study we measured brain endocannabinoid levels and [35S]GTPgammaS binding stimulated by the CB receptor agonist CP55,940 in rats undergoing withdrawal from subchronic administration of phencyclidine (PCP), a well-established pharmacological model of schizophrenia. We also investigated whether systemic application of the fatty-acid amide hydrolase (FAAH) inhibitor URB597 or CB1 receptor blockade by AM251 affected the following PCP-induced behavioural deficits reminiscent of schizophrenia-like symptoms: (1) working-memory impairment (cognitive deficit), (2) social withdrawal (negative symptom), and (3) hyperactivity in response to d-amphetamine challenge (positive symptoms). PCP-treated rats showed increased endocannabinoid levels in the nucleus accumbens and ventral tegmental area, whereas CB1 receptor expression and CP55,940-stimulated [35S]GTPgammaS binding were unaltered. URB597 reversed the PCP-induced social withdrawal but caused social withdrawal and working-memory deficits in saline-treated rats that were comparable to those observed after PCP treatment. Administration of AM251 ameliorated the working-memory deficit in PCP-treated rats, but impaired working memory in saline-injected controls. Taken together, these results suggest that FAAH inhibition may improve negative symptoms in PCP-treated rats but produce deleterious effects in untreated animals, possibly by disturbing endocannabinoid tone. A similar pattern (beneficial for schizophrenia-related cognitive deficits, but detrimental under normal conditions) accompanies CB1 receptor blockade.

Topics: Amidohydrolases; Amphetamine; Animals; Autoradiography; Behavior, Animal; Benzamides; Cannabinoid Receptor Modulators; Carbamates; Central Nervous System Stimulants; Cyclohexanols; Drug Administration Schedule; Drug Interactions; Enzyme Inhibitors; Guanosine 5'-O-(3-Thiotriphosphate); Hallucinogens; Interpersonal Relations; Male; Memory, Short-Term; Motor Activity; Neuropsychological Tests; Phencyclidine; Piperidines; Protein Binding; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Statistics, Nonparametric; Sulfur Isotopes; Time Factors

The endocannabinoid system plays a crucial role in the pathophysiology of obesity. However, the clinical use of cannabinoid antagonists has been recently stopped because of its central side-effects. The aim of this study was to compare the effects of a chronic treatment with the CB(1) cannabinoid antagonist rimonabant or the CB(1) inverse agonist taranabant in diet-induced obese female rats to clarify the biological consequences of CB(1) blockade at central and peripheral levels. As expected, chronic treatment with rimonabant and taranabant reduced body weight and fat content. Interestingly, a decrease in the number of CB(1) receptors and its functional activity was observed in all the brain areas investigated after chronic taranabant treatment in both lean and obese rats. In contrast, chronic treatment with rimonabant did not modify the density of CB(1) cannabinoid receptor binding, and decreased its functional activity to a lower degree than taranabant. Six weeks after rimonabant and taranabant withdrawal, CB(1) receptor density and activity recovered to basal levels. These results reveal differential adaptive changes in CB(1) cannabinoid receptors after chronic treatment with rimonabant and taranabant that could be related to the central side-effects reported with the use of these cannabinoid antagonists.

Topics: Amides; Analysis of Variance; Animals; Autoradiography; Benzoxazines; Body Weight; Brain; Cyclohexanols; Diet Fads; Disease Models, Animal; Eating; Female; Guanosine 5'-O-(3-Thiotriphosphate); International Cooperation; Morpholines; Naphthalenes; Obesity; Piperidines; Protein Binding; Pyrazoles; Pyridines; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Rimonabant; Sulfur Isotopes; Time Factors; Tomography Scanners, X-Ray Computed; Tritium; Whole Body Imaging

Bilateral olfactory bulbectomy (OBX) in rodents produces behavioral and neurochemical changes associated clinically with depression and schizophrenia. Most notably, OBX induces hyperlocomotion in response to the stress of exposure to a novel environment. We examined the role of the endocannabinoid system in regulating this locomotor response in OBX and sham-operated rats. In our study, OBX-induced hyperactivity was restricted to the first 3 min of the open field test, demonstrating the presence of novelty (0-3 min) and habituation (3-30 min) phases of the open field locomotor response. Levels of the endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide were decreased in the ventral striatum, a brain region deafferented by OBX, whereas cannabinoid receptor densities were unaltered. In sham-operated rats, 2-AG levels in the ventral striatum were negatively correlated with distance traveled during the novelty phase. Thus, low levels of 2-AG are reflected in a hyperactive open field response. This correlation was not observed in OBX rats. Conversely, 2-AG levels in endocannabinoid-compromised OBX rats correlated with distance traveled during the habituation phase. In OBX rats, pharmacological blockade of cannabinoid CB(1) receptors with either AM251 (1 mg kg(-1) i.p.) or rimonabant (1 mg kg(-1) i.p.) increased distance traveled during the habituation phase. Thus, blockade of endocannabinoid signaling impairs habituation of the hyperlocomotor response in OBX, but not sham-operated, rats. By contrast, in sham-operated rats, effects of CB(1) antagonism were restricted to the novelty phase. These findings suggest that dysregulation in the endocannabinoid system, and 2-AG in particular, is implicated in the hyperactive locomotor response induced by OBX. Our studies suggest that drugs that enhance 2-AG signaling, such as 2-AG degradation inhibitors, might be useful in human brain disorders modeled by OBX.

Topics: Animals; Arachidonic Acids; Autoradiography; Cannabinoid Receptor Modulators; Chromatography, High Pressure Liquid; Cyclohexanols; Densitometry; Dopamine; Endocannabinoids; Environment; Glycerides; Limbic System; Lipid Metabolism; Male; Mass Spectrometry; Motor Activity; Olfactory Bulb; Piperidines; Pyrazoles; Radiopharmaceuticals; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Rimonabant; Signal Transduction

Cannabinoid 1 (CB1) receptors have the ability to change conformation between active (R*) and inactive (R) receptor states. Herein, we further characterize these receptor states using series of saturation radioligand binding studies and their differential displacement binding by various CB1 receptor ligands. Binding experiments were carried out in naïve rat/dog whole brain membranes using radioligands [(3)H]CP55,940 (for R* state) & [(3)H]SR141716A (both R* and R states) and various agonist, antagonist & inverse agonist ligands at CB1 receptors. In the saturation binding experiments, of the total number of CB1 receptor binding sites (R* + R) in the rat and dog whole brain membranes, only about 18.3 and 11.6% were in the active (R*) state recognized by [(3)H]CP55,940, respectively. In the competitive binding studies, all the CB1 receptor agonists investigated had significantly very high affinity for the active R* state recognized by [(3)H]CP55,940 and lower affinity for the inactive R state mainly recognized by [(3)H]SR141716A in the presence of a non-hydrolyzable analogue of GTP [Gpp(NH)p]. In contrast, various CB1 receptor antagonists/inverse agonists had similar nanomolar affinities at both [(3)H]CP55,940 and [(3)H]SR141716A recognized binding states. These results clearly characterize the significant differences between the active R* and inactive R binding states of CB1 receptors in naive rat and dog brain. In addition, these results also demonstrates that the CB1 agonists and antagonists/inverse agonists can be differentiated by their relative affinities at active (R*) and inactive (R) binding states of the CB1 receptor.

Topics: Animals; Binding, Competitive; Brain; Cyclohexanols; Dogs; Drug Inverse Agonism; In Vitro Techniques; Ligands; Piperidines; Protein Binding; Pyrazoles; Radioligand Assay; Rats; Receptor, Cannabinoid, CB1; Rimonabant

Marijuana-dependent individuals report using marijuana to alleviate withdrawal, suggesting that pharmacotherapy of marijuana withdrawal could promote abstinence. To identify potential pharmacotherapies for marijuana withdrawal, this study first characterized rimonabant-induced Delta(9)-tetrahydrocannabinol (Delta(9)-THC) withdrawal in rhesus monkeys by using drug discrimination and directly observable signs. Second, drugs were examined for their capacity to modify cannabinoid withdrawal. Monkeys receiving chronic Delta(9)-THC (1 mg/kg/12 h s.c.) discriminated the cannabinoid antagonist rimonabant (1 mg/kg i.v.) under a fixed ratio schedule of stimulus-shock termination. The discriminative stimulus effects of rimonabant were dose-dependent (ED(50) = 0.25 mg/kg) and accompanied by head shaking. In the absence of chronic Delta(9)-THC treatment (i.e., in nondependent monkeys), a larger dose (3.2 mg/kg) of rimonabant produced head shaking and tachycardia. Temporary discontinuation of Delta(9)-THC treatment resulted in increased responding on the rimonabant lever, head shaking, and activity during the dark cycle. The rimonabant discriminative stimulus was attenuated fully by Delta(9)-THC (at doses larger than mg/kg/12 h) and the cannabinoid agonist CP 55940 [5-(1,1-dimethylheptyl)-2-[5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]phenol], and partially by the cannabinoid agonist WIN 55212-2 [(R)-(+)-[2, 3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate] and the alpha(2)-adrenergic agonist clonidine. In contrast, a benzodiazepine (diazepam) and monoamine agonist (cocaine) did not attenuate the rimonabant discriminative stimulus. Head shaking was attenuated by all test compounds. These results show that the discriminative stimulus effects of rimonabant in Delta(9)-THC-treated monkeys are a more pharmacologically selective measure of cannabinoid withdrawal than rimonabant-induced head shaking. These results suggest that cannabinoid and noncannabinoid (alpha(2)-adrenergic) agonists are potentially useful therapeutics for marijuana dependence inasmuch as they attenuate the subjective experience of Delta(9)-THC withdrawal.

Topics: Adrenergic Agonists; Animals; Behavior, Animal; Benzoxazines; Cannabinoid Receptor Agonists; Cannabinoid Receptor Antagonists; Clonidine; Cyclohexanols; Discrimination, Psychological; Dronabinol; Female; Heart Rate; Macaca mulatta; Male; Morpholines; Motor Activity; Naphthalenes; Piperidines; Pyrazoles; Rimonabant; Substance Withdrawal Syndrome

Hemopressin is a short, nine amino acid peptide (H-Pro-Val-Asn-Phe-Lys-Leu-Leu-Ser-His-OH) isolated from rat brain that behaves as an inverse agonist at the cannabinoid receptor CB(1), and is shown here to inhibit agonist-induced receptor internalization in a heterologous cell model. Since this peptide occurs naturally in the rodent brain, we determined its effect on appetite, an established central target of cannabinoid signaling. Hemopressin dose-dependently decreases night-time food intake in normal male rats and mice, as well as in obese ob/ob male mice, when administered centrally or systemically, without causing any obvious adverse side effects. The normal, behavioral satiety sequence is maintained in male mice fasted overnight, though refeeding is attenuated. The anorectic effect is absent in CB(1) receptor null mutant male mice, and hemopressin can block CB(1) agonist-induced hyperphagia in male rats, providing strong evidence for antagonism of the CB(1) receptor in vivo. We speculate that hemopressin may act as an endogenous functional antagonist at CB(1) receptors and modulate the activity of appetite pathways in the brain.

Topics: Analysis of Variance; Animals; Behavior, Animal; Benzoxazines; Chlorocebus aethiops; Circadian Rhythm; COS Cells; Cyclohexanols; Dose-Response Relationship, Drug; Drinking Behavior; Dronabinol; Drug Administration Routes; Eating; Food Deprivation; Green Fluorescent Proteins; Hemoglobins; Hyperphagia; Leptin; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Morpholines; Naphthalenes; Peptide Fragments; Piperidines; Protein Transport; Psychotropic Drugs; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Rimonabant; Time Factors; Transfection

Disruption of the substantial re-organization of the brain during adolescence may be induced by persistent abuse of marijuana. The aim of this study was to determine whether adolescent and adult rats exhibit differential adaptation of brain cannabinoid (CB(1)) receptors after repeated exposure to Delta(9)-tetrahydrocannabinol (THC).. Rats of both ages and sexes were dosed with 10 mg kg(-1) THC or vehicle twice daily for 9.5 days. Subsequently, CB(1) receptor function and density were assessed.. In all brain regions, THC treatment produced desensitization and down-regulation of CB(1) receptors. While the magnitude of down-regulation did not differ across groups, greater desensitization was evident in the brains of THC-treated female adolescent rats for most regions. Adolescent females showed greater desensitization than adult females in the prefrontal cortex, hippocampus, periaqueductal gray (PAG) and ventral midbrain. In contrast, adolescent males exhibited less desensitization in the prefrontal cortex, hippocampus and PAG, an effect opposite to that seen in females. With the exception of the PAG, sex differences were seen only in adolescents, with greater desensitization in the prefrontal cortex, striatum, hippocampus, PAG, and ventral midbrain of females.. These results suggest that the brains of adolescent females may be particularly vulnerable to disruption of CB(1) receptor signalling by marijuana abuse. Alternatively, increased desensitization may reflect protective adaptation. Given the extensive re-organization of the brain during adolescence, this disruption has potential long-term consequences for maturation of the endocannabinoid system.

Topics: Age Distribution; Animals; Brain; Cyclohexanols; Dronabinol; Drug Administration Schedule; Drug Tolerance; Female; Male; Piperidines; Protein Binding; Pyrazoles; Rats; Rats, Long-Evans; Receptor, Cannabinoid, CB1; Rimonabant; Sex Characteristics; Sexual Maturation

Drugs used clinically usually have a primary mechanism of action, but additional effects on other biological targets can contribute to their effects. A potentially useful additional target is the endocannabinoid metabolizing enzyme monoacylglycerol lipase (MGL). We have screened a range of drugs for inhibition of MGL and compared the observed potencies using different MGL enzyme assays.. MGL activity was screened using recombinant human MGL (cell lysates and purified enzyme) with 4-nitrophenyl acetate (NPA) as substrate. 2-Oleolyglycerol metabolism by rat cerebellar cytosolic MGL and by recombinant MGL was also investigated.. Among the 96 compounds screened in the NPA assay, troglitazone, CP55,940, N-arachidonoyl dopamine and AM404 inhibited NPA hydrolysis by the lysates with IC(50) values of 1.1, 4.9, 0.78 and 3.1µM, respectively. The potency for troglitazone is in the same range as its primary pharmacological activity, activation of peroxisome proliferator-activated receptor (PPAR) γ. Among PPARγ ligands, the potency order towards human MGL was troglitazone > ciglitazone > rosiglitazone > 15-deoxy-Δ(12,14) -prostaglandin J(2) ≈ CAY 10415 > CAY 10514. In contrast to the time-dependent inhibitor JZL184, the potency of troglitazone was dependent upon the enzyme assay system used. Thus, troglitazone inhibited rat cytosolic 2-oleoylglycerol hydrolysis less potently (IC(50) 41µM) than hydrolysis of NPA by the human MGL lysates.. 'Hits' in screening programmes for MGL inhibitors should be assessed in different MGL assays. Troglitazone may be a useful lead for the design of novel, dual action MGL inhibitors/PPARγ activators.

Topics: Animals; Arachidonic Acids; Benzodioxoles; Cannabinoid Receptor Modulators; Chromans; Cyclohexanols; Dopamine; Enzyme Assays; Glycerides; Humans; Monoacylglycerol Lipases; Nitrophenols; Piperidines; PPAR gamma; Rats; Rats, Sprague-Dawley; Rats, Wistar; Thiazolidinediones; Troglitazone

In the last decade, the neurovascular effects exerted by endocannabinoids (eCBs) have attracted growing interest, because they hold the promise to open new avenues of therapeutic intervention against major causes of death in Western society. Several actions of eCBs are mediated by type-1 (CB₁) or type-2 (CB₂) cannabinoid receptors, yet there is no clear evidence of the presence of these proteins in platelets. To demonstrate that CB₁ and CB₂ are expressed in human platelets, we analyzed their protein level by Western blotting and ELISA, visualized their cellular localization by confocal microscopy, and ascertained their functionality by binding assays. We found that CB₁, and to a lesser extent CB₂, are expressed in highly purified human platelets. Both receptor subtypes were predominantly localized inside the cell, thus explaining why they might remain undetected in preparations of plasma membranes. The identification of authentic CB₁ and CB₂ in human platelets supports the potential exploitation of selective agonists or antagonists of these receptors as novel therapeutics to combat neurovascular disorders. It seems remarkable that some of these substances have been already used in humans to treat disease states.

Topics: Adult; Binding, Competitive; Blood Platelets; Calcium; Camphanes; Cyclic AMP; Cyclohexanols; Drug Interactions; Enzyme-Linked Immunosorbent Assay; Female; Flow Cytometry; Gene Expression; Humans; Inositol 1,4,5-Trisphosphate; Integrin beta3; Male; Piperidines; Platelet Count; Protein Binding; Pyrazoles; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Rimonabant; Tritium; Young Adult

Folic acid (FA) is an essential micronutrient that is particularly important during pregnancy for normal placental and fetal development and growth. The placenta and the fetus become frequently exposed to drugs of abuse such as cannabinoids because of maternal use of these substances. The aim of this study was to investigate the influence of cannabinoids on the uptake of FA by BeWo cells. Acute treatment with anandamide (1-10 micromol/l) caused a 15% decrease in (3)H-FA uptake at pH 7.5. Moreover, tetrahydrocannabinol (THC) (1-10 micromol/l) caused a 30% increase, and AM630 (1 mumol/l) a 15% decrease in this parameter at pH 6.5. Neither the inhibitory effect of anandamide nor the stimulatory effect of THC were changed in the presence of cannabi- noid receptor type 1 or type 2 antagonists (AM251 and AM630, respectively). Chronic treatment (48 h) with THC (100 nmol/l) and AM251 (100 nmol/l) decreased the uptake of (3)H-FA by 20% at pH 7.5, and anandamide (1 micromol/l) and AM630 (10-500 nmol/l) increased it by 30%. Moreover, CP55,940 (10 nmol/l) increased the uptake of (3)H-FA by 30% at pH 6.5. RT-PCR analysis showed that the mRNA levels of the reduced folate transporter 1 increased by 9% after chronic treatment with AM630 (500 nmol/l). The mRNA levels of the proton-coupled folate transporter decreased by 17% and increased by 18% after chronic treatment with THC (0.1 mumol/l) and AM251 (100 nmol/l), respectively. In conclusion, (3)H-FA uptake by BeWo cells is significantly, although not very markedly, changed by several distinct CB receptor agonists and antagonists, both after acute and chronic exposure of the cells. The acute effects of cannabinoid receptor agonists do not seem to be mediated by the cannabinoid receptor, and with a few exceptions the chronic effects do not seem to be related to changes in the expression levels of FA transporters.

Topics: Arachidonic Acids; Carrier Proteins; Cell Culture Techniques; Cell Line, Tumor; Cyclohexanols; Dronabinol; Drug Administration Schedule; Endocannabinoids; Female; Folate Receptors, GPI-Anchored; Folic Acid; Humans; Hydrogen-Ion Concentration; Indoles; Membrane Transport Proteins; Piperidines; Placenta; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Receptors, Cell Surface

A significant number of youths use cigarettes, and more than half of the youths who smoke daily also use illicit drugs. The focus of these studies is on how exposure to nicotine affects subsequent responses to both nicotine and cannabinoids in adolescents compared with adults. We have shown previously that chronic treatment with nicotine produces sensitization to its locomotor-activating effects in female and adult rats but not male adolescent rats. To better understand the effects of nicotine on adolescent and adult rats, rats were injected with nicotine or saline for 7 days and, on day 8, either challenged with delta-9-tetrahydrocannabinol (Delta 9-THC) or the cannabinoid agonist CP 55,940 and tested for locomotor activity, or the brains were removed for quantitative autoradiography studies of the cannabinoid(1) receptor. A separate group of rats was treated with nicotine plus the cannabinoid antagonist AM 251 and then challenged with CP 55,940. In adolescent male rats, nicotine administration led to sensitization to the locomotor-decreasing effects of both Delta 9-THC and CP 55,940, but in adult male rats, the response to either drug was unchanged compared to controls. The effect of nicotine on CP 55,940-mediated locomotor activity was blocked by co-administration of AM 251 with the nicotine. Further, cannabinoid receptor density was increased in the prelimbic prefrontal cortex, ventral tegmental area, and select regions of the hippocampus in adolescent male rats pretreated with nicotine compared to vehicle-treated controls. There were no significant changes in cannabinoid receptor binding, however, in any of the brain regions examined in adult males pretreated with nicotine. The prelimbic prefrontal cortex and the hippocampus have been shown previously to be involved in stimulant reinforcement; thus it is possible that these changes contribute to the unique behavioral effects of chronic nicotine and subsequent drug administration in adolescents compared with adults.

Topics: Adolescent; Aging; Analgesics; Animals; Cannabinoids; Cyclohexanols; Dronabinol; Female; Humans; Male; Motor Activity; Nicotine; Nicotinic Agonists; Piperidines; Psychotropic Drugs; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptors, Cannabinoid

It is firmly established that the hippocampus, a brain region implicated in spatial learning, episodic memory, and consolidation, contains a high concentration of CB(1) receptors. Moreover, systemic and intrahippocampal administration of cannabinoid agonists have been shown to impair hippocampal-dependent memory tasks. However, the degree to which CB(1) receptors in the hippocampus play a specific functional role in the memory disruptive effects of marijuana or its primary psychoactive constituent Delta(9)-tetrahydrocannabinol (Delta(9)-THC) is unknown. This study was designed to determine whether hippocampal CB(1) receptors play a functional role in the memory disruptive effects of systemically administered cannabinoids, using the radial arm maze, a well characterized rodent model of working memory. Male Sprague-Dawley rats were implanted with bilateral cannulae aimed at the CA1 region of the dorsal hippocampus. The CB(1) receptor antagonist, rimonabant, was delivered into the hippocampus before to a systemic injection of either Delta(9)-THC or the potent cannabinoid analog, CP-55,940. Strikingly, intrahippocampal administration of rimonabant completely attenuated the memory disruptive effects of both cannabinoids in the radial arm maze task, but did not affect other pharmacological properties of cannabinoids, as assessed in the tetrad assay (that is, hypomotility, analgesia, catalepsy, and hypothermia). Infusions of rimonabant just dorsal or ventral to the hippocampus did not prevent Delta(9)-THC-induced memory impairment, indicating that its effects on mnemonic function were regionally selective. These findings provide compelling evidence in support of the view that hippocampal CB(1) receptors play a necessary role in the memory disruptive effects of marijuana.

Topics: Analysis of Variance; Animals; Catheterization; Central Nervous System Agents; Cyclohexanols; Dronabinol; Hippocampus; Male; Maze Learning; Memory Disorders; Memory, Short-Term; Microinjections; Piperidines; Psychotropic Drugs; Pyrazoles; Random Allocation; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Rimonabant

Drugs targeting brain kappa-opioid receptors produce profound alterations in mood. In the present study we investigated the possible anxiolytic- and antidepressant-like effects of the kappa-opioid receptor agonist salvinorin A, the main active ingredient of Salvia divinorum, in rats and mice.. Experiments were performed on male Sprague-Dawley rats or male Albino Swiss mice. The anxiolytic-like effects were tested by using the elevated plus maze, in rats. The antidepressant-like effect was estimated through the forced swim (rats) and the tail suspension (mice) test. kappa-Opioid receptor involvement was investigated pretreating animals with the kappa-opioid receptor antagonist, nor-binaltorphimine (1 or 10 mgxkg(-1)), while direct or indirect activity at CB(1) cannabinoid receptors was evaluated with the CB(1) cannabinoid receptor antagonist, N-(piperidin-1-yl) -5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251, 0.5 or 3 mgxkg(-1)), binding to striatal membranes of naïve rats and assay of fatty acid amide hydrolase in prefrontal cortex, hippocampus and amygdala.. Salvinorin A, given s.c. (0.001-1000 microgxkg(-1)), exhibited both anxiolytic- and antidepressant-like effects that were prevented by nor-binaltorphimine or AM251 (0.5 or 3 mgxkg(-1)). Salvinorin A reduced fatty acid amide hydrolase activity in amygdala but had very weak affinity for cannabinoid CB(1) receptors.. The anxiolytic- and antidepressant-like effects of Salvinorin A are mediated by both kappa-opioid and endocannabinoid systems and may partly explain the subjective symptoms reported by recreational users of S. divinorum.

Topics: Amidohydrolases; Animals; Anti-Anxiety Agents; Antidepressive Agents; Behavior, Animal; Binding, Competitive; Brain; Cyclohexanols; Diterpenes, Clerodane; Dose-Response Relationship, Drug; Emotions; Injections, Subcutaneous; Male; Mice; Models, Animal; Motor Activity; Naltrexone; Narcotic Antagonists; Piperidines; Pyrazoles; Pyrrolidines; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Receptors, Opioid, kappa; Salvia; Swimming

The cannabinoid CB(1) receptor antagonist/inverse agonist rimonabant (SR 141716) has been shown to block reinforcing and rewarding effects of nicotine. Research has not investigated whether the cannabinoid system is involved in the interoceptive stimulus effects of nicotine functioning as a conditional stimulus (CS).. We examined the effects of rimonabant and the CB(1/2) receptor agonist, CP 55,940, on responding evoked by a nicotine CS in rats. Additionally, we determined whether CP 55,940 functioned as a CS or a Pavlovian positive drug feature. Pavlovian discrimination training involved intermixed nicotine (0.2 mg base/kg) and saline sessions with intermittent access to water only on nicotine. Antagonism tests with rimonabant (0.1-3 mg/kg) and substitution tests with CP 55,940 (0.003-0.1 mg/kg) followed. An effective dose of CP 55,940 was tested against the nicotine generalization curve. A separate group received CS training with CP 55,940 (0.01 mg/kg). Two other groups were trained using CP 55,940 (0.01 or 0.03 mg/kg) as a positive drug feature in which a brief light CS signaled access to water only on CP 55,940 sessions.. Rimonabant blocked nicotine-evoked responding. CP 55,940 partially substituted for nicotine and enhanced responding to lower nicotine doses. Overall, CP 55,940 did not acquire control of conditioned responding in either Pavlovian drug discrimination task.. The cannabinoid system was involved in the CS effects of nicotine. This finding is counter to the operant drug discrimination research with nicotine as a discriminative stimulus, warranting further research into this possible dissociation.

Topics: Animals; Conditioning, Classical; Cyclohexanols; Discrimination Learning; Drug Interactions; Male; Nicotine; Piperidines; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Rimonabant

Endocannabinoids have a variety of effects by acting through cannabinoid 1 (CB1) receptors located throughout the brain. However, since CB1 receptors are located presynaptically, and because the strength of downstream coupling varies with brain region, expression studies alone do not provide a firm basis for interpreting sites of action. Likewise, to date most functional studies have used high doses of drugs, which can bias results toward non-relevant adverse effects, and which mask more behaviourally-relevant actions. Here we use a low, orexigenic dose of the full CB1 agonist, CP55940, to map responsive brain regions using the complementary techniques of pharmacological-challenge functional magnetic resonance imaging (phMRI) and immediate-early gene activity. Areas of interest demonstrate a drug interaction when the CB1 receptor inverse agonist, rimonabant, is co-administered. This analysis highlights the corticostriatal-hypothalamic pathway, which is central to the motivational drive to eat.

Topics: Animals; Brain; Brain Mapping; Cannabinoids; Central Nervous System Agents; Cyclohexanols; Drug Interactions; Eating; Genes, Immediate-Early; Hyperphagia; Immunohistochemistry; Magnetic Resonance Imaging; Male; Oxygen; Piperidines; Proto-Oncogene Proteins c-fos; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Rimonabant

Considerable preclinical research has demonstrated the efficacy of Delta(9)-tetrahydrocannabinol (Delta(9)-THC), the primary psychoactive constituent of Cannabis sativa, in a wide variety of animal models of pain, but few studies have examined other phytocannabinoids. Indeed, other plant-derived cannabinoids, including cannabidiol (CBD), cannabinol (CBN), and cannabichromene (CBC) elicit antinociceptive effects in some assays. In contrast, tetrahydrocannabivarin (THCV), another component of cannabis, antagonizes the pharmacological effects of Delta(9)-THC. These results suggest that various constituents of this plant may interact in a complex manner to modulate pain. The primary purpose of the present study was to assess the antinociceptive effects of these other prevalent phytocannabinoids in the acetic acid stretching test, a rodent visceral pain model. Of the cannabinoid compounds tested, Delta(9)-THC and CBN bound to the CB(1) receptor and produced antinociceptive effects. The CB(1) receptor antagonist, rimonabant, but not the CB(2) receptor antagonist, SR144528, blocked the antinociceptive effects of both compounds. Although THCV bound to the CB(1) receptor with similar affinity as Delta(9)-THC, it had no effects when administered alone, but antagonized the antinociceptive effects of Delta(9)-THC when both drugs were given in combination. Importantly, the antinociceptive effects of Delta(9)-THC and CBN occurred at lower doses than those necessary to produce locomotor suppression, suggesting motor dysfunction did not account for the decreases in acetic acid-induced abdominal stretching. These data raise the intriguing possibility that other constituents of cannabis can be used to modify the pharmacological effects of Delta(9)-THC by either eliciting antinociceptive effects (i.e., CBN) or antagonizing (i.e., THCV) the actions of Delta(9)-THC.

Topics: Acetic Acid; Analgesics; Animals; Anti-Obesity Agents; Camphanes; Cannabinoids; Cyclohexanols; Dose-Response Relationship, Drug; Dronabinol; Male; Mice; Mice, Inbred ICR; Motor Activity; Pain; Pain Measurement; Piperidines; Pyrazoles; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Rimonabant; RNA, Messenger

The cannabinoid receptor 1 (CB(1)) and CB(2) cannabinoid receptors, associated with drugs of abuse, may provide a means to treat pain, mood, and addiction disorders affecting widespread segments of society. Whether the orphan G-protein coupled receptor GPR55 is also a cannabinoid receptor remains unclear as a result of conflicting pharmacological studies. GPR55 has been reported to be activated by exogenous and endogenous cannabinoid compounds but surprisingly also by the endogenous non-cannabinoid mediator lysophosphatidylinositol (LPI). We examined the effects of a representative panel of cannabinoid ligands and LPI on GPR55 using a beta-arrestin-green fluorescent protein biosensor as a direct readout of agonist-mediated receptor activation. Our data demonstrate that AM251 and SR141716A (rimonabant), which are cannabinoid antagonists, and the lipid LPI, which is not a cannabinoid receptor ligand, are GPR55 agonists. They possess comparable efficacy in inducing beta-arrestin trafficking and, moreover, activate the G-protein-dependent signaling of protein kinase CbetaII. Conversely, the potent synthetic cannabinoid agonist CP55,940 acts as a GPR55 antagonist/partial agonist. CP55,940 blocks GPR55 internalization, the formation of beta-arrestin GPR55 complexes, and the phosphorylation of ERK1/2; CP55,940 produces only a slight amount of protein kinase CbetaII membrane recruitment but does not stimulate membrane remodeling like LPI, AM251, or rimonabant. Our studies provide a paradigm for measuring the responsiveness of GPR55 to a variety of ligand scaffolds comprising cannabinoid and novel compounds and suggest that at best GPR55 is an atypical cannabinoid responder. The activation of GPR55 by rimonabant may be responsible for some of the off-target effects that led to its removal as a potential obesity therapy.

Topics: Analgesics; Arrestins; beta-Arrestins; Cannabinoid Receptor Agonists; Cannabinoids; Cell Line; Cyclohexanols; Humans; Ligands; Lysophospholipids; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Obesity; Phosphorylation; Piperidines; Protein Kinase C; Protein Kinase C beta; Pyrazoles; Receptors, Cannabinoid; Receptors, G-Protein-Coupled; Rimonabant

Brain injuries as well as neurodegenerative diseases, are associated with neuro-inflammation characterized by astroglial and microglial activation and/or proliferation. Recently, we reported that lipopolysaccharide (LPS)-activation of microglia inhibits junctional channels and promotes hemichannels, two connexin43 functions in astrocytes. This opposite regulation is mediated by two pro-inflammatory cytokines, interleukin-1 beta and tumor necrosis factor-alpha, released from activated microglia. Because cannabinoids (CBs) have anti-inflammatory properties and their receptors are expressed by glial cells, we investigated on primary cortical cultures the effects of CB agonists, methanandamide and synthetic CBs on (i) cytokines released from LPS-activated microglia and (ii) connexin43 functions in astrocytes subjected to pro-inflammatory treatments. We observed that CBs inhibited the LPS-induced release of interleukin-1 beta and tumor necrosis factor-alpha from microglia. Moreover, the connexin43 dual regulation evoked by the pro-inflammatory treatments, was prevented by CB treatments. Pharmacological characterizations of CB actions on astrocytic connexin43 channels revealed that these effects were mainly mediated through CB1 receptors activation, although non-CB1/CB2 receptors seemed to mediate the action of the methanandamide. Altogether these data demonstrate that in inflammatory situations CBs exert, through the activation of different sub-types of glial CB receptors, a regulation on two functions of connexin43 channels in astrocytes known to be involved in neuron survival.

Topics: Analgesics; Animals; Animals, Newborn; Arachidonic Acids; Astrocytes; Benzoxazines; Cannabinoids; Cells, Cultured; Cerebral Cortex; Connexin 43; Connexins; Culture Media, Conditioned; Cyclohexanols; Drug Interactions; Enzyme-Linked Immunosorbent Assay; Ethidium; Gene Expression Regulation; Interleukin-1beta; Lipopolysaccharides; Maximum Tolerated Dose; Mice; Microglia; Morpholines; Naphthalenes; Piperidines; Pyrazoles; Rimonabant; Tumor Necrosis Factor-alpha

Recent studies have indicated a role for the endocannabinoid system in ethanol-related behaviors. This study examined the effect of pharmacological activation, blockade, and genetic deletion of the CB(1) receptors on ethanol-drinking behavior in ethanol preferring C57BL/6J (B6) and ethanol nonpreferring DBA/2J (D2) mice. The deletion of CB(1) receptor significantly reduced the ethanol preference. Although the stimulation of the CB(1) receptor by CP-55,940 markedly increased the ethanol preference, this effect was found to be greater in B6 than in D2 mice. The antagonism of CB(1) receptor function by SR141716A led to a significant reduction in voluntary ethanol preference in B6 than D2 mice. A significant lower hypothermic and greater sedative response to acute ethanol administration was observed in both the strains of CB(1) -/- mice than wild-type mice. Interestingly, genetic deletion and pharmacological blockade of the CB(1) receptor produced a marked reduction in severity of handling-induced convulsion in both the strains. The radioligand binding studies revealed significantly higher levels of CB(1) receptor-stimulated G-protein activation in the striatum of B6 compared to D2 mice. Innate differences in the CB(1) receptor function might be one of the contributing factors for higher ethanol drinking behavior. The antagonists of the CB(1) receptor may have therapeutic potential in the treatment of ethanol dependence.

Topics: Alcohol-Induced Disorders, Nervous System; Alcoholism; Analgesics; Animals; Binding, Competitive; Brain; Cyclohexanols; Disease Models, Animal; Genetic Predisposition to Disease; Male; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Mice, Knockout; Piperidines; Pyrazoles; Receptor, Cannabinoid, CB1; Receptors, G-Protein-Coupled; Rimonabant; Species Specificity

Circadian activity rhythms in hamsters are entrained to the daily light:dark cycle by photic information arriving from the retina to the suprachiasmatic nucleus, the site of the master circadian pacemaker in mammals. The effects of light on adjusting the timing of the circadian pacemaker is modified, both positively and negatively, by a variety of transmitter systems, but the effects of endocannabinoids have not been reported. Therefore, in this study we evaluated cannabinoids specific for the cannabinoid type 1 receptor (CB(1)) for their ability to modulate light-induced phase advances in hamster circadian activity rhythms. All compounds were administered intraperitoneally. The CB(1) agonist CP55940 potently inhibited light-induced phase shifts with near 90% inhibition achieved with a dose of 0.125 mg/kg. The inhibitory effect of CP55940 was partially reversed by the CB(1) antagonist LY320135 and completely reversed with 1 mg/kg of the CB(1) antagonist AM 251. Neither LY320135 nor AM 251 had any effect on light-induced phase shifts when administered alone. Further evidence for CB(1) involvement in hamster circadian rhythms was provided by immunohistochemical detection of CB(1) receptors in four separate nuclei comprising the principal components of the hamster circadian system: the suprachiasmatic nucleus, intergeniculate leaflet of the thalamus, and dorsal and median raphe nuclei. Altogether these data indicate that the endocannabinoid system has the capability to modulate circadian rhythms in the hamster and cannabis use should be evaluated for adverse effects on circadian rhythms in humans.

Topics: Analgesics; Animals; Benzofurans; Cannabinoids; Circadian Rhythm; Cricetinae; Cyclohexanols; Dose-Response Relationship, Drug; Drug Interactions; Gene Expression Regulation; Male; Piperidines; Pyrazoles; Receptor, Cannabinoid, CB1

Recent investigations in our laboratory showed that voltage-gated sodium channels (VGSCs) in brain are sensitive to inhibition by various synthetic cannabinoids and endocannabinoids. The present experiments examined the effects of the cannabinoid-1 (CB1) receptor agonist CP-55,940 and ethyl arachidonate on [(3)H]batrachotoxinin A 20 alpha-benzoate ([(3)H]BTX-B]) binding and VGSC-dependent depolarization of the nerve membrane in synaptoneurosomes isolated from mouse whole brain. CP-55,940 acted as a full inhibitor of [(3)H]BTX-B binding and its IC(50) was established at 22.3 microM. At its maximum effect concentration, ethyl arachidonate achieved partial (approximately 70%) inhibition and was less effective than CP-55,940 as an inhibitor of binding (IC(50)=262.7 microM). The potent CB1 receptor antagonist AM251 (2 microM) had no significant effect on the displacement of [(3)H]BTX-B by either compound (P>0.05). Scatchard analyses showed that CP-55,940 and ethyl arachidonate reduce the binding of [(3)H]BTX-B by lowering its B(max) but ethyl arachidonate also increased the K(d) of radioligand binding. In kinetic experiments, CP-55,940 and ethyl arachidonate were found to boost the dissociation of [(3)H]BTX-B from VGSCs to rates that exceed the maximum velocity achievable by veratridine, indicating they operate as allosteric inhibitors of [(3)H]BTX-B binding. Neither compound was effective at changing the initial rate of association of [(3)H]BTX-B with sodium channels. CP-55,940 and ethyl arachidonate inhibited veratridine-dependent (TTX-suppressible) depolarization of the plasma membrane of synaptoneurosomes with IC(50)s of 3.2 and 50.1 microM respectively. These inhibitory effects were again not influenced by 2 microM AM251. Our data demonstrate that the potent cannabinoid receptor agonist CP-55,940 and the ethyl ester of arachidonic acid have the ability to associate with VGSCs and inhibit their function independently of effects on CB1 receptors. Binding data comparisons using mouse brain preparations indicate CP-55,940 is approximately 10,000 times more potent as a CB1 receptor ligand than a sodium channel ligand while ethyl arachidonate shows a much smaller differential. Ethyl arachidonate has been shown previously to be the principal metabolite of ethanol in the brains of intoxicated individuals and effects of this ester on VGSCs and CB1 receptors may contribute to the depressant effects of alcohol.

Topics: Animals; Arachidonic Acids; Batrachotoxins; Brain; Cannabinoid Receptor Agonists; Cyclohexanols; Mice; Piperidines; Pyrazoles; Sodium Channels; Veratridine

A variety of behavioral procedures have been developed to assess cannabinoid activity in mice; however, the feasibility of establishing Delta(9)-THC as a discriminative stimulus in mice has not been documented.. One goal was to establish Delta(9)-THC as a discriminative stimulus in mice; after having done so, another goal was to examine the in vivo mechanism of action of Delta(9)-THC with other cannabinoids and noncannabinoids.. C57BL/6J mice (n = 8) were trained to discriminate Delta(9)-THC (10 mg/kg i.p.) from vehicle while responding under a fixed ratio 30 schedule of food presentation.. Mice satisfied the discrimination criteria in 18-98 (median = 67) sessions and the discriminative stimulus effects of Delta(9)-THC were dose-dependent (ED(50) = 2.6 mg/kg). CP 55940 and WIN 55212-2 dose-dependently increased Delta(9)-THC-appropriate responding to 100% (ED(50) = 0.032 and 0.45 mg/kg, respectively), whereas methanandamide and a variety of noncannabinoids (cocaine, ethanol, and ketamine) produced a maximum of 34% Delta(9)-THC-appropriate responding. The cannabinoid CB(1) antagonist SR 141716A (rimonabant) surmountably antagonized the discriminative effects of Delta(9)-THC, CP 55940, and WIN 55212-2; methanandamide did not significantly modify the Delta(9)-THC discriminative stimulus.. The discriminative stimulus effects of Delta(9)-THC, CP 55940, and WIN 55212-2 are mediated by the same (i.e., CB(1)) receptors, whereas the effects of methanandamide or a metabolite of methanandamide are mediated at least in part by non-CB(1) receptors. The discriminative stimulus effects of Delta(9)-THC in mice could be used to evaluate mechanisms of cannabinoid activity with approaches (e.g., inducible knockouts) currently unavailable in nonmurine species.

Topics: Animals; Arachidonic Acids; Benzoxazines; Cannabinoids; Cyclohexanols; Data Interpretation, Statistical; Discrimination Learning; Discrimination, Psychological; Dose-Response Relationship, Drug; Dronabinol; Drug Interactions; Hallucinogens; Male; Mice; Mice, Inbred C57BL; Morpholines; Naphthalenes; Piperidines; Pyrazoles; Receptor, Cannabinoid, CB1; Reinforcement Schedule; Rimonabant

Endocannabinoids have been implicated in the mechanisms of implantation, maintenance of pregnancy, and parturition in women. Intrauterine prostaglandin production and actions are also critical in each of these mechanisms. Hence, we have evaluated the effects of cannabinoids on prostaglandin biosynthesis by human gestational membranes. Explants of term amnion and choriodecidua were established and treated with the endogenous endocannabinoids 2-arachidonoyl glycerol and anandamide, as well as the synthetic cannabinoid CP55,940, to determine their ability to modulate PGE(2) production. The explants were also treated with CP55,940 in the presence of either SR141716A (a potent and selective antagonist of the cannabinoid receptor CB1) or NS398 [a cyclooxygenase (COX)-2 inhibitor] to determine whether any observed stimulation of PGE(2) production was mediated through the CB1-receptor and/or COX-2 activity. All three cannabinoids caused a significant increase in PGE(2) production in the amnion but not in the choriodecidua. However, separated fetal (chorion) explants responded to cannabinoid treatment in a similar manner to amnion, whereas maternal (decidual) explants did not. The enhanced PGE(2) production caused by CP55,940 was abrogated by cotreatment with either SR141716A or NS398, illustrating that the cannabinoid action on prostaglandin production in fetal membranes is mediated by CB1 agonism and COX-2. Data from Western blotting show that cannabinoid treatment results in the upregulation of COX-2 expression. This study demonstrates a potential role for endocannabinoids in the modulation of prostaglandin production in late human pregnancy, with potentially important implications for the timing and progression of term and preterm labor and membrane rupture.

Topics: Adult; Amnion; Blotting, Western; Cannabinoid Receptor Modulators; Chorion; Cyclohexanols; Cyclooxygenase 2 Inhibitors; Cyclooxygenase Inhibitors; Decidua; Dinoprostone; Female; Humans; In Vitro Techniques; Nitrobenzenes; Piperidines; Placenta; Pregnancy; Prostaglandin-Endoperoxide Synthases; Prostaglandins; Pyrazoles; Receptor, Cannabinoid, CB1; Receptors, Cannabinoid; Rimonabant; Sulfonamides

Marijuana cannabinoid treatment increases Th2 activity, and previous reports showed that B cells express the highest level of CB(2) mRNA relative to other immune cells, suggesting that cannabinoids play a critical role in B cell activation and maturation. We previously reported evidence of Th2 biasing and class switching in cannabinoid-treated and antigen-challenged mice. We now explore the possibility that cannabinoids directly influence B cell antibody class switching. Mouse splenic B cells were purified by negative selection and cultured with IL4 and anti-CD40 in the presence or absence of the nonselective cannabinoid agonist, CP55940, or the CB(1) selective cannabinoid agonist, methanandamide, and analyzed at different days by flow cytometry for surface expression of either IgM or IgE. Cells treated with CP55940 showed an increase in expression of IgE by day 5 in culture; methanandamide had no effect. CP55940 also induced an increase in secreted IgE in culture supernatants as analyzed by ELISA. In addition, CB(2) receptors were increased on B cells after stimulation with IL-4 and anti-CD40, and the class switching effect of CP55940 was attenuated by the CB(2) antagonist, SR144528. These results suggest that cannabinoids bias toward Th2-type immunity by directly inducing B cell class switching from IgM to IgE through a mechanism involving CB(2) receptors.

Topics: Animals; B-Lymphocytes; Camphanes; Cannabinoids; Cyclohexanols; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Immunoglobulin Class Switching; Immunoglobulin E; Immunoglobulin M; In Vitro Techniques; Mice; Piperidines; Pyrazoles; Receptor, Cannabinoid, CB2; Rimonabant; Th2 Cells

The chemotactic response of murine peritoneal macrophages to RANTES/CCL5 was inhibited significantly following pretreatment with delta-9-tetrahydrocannabinol (THC), the major psychoactive component in marijuana. Significant inhibition of this chemokine directed migratory response was obtained also when the full cannabinoid agonist CP55940 was used. The CB2 receptor-selective ligand O-2137 exerted a robust inhibition of chemotaxis while the CB1 receptor-selective ligand ACEA had a minimal effect. The THC-mediated inhibition was reversed by the CB2 receptor-specific antagonist SR144528 but not by the CB1 receptor-specific antagonist SR141716A. In addition, THC treatment had a minimal effect on the chemotactic response of peritoneal macrophages from CB2 knockout mice. Collectively, these results suggest that cannabinoids act through the CB2 receptor to transdeactivate migratory responsiveness to RANTES/CCL5. Furthermore, the results suggest that the CB2 receptor may be a constituent element of a network of G protein-coupled receptor signal transductional systems, inclusive of chemokine receptors, that act coordinately to modulate macrophage migration.

Topics: Animals; Arachidonic Acids; Camphanes; Chemokine CCL5; Chemotaxis; Cyclohexanols; Dronabinol; Female; Macrophages, Peritoneal; Mice; Mice, Inbred C57BL; Mice, Knockout; Piperidines; Pyrazoles; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Receptors, CCR1; Receptors, CCR5; Receptors, G-Protein-Coupled; Rimonabant; RNA, Messenger; Signal Transduction

Impaired auditory gating and abnormal neuronal synchrony are indicators of dysfunctional information processing in schizophrenia patients and possible underlying mechanisms of their impaired sensory and cognitive functions. Because cannabinoid receptors and endocannabinoids have been linked to psychiatric disorders, including schizophrenia, the aim of this study was to evaluate the effects of cannabinoid-1 (CB1) receptor activation on sensory gating and neuronal oscillations in rats.. Auditory sensory gating has been recorded from the hippocampus and entorhinal cortex (EC) in anesthetized rats. Neuronal network oscillations were recorded from the hippocampus, medial septum, EC, and medial prefrontal cortex in anesthetized and freely moving rats. Effects of systemic administration of CB1 receptor agonist CP-55940 were evaluated on these parameters.. CP-55940 significantly disrupted auditory gating both in the hippocampus and EC in anesthetized rats. Theta field potential oscillations were disrupted in the hippocampus and EC, with simultaneous interruption of theta-band oscillations of septal neurons. Administration of the CB1 receptor antagonist AM-251 reversed both the agonist-induced gating deficit and the diminished oscillations. In freely moving rats, CP-55940 significantly reduced theta and gamma power in the hippocampus, whereas in the EC, only gamma power was attenuated. However, novelty-induced theta and gamma activities were significantly diminished by CP-55940 in both the hippocampus and EC.. Our data indicate that activation of CB1 receptors interferes with neuronal network oscillations and impairs sensory gating function in the limbic circuitry, further supporting the connection between cannabis abuse and increased susceptibility of developing schizophrenia spectrum disorders.

Topics: Acoustic Stimulation; Action Potentials; Animals; Biological Clocks; Brain; Cyclohexanols; Disease Models, Animal; Electroencephalography; Evoked Potentials, Auditory; Fourier Analysis; Gait Disorders, Neurologic; Male; Maximum Tolerated Dose; Neural Pathways; Neurons; Piperidines; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Wakefulness

In this study we evaluated the effects of the CB1/CB2 cannabinoid receptor agonist CP55, 940 (CP) on antigen-induced asthma-like reaction in sensitized guinea pigs and we tested the ability of the specific CB2 receptor antagonist SR144528 (SR) and CB1 receptor antagonist AM251 (AM) to interfere with the effects of CP. Ovalbumin-sensitized guinea pigs placed in a respiratory chamber were challenged with the antigen given by aerosol. CP (0.4 mg/kg b.wt.) was given i.p. 3 hrs before ovalbumin challenge. Sixty minutes before CP administration, some animals were treated i.p. with either AM, or SR, or both (0.1 mg/kg b.wt.). Respiratory parameters were recorded and quantified. Lung tissue specimens were then taken for histopathological and morphometric analyses and for eosinophilic major basic protein immunohistochemistry. Moreover, myeloperoxidase activity, 8-hydroxy-2-deoxyguanosine, cyclic adenosine monophosphate (cAMP) and guanosine monophosphate (cGMP) levels, and CB1 and CB2 receptor protein expression by Western blotting were evaluated in lung tissue extracts. In the bronchoalveolar lavage fluid, the levels of prostaglandin D2 and tumour necrosis factor-alpha TNF-alpha were measured. Ovalbumin challenge caused marked abnormalities in the respiratory, morphological and biochemical parameters assayed. Treatment with CP significantly reduced these abnormalities. Pre-treatment with SR, AM or both reverted the protective effects of CP, indicating that both CB1 and CB2 receptors are involved in lung protection. The noted treatments did not change the expression of cannabinoid receptor proteins, as shown by Western blotting. These findings suggest that targeting cannabinoid receptors could be a novel preventative therapeutic strategy in asthmatic patients.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Antigens; Asthma; Camphanes; Cyclic AMP; Cyclohexanols; Deoxyguanosine; DNA Damage; Guinea Pigs; Humans; Leukocytes; Lung; Male; Mast Cells; Models, Biological; Ovalbumin; Piperidines; Prostaglandin D2; Pyrazoles; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Tumor Necrosis Factor-alpha

The endogenous cannabinoid system plays a role in the regulation of energy homeostasis acting through central pathways, and its dysregulation may be implicated in the pathogenesis of obesity. Recent evidence is accumulating showing that the endogenous cannabinoid system is also present in peripheral tissues. The aim of this work was to investigate the effect of cannabinoids upon the intestinal absorption of glucose. For this, we investigated the effect of some cannabinoid receptor agonists and antagonists upon the apical uptake of 3H-2-deoxy-D-glucose by the human intestinal epithelial Caco-2 cells. Uptake of a low concentration of 3H-2-deoxy-D-glucose (1 micromol/l) was both cytochalasin B- and phloridzin-sensitive. The maximal inhibition obtained with each of these inhibitors was 50%, and their effect was not cumulative. On the other hand, uptake of a high concentration of 3H-2-deoxy-D-glucose (20 mmol/l) was partially inhibited by cytochalasin B (+/-20%) and phloridzin had no effect. We verified that neither the cannabinoid receptor agonists [tetrahydrocannabinol (1-10 micromol/l), anandamide (0.1-10 micromol/l) and CP 55,940 (5 nmol/l to 1 micromol/l)], nor the specific CB1 and CB2 antagonists [AM251 (10-500 nmol/l) and AM630 (50 nmol/l to 1 micromol/l), respectively] had a significant effect upon 3H-2-deoxy-D-glucose uptake by Caco-2 cells. This was true for both the uptake of a low (1 micromol/l) and of a high (20 mmol/l) concentration of 3H-2-deoxy-D-glucose. From these results, we may hypothesize that cannabinoids do not interfere with the intestinal GLUT2-mediated apical uptake of glucose.

Topics: Arachidonic Acids; Caco-2 Cells; Cannabinoid Receptor Agonists; Cannabinoid Receptor Antagonists; Cannabinoids; Cyclohexanols; Deoxyglucose; Dronabinol; Endocannabinoids; Humans; Indoles; Intestinal Absorption; Piperidines; Polyunsaturated Alkamides; Pyrazoles

A nonpsychoactive constituent of the cannabis plant, cannabidiol has been demonstrated to have low affinity for both cannabinoid CB1 and CB2 receptors. We have shown previously that cannabidiol can enhance electrically evoked contractions of the mouse vas deferens, suggestive of inverse agonism. We have also shown that cannabidiol can antagonize cannabinoid receptor agonists in this tissue with a greater potency than we would expect from its poor affinity for cannabinoid receptors. This study aimed to investigate whether these properties of cannabidiol extend to CB1 receptors expressed in mouse brain and to human CB2 receptors that have been transfected into CHO cells.. The [35S]GTPS binding assay was used to determine both the efficacy of cannabidiol and the ability of cannabidiol to antagonize cannabinoid receptor agonists (CP55940 and R-(+)-WIN55212) at the mouse CB1 and the human CB2 receptor.. This paper reports firstly that cannabidiol displays inverse agonism at the human CB2 receptor. Secondly, we demonstrate that cannabidiol is a high potency antagonist of cannabinoid receptor agonists in mouse brain and in membranes from CHO cells transfected with human CB2 receptors.. This study has provided the first evidence that cannabidiol can display CB2 receptor inverse agonism, an action that appears to be responsible for its antagonism of CP55940 at the human CB2 receptor. The ability of cannabidiol to behave as a CB2 receptor inverse agonist may contribute to its documented anti-inflammatory properties.

Topics: Animals; Anti-Inflammatory Agents; Benzoxazines; Brain; Camphanes; Cannabidiol; Cannabinoid Receptor Agonists; Cannabinoid Receptor Antagonists; Cell Membrane; CHO Cells; Cricetinae; Cricetulus; Cyclohexanes; Cyclohexanols; Dose-Response Relationship, Drug; Guanosine 5'-O-(3-Thiotriphosphate); Humans; In Vitro Techniques; Mice; Mice, Inbred C57BL; Mice, Knockout; Morpholines; Naphthalenes; Phenols; Piperidines; Protein Binding; Pyrazoles; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Receptors, Cannabinoid; Rimonabant; Transfection

Since endocannabinoids modulate reward processing and the stress response, we tested the hypothesis that endocannabinoids regulate stress-induced decreased sensitivity to natural reward. Restraint was used to produce stress-induced reductions in sucrose consumption and preference in male mice. Central cannabinoid receptor (CB(1)) signaling was modulated pharmacologically prior to the application of stress. The preference for sucrose over water was significantly decreased in mice exposed to restraint. Treatment of mice with a cannabinoid receptor agonist (CP55940) or fatty acid amide hydrolase inhibitor (URB597) attenuated, while the CB(1) receptor antagonist/inverse agonist, rimonabant (SR141716), enhanced, stress-induced decreases in sucrose preference. These data are consistent with a tonically active, stress-inhibitory role for the CB(1) receptor. Mice treated with 10 daily episodes of restraint showed reduced sucrose preference that was unaffected by CP55940 and URB597. However, rimonabant produced a greater reduction in sucrose preference on day 10 compared to day 1. These data suggest that on day 10, endocannabinoid signaling is maximally activated and essential for reward sensitivity. The findings of the present study indicate that the CB(1)/endocannabinoid signaling system is an important allostatic mediator that both modulates the responses of mice to stress and is itself modulated by stress.

Topics: Analysis of Variance; Animals; Behavior, Animal; Benzamides; Body Weight; Cannabinoid Receptor Modulators; Carbamates; Cyclohexanes; Cyclohexanols; Dose-Response Relationship, Drug; Drinking Behavior; Drug Interactions; Endocannabinoids; Food Deprivation; Food Preferences; Male; Mice; Mice, Inbred ICR; Phenols; Piperidines; Pyrazoles; Restraint, Physical; Reward; Rimonabant; Stress, Psychological; Sucrose; Time Factors; Water Deprivation

The cannabinoid-1 receptor (CB1R) has been implicated in the control of energy balance. To explore the pharmacological utility of CB1R inhibition for the treatment of obesity, we evaluated the efficacy of N-[(1S,2S)-3-(4-chlorophenyl)-2-(3-cyanophenyl)-1-methylpropyl]-2-methyl-2-[[5-(trifluoromethyl)pyridin-2-yl]oxy]propanamide (MK-0364) and determined the relationship between efficacy and brain CB1R occupancy in rodents. MK-0364 was shown to be a highly potent CB1R inverse agonist that inhibited the binding and functional activity of various agonists with a binding K(i) of 0.13 nM for the human CB1R in vitro. MK-0364 dose-dependently inhibited food intake and weight gain, with an acute minimum effective dose of 1 mg/kg in diet-induced obese (DIO) rats. CB1R mechanism-based effect was demonstrated for MK-0364 by its lack of efficacy in CB1R-deficient mice. Chronic treatment of DIO rats with MK-0364 dose-dependently led to significant weight loss with a minimum effective dose of 0.3 mg/kg (p.o.), or a plasma C(max) of 87 nM. Weight loss was accompanied by the loss of fat mass. Partial occupancy (30-40%) of brain CB1R by MK-0364 was sufficient to reduce body weight. The magnitude of weight loss was correlated with brain CB1R occupancy. The partial receptor occupancy requirement for efficacy was also consistent with the reduced food intake of the heterozygous mice carrying one disrupted allele of CB1R gene compared with the wild-type mice. These studies demonstrated that MK-0364 is a highly potent and selective CB1R inverse agonist and that it is orally active in rodent models of obesity.

Topics: Amides; Animals; Anti-Obesity Agents; Binding, Competitive; Body Temperature; Body Weight; CHO Cells; Colforsin; Cricetinae; Cricetulus; Cyclic AMP; Cyclohexanols; Dose-Response Relationship, Drug; Eating; Humans; Indoles; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Molecular Structure; Obesity; Piperidines; Pyridines; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Transfection

We have previously shown that the endocannabinoid anandamide and its metabolically stable analog (R)-methanandamide produce vasorelaxation in rabbit aortic ring preparations in an endothelium-dependent manner that could not be mimicked by other CB(1) cannabinoid receptor agonists (Am J Physiol 282: H2046-H2054, 2002). Here, we show that (R)-methanandamide and abnormal cannabidiol stimulated nitric oxide (NO) production in rabbit aortic endothelial cells (RAEC) in a dose-dependent manner but that other CB(1) and CB(2) receptor agonists, such as cis-3R-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4R-3(3-hydroxypropyl)-1R-cyclohexanol (CP55940) and (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl) pyrrolo-[1,2,3-d,e]-1,4-benzoxazin-6-yl]-1-naphthalenyl-methanone (WIN55212-2), failed to do so. CB(1) antagonists rimonabant [also known as SR141716; N-piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide] and 6-methoxy-2-(4-methoxyphenyl)benzo[b]-thien-3-yl][4-cyanophenyl]methanone (LY320135) and CB(2) antagonist N-[(1S)-endo-1,3,3,-trimethylbicyclo[2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR144528) failed to block (R)-methanandamide-mediated NO production in RAEC. However, anandamide receptor antagonist (-)-4-(3-3,4-trans-p-menthadien-(1,8)-yl)-orcinol (O-1918) blocked (R)-methanandamide-mediated NO production in RAEC. Reverse transcriptase-polymerase chain reaction and Western blot analyses failed to detect the CB(1) receptor in RAEC, making this a good model to study non-CB(1) responses to anandamide. (R)-Methanandamide produced endothelial nitric-oxide synthase (eNOS) phosphorylation via the activation of phosphoinositide 3-kinase-Akt signaling. Inhibition of G(i) signaling with pertussis toxin, or phosphatidylinositol 3-kinase activity with 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), resulted in a decrease in (R)-methanandamide-induced Akt phosphorylation and NO production. Results from this study suggest that in RAEC, (R)-methanandamide acts on a novel non-CB(1) and non-CB(2) anandamide receptor and signals through G(i) and phosphatidylinositol 3-kinase, leading to Akt activation, eNOS phosphorylation, and NO production.

Topics: Animals; Arachidonic Acids; Benzofurans; Benzoxazines; Camphanes; Cannabinoid Receptor Modulators; Cells, Cultured; Chromones; Cyclohexanols; Dose-Response Relationship, Drug; Endocannabinoids; Endothelial Cells; Enzyme Inhibitors; GTP-Binding Protein alpha Subunits, Gi-Go; Morpholines; Naphthalenes; Nitric Oxide; Pertussis Toxin; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Piperidines; Polyunsaturated Alkamides; Proto-Oncogene Proteins c-akt; Pyrazoles; Rabbits; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Resorcinols; Rimonabant; Signal Transduction

Cannabinoids have been shown to increase the extracellular levels of glutamate in vivo and in vitro, but no studies have evaluated the possible involvement of glial glutamate reuptake system. The present study investigates whether cannabinoids and endocannabinoid, anandamide have an effect on astroglial excitatory amino acid (EAA) transport. The kinetics of glutamate transport was studied in rat cortical astrocytes, using the radiolabeled, non-metabolized amino acid, D-[3H] aspartate in the absence or presence of cannabinoid receptor agonists. The results show that in vehicle controls the uptake of d-aspartate was rapid, sodium-dependent and saturated within the first 5 min, resulting in a K(m) 7.365+/-1.16 micromol/L (n=5) and the maximum velocity (V(max)) 1207+/-51 nmol/mg protein/min. Addition of the synthetic cannabinoid analog R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolol][1,2,3de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone (WIN 55,212-2; 3 micromol/L) increased the K(m) (26.25+/-4.84 micromol/L) without affecting the V(max) (1122+/-77 nmol/mg protein/min), suggesting the inhibition was competitive and reversible. Various other cannabinoid agonists also inhibited D-aspartate uptake in a dose-dependent and stereospecific manner. The cannabinoid inhibition of EAA transport was partially blocked by the cannabinoid type-1 (CB1) receptor antagonist N-(piperidin-1-yl-5(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamidehydrochloride (SR141716A; 100 nmol/L). The inhibitory effects of WIN 55,212-2, or its endogenous counterpart anandamide were reversed by 98,059, an inhibitor of mitogen-activated kinase (MAPK) kinase (MEK). These results suggest that cannabinoids and endocannabinoids may constitute a novel class of inhibitors of astroglial glutamate transport system.

Topics: Animals; Animals, Newborn; Arachidonic Acids; Aspartic Acid; Astrocytes; Benzoxazines; Biological Transport; Biomarkers; Cannabinoid Receptor Agonists; Cannabinoid Receptor Modulators; Cannabinoids; Cells, Cultured; Cerebral Cortex; Cyclohexanols; Dose-Response Relationship, Drug; Endocannabinoids; Excitatory Amino Acids; Glial Fibrillary Acidic Protein; Immunohistochemistry; Kinetics; L-Lactate Dehydrogenase; Morpholines; Naphthalenes; Piperidines; Polyunsaturated Alkamides; Proteins; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Rimonabant; Sodium; Time Factors

We investigated expression of cannabinoid receptors and the effects of the endogenous cannabinoid virodhamine and the synthetic agonist CP55,940 on cAMP accumulation and interleukin-8 (IL-8) release in human bronchial epithelial cells.. Human bronchial epithelial (16HBE14o(-)) cells were used. Total mRNA was isolated and cannabinoid receptor mRNAs were detected by RT-PCR. Expression of CB(1) and CB(2) receptor proteins was detected with Western blotting using receptor-specific antibodies. cAMP accumulation was measured by competitive radioligand binding assay. IL-8 release was measured by ELISA.. CB(1) and CB(2) receptor mRNAs and proteins were found. Both agonists concentration-dependently decreased forskolin-induced cAMP accumulation. This effect was inhibited by the CB(2) receptor antagonist SR144528, and was sensitive to Pertussis toxin (PTX), suggesting the involvement of CB(2) receptors and G(i/o)-proteins. Cell pretreatment with PTX unmasked a stimulatory component, which was blocked by the CB(1) receptor antagonist SR141716A. CB(2) receptor-mediated inhibition of cAMP production by virodhamine and CP55,940 was paralleled by inhibition of tumor necrosis factor-alpha (TNF-alpha) induced IL-8 release. This inhibition was insensitive to SR141716A. In the absence of agonist, SR144528 by itself reduced TNF-alpha induced IL-8 release.. Our results show for the first time that 16HBE14o(-) cells respond to virodhamine and CP55,940. CB(1) and CB(2) receptor subtypes mediated activation and inhibition of adenylyl cyclase, respectively. Stimulation of the dominant CB(2) receptor signalling pathway diminished cAMP accumulation and TNF-alpha-induced IL-8 release. These observations may imply that cannabinoids exert anti-inflammatory properties in airways by modulating cytokine release.

Topics: Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Analgesics; Arachidonic Acids; Blotting, Western; Bronchi; Camphanes; Cannabinoids; Cell Line; Colforsin; Cyclic AMP; Cyclohexanols; Dose-Response Relationship, Drug; Drug Antagonism; Epithelial Cells; Humans; Immunosuppressive Agents; Interleukin-8; Pertussis Toxin; Piperidines; Pyrazoles; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Reverse Transcriptase Polymerase Chain Reaction; Rimonabant; RNA, Messenger; Tumor Necrosis Factor-alpha

Rimonabant (Acomplia, SR141716A), a cannabinoid CB1 receptor inverse agonist, has recently been approved for the treatment of obesity. There are, however, concerns regarding its side effect profile. Developing a CB1 antagonist with a different pharmacological mechanism may lead to a safer alternative. To this end we have screened a proprietary small molecule library and have discovered a novel class of allosteric antagonist at CB1 receptors. Herein, we have characterized an optimized prototypical molecule, PSNCBAM-1, and its hypophagic effects in vivo.. A CB1 yeast reporter assay was used as a primary screen. PSNCBAM-1 was additionally characterized in [35S]-GTPgammaS, cAMP and radioligand binding assays. An acute rat feeding model was used to evaluate its effects on food intake and body weight in vivo.. In CB1 receptor yeast reporter assays, PSNCBAM-1 blocked the effects induced by agonists such as CP55,940, WIN55212-2, anandamide (AEA) or 2-arachidonoyl glycerol (2-AG). The antagonist characteristics of PSNCBAM-1 were confirmed in [35S]-GTPgammaS binding and cAMP assays and was shown to be non-competitive by Schild analyses. PSNCBAM-1 did not affect CB2 receptors. In radioligand binding assays, PSNCBAM-1 increased the binding of [3H]CP55,940 despite its antagonist effects. In an acute rat feeding model, PSNCBAM-1 decreased food intake and body weight.. PSNCBAM-1 exerted its effects through selective allosteric modulation of the CB1 receptor. The acute effects on food intake and body weight induced in rats provide a first report of in vivo activity for an allosteric CB1 receptor antagonist.

Topics: Allosteric Regulation; Animals; Appetite Depressants; Cell Line; Cell Membrane; Cerebral Cortex; Cyclic AMP; Cyclohexanols; Dose-Response Relationship, Drug; Eating; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Male; Molecular Structure; Phenylurea Compounds; Piperidines; Pyrazoles; Pyridines; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Rimonabant; Saccharomyces cerevisiae; Silicone Elastomers; Sulfur Radioisotopes; Weight Gain

A putative novel cannabinoid receptor mediates vasorelaxation to anandamide and abnormal-cannabidiol and is blocked by O-1918 and by high concentrations of rimonabant. This study investigates VSN16, a novel water-soluble agonist, as a vasorelaxant potentially acting at non-CB1, non-CB2 cannabinoid receptors in the vasculature.. VSN16 and some analogues were synthesized and assayed for vasodilator activity in the rat third generation mesenteric artery using wire myography. Also carried out with VSN16 were haemodynamic studies in conscious rats and binding studies to CB1 receptors of rat cerebellum.. VSN16 relaxed mesenteric arteries in an endothelium-dependent manner. The vasorelaxation was antagonized by high concentrations of the classical cannabinoid antagonists, rimonabant and AM 251, as well as by O-1918, an antagonist at the abnormal-cannabidiol receptor but not at CB1 or CB2 receptors. It did not affect [3H]CP55,940 binding to CB1 receptors in rat cerebellum. The vasorelaxation was not pertussis toxin-sensitive but was reduced by inhibition of nitric oxide synthesis, Ca(2+)-sensitive K+ channels (KCa) and TRPV1 receptors. In conscious rats VSN16 transiently increased blood pressure and caused a longer-lasting increase in mesenteric vascular conductance. Structure-activity studies on vasorelaxation showed a stringent interaction with the target receptor.. VSN16 is an agonist at a novel cannabinoid receptor of the vasculature. It acts on the endothelium to release nitric oxide and activate KCa and TRPV1. As it is water-soluble it might be useful in bringing about peripheral cannabinoid-like effects without accompanying central or severe cardiovascular responses.

Topics: Animals; Apamin; Benzamides; Cannabinoid Receptor Agonists; Cannabinoid Receptor Antagonists; Cannabinoids; Cerebellum; Charybdotoxin; Cyclohexanols; Dose-Response Relationship, Drug; Endothelium, Vascular; In Vitro Techniques; Indomethacin; Male; Mesenteric Arteries; Molecular Structure; NG-Nitroarginine Methyl Ester; Pertussis Toxin; Piperidines; Pyrazoles; Radioligand Assay; Rats; Rats, Wistar; Receptors, Cannabinoid; Resorcinols; Rimonabant; Tritium; TRPV Cation Channels; Vascular Resistance; Vasodilation

Trabecular meshwork (TM) is an ocular tissue involved in the regulation of aqueous humour outflow and intraocular pressure (IOP). CB1 receptors (CB1) are present in TM and cannabinoid administration decreases IOP. CB1 signalling was investigated in a cell line derived from human TM (hTM).. CB1 signalling was investigated using ratiometric Ca2+ imaging, western blotting and infrared In-Cell Western analysis.. WIN55212-2, a synthetic aminoalkylindole cannabinoid receptor agonist (10-100 microM) increased intracellular Ca2+ in hTM cells. WIN55,212-2-mediated Ca2+ increases were blocked by AM251, a CB1 antagonist, but were unaffected by the CB2 antagonist, AM630. The WIN55,212-2-mediated increase in [Ca2+]i was pertussis toxin (PTX)-insensitive, therefore, independent of Gi/o coupling, but was attenuated by a dominant negative Galpha(q/11) subunit, implicating a Gq/11 signalling pathway. The increase in [Ca2+]i was dependent upon PLC activation and mobilization of intracellular Ca2+ stores. A PTX-sensitive increase in extracellular signal-regulated kinase (ERK1/2) phosphorylation was also observed in response to WIN55,212-2, indicative of a Gi/o signalling pathway. CB1-Gq/11 coupling to activate PLC-dependent increases in Ca2+ appeared to be specific to WIN55,212-2 and were not observed with other CB1 agonists, including CP55,940 and methanandamide. CP55940 produced PTX-sensitive increases in [Ca2+]i at concentrations>or=15 microM, and PTX-sensitive increases in ERK1/2 phosphorylation.. This study demonstrates that endogenous CB1 couples to both Gq/11 and Gi/o in hTM cells in an agonist-dependent manner. Cannabinoid activation of multiple CB1 signalling pathways in TM tissue could lead to differential changes in aqueous humour outflow and IOP.

Topics: Arachidonic Acids; Benzoxazines; Blotting, Western; Calcium Signaling; Cannabinoid Receptor Agonists; Cannabinoid Receptor Antagonists; Cell Line; Cells, Cultured; Cyclohexanols; Dose-Response Relationship, Drug; Humans; Mitogen-Activated Protein Kinases; Morpholines; Naphthalenes; Piperidines; Pyrazoles; Receptors, Cannabinoid; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Time Factors; Trabecular Meshwork; Type C Phospholipases

In the terrestrial snail a direct monosynaptic glutamatergic connection between the primary sensory neuron and a premotor interneuron involved in withdrawal behaviour can be functionally identified using electrophysiological techniques. We investigated the involvement of cannabinoids in regulation of this synaptic contact. The results demonstrate that the specific binding sites for agonists to mammalian type 1 cannabinoid receptors (CB1Rs) exist in the snail's nervous system. Application of a synthetic cannabinoid agonist anandamide selectively changed the efficacy of synaptic contacts between the identified neurons. A decrease in the long-term synaptic facilitation of the synaptic contact elicited by high-frequency nerve tetanization in the presence of cannabinoid agonist anandamide was observed, suggesting a possible role of endocannabinoids in regulation of plasticity at this synaptic site. The selective antagonist of CB1Rs [N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide] AM251 bath application was changing the efficacy of the synaptic contact only when the postsynaptic neuron had been intracellularly activated before its application. This observation implies an involvement of endocannabinoids in plasticity phenomena induced by activity in the postsynaptic target. Additional support of endocannabinoid involvement in synaptic function at this site was given by experiments in which AM251 blocked the short-term suppression of synaptic excitation evoked by low-frequency nerve tetanization, a phenomenon qualitatively similar to cannabinoid-dependent synaptically evoked suppression of excitation demonstrated in the mammalian nervous system. The results of the present study suggest an involvement of cannabinoids in the regulation of synaptic efficacy. Further, anandamide could be a candidate for an endogenous neuromessenger involved in plasticity processes.

Topics: Animals; Arachidonic Acids; Cannabinoid Receptor Modulators; Cannabinoids; Cyclohexanols; Dose-Response Relationship, Radiation; Electric Stimulation; Endocannabinoids; Excitatory Postsynaptic Potentials; Nervous System; Neurons; Patch-Clamp Techniques; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB1; Snails; Synapses; Tritium

The current experiments were designed to study the antinociceptive effects of intrathecal (i.t.) administration of cannabinoid CB1 receptor and 2-adrenoceptor drugs in the nociceptive processing and also their receptor interactions. Different doses of a cannabinoid receptor agonist, CP 55,940, and an 2-adrenoceptor agonist, clonidine induced a dose-dependent antinociception in both phases of the formalin test.CP 55,940-induced antinociception was reduced by pretreatment of a selective cannabinoid CB1 receptor antagonist, SR 141716A, but not by pretreatment with an 2-adrenoceptor antagonist, yohimbine in both phases of the test. However, yohimbine and SR 141716A attenuated the antinociception induced by clonidine in the early phase but not in the late phase of the test. While SR 141716A by itself did not influence pain behaviour, the reversal effect of clonidine by SR 141716A indicate that clonidine stimulate the release of endocannabinoid(s). In conclusion, our findings may suggest that: (1) spinal cannabinoid and 2-adrenoceptor systems are able to induce antinociception in both phases of formalin test, and (2) the cannabinoid system may be involved in the antinociception induced by adrenoceptors in the early phase.

Topics: Adrenergic alpha-Agonists; Adrenergic alpha-Antagonists; Analgesics; Animals; Cyclohexanols; Formaldehyde; Injections, Spinal; Male; Pain Measurement; Piperidines; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Receptors, Adrenergic, alpha-2; Receptors, Cannabinoid; Rimonabant; Spinal Cord; Yohimbine

A series of 1,2,4-triazole-3-carboxamides has been prepared from alkyl-1,2,4-triazole-3-carboxylates under mild conditions. The ability of these triazoles to displace [3H]-CP55940 from CB1 cannabinoid receptor was measured. However, they showed only poor to moderate binding affinities, indicating that substitution of the C-4 pyrazole atom of the CB1 reference compound SR141716 by a nitrogen atom results in loss of affinity. Further investigations for functionality indicated that the compound 6a exhibited significant cannabinoid antagonistic properties in the mouse vas deferens functional assay. This leads us to the conclusion that 6a binds at a different CB1 binding site or at a new cannabinoid receptor subtype.

Topics: Animals; Binding Sites; Cyclohexanols; Male; Mice; Piperidines; Pyrazoles; Receptor, Cannabinoid, CB1; Rimonabant; Structure-Activity Relationship; Triazoles; Vas Deferens

To investigate how specific amino acid residues affect human cannabinoid CB1 receptor binding and activation, CHO cell lines stably expressing wild type and the phenylalanine 200 to alanine mutant of human cannabinoid CB1 receptor (F200A) were examined. AM2233 functions as an agonist at the wild type receptor (EC50=0.93 nM), but behaves as an inverse agonist at F200A (EC50=4.8 nM). The F200A mutant has significantly lower forskolin-stimulated basal cAMP accumulation than that of the wild type, indicating that the F200A mutant possesses higher constitutive activity. F200 doesn't contribute substantially to the high affinity binding of AM2233 at human cannabinoid CB1 receptor. CP55940, HU-210 and Win55212-2 still function as agonists at the F200A mutant, with similar efficacy, potency, and apparent binding affinity for both wild type human cannabinoid CB1 receptor and F200A mutant. These data indicate that the phenylalanine 200 residue in human cannabinoid CB1 receptor is involved in the receptor activation induced by a specific class of agonists, and supports a model of agonist-structure-dependent conformational changes.

Topics: Alanine; Amino Acid Substitution; Animals; Binding Sites; Binding, Competitive; CHO Cells; Colforsin; Cricetinae; Cricetulus; Cyclic AMP; Cyclohexanols; Dose-Response Relationship, Drug; Gene Expression; Humans; Indoles; Molecular Structure; Pertussis Toxin; Phenylalanine; Piperidines; Pyrazoles; Receptor, Cannabinoid, CB1; Tritium

Previous reports have described modulation of noradrenergic activity by cannabinoid receptors. The aim of the present research was to examine the effect of two synthetic cannabinoid CB1/CB2 receptor agonists, R-(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)-methyl]pyrrolol-[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl) methanone (WIN 55212-2) and (-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl) cyclohexanol (CP 55940), on the spontaneous activity of locus coeruleus noradrenergic neurons by single-unit extracellular recordings in vivo and in vitro. In anaesthetized rats, intravenous administrations of WIN 55212-2 (31.3-500 microg/kg) or CP 55940 (31.3-500 microg/kg) increased the firing rate of locus coeruleus neurons in a dose-dependent manner. The stimulatory effect of WIN 55212-2 was blocked by pretreatment with the cannabinoid CB1 receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR 141716A; 2 mg/kg). Paradoxically, local administration of WIN 55212-2 (8.3-31.3 pmol) into the locus coeruleus and intracerebroventricular injections of WIN 55212-2 (10-20 microg) or CP 55940 (20-40 microg) failed to change the spontaneous firing rate of locus coeruleus neurons. Likewise, in rat brain slice preparations perfusion with WIN 55212-2 (10 microM) or CP 55940 (10-30 microM) did not specifically affect the spontaneous firing rate of locus coeruleus cells. Therefore, we conclude that synthetic cannabinoids increase the spontaneous firing activity of noradrenergic neurons in the rat locus coeruleus through cannabinoid CB1 receptors. This stimulation appears to be indirectly induced via a receptor mechanism probably located at the peripheral level.

Topics: Action Potentials; Animals; Benzoxazines; Cannabinoids; Cyclohexanols; Dose-Response Relationship, Drug; In Vitro Techniques; Injections, Intravenous; Injections, Intraventricular; Kinetics; Locus Coeruleus; Male; Morpholines; Naphthalenes; Neurons; Norepinephrine; Piperidines; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Rimonabant

Several clinical studies have shown that alterations in the cannabinoid system in the brain may be associated with schizophrenia. Although evidence points towards an antipsychotic potential for cannabinoid antagonists, experimental studies have shown inconsistent behavioural effects of cannabinoid ligands within and across species. The aim of the present study was to explore these contradictory findings in a non-human primate model, predictive of antipsychotic efficacy in humans. The effects of the cannabinoid CB1 receptor antagonist SR141716A and the CB1 receptor agonist CP55,940 were explored in an d-amphetamine-based Cebus monkey model of psychosis. The monkeys were sensitive to extrapyramidal side effects (EPS), and the side-effect profiles of the drugs were explored as well. SR141716A (0.1, 0.25, 0.375, 0.5 and 0.75 mg/kg) and CP55,940 (0.0025, 0.005 and 0.01 mg/kg) were administered by subcutaneous injection alone and in combination with d-amphetamine (0.25mg/kg). SR141716A (0.1-0.5mg/kg) reduced d-amphetamine-induced arousal, while CP55,940 had no significant effect upon d-amphetamine-induced behaviours. No EPS were observed with either of these compounds. These data suggest that cannabinoid CB1 antagonists such as SR141716A may have limited antipsychotic potential in man as to positive symptoms. SR141716A administered alone induced anxiolytic-like behaviour, whereas administration of CP55,940 alone showed anxiogenic properties.

Topics: Animals; Anti-Anxiety Agents; Arousal; Behavior, Animal; Cebus; Central Nervous System Stimulants; Cyclohexanols; Dextroamphetamine; Drug Interactions; Female; Male; Piperidines; Pyrazoles; Receptor, Cannabinoid, CB1; Rimonabant; Stereotyped Behavior

Sphingosine-1-phosphate (S1P) and cannabinoid receptors are G-protein-coupled receptors that mediate the effects of S1P and endocannabinoids, respectively. Cannabinoid receptors also mediate the effects of Delta9-tetrahydrocannabinol, the primary psychoactive ingredient in marijuana, whereas S1P receptors contribute to the immunosuppressant effects of 2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol (FTY720). FTY720 is a sphingosine analog that can prevent renal graft rejections and suppress a variety of autoimmune disorders in animal models and clinical trials. We now report that both FTY720 and sphingosine interact with CB1 but not CB2 cannabinoid receptors. FTY720 and sphingosine inhibited the binding of the CB1-selective antagonist [3H]N-(piperidinyl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide ([3H]SR141716A) and the cannabinoid agonist [3H](-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol ([3H]CP55,940) in a concentration-dependent manner in both CB1-expressing cell lines and mouse cerebellum. However, these compounds did not significantly alter [3H]CP55,940 binding to CB2 receptors. In G-protein activation assays, FTY720 and sphingosine inhibited the maximal stimulation of guanosine 5'-O-(3-[35S]thio)triphosphate binding by the cannabinoid agonist R-(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone mesylate (WIN55,212-2) in a concentration-dependent manner, and this antagonist effect was not mimicked by S1P. FTY720 and sphingosine also inhibited activation of extracellular signal-regulated kinases 1 and 2 and Akt by WIN55,212-2 in intact Chinese hamster ovary (CHO) cells expressing CB1 receptors and attenuated WIN55,212-2-stimulated internalization of a fluorescence-tagged CB1 receptor in CHO cells. Moreover, both FTY720 and sphingosine produced rightward shifts in the concentration-effect curves of cannabinoid agonists for G-protein activation, indicating that they act as competitive CB1 antagonists. These results suggest that the CB1 receptor could be a novel target of FTY720 and that sphingosine could be an endogenous CB11 antagonist.

Topics: Animals; Benzoxazines; Binding, Competitive; Cell Line; CHO Cells; Cricetinae; Cricetulus; Cyclohexanes; Cyclohexanols; Endocytosis; Enzyme Activation; Fingolimod Hydrochloride; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Immunosuppressive Agents; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Morpholines; Naphthalenes; Phenols; Piperidines; Propylene Glycols; Proto-Oncogene Proteins c-akt; Pyrazoles; Radioligand Assay; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Rimonabant; Sphingosine

The present study evaluates the pharmacological profile of the new neutral cannabinoid CB1 receptor antagonist 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-3-hexyl-1H-1,2,4-triazole -LH-21- on feeding behavior and alcohol self-administration in rats, two behaviors inhibited by cannabinoid CB1 receptor antagonists. Administration of LH-21 (0.03, 0.3 and 3 mg/kg) to food-deprived rats resulted in a dose-dependent inhibition of feeding. Subchronic administration of LH-21 reduced food intake and body weight gain in obese Zucker rats. Acute effects on feeding were not associated with anxiety-like behaviors, or induction of complex motor behaviors such as grooming or scratching sequences, usually observed after central administration of cannabinoid receptor blockers with inverse agonist properties. LH-21 did not markedly reduce alcohol self-administration (30% reduction observed only at a high dose of 10 mg/kg). This pharmacological pattern partially overlaps that of the reference cannabinoid CB1 receptor antagonist N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methylpyrazole-3-carboxamide, SR141716A, (0.3, 1 and 3 mg/kg) that reduced feeding and alcohol self-administration with similar efficacy. In vitro analysis of blood-brain barrier permeability using a parallel artificial membrane permeation assay demonstrated that LH-21 has lower permeation through membranes than SR141716A. That was confirmed in vivo by studies showing lower potency of peripherally injected LH-21 when compared to SR141716A to antagonize motor depression induced by intracerebroventricular administration of the CB1 agonist CP55,940. The neutral antagonist profile and the lower penetration into the brain of LH-21 favour this class of antagonists with respect to reference inverse agonists for the treatment of obesity because they potentially will display reduced side effects.

Topics: Animals; Anti-Obesity Agents; Blood-Brain Barrier; Brain; Cannabinoid Receptor Agonists; Cannabinoid Receptor Antagonists; Cyclohexanes; Cyclohexanols; Eating; Ethanol; Feeding Behavior; Male; Maze Learning; Membranes, Artificial; Motor Activity; Permeability; Phenols; Piperidines; Pyrazoles; Rats; Rats, Wistar; Rats, Zucker; Rimonabant; Self Administration; Triazoles

Whether cannabinoids act as neuroprotectants or, on the contrary, even worsen neuronal damage after cerebral ischemia is currently under discussion. We have previously shown that treatment with the cannabinoid (CB1) receptor antagonist SR141716A reduces infarct volume by approximately 40% after experimental stroke. Since it is suggested that SR141716A may exert neuroprotection besides its cannabinoid receptor-blocking effect, we addressed the question whether SR141716A may act via modulation of postischemic ligand binding to excitatory NMDA and/or alpha-amino-3-hydroxy-5-methyl-4-isoxazole-proprionic acid (AMPA) receptors. For this purpose, rats (n = 12) were treated with either intravenous saline (control) or CB1 receptor antagonist SR141716A (1 mg/kg) 30 min after permanent middle cerebral artery occlusion. Five hours after ischemia, quantitative receptor autoradiography was performed using [(3)H]CP 55,940, [(3)H]MK-801, and [(3)H]AMPA for labeling of CB1, NMDA, and AMPA receptors, respectively. Ligand binding was analyzed within the infarct core, cortical penumbra, and corresponding areas of the contralateral hemisphere and compared to that of sham-operated rats (n = 5). Both in ischemic controls and SR141716A-treated rats [(3)H]CP 55,940 ligand binding was not specifically regulated in the cortical penumbra or contralateral cortex. Importantly, reduced infarct volumes in SR141716A-treated rats were associated with maintained [(3)H]MK-801 binding to excitotoxic NMDA receptors in the penumbra, compared to a decrease in the control group. In summary, our data suggest that SR141716A may possess additional intrinsic neuroprotective properties independent of receptor-coupled pathways or due to action as a partial agonist.

Topics: Animals; Autoradiography; Binding, Competitive; Brain Ischemia; Cyclohexanols; Dizocilpine Maleate; Down-Regulation; Excitatory Amino Acid Antagonists; Male; Neuroprotective Agents; Piperidines; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate; Rimonabant

Activation of a G-protein-coupled receptor involves changes in specific microdomain interactions within the transmembrane region of the receptor. Here, we have focused on the role of L207, proximal to the DRY motif of the human cannabinoid receptor 1 in the interconversion of the receptor resting and active states. Ligand binding analysis of the mutant receptor L207A revealed an enhanced affinity for agonists (three- to six-fold) and a diminished affinity for inverse agonists (19- to 35-fold) compared to the wild-type receptor, properties characteristic of constitutive activity. To further examine whether this mutant adopts a ligand-independent, active form, treatment with GTPgammaS was used to inhibit G protein coupling. Under these conditions, the L207A receptor exhibited a 10-fold increase in affinity for the inverse agonist SR141716A, consistent with a shift away from an enhanced precoupled state. Analysis of the cellular activity of the L207A receptor showed elevated basal cyclic AMP accumulation relative to the wild type that is inhibited by SR141716A, consistent with receptor-mediated Gs precoupling. Using toxins to selectively abrogate Gs or Gi coupling, we found that CP55940 nonetheless induced only a Gi response suggesting a strong preference of this ligand-bound form for Gi in this system. Molecular dynamics simulations reveal that the single residue change of L207A impacts the association of TM3 and TM6 in the receptor by altering hydrophobic interactions involving L207, the salt bridge involving the Arg of the DRY motif, and the helical structure of TM6, consistent with events leading to activation. The structural alterations parallel those observed in models of a mutant CB(1) receptor T210I, with established constitutive activity (D'Antona, A.M., Ahn, K.H. and Kendall, D.A., 2006. Mutations of CB1 T210 produce active and inactive receptor forms: correlations with ligand affinity, receptor stability, and cellular localization. Biochemistry, 45, 5606-5617).

Topics: Amino Acid Motifs; Amino Acid Sequence; Amino Acids; Binding, Competitive; Cannabinoids; Cell Line; Cyclic AMP; Cyclohexanes; Cyclohexanols; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Ligands; Molecular Structure; Mutation; Phenols; Piperidines; Pyrazoles; Radioligand Assay; Receptor, Cannabinoid, CB1; Rimonabant

Cannabinoids are known to possess both anti-inflammatory and neuroprotective effects. In the present study, we have investigated the ability of cannabinoids to inhibit the transmigration of neutrophils in response to chemotaxic stimuli. The cannabinoid receptor agonist WIN 55,212-2 ((R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)-pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate) significantly decreased the number of migrating neutrophils across a monolayer of tumour necrosis factor alpha (TNF-alpha) activated ECV304 cells at concentrations >or=1 microM. In contrast, the agonists HU210 and CP 55,940 (0.01-1 microM) and the endocannabinoid anandamide (0.1-10 microM) were without significant effect on the response to TNF-alpha. The ability of WIN 55,212-2 to reduce the neutrophil transmigration was still seen in the presence of the cannabinoid CB(1) receptor antagonist/inverse agonist AM251 (0.1-1 microM) and the cannabinoid CB(2) receptor antagonist/inverse agonist AM630 (0.1-1 microM). TNF-alpha treatment of ECV304 cells caused release of interleukin-8 (IL-8), but WIN 55,212-2 did not affect either the ability of neutrophils to migrate across chemotaxis plates in response to an IL-8 stimulus, or to change the percentage of CXC 1 and CXC 2 receptors expressed by the neutrophils. WIN 55,212-2 at a concentration of 1 microM, but not at lower concentrations, produced a significant inhibition of IL-8 release from ECV304 cells in response to TNF-alpha-stimulation. Thus WIN 55,212-2 reduces the transmigration of neutrophils across a monolayer of TNF-alpha-activated ECV304 cells by an indirect action upon the release of IL-8 and/or other chemokine release from the ECV304 cells, and that this effect is brought about mainly by a cannabinoid CB receptor-independent mechanism.

Topics: Analysis of Variance; Arachidonic Acids; Benzoxazines; Cannabinoid Receptor Agonists; Cannabinoid Receptor Antagonists; Cell Line; Cell Movement; Chemotaxis; Cyclohexanols; Dose-Response Relationship, Drug; Dronabinol; Endocannabinoids; Endothelial Cells; Flow Cytometry; Humans; Indoles; Interleukin-8; L-Lactate Dehydrogenase; Morpholines; Naphthalenes; Neutrophils; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptors, Cannabinoid; Receptors, Interleukin-8A; Receptors, Interleukin-8B; Thapsigargin; Tumor Necrosis Factor-alpha

Cannabinoid CB(1) receptors are hypothesized to mediate the discriminative stimulus effects of cannabinoids. This study characterized a Delta(9)-tetrahydrocannabinol (Delta(9)-THC; 0.1 mg/kg i.v.) discriminative stimulus and examined antagonism of cannabinoid agonists in rhesus monkeys. High levels of responding on the Delta(9)-THC lever were produced by cannabinoid agonists with the following rank order potency: CP 55940 [(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol] > Delta(9)-THC = WIN 55212-2 [(+)-[2,3-dihydro-5-methyl-3[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone mesylate salt] > arachidonylcyclopropylamide = (R)-methanandamide. A CB(2)-selective agonist, AM 1241 [(R)-3-(2-iodo-5-nitrobenzoyl)-1-(1-methyl-2-piperidinylmethyl)-1H-indole], and noncannabinoids (cocaine, ketamine, midazolam, and morphine) did not produce high levels of Delta(9)-THC lever responding. The CB(1)-selective antagonist SR 141716A [N-(piperidin-1-yl)-1-(2,4-dichlorophenyl)-5-(4-chlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide] surmountably antagonized the discriminative stimulus effects of Delta(9)-THC and CP 55940, and Schild analysis was consistent with a simple, competitive interaction (apparent pA(2) values were 6.1 and 6.7, respectively). SR 141716A surmountably antagonized WIN 55212-2; however, larger doses disrupted responding, precluding Schild analysis. The CB(1)-selective antagonist AM 251 surmountably antagonized Delta(9)-THC, CP 55940, and WIN 55212-2, and Schild analysis was consistent with a simple, competitive interaction (apparent pA(2) values were 6.3, 6.1, and 6.2, respectively). The CB(2)-selective antagonist SR 144528 [N-[(1S)-endo-1,3,3-trimethylbicyclo(2.2.1)heptan-2-yl]5-(4-chloro-3-methyl-phenyl)-1-(4-methylbenzyl)pyrazole-3-carboxamide] did not modify the Delta(9)-THC discriminative stimulus. These results demonstrate that the discriminative stimulus effects of Delta(9)-THC are selective for cannabinoid activity, and the results of Schild analysis suggest that the same receptors mediate the discriminative stimulus effects of Delta(9)-THC, CP 55940, and WIN 55212-2. CB(2) receptors do not seem to mediate the discriminative stimulus effects of cannabinoid agonists. Schild analysis has the potential for identifying receptor subtypes that mediate the in vivo effects of cannabinoid agonists.

Topics: Analgesics, Opioid; Anesthetics, Dissociative; Anesthetics, Intravenous; Anesthetics, Local; Animals; Benzoxazines; Camphanes; Cannabinoid Receptor Agonists; Cannabinoid Receptor Antagonists; Cannabinoids; Cocaine; Cyclohexanols; Discrimination Learning; Dose-Response Relationship, Drug; Dronabinol; Female; Hallucinogens; Ketamine; Macaca mulatta; Male; Midazolam; Morphine; Morpholines; Naphthalenes; Piperidines; Pyrazoles; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2

Drug discrimination can be used to examine tolerance and dependence in agonist-treated animals by establishing an appropriate antagonist as a discriminative stimulus.. Establish intravenous SR 141716A as a discriminative stimulus in four rhesus monkeys pretreated with a relatively small dose of Delta9-tetrahydrocannabinol (Delta9-THC).. Rhesus monkeys received i.v. Delta9-THC (0.32 mg/kg) and discriminated i.v. SR 141716A (1 mg/kg) from vehicle while responding under a fixed ratio (FR) 5 schedule of stimulus-shock termination.. The discriminative stimulus effects of SR 141716A were dose-dependent (ED50=0.33 mg/kg) and were mimicked by the CB1 antagonist AM 251 (ED50=0.98 mg/kg), but not by a benzodiazepine (midazolam) or an N-methyl-D-aspartate antagonist (ketamine). An additional dose (0.32 mg/kg in addition to 0.32 mg/kg administered before the session) of Delta9-THC shifted the SR 141716A dose-effect curve 3-fold rightward. Omitting Delta9-THC before test sessions resulted in responding on the SR 141716A lever that was attenuated by subsequent administration of Delta9-THC (ED50=0.13 mg/kg), CP 55940 (ED50=0.013 mg/kg), and WIN 55212-2 (ED50=0.35 mg/kg); midazolam and ketamine did not attenuate responding on the SR 141716A lever. SR 141716A (1 mg/kg) shifted the Delta9-THC and CP 55940 dose-effect curves 3.4-fold rightward; the WIN 55212-2 dose-effect curve was not significantly modified by a dose of 1 mg/kg of SR 141716A.. SR 141716A can be established as a discriminative stimulus in animals pretreated with Delta9-THC, and this assay is selective for cannabinoid activity. Differential antagonism of cannabinoids by SR 141716A might indicate that the mechanism of action of WIN 55212-2 is not identical to other cannabinoids. This study demonstrates that, under the appropriate conditions, drug discrimination has utility for examining cannabinoid dependence and withdrawal.

Topics: Animals; Benzoxazines; Cannabinoids; Conditioning, Operant; Cyclohexanols; Discrimination Learning; Dose-Response Relationship, Drug; Dronabinol; Drug Interactions; Female; Hallucinogens; Humans; Injections, Intravenous; Injections, Subcutaneous; Ketamine; Macaca mulatta; Male; Midazolam; Morpholines; Naphthalenes; Piperidines; Pyrazoles; Receptor, Cannabinoid, CB1; Rimonabant; Time Factors

Series of thiazoles, triazoles, and imidazoles were designed as bioisosteres, based on the 1,5-diarylpyrazole motif that is present in the potent CB(1) receptor antagonist rimonabant (SR141716A, 1). A number of target compounds was synthesized and evaluated in cannabinoid (hCB(1) and hCB(2)) receptor assays. The thiazoles, triazoles, and imidazoles elicited in vitro( )()CB(1) antagonistic activities and in general exhibited considerable CB(1) vs CB(2) receptor subtype selectivities, thereby demonstrating to be cannabinoid bioisosteres of the original diarylpyrazole class. Some key representatives in the imidazole series showed potent pharmacological in vivo activities after oral administration in both a CB agonist-induced hypotension model and a CB agonist-induced hypothermia model. Molecular modeling studies showed a close three-dimensional structural overlap between the key compound 62 and rimonabant. A structure-activity relationship (SAR) study revealed a close correlation between the biological results in the imidazole and pyrazole series.

Topics: Administration, Oral; Animals; CHO Cells; Cricetinae; Cricetulus; Cyclohexanols; Hypotension; Hypothermia; Imidazoles; Mice; Models, Molecular; Molecular Conformation; Piperidines; Pyrazoles; Radioligand Assay; Rats; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Rimonabant; Stereoisomerism; Structure-Activity Relationship; Thiazoles; Triazoles

1 Delta9-tetrahydrocannabinol (THC) produces varying effects in mesenteric arteries: vasorelaxation (third-order branches, G3), modest vasorelaxation (G2), no effect (G1) and vasoconstriction (the superior mesenteric artery, G0). 2 In G3, vasorelaxation to THC was inhibited by pertussis toxin, but was unaffected by the CB1 receptor antagonist, AM251 (1 microM), incubation with the TRPV1 receptor agonist capsaicin (10 microM, 1 h), the TRPV1 receptor antagonist capsazepine (10 microM) or de-endothelialisation. 3 In G3, vasorelaxation to THC was inhibited by high K+ buffer, and by the following K+ channel inhibitors: charybdotoxin (100 nM), apamin (500 nM) and barium chloride (30 microM), but not by 4-aminopyridine, glibenclamide or tertiapin. 4 In G3, THC (10 and 100 microM) inhibited the contractile response to Ca2+ in a Ca2+-free, high potassium buffer, indicating that THC blocks Ca2+ influx. 5 In G0, the vasoconstrictor responses to THC were inhibited by de-endothelialisation and SR141716A (100 nM), but not by the endothelin (ET(A)) receptor antagonist FR139317 (1 microM).THC (1 and 10 microM) antagonised vasorelaxation to anandamide in G3 but not G0. THC did not antagonise the noncannabinoid verapamil, capsaicin or the CB1 receptor agonist CP55,940. 6 THC (10 and 100 microM) inhibited endothelium-derived relaxing factor (EDHF)-mediated responses to carbachol in a manner similar to the gap junction inhibitor 18alpha-glycyrrhetinic acid. 7 These data show that THC causes vasorelaxation through activation of K+ channels and inhibition of Ca2+ channels, and this involves non-CB1, non-TRPV1 but G-protein-coupled receptors. In G0, THC does not cause relaxation and at high concentrations causes contractions. Importantly, THC antagonises the effects of anandamide, possibly through inhibition of EDHF activity.

Topics: Animals; Apamin; Arachidonic Acids; Azepines; Barium Compounds; Biological Factors; Calcium; Cannabinoid Receptor Modulators; Capsaicin; Charybdotoxin; Chlorides; Cyclohexanols; Dose-Response Relationship, Drug; Dronabinol; Drug Interactions; Endocannabinoids; Endothelium, Vascular; Female; In Vitro Techniques; Indoles; Male; Mesenteric Arteries; Pertussis Toxin; Piperidines; Polyunsaturated Alkamides; Potassium Channel Blockers; Potassium Channels; Pyrazoles; Rats; Rats, Wistar; Rimonabant; Vasodilation; Verapamil

Prostanoids and cannabinoids have ocular hypotensive and neuroprotective properties. The effect of the prostanoid AH13205 (EP2), the thromboxane-mimetic U46619, the cannabinoid (CB) agonists WIN55212-2 and CP 55,940, endothelin-1 (ET-1) and 8-bromo-cAMP on the membrane currents of trabecular meshwork (TM) cells were measured using the patch-clamp technique and compared to their effects on TM contractility. Previous studies show relaxation of TM to AH 13205 and other substances that elevate cAMP, while U46619 and endothelin-1 contract TM. This study shows that after contraction (100%) with carbachol (10(-6)m), the CB agonist CP 55,940 dose-dependently reduced contractility to 83+/-4% (n=9) (10(-6)m) and 61+/-10%, (n=7) (10(-5)m). In the presence of both the CB1 antagonist AM251 (10(-6)m) and CP 55,940 (10(-5)m), the contractile response to carbachol reached 84+/-3% (n=6) of the original level. In patch-clamp experiments, membrane permeable 8-bromo-cAMP (10(-4)m) had no effect on currents of TM cells. In contrast, AH 13205 and two cannabinoids reversibly enhanced outward current through high-conductance Ca(2+)-activated K(+) channels (BKCa, BK, maxi-K) to the following values (in % of the initial value at 100 mV): AH 13205 (10(-5)m): 200+/-28% (n=6), CP 55,940 (10(-6)m): 196+/-33% (n=7), CP 55,940 (10(-5)m): 484+/-113% (n=7), WIN55212-2 (10(-5)m): 205+/-41% (n=10). Iberiotoxin (10(-7)m) completely blocked these responses. The current response to CP 55,940 (10(-5)m) could be partially blocked by the CB1 antagonist AM251 (10(-6)m). Conversely, the contractile agents in this study either caused a transient reduction in outward current (ET-1(5x10(-8)m)) or had no effect (U46619 (10(-6)m)). We conclude that stimulation of EP2 and CB1 receptors in TM is coupled to the activation of BKCa channels via a non-diffusible second messenger cascade. This effect may contribute to the relaxant activity of EP2 and CB1 agonists in isolated TM strips, modulating ocular outflow.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; 8-Bromo Cyclic Adenosine Monophosphate; Adult; Aged; Animals; Benzoxazines; Calcium; Calcium Channel Blockers; Cannabinoids; Carbachol; Cattle; Cells, Cultured; Cholinergic Agonists; Cyclic AMP; Cyclohexanols; Endothelin-1; Humans; In Vitro Techniques; Ion Channel Gating; Middle Aged; Morpholines; Naphthalenes; Patch-Clamp Techniques; Peptides; Piperidines; Potassium Channels, Calcium-Activated; Prostanoic Acids; Pyrazoles; Receptor, Cannabinoid, CB1; Receptors, Prostaglandin E; Stimulation, Chemical; Trabecular Meshwork

Antinociceptive effects of cannabinoids are mediated, in part, at the spinal level. Cannabinoid CB1 receptors are co-localized with dorsal horn interneurons containing gamma-aminobutyric acid (GABA). In this study, we investigated the interaction between intrathecally administered cannabinoid and GABA(B) receptor agonists and antagonists in the modulation of formalin-induced pain at the spinal level. Intrathecal pretreatment of rats with a cannabinoid receptor antagonist [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1-H-pyrazole-3-carboxamide] (SR141716A, 30 microg) decreased the analgesic effect of the intrathecal administration of the GABA(B) receptor agonist, baclofen (0.125 microg and 0.25 microg). Intrathecal administration of the GABA(B) receptor antagonist, saclofen (30 microg), 10 min before administration of the cannabinoid receptor agonist (-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)-phenyl]-trans-4-(3-hydroxy-propyl)-cyclohexano (CP55940), did not affect the analgesia produced by the cannabinoid receptor agonist. Our results confirm that intrathecal administration of cannabinoid and GABA(B) receptor agonists have analgesic effects and that spinal antinociceptive effects of GABA(B) receptor agonists are likely through endocannabinoid modulation.

Topics: Analysis of Variance; Animals; Baclofen; Cyclohexanols; Dose-Response Relationship, Drug; Drug Interactions; Formaldehyde; GABA Agonists; GABA Antagonists; GABA-B Receptor Agonists; GABA-B Receptor Antagonists; Injections, Spinal; Male; Pain; Piperidines; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Receptors, GABA-B; Rimonabant; Signal Transduction; Spinal Cord

Exogenously administered cannabinoids are neuroprotective in several different cellular and animal models. In the current study, two cannabinoid CB1 receptor ligands (WIN 55,212-2, CP 55,940) markedly reduced hippocampal cell death, in a time-dependent manner, in cultured neurons subjected to high levels of NMDA (15 microM). WIN 55,212-2 was also shown to inhibit the NMDA-induced increase in intracellular calcium concentration ([Ca2+](i)) indicated by FURA-2 fluorescence imaging in the same cultured neurons. Changes in [Ca2+](i) occurred with similar concentrations (25-100 nM) and in the same time-dependent manner (pre-exposure 1-15 min) as CB1 receptor mediated neuroprotective actions. Both effects were blocked by the CB1 receptor antagonist SR141716A. An underlying mechanism was indicated by the fact that (1) the NMDA-induced increase in [Ca2+](i) was inhibited by ryanodine, implicating a ryanodine receptor (RyR) coupled intracellular calcium channel, and (2) the cannabinoid influence involved a reduction in cAMP cAMP-dependent protein kinase (PKA) dependent phosphorylation of the same RyR levels that regulate channel. Moreover the time course of CB1 receptor mediated inhibition of PKA phosphorylation was directly related to effective pre-exposure intervals for cannabinoid neuroprotection. Control studies ruled out the involvement of inositol-trisphosphate (IP3) pathways, enhanced calcium reuptake and voltage sensitive calcium channels in the neuroprotective process. The results suggest that cannabinoids prevent cell death by initiating a time and dose dependent inhibition of adenylyl cyclase, that outlasts direct action at the CB1 receptor and is capable of reducing [Ca2+](i) via a cAMP/PKA-dependent process during the neurotoxic event.

Topics: Animals; Benzoxazines; Calcium; Cell Culture Techniques; Cell Death; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclohexanols; Dantrolene; Drug Interactions; Estrenes; Fetus; Hippocampus; Macrocyclic Compounds; Morpholines; N-Methylaspartate; Naphthalenes; Neurons; Neuroprotective Agents; Okadaic Acid; Oxazoles; Piperidines; Pyrazoles; Pyrrolidinones; Rats; Rats, Inbred Strains; Receptor, Cannabinoid, CB1; Rimonabant; Ryanodine; Thionucleotides

Rats with a previous history of heroin self-administration were studied to assess interactions occurring between cannabinoids and opioids in an animal model of reinstatement of heroin-seeking behaviour. Rats were trained to self-administer heroin and after a long-term extinction were primed with one of the following non-contingent non-reinforced drug administrations: saline (or vehicle), heroin, synthetic cannabinoid CB(1) receptor agonists (WIN 55,212-2 or CP 55,940), opioid antagonist (naloxone) or CB(1) antagonist (SR 141716A), alone or in combination. After primings, lever-pressing activity was recorded and compared to those observed during previous phases of training and extinction. Results of this study showed that (i) priming injections of heroin (0.1 mg/kg) as well as CB(1) agonists WIN 55,212-2 (0.15 or 0.30 mg/kg) and CP 55,940 (0.05 or 0.1 mg/kg) completely restore heroin-seeking behaviour; (ii) primings of naloxone (1 mg/kg) and SR 141716A (0.3 mg/kg) had no effect when administered alone; (iii) heroin-induced reinstatement was fully prevented by pre-treatment with either naloxone or SR 141716A; (iv) pre-treatment with SR 141716A significantly reduced WIN 55,212-2 and CP 55,940 priming effects. These results suggest that cannabinoid CB(1) receptors play an important role in the mechanisms underlying relapse to heroin-seeking and depict CB(1) antagonists as possible therapeutic agents for use in the prevention of relapse to heroin abuse.

Topics: Animals; Behavior, Addictive; Benzoxazines; Conditioning, Operant; Cyclohexanols; Drug Interactions; Extinction, Psychological; Heroin; Heroin Dependence; Male; Morpholines; Naloxone; Naphthalenes; Piperidines; Pyrazoles; Rats; Rats, Inbred Strains; Receptor, Cannabinoid, CB1; Rimonabant; Self Administration

Little is known about the mechanism of action behind the orexigenic activity of cannabinoids. Neuropeptide Y (NPY) is one of the most potent orexigenic factors and is a key mediator in the hypothalamic control of food intake. We examined the effect of cannabinoids on NPY release using a rat hypothalamic explant model. The cannabinoid agonists anandamide (AEA) and CP55,940 both significantly augmented resting and KCl-evoked NPY release. AM251, a cannabinoid receptor antagonist, blocked the augmentation of NPY release elicited by AEA and CP55,940. Additionally, AM251 administered alone, in the absence of exogenous cannabinoid agonists, inhibited NPY release demonstrating the role of endogenous cannabinoids in NPY release. Combined, these findings demonstrate that cannabinoids augment NPY release in the hypothalamus and that this may be a potential mechanism behind the orexigenic activity of cannabinoids.

Topics: Analgesics; Animals; Arachidonic Acids; Cannabinoid Receptor Agonists; Cannabinoid Receptor Antagonists; Cannabinoids; Cyclohexanols; Dose-Response Relationship, Drug; Endocannabinoids; Hypothalamus; Male; Neuropeptide Y; Organ Culture Techniques; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Sprague-Dawley

An extended series of alkyl carboxamide analogs of N-(piperidinyl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl- 1H-pyrazole-3-carboxamide (SR141716; 5) was synthesized. Each compound was tested for its ability to displace the prototypical cannabinoid ligands ([3H]CP-55,940, [3H]2; [3H]SR141716, [3H]5; and [3H]WIN55212-2, [3H]3), and selected compounds were further characterized by determining their ability to affect guanosine 5'-triphosphate (GTP)-gamma-[35S] binding and their effects in the mouse vas deferens assay. This systematic evaluation has resulted in the discovery of novel compounds with unique binding properties at the central cannabinoid receptor (CB1) and distinctive pharmacological activities in CB1 receptor tissue preparations. Specifically, compounds with nanomolar affinity which are able to fully displace [3H]5 and [3H]2, but unable to displace [3H]3 at similar concentrations, have been synthesized. This selectivity in ligand displacement is unprecedented, in that previously, compounds in every structural class of cannabinoid ligands had always been shown to displace each of these radioligands in a competitive fashion. Furthermore, the selectivity of these compounds appears to impart unique pharmacological properties when tested in a mouse vas deferens assay for CB1 receptor antagonism.

Topics: Amides; Animals; Binding Sites; Brain; Cell Membrane; Cyclohexanols; Humans; Mice; Piperidines; Pyrazoles; Rats; Receptor, Cannabinoid, CB1; Rimonabant

In the present study, we investigated the effects of the cannabinoid receptor agonist CP55,940 on excitatory and inhibitory synaptic transmission in the rat supraoptic nucleus. Whole-cell patch clamp recordings were performed on supraoptic neurones in in vitro brain slice preparations. CP55,940 significantly reduced the frequency of spontaneous excitatory and inhibitory postsynaptic currents in a concentration-dependent manner. These changes were potently reversed by the CB1 receptor antagonist AM251. The results indicate that cannabinoids modulate the activity of magnocellular neurosecretory neurones by presynaptic inhibition of both excitatory and inhibitory synaptic transmission.

Topics: Anesthetics, Local; Animals; Cannabinoids; Cyclohexanols; Excitatory Postsynaptic Potentials; In Vitro Techniques; Male; Membrane Potentials; Neurons, Afferent; Patch-Clamp Techniques; Piperidines; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Receptors, Presynaptic; Supraoptic Nucleus; Synapses; Tetrodotoxin

We investigated the pharmacology of three novel compounds, Org 27569 (5-chloro-3-ethyl-1H-indole-2-carboxylic acid [2-(4-piperidin-1-yl-phenyl)-ethyl]-amide), Org 27759 (3-ethyl-5-fluoro-1H-indole-2-carboxylic acid [2-94-dimethylamino-phenyl)-ethyl]-amide), and Org 29647 (5-chloro-3-ethyl-1H-indole-2-carboxylic acid (1-benzyl-pyrrolidin-3-yl)-amide, 2-enedioic acid salt), at the cannabinoid CB1 receptor. In equilibrium binding assays, the Org compounds significantly increased the binding of the CB1 receptor agonist [3H]CP 55,940 [(1R,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol], indicative of a positively cooperative allosteric effect. The same compounds caused a significant, but incomplete, decrease in the specific binding of the CB1 receptor inverse agonist [3H]SR 141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride], indicative of a limited negative binding cooperativity. Analysis of the data according to an allosteric ternary complex model revealed that the estimated affinity of each Org compound was not significantly different when the radioligand was [3H]CP 55,940 or [3H]SR 141716A. However, the estimated cooperatively factor for the interaction between modulator and radioligand was greater than 1 when determined against [3H]CP 55,940 and less than 1 when determined against [3H]SR 141716A. [3H]CP 55,940 dissociation kinetic studies also validated the allosteric nature of the Org compounds, because they all significantly decreased radioligand dissociation. These data suggest that the Org compounds bind allosterically to the CB1 receptor and elicit a conformational change that increases agonist affinity for the orthosteric binding site. In contrast to the binding assays, however, the Org compounds behaved as insurmountable antagonists of receptor function; in the reporter gene assay, the guanosine 5'-O-(3-[35S]thio)triphosphate binding assay and the mouse vas deferens assay they elicited a significant reduction in the Emax value for CB1 receptor agonists. The data presented clearly demonstrate, for the first time, that the cannabinoid CB1 receptor contains an allosteric binding site that can be recognized by synthetic small molecule ligands.

Topics: Allosteric Regulation; Animals; Binding Sites; Cyclohexanols; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Male; Mice; Piperidines; Pyrazoles; Receptor, Cannabinoid, CB1; Rimonabant; Vas Deferens

It is well established that Cannabis sativa can increase appetite, particularly for sweet and palatable foods. In laboratory animals, cannabinoid CB1 receptor antagonism decreases motivation for palatable foods, and most recently, the CB1 receptor antagonist SR141716A, or rimonabant (Acomplia), was reported to produce weight loss in obese human subjects. Indeed, the endocannabinoid system plays a select role in the rewarding properties of palatable foods, and this is well characterized in laboratory animals with sweet sucrose solutions. In the present study, CB1 knockout mice (CB1 KO) and wild-type littermate mice (WT) were trained to respond for a complex sweet as well as a pure fat reinforcer under a progressive ratio (PR) schedule, to determine whether motivation to consume different palatable foods is tonically regulated by CB1 receptors. To assess sweet reinforcement, several concentrations of the liquid nutritional drink, Ensure, were presented under the PR schedule. For fat reinforcement, several concentrations of corn oil (emulsified in 3% xanthan gum) were made available. Additionally, to compare the result of genetic invalidation of the CB1 receptor to antagonism of the CB1 receptor system, the effect of SR141716A (3.0 mg/kg) on responding for Ensure and corn oil were also assessed using the PR schedule. We also assessed the effect of the CB1 agonist CP-55940 (30 microg/kg) on responding for Ensure and corn oil. CB1 KOs took significantly longer to acquire operant responding maintained by Ensure, and responding for Ensure under the PR schedule was significantly reduced in CB1 KOs as well as in WTs pretreated with SR141716A, as compared to WT controls. Additionally, pretreatment with the CB1 agonist CP-55940 increased responding for Ensure. In contrast, responding for corn oil during acquisition and under the PR schedule was not significantly different in CB1 KOs versus wild-type mice. However, SR141716A did reduce responding for corn oil in WTs, and CP-55940 significantly increased responding for corn oil. Taken together, these results suggest that CB1 receptors are preferentially involved in the reinforcing effects of a complex sweet, as compared to a pure fat, reinforcer. These data also suggest, however, that antagonism of CB1 receptors with SR141716A is sufficient to attenuate the reinforcing effect of Ensure and corn oil, while activation of the central CB1 system is sufficient to enhance Ensure and corn oil reinforcement.

Topics: Animals; Conditioning, Operant; Corn Oil; Cyclohexanols; Dietary Fats; Dietary Sucrose; Feeding Behavior; Female; Food, Formulated; Gene Deletion; Genotype; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Piperidines; Pyrazoles; Receptor, Cannabinoid, CB1; Reinforcement, Psychology; Rimonabant; Taste; Time Factors

Newly developed cannabinoids may hold the promise of the development of useful and safe drugs. This study aimed to investigate the behavioral effects of the novel 1',1'-dithiolane delta8-HC analogue AMG-3, a cannabinomimetic molecule with high affinity for CB1/CB2 receptors. This analog was chosen for its binding affinity to these receptors, which is higher than that reported for delta8-tetrahydrocannabinol (delta8-THC). Behavioral responses were assessed after the administration of AMG-3 (1, 2, 4, 8 mg/kg, i.p.) in the open field, on the bar test, on the hot plate and in the intracranial self-stimulation procedure. AMG-3 increased the reactivity time on the hot plate in a dose- and time-dependent manner, indicating a long-lasting analgesic effect (at least 24 h). The substance was found dose-dependently to decrease spontaneous motor activity and to induce catalepsy, particularly at the highest dose (8 mg/kg). AMG-3 did not affect the rewarding value of intracranial self-stimulation, except to increase the reward threshold at the highest dose (8 mg/kg). The effects of the highest dose of AMG-3 on spontaneous activity and on the self-stimulation paradigm were completely reversed by pre-treatment with the CB1 receptor antagonist AM-251. These findings indicate that the administration of AMG-3 to rats elicits a specific behavioral profile, most probably associated with the activation of CB1 receptors and without effects indicating abuse potential.

Topics: Animals; Behavior, Animal; Binding, Competitive; Cannabinoids; Catalepsy; Cell Membrane; Cerebral Cortex; Cyclohexanols; Dose-Response Relationship, Drug; Male; Molecular Structure; Motor Activity; Pain; Pain Measurement; Piperidines; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Time Factors; Tritium

Central nervous system responses to cannabis are primarily mediated by CB(1) receptors, which couple preferentially to G(i/o) G proteins. Here, we used calcium photometry to monitor the effect of CB(1) activation on intracellular calcium concentration. Perfusion with 5 microM CB(1) aminoalkylindole agonist, WIN55,212-2 (WIN), increased intracellular calcium by several hundred nanomolar in human embryonic kidney 293 cells stably expressing CB(1) and in cultured hippocampal neurons. The increase was blocked by coincubation with the CB(1) antagonist, SR141716A, and was absent in nontransfected human embryonic kidney 293 cells. The calcium rise was WIN-specific, being essentially absent in cells treated with other classes of cannabinoid agonists, including Delta(9)-tetrahydrocannabinol, HU-210, CP55,940, 2-arachidonoylglycerol, methanandamide, and cannabidiol. The increase in calcium elicited by WIN was independent of G(i/o), because it was present in pertussis toxin-treated cells. Indeed, pertussis toxin pretreatment enhanced the potency and efficacy of WIN to increase intracellular calcium. The calcium increases appeared to be mediated by G(q) G proteins and phospholipase C, because they were markedly attenuated in cells expressing dominant-negative G(q) or treated with the phospholipase C inhibitors U73122 and ET-18-OCH(3) and were accompanied by an increase in inositol phosphates. The calcium increase was blocked by the sarco/endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin, the inositol trisphosphate receptor inhibitor xestospongin D, and the ryanodine receptor inhibitors dantrolene and 1,1'-diheptyl-4,4'-bipyridinium dibromide, but not by removal of extracellular calcium, showing that WIN releases calcium from intracellular stores. In summary, these results suggest that WIN stabilizes CB(1) receptors in a conformation that enables G(q) signaling, thus shifting the G protein specificity of the receptor.

Topics: Analgesics; Animals; Arachidonic Acids; Benzoxazines; Calcium; Cannabinoids; Cell Line; Cyclohexanols; Cytoplasm; DNA, Complementary; Dronabinol; Endocannabinoids; Endoplasmic Reticulum; Excitatory Amino Acid Antagonists; Fluorescent Dyes; Fura-2; Glycerides; GTP-Binding Protein alpha Subunits, Gq-G11; Hippocampus; Humans; Immunosuppressive Agents; Morpholines; Naphthalenes; Neurons; Pertussis Toxin; Piperidines; Protein Binding; Protein Conformation; Pyrazoles; Rats; Receptor, Cannabinoid, CB1; Rimonabant; Ryanodine; Time Factors; Type C Phospholipases

1. The present study investigated the binding characteristics of various ligands to cannabinoid CB(1) receptors in human neocortex and amygdala. In addition, the functionality of CB(1) receptors in the human neocortex was assessed by examining the effects of CB(1) receptor ligands on evoked [(3)H]-dopamine (DA) release in superfused brain slices and on synaptosomal cAMP accumulation. 2. Saturation-binding assays in human neocortical and amygdala synaptosomes using a radiolabelled cannabinoid receptor agonist ([(3)H]-CP55.940) revealed pK(d) values of 8.96 and 8.63, respectively. The numbers of binding sites (B(max)) were 3.99 and 2.67 pmol (mg protein)(-1), respectively. 3. Various cannabinoid receptor ligands inhibited [(3)H]-CP55.940 binding with rank order potencies corresponding to those of previous studies in animal tissues. 4. Electrically evoked [(3)H]-DA release from human neocortical slices was inhibited by CP55.940 (IC(50) 6.76 nm, I(max) 65%) and strongly enhanced by the cannabinoid receptor antagonist AM251. However, [(3)H]-DA release was not influenced in rat neocortex. In human tissue, the estimated endocannabinoid concentration in the biophase of the release-modulating CB(1) receptors was 1.07 nm, expressed in CP55.940 units. 5. K(+)-evoked [(3)H]-DA release in the presence of tetrodotoxin (TTX) was strongly inhibited by CP55.940 in humans, but not in rats. 6. In human tissue, CP55.940 inhibited forskolin-stimulated cAMP accumulation (IC(50) 20.89 nm, I(max) 35%). AM251 blocked this effect and per se increased forskolin-stimulated cAMP accumulation by approximately 20%. 7. In conclusion, cannabinoids modulate [(3)H]-DA release and adenylyl cyclase activity in the human neocortex. CB(1) receptors are located on dopaminergic nerve terminals and seem to be tonically activated by endocannabinoids.

Topics: Adenylyl Cyclases; Amygdala; Animals; Arachidonic Acids; Benzoxazines; Binding Sites; Colforsin; Cyclic AMP; Cyclohexanols; Dopamine; Dronabinol; Electric Stimulation; Endocannabinoids; Female; Humans; Ligands; Male; Morpholines; Naphthalenes; Neocortex; Piperidines; Polyunsaturated Alkamides; Potassium; Pyrazoles; Rats; Receptor, Cannabinoid, CB1; Synaptosomes; Tetrodotoxin; Tritium

Both cannabinoid CB1 receptor agonists, such as delta-tetrahydrocannabinol (delta-THC), CP 55,940 and WIN 55,212-2, and the antagonist/inverse agonist SR141716A, dose-dependently suppress operant behavior. The present study investigated to what extent combined i.p. application of SR141716A with these cannabinoids resulted in mutually antagonistic effects, in additive effects, or in no interactive effects on operant responding in rats trained in a fixed-ratio 10, food-reinforced 10-min procedure. Pretreatment with SR141716A either had no effect on (at 0.3-1mg/kg), or partially blocked (at 3 mg/kg), the inhibitory effects on responding induced by delta-THC (3-5 mg/kg) and CP 55,940 (0.03-0.2 mg/kg). Interestingly, while 3 mg/kg SR141716A induced moderate inhibitory effects on operant responding, its combination with either agonist resulted in the same level of inhibitory activity on responding as that obtained by SR141716A when tested alone. Pretreatment with a low dose of CP 55,940 (0.01 mg/kg) or WIN 55,212-2 (0.3 mg/kg) did not affect response inhibition induced by SR141716A. Combination of SR141716A (0.5 and 1mg/kg) with delta-THC (3 mg/kg) resulted in the same level of response inhibition, independently of whether SR141716A was given 5 min before or 15 min after delta-THC. Although alternative explanations are conceivable, the data may indicate that SR141716A is a partial agonist at those cannabinoid receptors mediating the response-rate suppressive effects of cannabinoids.

Topics: Animals; Appetitive Behavior; Benzoxazines; Cannabinoids; Conditioning, Operant; Cyclohexanols; Dose-Response Relationship, Drug; Dronabinol; Drug Interactions; Excitatory Amino Acid Antagonists; Male; Morpholines; Motivation; Naphthalenes; Piperidines; Premedication; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Reinforcement Schedule; Rimonabant

Cannabinoid-MDMA interactions were examined in male Wistar rats. MDMA (4 x 5 mg/kg or 2 x 10 mg/kg over 4 h on each of 2 days) was administered with or without Delta 9-tetrahydrocannabinol (THC) (4 x 2.5 mg/kg), the synthetic cannabinoid receptor agonist CP 55,940 (2 x 0.1 or 0.2 mg/kg) or the cannabinoid receptor antagonist SR 141716 (2 x 5 mg/kg). Co-administered Delta 9-THC and CP 55,940 but not SR 141716 prevented MDMA-induced hyperthermia, causing a powerful hypothermia. Co-administered Delta 9-THC, CP 55,940 and SR 141716 all tended to decrease MDMA-induced hyperactivity. Co-administered Delta 9-THC provided protection against the long-term increases in anxiety seen in the emergence test, but not the social interaction test, 6 weeks after MDMA treatment. Co-administered Delta 9-THC and CP 55,940, but not SR 141716, partly prevented the long-term 5-HT and 5-HIAA depletion caused by MDMA in various brain regions. SR 141716 administered with CP 55,940 and MDMA prevented the hypothermic response to the CP 55,940/MDMA combination but did not alter the CP 55,940 attenuation of MDMA-induced 5-HT depletion. These results suggest a partial protective effect of co-administered cannabinoid receptor agonists on MDMA-induced 5-HT depletion and long-term anxiety. This action appears to operate independently of cannabinoid CB1 receptors.

Topics: Animals; Cannabinoids; Cyclohexanols; Fever; Male; Motor Activity; N-Methyl-3,4-methylenedioxyamphetamine; Piperidines; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Rimonabant; Serotonin

The roles of the two cannabinoid receptor subtypes, CB-1 and CB-2, have not been clarified in cannabinoid-mediated analgesia. We investigated the efficacy of the non-selective cannabinoid receptor agonist CP55,940 in the modulation of responses in the rat to both acute pain (tail flick) and neuropathic pain (tactile allodynia following chronic L5/6 spinal nerve ligation). Responses were also assessed in the presence of the CB-1 antagonist SR141716A (SR1) and the CB-2 antagonist SR144528 (SR2). CP55,940 attenuated tactile allodynia (ED(50) 0.04 mg/kg i.t. (95% CI 0.032-0.044 mg/kg), 0.12 mg/kg i.p. (95% CI 0.10-0.15 mg/kg)) and induced thermal antinociception (ED(50) tail flick 0.07 mg/kg i.t. (95% CI 0.05-0.10 mg/kg), 0.17 mg/kg i.p. (95% CI 0.11-0.26 mg/kg)). SR1 0.5 mg/kg i.t. attenuated the antinociceptive effect of CP55,940 in both modalities. However, SR1 1.0 mg/kg i.p. decreased tail flick latency but had no effect on tactile allodynia antinociception. In contrast, SR2 1.0 mg/kg i.p. significantly decreased the effect of i.p. CP55,940 on both tail flick antinociception and tactile allodynia (P<0.005). The combination of SR1 and SR2 (i.p.) had an additive effect in decreasing the antinociception induced by CP55,940 on tail flick responses (P<0.005). These results suggest a role for CB-2 receptor-mediated antinociception in both acute and neuropathic pain in addition to centrally located CB-1 mechanisms.

Topics: Analgesics; Analysis of Variance; Animals; Behavior, Animal; Camphanes; Cyclohexanols; Dose-Response Relationship, Drug; Drug Administration Routes; Drug Interactions; Ligation; Male; Nociceptors; Pain; Pain Measurement; Piperidines; Pyrazoles; Rats; Rats, Sprague-Dawley; Reaction Time; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Rimonabant; Spinal Nerves; Time Factors

We studied the effect of cannabinoids on the activity of N-methyl-d-aspartate (NMDA) receptors in the locus coeruleus from rat brain slices by single-unit extracellular recordings. As expected, NMDA (100 microM) strongly excited (by nine fold) the cell firing activity of the locus coeruleus. Perfusion with the endocannabinoid anandamide (1 and 10 microM) or the anandamide transport inhibitor AM 404 (30 microM) enhanced the NMDA-induced excitation of locus coeruleus neurons. Similarly, the synthetic agonists R(+)-WIN 55212-2 (10 microM) and CP 55940 (30 microM) enhanced the effect of NMDA. In the presence of the CB(1) receptor antagonists SR 141716A (1 microM) or AM 251 (1 microM), the enhancement induced by anandamide (10 microM) was blocked. Our results suggest that cannabinoids modulate the activity of NMDA receptors in the locus coeruleus through CB(1) receptors.

Topics: Action Potentials; Analgesics; Animals; Arachidonic Acids; Benzoxazines; Calcium Channel Blockers; Cannabinoids; Cyclohexanols; Drug Interactions; Electrophysiology; Endocannabinoids; Excitatory Amino Acid Agonists; In Vitro Techniques; Locus Coeruleus; Male; Morpholines; N-Methylaspartate; Naphthalenes; Neurons; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Rimonabant

The present study investigated the effects of different classes of cannabinoid (CB) receptor ligands on sensory neurotransmission in the rat isolated mesenteric arterial bed. Electrical field stimulation of the mesenteric bed evoked frequency-dependent vasorelaxation due to the activation of capsaicin-sensitive sensory nerves and release of calcitonin gene-related peptide (CGRP). The CB(1)/CB(2) cannabinoid agonists WIN55,212 [(R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone] and CP55,940 [(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl) cyclohexanol] (0.01-1 microM) attenuated sensory neurogenic relaxation in a concentration-dependent manner. At 0.1 microM, WIN55,212 and CP55,940 were largely ineffective in the presence of the CB(1) antagonists SR141716A [N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichloro phenyl)-4-methyl-3-pyrazole-carboxamide] and LY320135 [[6-methoxy-2-(4-methoxyphenyl)benzo[b]-thien-3-yl][4-cyanophenyl] methanone] (1 microM), but their inhibitory actions remained in the presence of the CB(2)-selective antagonist SR144528 [N-[1S)-endo-1,3,3,-trimetyl bicyclo [2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide] (1 microM). The CB(1)/CB(2) agonist Delta(9)-tetrahydrocannabinol (THC) (1 microM) attenuated sensory neurogenic relaxations, as did the CB(2) agonist JWH-015 [(2-methyl-1-propyl-1H-indol-3-yl)-1-naphthalenylmethanone]. The inhibitory actions of both THC and JWH-015 were still evident in the presence of SR141716A (1 microM) and SR144528 (1 microM). None of the cannabinoid agonists investigated had an effect on vasorelaxation elicited by exogenous CGRP, indicating a prejunctional mechanism. These data demonstrate that different classes of cannabinoid agonists attenuate sensory neurotransmission via a prejunctional site and provide evidence for mediation by a CB(1) and/or a non-CB(1)/CB(2) receptor.

Topics: Animals; Benzofurans; Benzoxazines; Calcitonin Gene-Related Peptide; Camphanes; Cannabinoids; Capsaicin; Cyclohexanols; Dronabinol; Electric Stimulation; Indoles; Male; Mesenteric Arteries; Morpholines; Naphthalenes; Piperidines; Pyrazoles; Rats; Rats, Wistar; Receptors, Cannabinoid; Rimonabant; Vasodilation

Marijuana is known to have psychotropic effects in humans. In this study, we used rat models of sensorimotor gating, hyperactivity and stereotypy to explore whether CB(1) receptor stimulation or blockade induces behavioral changes consistent with psychotomimetic or antipsychotic agents, respectively.. We determined whether (a) the cannabinoid agonist CP 55940 decreased pre-pulse inhibition (PPI) as might be expected from a psychotomimetic agent, and (b) the selective CB(1) receptor antagonist, SR 141716A, had any effect on PPI on its own or following disruptions by psychotomimetic agents. In addition, we investigated the effects of SR 141716A on elevated levels of hyperactivity and stereotypy elicited by d-amphetamine.. These studies were conducted in rats using standard methodologies for determination of PPI following acoustic stimuli, and d-amphetamine-induced hyperactivity and stereotypies.. Decreased startle responses to 120 dB stimuli were observed in rats treated with CP 55940 (0.1 mg/kg IP) in the absence and presence of a 73 dB pre-pulse. These effects were reversed by SR 141716A (5 and 10 mg/kg, respectively). SR 141716A (0.1, 5, 10 mg/kg) had no effect on PPI on its own or following disruptions by apomorphine, d-amphetamine or MK-801. Conversely, in separate experiments different antipsychotic agents reversed disruptions in PPI induced by d-amphetamine (haloperidol), apomorphine (haloperidol or clozapine) or MK-801 (clozapine or olanzapine). In addition, unlike haloperidol, SR 141716A (5 mg/kg) did not reverse d-amphetamine-mediated increases in hyperactivity or stereotypy.. The CP 55940-mediated decreases in startle amplitude confound assessment of the effects of CB(1) receptor activation on PPI. The failure of SR 141716A to reverse disruptions in PPI, hyperactivity or stereotypy induced by non-cannabinoid psychotomimetic agents suggests that blockade of the CB(1) receptor on its own is not sufficient for antipsychotic therapy.

Topics: Animals; Antipsychotic Agents; Apomorphine; Arousal; Attention; Behavior, Animal; Cyclohexanols; Dextroamphetamine; Dizocilpine Maleate; Dose-Response Relationship, Drug; Hallucinogens; Male; Motor Activity; Piperidines; Psychoses, Substance-Induced; Pyrazoles; Rats; Rats, Wistar; Receptors, Cannabinoid; Receptors, Drug; Reflex, Startle; Rimonabant; Stereotyped Behavior

We have investigated the effects of cannabinoid agonists and antagonists on tumour necrosis factor-alpha (TNF-alpha)-induced secretion of interleukin-8 from the colonic epithelial cell line, HT-29. The cannabinoid receptor agonists [(-)-3-[2-hydroxy-4-(1,1-dimethyl-heptyl)-phenyl]4-[3-hydroxypropyl]cyclo-hexan-1-ol] (CP55,940); Delta-9-tetrahydrocannabinol; [R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl) methyl] pyrrolo[1,2,3-de]1,4-benzoxazin-6-yl](1-naphthyl) methanone mesylate] (WIN55,212-2) and 1-propyl-2-methyl-3-naphthoyl-indole (JWH 015) inhibited TNF-alpha induced release of interleukin-8 in a concentration-dependent manner. The less active enantiomer of WIN55212-2, [S(-)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]1,4-benzoxazin-6-yl](1-naphthyl) methanone mesylate (WIN55212-3), and the cannabinoid CB(1) receptor agonist arachidonoyl-2-chloroethylamide (ACEA) had no significant effect on TNF-alpha-induced release of interleukin-8. The cannabinoid CB(1) receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1,4-pyrazole-3-carboxamide hydrochloride (SR141716A; 10(-6) M) antagonised the inhibitory effect of CP55,940 (pA(2)=8.3+/-0.2, n=6) but did not antagonise the inhibitory effects of WIN55212-2 and JWH 015. The cannabinoid CB(2) receptor antagonist N-(1,S)-endo1,3,3-trimethylbicyclo(2,2,1)heptan-2-yl)-5(4-chloro-3-methyl-phenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR144528; 10(-6) M) antagonised the inhibitory effects of CP55,940 (pA(2)=8.2+/-0.8, n=6), WIN55212-2 (pA(2)=7.1+/-0.3, n=6) and JWH 015 (pA(2)=7.6+/-0.3, n=6), respectively. Western immunoblotting of HT-29 cell lysates revealed a protein with a size that is consistent with the presence of cannabinoid CB(2) receptors. We conclude that in HT-29 cells, TNF-alpha-induced interleukin-8 release is inhibited by cannabinoids through activation of cannabinoid CB(2) receptors.

Topics: Arachidonic Acids; Benzoxazines; Camphanes; Cannabinoids; Cell Survival; Cyclohexanols; Dose-Response Relationship, Drug; HT29 Cells; Humans; Immunoblotting; Indoles; Interleukin-8; Kinetics; Morpholines; Naphthalenes; Piperidines; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Tumor Necrosis Factor-alpha

Dibenzopyran (Delta(9)-tetrahydrocannabinol) and aminoalkylindole [R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrolol[1,2,3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl) methanone mesylate; (WIN55,212-2)] cannabinoids suppress vomiting produced by cisplatin via cannabinoid CB(1) receptors. This study investigates the antiemetic potential of the "nonclassical" cannabinoid CP55,940 [1alpha,2beta-(R)-5alpha]-(-)-5-(1,1-dimethyl)-2-[5-hydroxy-2-(3-hydroxypropyl) cyclohexyl-phenol] against cisplatin-induced vomiting and assesses the presence and functionality of cannabinoid CB(1) receptors in the least shrew (Cryptotis parva) brain. CP55,940 (0.025-0.3 mg/kg) reduced both the frequency of cisplatin-induced emesis (ID(50)=0.025 mg/kg) and the percentage of shrews vomiting (ID(50)=0.09 mg/kg). CP55,940 also suppressed shrew motor behaviors (ID(50)=0.06- 0.21 mg/kg) at such doses. The antiemetic and motor-suppressant actions of CP55,940 were countered by SR141716A [N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methylpyrazole-3-carboxamide], indicating both effects are cannabinoid CB(1) receptor-mediated. Autoradiographic studies with [3H]-SR141716A and [35S]-GTPgammaS binding revealed that the distribution of the cannabinoid CB(1) receptor and its activation pattern are similar to rodent brain and significant levels are present in brain loci (e.g., nucleus tractus solitarius (NTS)) that control emesis. The affinity rank order of structurally diverse cannabinoid ligands for cannabinoid CB(1) receptor in shrew brain is similar to rodent brain: HU-210=CP55,940=SR141716A>/=WIN55,212-2>/=delta-9-tetrahydrocannabinol>methanandamide=HU-211=cannabidiol=2-arachidonoylglycerol. This affinity order is also similar and is highly correlated to the cannabinoid EC(50) potency rank order for GTPgammaS stimulation except WIN55,212-2 and delta-9-tetrahydrocannabinol potency order were reversed. The affinity and the potency rank order of tested cannabinoids were significantly correlated with their antiemetic ID(50) potency order against cisplatin-induced vomiting (CP55,940>WIN55,212-2=delta-9-tetrahydrocannabinol) as well as emesis produced by 2-arachidonoylglycerol or SR141716A (CP55,940>WIN55,212-2>delta-9-tetrahydrocannabinol).

Topics: Animals; Antiemetics; Benzoxazines; Binding, Competitive; Brain; Cisplatin; Cyclohexanols; Dose-Response Relationship, Drug; Dronabinol; Female; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Injections, Intraperitoneal; Injections, Subcutaneous; Male; Morpholines; Motor Activity; Naphthalenes; Piperidines; Pyrazoles; Radioligand Assay; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Shrews; Sulfur Radioisotopes; Tritium; Vomiting

Recently, the gastrointestinal pharmacology of cannabinoid CB(1) receptors has been extensively explored. We employed western blotting and immunohistochemistry techniques to study the distribution of the cannabinoid CB(1) receptor protein in the mouse gastroenteric tract. The cannabinoid CB(1) receptor peptide was detected by western blotting only in its glycosylated form (63 kDa) with a significant differential distribution. The highest levels of expression were detected in the stomach and in the colon, while the pyloric valve was devoid of any cannabinoid CB(1) receptor protein. The immunohistochemical study showed intense cannabinoid CB(1) receptor immunoreactivity in ganglia subadjacent to the gastric epithelium and in the smooth muscle layers of both the small and large intestine. Only the small intestine showed (-)-3-[2-hydroxyl-4-(1,1-dimethylheptyl)-phenyl]-4-(3-hydroxylpropyl) cyclohexan-1-ol) ([3H]CP 55,940) specific binding (27%). These receptors mediated pharmacologically significant effects since the cannabinoid CB(1) receptor agonist R(-)-7-hydroxy-delta-6-tetra-hydrocannabinol-dimethylheptyl (HU 210) dose dependently inhibited gastrointestinal transit up to 70%, while the cannabinoid CB(1) receptor antagonist N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methylpyrazole-3-carboxamide (SR 141716A) increased gastrointestinal transit. Moreover, the dose of 0.3 microg/kg of HU 210, devoid per se of any activity on mouse intestinal propulsion, blocked the increased gastroenteric transit induced by the cannabinoid CB(1) antagonist SR 141716A.

Topics: Animals; Antiemetics; Binding, Competitive; Blotting, Western; Cyclohexanols; Digestive System; Dose-Response Relationship, Drug; Dronabinol; Gastric Mucosa; Gastrointestinal Transit; Immunohistochemistry; Intestine, Large; Intestine, Small; Male; Mice; Piperidines; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Stomach; Tritium

Cannabinoids, whether plant-derived, synthetic or endogenous, have been shown to stimulate appetite in the adult organism. We have reported previously that cannabinoid receptors play a critical role during the early suckling period: The selective cannabinoid CB(1) receptor antagonist N-(piperidiny-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141617A) permanently prevented milk ingestion in a dose-dependent manner, when administered to (Sabra, albino) mouse pups, within 1 day of birth. As a consequence, these pups died within the first week of life. We now generalize this finding to a different strain of mice (C57BL/6). Further, we show that cannabinoid CB(1) receptor blockade (20 mg/kg SR141716A) must occur within 24 h after birth as injection of SR141716A into 2- or 5-day-old pups had a much smaller effect or no effect at all, respectively. Cannabinoid CB(1) receptor knockout mice did not ingest milk on the first day of life, similarly to SR141716A-treated normal pups, as measured by the appearance of "milkbands". However, the knockout pups started to display milkbands from day 2 of life. Survival rates of cannabinoid CB(1) receptor knockout mice were affected significantly, but to a lesser extent than normal pups, by the administration of SR141716A. Daily administration of the endocannabinoid 2-arachidonoyl glycerol, or the synthetic agonists (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (WIN55,212-2, 5 mg/kg) or (-)-cis-3-[2-Hydroxy4-(1,1-dimethylheptyl) phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol (CP55,940, 5 or 20 mg/kg) did not promote survival or weight gain in CB(1)(-/-) pups. Our data support previous evidence for a critical role of cannabinoid CB(1) receptors for the initiation of suckling. Further, the present observations support the existence of an unknown cannabinoid receptor, with partial control over milk ingestion in newborns. Our data also suggest that the CB(1)(-/-) neonates possess a compensatory mechanism which helps them overcome the lack of cannabinoid CB(1) receptors.

Topics: Animals; Animals, Newborn; Animals, Suckling; Arachidonic Acids; Behavior, Animal; Benzoxazines; Cannabinoid Receptor Modulators; Cyclohexanols; Dose-Response Relationship, Drug; Drug Administration Schedule; Eating; Endocannabinoids; Female; Glycerides; Injections, Subcutaneous; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Milk; Morpholines; Naphthalenes; Piperidines; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Survival Rate; Time Factors

We have recently reported that the administration of AM404, an inhibitor of the endocannabinoid re-uptake process, which also has affinity for the vanilloid VR1 receptors, is able to reduce hyperkinesia, and causes recovery from neurochemical deficits, in a rat model of Huntington's disease (HD) generated by bilateral intrastriatal injections of 3-nitropropionic acid (3NP). In the present study, we wanted to explore the mechanism(s) by which AM404 produces its antihyperkinetic effect in 3NP-lesioned rats by employing several experimental approaches. First, we tried to block the effects of AM404 with selective antagonists for the CB1 or VR1 receptors, i.e. SR141716A and capsazepine, respectively. We found that the reduction caused by AM404 of the increased ambulation exhibited by 3NP-lesioned rats in the open-field test was reversed when the animals had been pre-treated with capsazepine but not with SR141716A, thus suggesting a major role of VR1 receptors in the antihyperkinetic effects of AM404. However, despite the lack of behavioral effects of the CB1 receptor antagonist, the pretreatment with this compound abolished the recovery of neurochemical [gamma-aminobutyric acid (GABA) and dopamine] deficits in the caudate- putamen caused by AM404, as also did capsazepine. In a second group of studies, we wanted to explore the potential antihyperkinetic effects of various compounds which, compared to AM404, exhibit more selectivity for either the endovanilloid or the endocannabinoid systems. First, we tested VDM11 or AM374, two selective inhibitors or the endocannabinoid re-uptake or hydrolysis, respectively. Both compounds were mostly unable to reduce hyperkinesia in 3NP-lesioned rats, although VDM11 produced a certain motor depression, and AM374 exhibited a trend to stimulate ambulation, in control rats. We also tested the effects of selective direct agonists for VR1 (capsaicin) or CB1 (CP55,940) receptors. Capsaicin exhibited a strong antihyperkinetic activity and, moreover, was able to attenuate the reductions in dopamine and GABA transmission provoked by the 3NP lesion, whereas CP55,940 had also antihyperkinetic activity but was unable to cause recovery of either dopamine or GABA deficits in the basal ganglia. In summary, our data indicate a major role for VR1 receptors, as compared to CB1 receptors, in the antihyperkinetic effects and the recovery of neurochemical deficits caused in 3NP-lesioned rats by compounds that activate both CB1 and VR1 receptors,

Topics: 3,4-Dihydroxyphenylacetic Acid; Amino Acids, Neutral; Animals; Arachidonic Acids; Basal Ganglia; Cannabinoid Receptor Modulators; Capsaicin; Corpus Striatum; Cyclohexanols; Disease Models, Animal; Dopamine; Endocannabinoids; gamma-Aminobutyric Acid; Huntington Disease; Hyperkinesis; Male; Motor Activity; Nitro Compounds; Palmitates; Piperidines; Propionates; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptors, Cannabinoid; Receptors, Drug; Rimonabant

The effects of a range of cannabinoid receptor agonists and antagonists on phytohaemagglutinin-induced secretion of interleukin-2 from human peripheral blood mononuclear cells were investigated. The nonselective cannabinoid receptor agonist WIN55212-2 ((R)-(+)-[2,3-dihydro-5-methyl-3-[4-morpholinylmethyl]pyrrolo[1,2,3-de]1,4-benzoxazin-6-yl](1-naphthyl) methanone mesylate) and the selective cannabinoid CB(2) receptor agonist JWH 015 ((2-methyl-1-propyl-1H-indol-3-yl)-1-napthalenylmethanone) inhibited phytohaemagglutinin (10 microg/ml)-induced release of interleukin-2 in a concentration-dependent manner (IC(1/2max), WIN55212-2=8.8 x 10(-7) M, 95% confidence limits (C.L.)=2.2 x 10(-7)-3.5 x 10(-6) M; JWH 015=1.8 x 10(-6) M, 95% C.L.=1.2 x 10(-6)-2.9 x 10(-6) M, n=5). The nonselective cannabinoid receptor agonists CP55,940 ((-)-3-[2-hydroxy-4-(1,1-dimethyl-hepthyl)-phenyl]4-[3-hydroxypropyl]cyclo-hexan-1-ol), Delta(9)-tetrahydrocannabinol and the selective cannabinoid CB(1) receptor agonist ACEA (arachidonoyl-2-chloroethylamide) had no significant (P>0.05) inhibitory effect on phytohaemagglutinin-induced release of interleukin-2. Dexamethasone significantly (P<0.05) inhibited phytohaemagglutinin-induced release of interleukin-2 in a concentration-dependent manner (IC(1/2max)=1.3 x 10(-8) M, 95% C.L.=1.4 x 10(-9)-3.2 x 10(-8) M). The cannabinoid CB(1) receptor antagonist SR141716A (N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride) (10(-6) M) did not antagonise the inhibitory effect of WIN55212-2 whereas the cannabinoid CB(2) receptor antagonist SR144528 (N-(1,S)-endo-1,3,3-trimethyl bicyclo(2,2,1)heptan-2-yl)-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide) antagonised the inhibitory effect of WIN55212-2 (pA(2)=6.3+/-0.1, n=5). In addition, CP55,940 (10(-6) M) and Delta(9)-tetrahydrocannabinol (10(-6) M) also antagonised the inhibitory effects of WIN55212-2 (pA(2)=6.1+/-0.1, n=5 and pA(2)=6.9+/-0.2, n=5). In summary, WIN55,212-2 and JWH 015 inhibited interleukin-2 release from human peripheral blood mononuclear cells via the cannabinoid CB(2) receptor. In contrast, CP55,940 and Delta(9)-tetrahydrocannabinol behaved as partial agonists/antagonists in these cells.

Topics: Arachidonic Acids; Benzoxazines; Camphanes; Cell Survival; Cyclohexanols; Dose-Response Relationship, Drug; Dronabinol; Humans; Indoles; Interleukin-2; Leukocytes, Mononuclear; Morpholines; Naphthalenes; Phytohemagglutinins; Piperidines; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant

Three experiments examined the influence of pre-exposure to the cannabinoid receptor agonist CP 55940 ((-)-cis-3-(2-hydroxy-4-(1,1-dimethylheptyl)phenyl)-trans-4-(3-hydroxypropyl)cyclohexanol) on the sensitization of morphine-induced locomotor hyperactivity and self-administration in Lewis rats. In Experiment 1, rats received daily injections of vehicle or CP 55940 (0.1 mg/kg for 7 days then 0.2 mg/kg for a further 7 days). Four weeks later, the locomotor response to morphine (10 mg/kg s.c.) was tested once per day over a 3-h period for 14 consecutive days. Rats given morphine showed hypoactivity during the first hour following morphine but hyperactivity during the second and third hours. A progressive increase in hyperactivity to morphine was seen over the 14 days of administration, which was significantly greater in rats pre-treated with CP 55940. In Experiment 2, rats were given morphine (10 mg/kg) once a day for 14 days in combination with either vehicle, CP 55940 (0.1 mg/kg) or the cannabinoid CB(1) receptor antagonist SR 141716 (N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride) (3 mg/kg). Both CP 55940 and SR 141716 initially inhibited the hyperactive response to morphine, but these effects gradually wore off and by the end of 14 days, hyperactivity was similar in all morphine-treated groups. When tested 3 weeks later for their response to morphine (10 mg/kg) given alone, rats previously given the morphine/CP 55940 combination, but not the SR 141716/morphine combination, showed a greater locomotor stimulation than those previously exposed to morphine only. In Experiment 3, rats were pre-exposed to CP 55940 or vehicle for 14 days and were subsequently trained to self-administer morphine intravenously (1 mg/kg per lever press) for 14 days. Rats pre-exposed to CP 55940 self-administered a significantly greater number of morphine infusions than vehicle pre-exposed rats. However, both active and inactive ('dummy') lever presses were increased by cannabinoid pre-treatment. Overall, these results suggest that cannabinoid pre-exposure can lead to an exaggeration of morphine-induced hyperactivity and may alter the reinforcing effects of morphine in Lewis rats. The implications for 'gateway' theories of cannabinoid effects in humans are discussed.

Topics: Analgesics; Animals; Behavior, Animal; Cannabinoids; Cyclohexanols; Drug Synergism; Male; Morphine; Motor Activity; Piperidines; Pyrazoles; Rats; Rats, Inbred Lew; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Self Administration; Time Factors

Our objective was to identify the sites of interaction of cannabinoids with cardiovascular sympathetic regulation in the rat. Effects on sympathetic tone were first determined in anaesthetised animals following i.v. administration of the drugs. Central effects were evaluated in anaesthetised rats receiving microinjections of cannabinoids into brain stem nuclei. Peripheral effects were identified in pithed rats with electrically stimulated sympathetic outflow. In anaesthetised and artificially ventilated rats, i.v. injection of the cannabinoid agonists WIN55212-2 and CP55940 decreased mean arterial pressure, heart rate and the plasma noradrenaline concentration. These effects were antagonized by the CB(1) cannabinoid receptor antagonist SR141716A. The bradycardia was abolished by the muscarinic acetylcholine receptor antagonist methylatropine. The decreases in mean arterial pressure and heart rate caused by cannabinoids in ventilated rats were much less pronounced than in spontaneously breathing rats. Microinjection of WIN55212-2 into the nucleus tractus solitarii had no effect. Microinjected into the rostral ventrolateral medulla oblongata, WIN55212-2 lowered mean arterial pressure slightly without changing other parameters. In pithed rats, WIN55212-2 inhibited the increases in mean arterial pressure, heart rate and the plasma noradrenaline concentration evoked by electrical stimulation of the sympathetic outflow. Our results show that activation of CB(1) cannabinoid receptors induces sympathoinhibition and enhancement of cardiac vagal tone, leading to hypotension and bradycardia. Presynaptic inhibition of noradrenaline release from terminals of postganglionic sympathetic neurons is the major component of the sympathoinhibition, but an effect in the rostral ventrolateral medulla oblongata may also contribute. The cannabinoid-evoked cardiovascular depression depends strongly on the respiratory state of the animals.

Topics: Animals; Atropine Derivatives; Benzoxazines; Bradycardia; Cannabinoids; Cardiovascular System; Cyclohexanols; Disease Models, Animal; Dose-Response Relationship, Drug; Hypotension; Male; Medulla Oblongata; Microinjections; Morpholines; Naphthalenes; Norepinephrine; Piperidines; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Rimonabant; Sympathetic Fibers, Postganglionic; Sympathetic Nervous System

Like other G-protein coupled receptors with hydrophobic ligands, the human cannabinoid receptor 1 (CB1) is thought to bind its ligands within the transmembrane region of the receptor. However, for some of these receptors the extracellular loops (ECs) have also been shown to play a role in ligand recognition and selectivity. We have taken a mutagenesis approach to examine the role of the amino terminus, EC1, and EC3 of CB1 in ligand binding. Eight mutant receptors, each with a dipeptide insertion, were constructed, expressed, and evaluated for binding to the cannabinoid ligands (-)-cis-3[2-hydroxy-4-(1',1'-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol (CP 55,940) and N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR 141716A). Mutants with insertions in the membrane distal region of the amino terminus or EC3 maintained affinity for both ligands. Those with insertions in the membrane proximal region of the amino terminus or EC1 exhibited a loss of affinity for CP 55,940 while retaining wild-type affinity for SR 141716A. Representative mutants were analyzed for agonist-induced inhibition of cyclic AMP accumulation, and the results indicated that G-protein coupling remained intact. Alanine substitution mutants were made to address whether it was the perturbation of the overall structure of the region or the displacement of particular side chains that was responsible for the loss of CP 55,940 binding. We conclude that a structurally intact EC1, but not the comparably short EC3, is essential for high-affinity CP 55,940 binding.

Topics: Amino Acid Sequence; Animals; Binding, Competitive; Cells, Cultured; CHO Cells; Cricetinae; Cyclohexanols; Dose-Response Relationship, Drug; Humans; Molecular Sequence Data; Piperidines; Protein Conformation; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant

1. There is considerable interest in novel therapies for cough, since currently used agents such as codeine have limited beneficial value due to the associated side effects. Sensory nerves in the airways mediate the cough reflex via activation of C-fibres and RARs. Evidence suggests that cannabinoids may inhibit sensory nerve-mediated responses. 2. We have investigated the inhibitory actions of cannabinoids on sensory nerve depolarisation mediated by capsaicin, hypertonic saline and PGE2 on isolated guinea-pig and human vagus nerve preparations, and the cough reflex in conscious guinea-pigs. 3. The non-selective cannabinoid (CB) receptor agonist, CP 55940, and the selective CB2 agonist, JWH 133 inhibited sensory nerve depolarisations of the guinea-pig vagus nerve induced by hypertonic saline, capsaicin and PGE2. These responses were abolished by the CB2 receptor antagonist SR144528, and unaffected by the CB1 antagonist SR141716A. Similarly, JWH 133 inhibited capsaicin-evoked nerve depolarisations in the human vagus nerve, and was prevented by SR144528. 4. Using a guinea-pig in vivo model of cough, JWH 133 (10 mg kg-1, i.p., 20 min) significantly reduced citric acid-induced cough in conscious guinea pigs compared to those treated with the vehicle control. 5. These data show that activation of the CB2 receptor subtype inhibits sensory nerve activation of guinea-pig and human vagus nerve, and the cough reflex in guinea-pigs, suggesting that the development of CB2 agonists, devoid of CB1-mediated central effects, will provide a new and safe antitussive treatment for chronic cough.

Topics: Animals; Camphanes; Cannabinoids; Capsaicin; Consciousness; Cough; Cyclohexanols; Dinoprost; Dose-Response Relationship, Drug; Guinea Pigs; Humans; Hypertonic Solutions; In Vitro Techniques; Male; Middle Aged; Neurons, Afferent; Piperidines; Pyrazoles; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Reflex; Rimonabant; Vagus Nerve

The cannabinoid CB(1) receptor transmembrane helix (TMH) 3-4-5-6 region includes an aromatic microdomain comprised of residues F3.25, F3.36, W4.64, Y5.39, W5.43, and W6.48. In previous work, we have demonstrated that aromaticity at position 5.39 in CB(1) is crucial for proper function of CB(1). Modeling studies reported here suggest that in the inactive state of CB(1), the binding site of the CB(1) inverse agonist/antagonist SR141716A is within the TMH3-4-5-6 aromatic microdomain and involves direct aromatic stacking interactions with F3.36, Y5.39, and W5.43, as well as hydrogen bonding with K3.28. Further, modeling studies suggest that in the active state of CB(1), the CB agonist WIN55,212-2 binds in this same aromatic microdomain, with direct aromatic stacking interactions with F3.36, W5.43, and W6.48. In contrast, in the binding pocket model, the CB agonist anandamide binds in the TMH2-3-6-7 region in which hydrogen bonding and C-H.pi interactions appear to be important. Only one TMH3 aromatic residue, F3.25, was found to be part of the anandamide binding pocket. To probe the importance of the TMH3-4-5-6 aromatic microdomain to ligand binding, stable transfected cell lines were created for single-point mutations of each aromatic microdomain residue to alanine. Improper cellular expression of the W4.64A was observed and precluded further characterization of this mutation. The affinity of the cannabinoid agonist CP55,940 was unaffected by the F3.25A, F3.36A, W5.43A, or W6.48A mutations, making CP55,940 an appropriate choice as the radioligand for binding studies. The binding of SR141716A and WIN55,212-2 were found to be affected by the F3.36A, W5.43A, and W6.48A mutations, suggesting that these residues are part of the binding site for these two ligands. Only the F3.25A mutation was found to affect the binding of anandamide, suggesting a divergence in binding site regions for anandamide from WIN55,212-2, as well as SR141716A. Taken together, these results support modeling studies that identify the TMH3-4-5-6 aromatic microdomain as the binding region of SR141716A and WIN55,212-2, but not of anandamide.

Topics: Benzoxazines; Binding Sites; Cyclohexanols; Humans; Immunochemistry; Ligands; Models, Molecular; Morpholines; Naphthalenes; Piperidines; Protein Structure, Secondary; Protein Structure, Tertiary; Pyrazoles; Radioligand Assay; Receptor, Cannabinoid, CB1; Rimonabant

In this study we investigated the effect of cannabinoids on [3H]glutamate release from hippocampal synaptosomes of rat and CB1-null mutant mouse. In the rat, cannabinoid receptor agonists, i.e. CP55,940 (EC50, 0.84 microm), WIN55,212-2 (EC50, 3.47 microm), ACEA (EC50, 17.8 microm), and R-(+)-methanandamide (EC50, 19.8 microm) concentration-dependently inhibited the 25-mm-K+ depolarization-evoked release of [3H]glutamate and, among them, WIN55,212-2 displayed the greatest efficacy. The CB1 receptor antagonists SR141716A (1-5 microm) and AM251 (1 microm) and the VR1 vanilloid receptor antagonist capsazepine (10 microm) did not antagonize the effect of the agonists. SR141716A by itself attenuated the evoked [3H]glutamate release. WIN55,212-2 inhibited the release of [3H]glutamate in CB1 -/- mice as well. These data demonstrate that the action of cannabinoids on glutamate release in the hippocampus is pharmacologically distinct and independent from the cloned CB1 receptor.

Topics: Analgesics; Animals; Arachidonic Acids; Benzoxazines; Cannabinoids; Capsaicin; Chromatography, High Pressure Liquid; Cyclohexanols; Dose-Response Relationship, Drug; Drug Interactions; Glutamic Acid; Hippocampus; Male; Morpholines; Naphthalenes; Piperidines; Pyrazoles; Radioactivity; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Rimonabant; Synaptosomes; Tritium

Cannabinoids, as a result of their ability to activate cannabinoid CB1 receptors, have been shown to possess neuroprotective properties in vivo. In vitro studies into neuroprotective effects mediated by CB1 receptors have in general used primary neuronal cultures derived from embryonic rodents. In the present study, we have investigated whether embryonic chick telencephalon primary cultures in serum-free medium are a useful alternative for such in vitro studies. The CB agonist CP 55940 reduced the cAMP response to 5 microM forskolin by 40 and 50% at concentrations of 3 nM and 30 nM, respectively. This reduction was blocked by the CB1 receptor antagonist AM251, indicating the presence of functional CB1 receptors in the cultures. Incubation of the cultures with glutamate (100 microM or 1 mM) for 1 h followed by medium change and incubation for 24 h produced a release of the cytoplasmic enzyme lactate dehydrogenase into the medium. This release was prevented by MK-801 confirming the central role of NMDA receptors in the glutamate toxicity. However, 3-30 nM CP 55940 did not produce any neuroprotection in this model regardless as to whether dibutyryl cyclic AMP was added to the culture medium. The endocannabinoid anandamide was also without effect when added either per se or together with the related N-acyl ethanolamines palmitoylethanolamide, oleoylethanolamide and stearoylethanolamide (at relative concentrations matching those seen in rat brain after excitotoxic insult). It is concluded that embryonic chick neurons in primary serum-free culture are not a useful model for the study of neuroprotective effects mediated by CB1 receptors in vitro.

Topics: Animals; Arachidonic Acids; Cannabinoids; Cells, Cultured; Chick Embryo; Colforsin; Cyclic AMP; Cyclohexanols; Dizocilpine Maleate; Dose-Response Relationship, Drug; Drug Combinations; Endocannabinoids; Excitatory Amino Acid Antagonists; Glutamic Acid; L-Lactate Dehydrogenase; Models, Animal; Neurons; Pipecolic Acids; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB1; Telencephalon

I.c.v. self-administration of MDMA (0.01-2 micro g per infusion), alone and in combination with CP 55,940 (0.4 micro g infusion(-1)), was studied on an operant responding procedure. On the basis of individual preference for one of two levers, developed during training, rats were allowed to self-administer vehicle from the preferred lever and MDMA from the other. Pressings on the MDMA associated-lever, except for the maximal unit dose, progressively increased. The combination of CP 55,940 with MDMA (1 micro g infusion(-1)) reduced the number of drug-associated lever pressings compared to the single drugs. Pre-treatment with SR 141716A (0.5 mg kg(-1) i.p.), 15 min before each daily session, significantly increased MDMA self-administration. These findings suggest that MDMA self-administration is under endogenous tonic control by the endocannabinoid system.

Topics: Animals; Cannabinoid Receptor Modulators; Cannabinoids; Conditioning, Operant; Cyclohexanols; Dose-Response Relationship, Drug; Endocannabinoids; Fatty Acids, Unsaturated; Hallucinogens; Injections, Intraventricular; Male; N-Methyl-3,4-methylenedioxyamphetamine; Piperidines; Pyrazoles; Rats; Rats, Wistar; Reinforcement, Psychology; Rimonabant; Self Administration

We studied the delay in gastric emptying and gastrointestinal transit induced by the cannabinoid receptor agonists (+)-WIN 55,212-2 (R(+)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl)methanone mesylate) and CP 55,940 ((-)-cis-3[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl) cyclohexanol), as prevented by the selective cannabinoid CB(1)-receptor antagonist SR141716 ((N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methylpyrazole-3-carboxamide)) in rats after systemic or central drug administration. Oral SR141716 showed comparable potency (ID(50) range 1.0-3.9 mg/kg) in antagonizing gastric emptying and gastrointestinal transit delay by (+)-WIN 55,212-2 or CP 55,940. Gastric emptying and gastrointestinal transit delay after intracerebroventricular (i.c.v.) (+)-WIN 55,212-2 was prevented by oral or i.c.v. SR141716, but i.c.v. SR141716 did not significantly reduce the effect of i.p. (+)-WIN 55,212-2. Pertussis toxin prevented the delaying action of i.c.v. (+)-WIN 55,212-2 on both gastric emptying and gastrointestinal transit, but had no effect on (+)-WIN 55,212-2 i.p. These findings are consistent with a primary role of peripheral cannabinoid CB(1) receptor mechanisms in gastrointestinal transit delay by specific agonists.

Topics: Administration, Oral; Analysis of Variance; Animals; Benzoxazines; Cannabinoids; Cyclohexanols; Dose-Response Relationship, Drug; Gastric Emptying; Gastrointestinal Transit; Injections, Intraventricular; Male; Morpholines; Naphthalenes; Piperidines; Pyrazoles; Rats; Receptors, Cannabinoid; Receptors, Drug; Rimonabant

The central cannabinoid receptor (CB(1)) antagonist, SR-141716A, has been used extensively to ascertain that cannabinoids interact with the CB(1) receptor. SR-141716A has been shown to produce effects opposite of cannabinoids when administered alone. It has been theorized that SR-141716A may act as an inverse agonist at the CB(1) receptor or by disinhibiting an endogenous cannabinoid tone. In an effort to ascertain the exact interaction between SR-141716A and the CB(1) receptor, we have conducted a structure-activity relationship study to compare CB(1) receptor affinity of SR-141716A analogs with their ability to produce an increase in locomotor activity. SR-141716A produced a significant increase in locomotor activity in mice within the first hour of administration. Twenty SR-141716A analogs from five different chemical series were also tested. Our data implicate particular regions of the SR-141716A molecule that may be involved in stimulation and depression of locomotor activity. When the K(I) of the analogs was plotted against the percent stimulation that each analog produced, it is evident that there is no correlation between the ability of the analogs to stimulate locomotor activity and their affinity for the CB(1) receptor. [35S]GTPgammaS binding data indicate that SR-141716A and five of the analogs are inverse agonists. However, none of the analogs demonstrating inverse agonism produce stimulation of locomotor activity. It is therefore concluded that the SR-141716A-induced stimulation in locomotor activity is not the result of inverse agonist activity at the CB(1) receptor or by disinhibition of an endogenous tone.

Topics: Animals; Cyclohexanols; Guanosine 5'-O-(3-Thiotriphosphate); Male; Mice; Mice, Inbred ICR; Motor Activity; Piperidines; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Stimulation, Chemical; Structure-Activity Relationship

Cannabinoids, including the endogenous cannabinoid or endocannabinoid, anandamide, modulate several gastrointestinal functions. To date, the gastrointestinal effects of the second putative endocannabinoid 2-arachidonoylglycerol (2-AG) have not been studied. In the present study using a shrew (Cryptotis parva) emetic model, 2-AG (0.25-10 mg/kg, i.p.) potently and dose-dependently increased vomiting frequency (ED(50) = 1.13 mg/kg) and the number of animals vomiting (ED(50) = 0.48 mg/kg). In contrast, neither anandamide (2.5-20 mg/kg) nor methanandamide (5-10 mg/kg) induced a dose-dependent emetogenic response, but both could partially block the induced emetic effects. Delta(9)-Tetrahydrocannabinol and its synthetic analogs reduced 2-AG-induced vomiting with the rank order potency: CP 55,940 > WIN 55,212-2 > Delta(9)-tetrahydrocannabinol. The nonpsychoactive cannabinoid, cannabidiol, was inactive. Nonemetic doses of SR 141716A (1-5 mg/kg) also blocked 2-AG-induced vomiting. The 2-AG metabolite arachidonic acid also caused vomiting. Indomethacin, a cyclooxygenase inhibitor, blocked the emetogenic effects of both arachidonic acid and 2-AG. CP 55,940 also blocked the emetic effects of arachidonic acid. 2-AG (0.25-10 mg/kg) reduced spontaneous locomotor activity (ED(50) = 11 mg/kg) and rearing frequency (ED(50) = 4.3 mg/kg) in the shrew, whereas such doses of both anandamide and methanandamide had no effect on locomotor parameters. The present study indicates that: 1) 2-AG is an efficacious endogenous emetogenic cannabinoid involved in vomiting circuits, 2) the emetic action of 2-AG and the antiemetic effects of tested cannabinoids are mediated via CB(1) receptors, and 3) the emetic effects of 2-AG occur in lower doses relative to its locomotor suppressant actions.

Topics: Animals; Antiemetics; Arachidonic Acids; Cannabinoid Receptor Modulators; Cannabinoids; Cyclohexanols; Dose-Response Relationship, Drug; Dronabinol; Emetics; Endocannabinoids; Female; Glycerides; Humans; Male; Motor Activity; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Shrews

The purpose of this study was to investigate the role of the central cannabinoid receptor (CB(1)) in mediating the actions of the endogenous cannabinoid agonist anandamide and the synthetic cannabinoid CP-55940. Activation of primary mouse astrocyte cultures by exposure to bacterial lipopolysaccharide (LPS) caused a marked (approximately tenfold) increase in nitric oxide (NO) release. Coincubation with the cannabinoid agonists anandamide or CP-55940 markedly inhibited release of NO (-12% to -55%). This effect was abolished by SR-141716A (1 microM), a CB1 receptor antagonist. SR-141716A alone also significantly increased NO release in response to LPS, suggesting that endogenous cannabinoids modify inflammatory responses. In contrast, coincubation with the CB2 receptor antagonist SR-144528 (1 microM) abolished the inhibitory effects of the endogenous cannabinoid anandamide on LPS-induced NO release, although this may reflect nonspecific effects of this ligand or cannabinoid actions through atypical receptors of anandamide. We also showed that endogenous or synthetic cannabinoids inhibit LPS-induced inducible NO synthase expression (mRNA and protein) in astrocyte cultures. These results indicate that CB1 receptors may promote antiinflammatory responses in astrocytes.

Topics: Analgesics; Animals; Arachidonic Acids; Astrocytes; Calcium Channel Blockers; Camphanes; Cannabinoids; Cells, Cultured; Cyclohexanols; Dronabinol; Endocannabinoids; Gene Expression; Lipopolysaccharides; Mice; Mice, Inbred Strains; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant

The peripubertal period appears to be critical in relation to the abuse of cannabinoids and opioids in humans. However there is little information about the acute effects of cannabinoids and their interactions with opioids in young experimental animals. We have studied the effects of the cannabinoid agonist CP 55,940 (0.1, 0.2, 0.4 and 0.6 mg/kg) on the nociceptive responses (tail immersion test) and holeboard activity of 40-day-old rats, and the involvement of the CB(1) receptor (antagonism by SR 141716A, 3 mg/kg). The implication of the opioid system was evaluated using the opioid antagonist naloxone (1 mg/kg) and a combined treatment with subeffective doses of CP 55,940 (0.1 mg/kg) and morphine (1 mg/kg). The effects of CP 55,940 on the serum corticosterone levels (radioimmunoassay) and on the dopamine and DOPAC contents of discrete brain regions (high-performance liquid chromatography) were also assessed. The antinociceptive effect of CP 55,940 was of a similar magnitude at all the doses used. The results show the involvement of the CB(1) receptor. The cannabinoid agonist significantly depressed the holeboard activity in a dose-dependent manner. The results indicate that the CB(1) receptor is involved in the effects on motor activity but not in the effects on the exploratory activity. The behavioural effects of CP 55,940 were modulated by morphine. The cannabinoid agonist (0.6 mg/kg) induced a CB(1)-mediated increase in the serum corticosterone levels, but no effect on the dopaminergic systems of either the striatum or the limbic forebrain was found.

Topics: Analgesics; Animals; Behavior, Animal; Cannabinoids; Corticosterone; Cyclohexanols; Dopamine; Dose-Response Relationship, Drug; Drug Interactions; Female; Grooming; Male; Marijuana Abuse; Morphine; Motor Activity; Naloxone; Narcotic Antagonists; Neostriatum; Neurosecretory Systems; Nociceptors; Pain Measurement; Piperidines; Pyrazoles; Rats; Rats, Wistar; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Sex Characteristics

Cannabinoid CB1 receptors occur as presynaptic receptors producing inhibition of neurotransmitter release. To elucidate their physiological role, experiments on tissues from CB1 receptor knockout mice would be helpful. We studied whether CB1 receptor-mediated inhibition of acetylcholine release is detectable in the brain of NMRI mice and of CD-1 and C57BL/6J mice (the latter two strains representing the wild-type strains of the two CB1 receptor knockout mouse models). Brain slices preincubated with [3H]choline were superfused and tritium overflow was evoked electrically (3 Hz) or by introduction of Ca2+ into Ca2+-free K+-rich medium (35 mM) containing tetrodotoxin. The eletrically evoked tritium overflow from NMRI mouse hippocampal slices was inhibited (maximally by 60%) by the cannabinoid receptor agonists CP-55,940 and WIN 55,212-2 but not affected by WIN 55,212-3 (the inactive enantiomer of WIN 55,212-2; pEC50=7.9, 7.4 and <5.5). The concentration-response curve of WIN 55,212-2 was shifted to the right by the CB1 receptor antagonist SR 141716 (apparent pA2=8.6). Compared to hippocampal slices from NMRI mice, WIN 55,212-2 1 microM inhibited the electrically evoked overflow (1) from cortical slices from NMRI mice to a lesser extent and from striatal slices not at all, (2) from hippocampal slices from CD-1 and C57BL/6J mice to an identical extent and (3) from hippocampal slices from Sprague-Dawley rats to at least the same extent. SR 141716 0.32 microM abolished the effect of WIN 55,212-2 1 microM in hippocampal slices from NMRI, CD-1 and C57BL/6J mice and in cortical slices from NMRI mice. The electrically evoked tritium overflow from NMRI mouse hippocampal slices was also inhibited by the muscarinic receptor agonist oxotremorine (maximum effect of 85%; pEC50=6.5) and this effect was antagonized by the muscarinic receptor antagonist AF-DX 384 (apparent pA2=8.3). The Ca2+-evoked tritium overflow from NMRI mouse hippocampal slices was inhibited by WIN 55,212-2 in a manner sensitive to SR 141716. In conclusion, the cholinergic axon terminals of the NMRI mouse hippocampus are endowed with presynaptic CB1 receptors. Such receptors are also detectable in the hippocampus of CD-1 and C57BL/6J mice. The maximum extent of the CB1 receptor-mediated inhibition of acetylcholine release is lower than the maximum effect mediated via the autoreceptor.

Topics: Acetylcholine; Animals; Benzoxazines; Brain; Calcium; Choline; Cyclohexanols; Dose-Response Relationship, Drug; Electric Stimulation; Hippocampus; In Vitro Techniques; Male; Mice; Mice, Inbred C57BL; Mice, Inbred Strains; Morpholines; Naphthalenes; Oxotremorine; Piperidines; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Tritium

The effect of CP 55,940 [(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclo-hesanol], heroin and etonitazene on intracerebroventricular (i.c.v.) self-administration in a free-choice procedure was evaluated in rats. Animals were trained in 1-h daily sessions with a continuous reinforcement schedule to press two active levers to obtain the vehicle of each drug. Then, when a stable baseline was reached, each drug could be self-administered by pressing the lever found to be less preferred during training, while the vehicle came from the other. The number of bar pressings associated with the delivery of increasing unit doses of CP 55,940 (0.1, 0.2, 0.4, 0.8, 1.6 microg/2 microl/infusion), heroin (0.125, 0.25, 0.5, 1, 2 microg/2 microl/infusion) or etonitazene (0.1--0.2--0.5--1 microg/ 2 microl/infusion) and with the delivery of the corresponding vehicle was fitted by symmetrical parabolas. The mean drug intake was linearly related to the log of self-administered drugs. Pretreatment with SR141716A [N-piperidino-5-(4-chlorophenyl)1-(2,4-dichloro-phenyl)-4-methylpyrazole-3-carboxamide] (0.5 mg/kg) or naloxone HCl (2 mg/kg/i.p.) 15 min before each daily session reduced the self-administration of both CP 55,940 and heroin. The combination of CP 55,940 with heroin or etonitazene reduced the number of drug-associated lever pressings compared to that obtained with the maximal reinforcing unit dose of each drug alone. These findings suggest there may be a strong interaction between opioids and the cannabinoid system.

Topics: Analgesics; Analgesics, Opioid; Animals; Benzimidazoles; Conditioning, Operant; Cyclohexanols; Heroin; Male; Naloxone; Narcotic Antagonists; Narcotics; Piperidines; Pyrazoles; Rats; Rats, Wistar; Receptors, Cannabinoid; Receptors, Drug; Reinforcement Schedule; Rimonabant; Self Administration

1. The interaction between the cannabinoid agonists, WIN 55,212-2 or CP 55,940 with the CB(1) receptor-selective antagonists, SR141716A or LY320135 was investigated using the rat electrically-stimulated vas deferens bioassay. 2. Tissues were stimulated by single-field pulses (150 V, 0.5 ms) delivered every 30 mins. In the presence of nifedipine (3 microM), agonists elicited a concentration-dependent inhibition of the contractile response, with pEC(50) values of 7.93 and 6.84 for WIN 55,212-2 and CP 55,940, respectively. 3. SR141716A and LY320135 caused parallel dextral displacements of the agonist concentration-response curves. However, the shift of the agonist curves by either antagonist was accompanied by a concentration-dependent enhancement of basal (agonist-independent) tissue contraction. 4. Addition of the amidase inhibitor, phenylmethylsulphonylfluoride (200 microM), resulted in a significant reduction of the basal twitch response, an effect consistent with the presence of tonic receptor activation mediated by the endogenous cannabinoid, anandamide. 5. In light of these findings, we propose a theoretical model of competitive agonist-antagonist interaction in the presence of endogenous agonist tone that was used to derive an optimized analytical approach for the determination of antagonist potency estimates under conditions of tonic receptor activation. 6. This approach yielded pK(B) estimates for SR141716A and LY320135 that were in good agreement with their activity at cannabinoid CB(1) receptors. 7. It is concluded that the rat vas deferens contains prejunctional cannabinoid CB(1) receptors that are under tonic activation from endogenous substances; under these conditions our analytical approach is preferable to the standard methods for the determination of antagonist potency.

Topics: Animals; Benzofurans; Benzoxazines; Calcium Channel Blockers; Cyclohexanols; Electric Stimulation; In Vitro Techniques; Male; Morpholines; Naphthalenes; Piperidines; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Vas Deferens

The purpose of these studies was to characterize the effects of agonists of the CB(1) cannabinoid receptor on cerebellar function in mice. We used two measures specific for cerebellar function: gait analysis and the bar cross test. CB(1) receptor agonists CP55940, Win 55212-2, Delta(9)-tetrahydrocannabinol, arachidonylethanolamide (AEA), and two AEA analogs with high affinity for the CB(1) receptor (arachidonyl-2-chloroethylamide and arachidonylcyclopropylamide) all produced increases in gait width, a measure of truncal ataxia. All of the CB(1) agonists tested significantly increased the number of slips on the bar cross test, which is consistent with motor incoordination. Pretreatment with the CB(1) receptor antagonist SR141716 attenuated both the change in gait width and number of slips induced by CP55940 and AEA. Neither cannabidiol nor Win 55212-3 affected these measures, further evidence that this effect is mediated by the CB(1) receptor. Pretreatment with the dopamine receptor agonists apomorphine or bromocriptine did not attenuate the diminished performance on the bar cross or the gait abnormality induced by CP55940. These data indicate that the assays used in this study are specific for cerebellar-mediated behavioral deficits, and that these deficits are not mediated by the basal ganglia or cannabinoid-induced alterations in nigrostriatal dopaminergic transmission. Other well known effects of cannabinoids in mice, such as hyperreflexia exemplified by jumping or "popcorn" behavior and postural hypotonia are discussed in relationship to cerebellar dysfunction and a working model of the effects of CB(1) receptor activation on cerebellar circuitry is presented.

Topics: Animals; Behavior, Animal; Benzoxazines; Calcium Channel Blockers; Cannabinoids; Cerebellar Diseases; Cyclohexanols; Dopamine Agonists; Gait; Male; Mice; Mice, Inbred ICR; Morpholines; Naphthalenes; Piperidines; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant

The pharmacological properties of brain cannabinoid receptors were investigated in brains of 35 day-old chickens, since little is known about the avian cannabinoid system. The cannabinoid1 receptor-selective antagonist ligand [3H]SR 141716A bound to chicken brain membranes with K(D) and Bmax values of 0.92+/-0.28 nM and 790+/-58 fmol/mg protein, respectively. The binding was inhibited by CP 55,940 with a pI50 value of 7.63+/-0.14 and by a series of compounds with the order of potency CP 55,940>R(+)WIN 55,212-2>R-1 methanandamide approximately DAK. S(-)WIN 55,212-3 and AM404 were without inhibitory effect at 1 microM. Similar results were found for rat brain membranes. For both rat and chicken brain membranes, addition of the non-hydrolysable GTP analogues Gpp[NH]p and GTPgammaS shifted the CP 55,940 inhibition curve to the right, consistent with an intact coupling to G-proteins in the preparations. Fatty acid amidohydrolase in chicken brain membranes was less sensitive to inhibition by phenylmethylsulphonyl fluoride and arachidonoyl serotonin than its rodent equivalent. However, when fatty acid amidohydrolase activity in the preparations was reduced by use of a lower assay membrane concentration, anandamide was found to inhibit the binding of [3H]SR 141716A to chicken membranes with a pI50 value of 6.39+/-0.16. Using a novel antibody raised to amino acids 346-359 from the C-terminal tail of the human cannabinoid2 receptor, it was found that embryonic chick brain tissue (and embryonic chick neurones in primary culture) expressed a approximately 53 kDa immunoreactive band. This immunoreactivity, which was prevented by preincubation of the antibody with the immunising peptide, was also seen in cells expressing the recombinant human cannabinoid, receptor, but was not seen in adult chicken brain homogenates or in rat cerebellar homogenates. However, a "classical" cannabinoid2-receptor component of [3H]WIN 55212-2 binding (i.e. a fraction inhibited by low concentrations of the cannabinoid2-receptor-selective antagonist SR 144528) was not found.

Topics: Amidohydrolases; Animals; Benzoxazines; Blotting, Western; Brain; Cell Membrane; Chick Embryo; Cyclohexanols; Drug Interactions; Ligands; Morpholines; Naphthalenes; Piperidines; Protein Binding; Pyrazoles; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Species Specificity

We have examined the effects of cannabinoid agonists on hyperalgesia in a model of neuropathic pain in the rat and investigated the possible sites of action. The antihyperalgesic activity of the cannabinoids was compared with their ability to elicit behavioural effects characteristic of central cannabinoid activity. WIN55,212-2 (0.3-10 mg kg(-1)), CP-55,940 (0.03-1 mg kg(-1)) and HU-210 (0.001-0.03 mg kg(-1)) were all active in a 'tetrad' of tests consisting of tail-flick, catalepsy, rotarod and hypothermia following subcutaneous administration, with a rank order of potency in each of HU-210 > CP-55,940 > WIN55,212-2. The effects of WIN55,212-2 in each assay were blocked by the Cannabinoid1 (CB1) antagonist SR141716A. In the partial sciatic ligation model of neuropathic pain WIN55,212-2, CP-55,940 and HU-210 produced complete reversal of mechanical hyperalgesia within 3 h of subcutaneous administration with D50 values of 0.52, 0.08 and 0.005 mg kg(-1), respectively. In this model WIN55,212-2 was also effective against thermal hyperalgesia and mechanical allodynia. WIN55,212-2 produced pronounced reversal of mechanical hyperalgesia following intrathecal administration that was blocked by the CB1 antagonist SR141716A. Following intraplantar administration into the ipsilateral hindpaw, WIN55,212-2 produced up to 70% reversal of mechanical hyperalgesia, although activity was also observed at high doses following injection into the contralateral paw. The antihyperalgesic effect of WIN55,212-2 injected into the ipsilateral paw was blocked by subcutaneously administered SR141716A, but was not affected by intrathecally administered SR141716A. These data show that cannabinoids are highly potent and efficacious antihyperalgesic agents in a model of neuropathic pain. This activity is likely to be mediated via an action in both the CNS and in the periphery.

Topics: Analgesics; Animals; Benzoxazines; Cannabinoids; Cyclohexanols; Disease Models, Animal; Dronabinol; Hyperalgesia; Morpholines; Naphthalenes; Pain Measurement; Piperidines; Pyrazoles; Rats; Rats, Wistar; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Sciatica

The two inbred strains of mice C57BL/6 (alcohol-preferring) and DBA/2 (alcohol-avoiding) mice have been shown to differ significantly in their preference for alcohol (EtOH). We have previously demonstrated the differences in the density and the affinity of cannabinoid (CB1) receptors in the brains of the two inbred C57BL/6 and DBA/2 mouse strains. In the present study, we investigated the CB1 receptor agonist-stimulated guanosine-5'-O-(3-[(35)S]thio)-triphosphate ([(35)S]GTPgammaS) binding in plasma membranes (PM) from C57BL/6 and DBA/2 mice. The results indicate that the net CP55,940-stimulated [(35)S]GTPgammaS binding was increased with increasing concentrations of CB1 receptor agonists and GDP. The net CB1 receptor agonist (WIN55,212-2 or HU-210 or CP55,940)-stimulated [(35)S]GTPgammaS binding was reduced significantly (-10% to -12%, P < 0.05) in PM from DBA/2 mice; no significant differences were observed in basal [(35)S]GTPgammaS binding among these strains. Nonlinear regression analysis of net CP55,940-stimulated [(35)S]GTPgammaS binding showed that the B(max) of cannabinoid agonist-stimulated binding was significantly reduced (-24%) in DBA/2 mice (B(max) = 12.43 +/- 0.64 for C57BL/6 and 9.46 +/- 0.98 pmol/mg protein for DBA/2; P < 0.05) without any significant changes in the G protein affinity. The pharmacological specificity of CP55,940-stimulated [(35)S]GTPgammaS binding was examined with CB1 receptor antagonist SR141716A, and these studies indicated that CP55,940-stimulated [(35)S]GTPgammaS binding was blocked by SR141716A, with a decrease in the IC(50) values in the PM from the DBA/2 mouse strain. These results suggest that a signal transduction pathway(s) downstream from the CB1 receptor system may play an important role in controlling the voluntary EtOH consumption by these strains of mice.

Topics: Analgesics; Animals; Brain; Cannabinoids; Cyclohexanols; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Male; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Piperidines; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Signal Transduction; Species Specificity

Cannabinoids appear atypical as drugs of abuse since controversial data exist concerning the ability to lower the thresholds for electrical self-stimulation (Stark and Dews, 1980; Gardner et al., 1988; Gardner, 1992) and to support self-administration (Martellotta et al., 1998; Tanda et al., 2000) or conditioned place preference in animals (Lepore et al., 1995; Parker and Gillies, 1995; McGregor et al., 1996; Sañudo-Peña et al., 1997; Chaperon et al., 1998; Hutcheson et al., 1998; Mallet and Beninger, 1998; Cheer et al., 2000; Valjent and Maldonado, 2000). Opioids and cannabinoids share some pharmacological properties (Manzanares et al., 1999). The most interactions were found in antinociception (Welch and Stevens, 1992; Smith et al., 1994) and, to a lesser extent, in drug reinforcement (Chen et al., 1990; Vela et al., 1995; Tanda et al., 1997). In the present study we asked whether: (1) a potent synthetic cannabinoid receptor agonist, [(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptil)-phenyl]-trans-4-(3-hydroxy propyl) cyclohexanol] (CP 55,940) (from 10 to 40 microg/kg), which binds to the brain cannabinoid receptors with high affinity (Herkenham et al., 1991), would induce conditioned place preference, in comparison with heroin (from 0.1 to 5 mg/kg); (2) what type of receptor was involved; (3) what kind of interaction there was between the two drugs, when given in combination, on reward. CP 55,940 elicited a conditioned place preference only at a dose of 20 microg/kg similar in intensity to that of heroin (2 mg/kg). The reinforcing properties of the cannabinoid agonist were fully antagonised by pretreatment with the brain cannabinoid receptor-1 (CB(1)) antagonist, [N-piperidino-5-(4-chlorophenyl) 1-(2,4-dichloro-phenyl)-4-methyl pyrazole-3-carboxamide hydrochloride] (SR 141716A) and naloxone. The combination of CP 55,940 and heroin, at the reinforcing doses, led to a reward which did not show any additive effect. Taken together these findings are important for understanding how the cannabinoids produce reward and the interconnection of the opioid and cannabinoid system in the motivation.

Topics: Analgesics; Analgesics, Opioid; Animals; Brain; Conditioning, Psychological; Cyclohexanols; Dose-Response Relationship, Drug; Drug Interactions; Heroin; Male; Naloxone; Narcotic Antagonists; Opioid Peptides; Piperidines; Pyrazoles; Rats; Rats, Wistar; Receptors, Cannabinoid; Receptors, Drug; Reward; Rimonabant

1. In the absence of indomethacin, anandamide did not contract the guinea-pig bronchus at concentrations up to 100 microM. In the presence of indomethacin (10 microM), anandamide induced concentration-related contractions with a pEC(50) value of 5.18+/-0.11. It was significantly less potent than capsaicin (pEC(50) 7.01+/-0.1). The anandamide uptake inhibitor AM404, produced only a 14.1+/-3.22% contraction at 100 microM. All experiments were conducted in the presence of PMSF (20 microM). 2. The vanilloid receptor antagonist, capsazepine (10 microM), significantly attenuated the contractile effect of anandamide, the response to 100 microM anandamide being 40.53+/-7.04% in the presence of vehicle and 1.57+/-8.93% in the presence of 10 microM capsazepine. The contractile actions of anandamide and AM404 were markedly enhanced by the peptidase inhibitor thiorphan. 3. The log concentration-response curve of anandamide was unaltered by the CB1 receptor antagonist, SR141716A. The pEC(50) values for anandamide were 4.88+/-0.08 and 5.17+/-0.19 in the presence of vehicle and SR141716A (1 microM) respectively. 4. The lipoxygenase inhibitors 5,8,11,14-eicosatetraynoic acid (ETYA) and 5,8,11 eicosatriynoic acid (ETI) reduced the effect of 100 microM anandamide from 34.7+/-1.9% (vehicle) to 7.7+/-5% (ETYA, 10 microM) and from 41.85+/-4.25% (n=6) (vehicle) to 10.31+/-3.54 (n=6) (ETI, 20 microM). Neither inhibitor significantly affected contraction of the tissue by substance P. 5. This study provides evidence that anandamide acts on vanilloid receptors in the guinea-pig isolated bronchus. These data raise the possibility that the contractile action of anandamide may be due, at least in part, to lipoxygenase metabolites of this fatty acid amide that are vanilloid receptor agonists.

Topics: 5,8,11,14-Eicosatetraynoic Acid; Animals; Arachidonic Acids; Bronchi; Calcium Channel Blockers; Capsaicin; Cyclohexanols; Dose-Response Relationship, Drug; Endocannabinoids; Fatty Acids, Unsaturated; Guinea Pigs; Hydrazines; In Vitro Techniques; Indomethacin; Lipoxygenase; Muscle Contraction; Oxazepines; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptors, Drug; Rimonabant; Thiorphan

1. The effects of anandamide on [3H]-acetylcholine release and muscle contraction were studied on the myenteric plexus-longitudinal muscle preparation of the guinea-pig ileum preincubated with [3H]-choline. 2. Anandamide increased both basal [3H]-acetylcholine release (pEC(50) 6.3) and muscle tone (pEC(50) 6.3). The concentration-response curves for anandamide were shifted to the right by 1 microM capsazepine (pK(B) 7.5 and 7.6), and by the combined blockade of NK1 and NK3 tachykinin receptors with the antagonists CP99994 plus SR142801 (each 0.1 microM). The CB1 and CB2 receptor antagonists, SR141716A (1 microM) and SR144528 (30 nM), did not modify the facilitatory effects of anandamide. 3. Anandamide inhibited the electrically-evoked release of [3H]-acetylcholine (pEC(50) 5.8) and contractions (pEC(50) 5.2). The contractile response to the muscarinic agonist methacholine was not significantly affected by 10 microM anandamide. 4. The inhibitory effects of anandamide were not changed by either capsazepine (1 microM), SR144528 (30 nM) or CP99994 plus SR142801 (each 0.1 microM). SR141716A (1 microM) produced rightward shifts in the inhibitory concentration-response curves for anandamide yielding pK(B) values of 6.6 and 6.2. 5. CP55940 inhibited the evoked [3H]-acetylcholine release and contractions, and SR141716A (0.1 microM) shifted the concentration-response curves of CP55940 to the right with pK(B) values of 8.4 and 8.9. 6. The experiments confirm the existence of release-inhibitory CB1 receptors on cholinergic myenteric neurones. We conclude that anandamide inhibits the evoked acetylcholine release via stimulation of a receptor that is different from the CB1 and CB2 receptor. Furthermore, anandamide increases basal acetylcholine release via stimulation of vanilloid receptors located at primary afferent fibres.

Topics: Acetylcholine; Animals; Arachidonic Acids; Camphanes; Capsaicin; Cyclohexanols; Dose-Response Relationship, Drug; Electric Stimulation; Endocannabinoids; Guinea Pigs; Ileum; In Vitro Techniques; Male; Methacholine Chloride; Muscle Contraction; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant

The effect of CP55,940, a presumed CB1/CB2 cannabinoid receptor agonist, on intracellular free Ca2+ levels ([Ca2+]i) in Madin-Darby canine kidney cells was examined by using the fluorescent dye fura-2 as a Ca2+ indicator. CP55,940 (2-50 microM) increased [Ca2+]i concentration-dependently with an EC50 of 8 microM. The [Ca2+]i signal comprised an initial rise and a sustained phase. Extracellular Ca2+ removal decreased the maximum [Ca2+]i signals by 32+/-12%. CP55,940 (20 microM)-induced [Ca2+]i signal was not altered by 5 microM of two cannabinoid receptor antagonists, AM-251 and AM-281. CP55,940 (20 microM)-induced [Ca2+]i increase in Ca2+-free medium was inhibited by 86+/-3% by pretreatment with 1 microM thapsigargin, an endoplasmic reticulum Ca2+ pump inhibitor. Conversely, pretreatment with 20 microM CP55,940 in Ca2+-free medium for 6 min abolished thapsigargin-induced [Ca2+]i increases. CP55,940 (20 microM)-induced intracellular Ca2+ release was not inhibited when inositol 1,4,5-trisphosphate formation was abolished by suppressing phospholipase C with 2 microM U73122. Collectively, this study shows that CP,55940 induced significant [Ca2+]i increases in canine renal tubular cells by releasing stored Ca2+ from the thapsigargin-sensitive pools in an inositol 1,4,5-trisphosphate-independent manner, and also by causing extracellular Ca2+ entry. The CP55,940's action appears to be dissociated from stimulation of cannabinoid receptors.

Topics: Animals; Calcium; Cannabinoids; Cell Line; Cyclohexanols; Dogs; Estrenes; Extracellular Space; Indicators and Reagents; Inositol 1,4,5-Trisphosphate; Kidney; Morpholines; Piperidines; Pyrazoles; Pyrrolidinones; Receptors, Cannabinoid; Receptors, Drug

The aim of the present study was to examine the effects of the cannabinoid agonist CP 55,940 and the antagonist SR 141716A, alone and in combination, on rat exploratory and anxiety-like behaviour in the holeboard and elevated plus-maze tests. A further aim was to evaluate the effects of these treatments on hypothalamic neurotransmitters. Animals treated with CP 55,940 doses of 0.125 and 0.1 mg/kg exhibited less exploration and an increase in anxiety-like behaviour accompanied by great motor inhibition. No hypoactivity was seen at 0.075 mg/kg dosage, but anxiety and neophobic responses persisted, indicating independent and specific effects. Motor activity effects induced by CP 55,940 were reversed by pretreatment with SR 141716A (3 mg/kg). Surprisingly, when administered on its own, the antagonist also induced a reduction in exploratory parameters and an increase in anxiety-like responses. These apparently similar effects might be caused by different neural mechanisms. Finally, CP 55,940 increased hypothalamic dopamine and serotonin levels. These increases might be involved in the activation of the hypothalamic-pituitary-adrenal axis described for cannabinoids.

Topics: Analgesics; Animals; Anxiety; Cannabinoids; Cyclohexanols; Drug Therapy, Combination; Exploratory Behavior; Hypothalamus; Male; Motor Activity; Neurotransmitter Agents; Piperidines; Pyrazoles; Rats; Rats, Wistar; Rimonabant

Feline immunodeficiency virus (FIV) infection causes a widespread natural immunodeficiency syndrome in cats that is considered a suitable animal model for studying human immunodeficiency virus (HIV) infection and pathogenesis. Short term cultures of bone marrow derived feline macrophages stimulated with recombinant feline interferon-gamma (r-IFN-gamma) and lipopolysaccharide (LPS) were shown to produce nitric oxide. Feline macrophages were shown to express cannabinoid receptors, and nitric oxide production decreased after in vitro exposure to synthetic cannabinoid CP-55940. Both cannabinoid receptors, CB1 and CB2, were involved in this process, since the inhibition was reversed by selective cannabinoid antagonists for both of these receptors.

Topics: Animals; Bone Marrow Cells; Camphanes; Cannabinoids; Cats; Cyclohexanols; Disease Models, Animal; Feline Acquired Immunodeficiency Syndrome; Histocytochemistry; Immunosuppressive Agents; Macrophages; Nitric Oxide; Phagocytosis; Piperidines; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant

1. We have studied the effect of cannabinoid agonists (CP 55,940 and cannabinol) on intestinal motility in a model of intestinal inflammation (induced by oral croton oil in mice) and measured cannabinoid receptor expression, endocannabinoids (anandamide and 2-arachidonylglycerol) and anandamide amidohydrolase activity both in physiological and pathophysiological states. 2. CP 55,940 (0.03 - 10 nmol mouse(-1)) and cannabinol (10 - 3000 nmol mouse(-1)) were more active in delaying intestinal motility in croton oil-treated mice than in control mice. These inhibitory effects were counteracted by the selective cannabinoid CB(1) receptor antagonist SR141716A (16 nmol mouse(-1)). SR141716A (1 - 300 nmol mouse(-1)), administered alone, increased intestinal motility to the same extent in both control and croton oil-treated mice. 3. Croton oil-induced intestinal inflammation was associated with an increased expression of CB(1) receptor, an unprecedented example of up-regulation of cannabinoid receptors during inflammation. 4. High levels of anandamide and 2-arachidonylglycerol were detected in the small intestine, although no differences were observed between control and croton oil-treated mice; by contrast anandamide amidohydrolase activity increased 2 fold in the inflamed small intestine. 5. It is concluded that inflammation of the gut increases the potency of cannabinoid agonists possibly by 'up-regulating' CB(1) receptor expression; in addition, endocannabinoids, whose turnover is increased in inflamed gut, might tonically inhibit intestinal motility.

Topics: Analgesics; Animals; Cannabinoid Receptor Modulators; Cannabinoids; Cannabinol; Croton Oil; Cyclohexanols; Dermatologic Agents; Disease Models, Animal; Dose-Response Relationship, Drug; Gastrointestinal Motility; Inflammatory Bowel Diseases; Injections, Intraperitoneal; Injections, Intraventricular; Male; Mice; Mice, Inbred ICR; Piperidines; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant

1. The objective of the present study was to analyse the peripheral effects of cannabinoids on adrenaline release from adrenal chromaffin cells. 2. In pithed rabbits with electrically stimulated sympathetic outflow, intravenous injection of the cannabinoid receptor agonists WIN55212-2 and CP55940 (5, 50 and 500 microg x kg(-1)) markedly lowered the plasma adrenaline concentration. The effect of WIN55212-2 was attenuated by the selective CB1 cannabinoid receptor antagonist SR141716A (500 microg x kg(-1)). WIN55212-3 (same doses as WIN55212-2), the enantiomer of WIN55212-2 lacking affinity for cannabinoid receptors, had no effect on the plasma adrenaline concentration. 3. In rabbit isolated adrenal glands, the release of adrenaline elicited by electrical stimulation was measured by fast cyclic voltammetry. Electrically-evoked adrenaline release was inhibited by WIN55212-2 (0.3, 1, 3 and 10 microM) and this effect was antagonized by SR141716A (1 microM). The non-cholinergic component of adrenaline release observed after blockade of nicotinic (by hexamethonium 100 microM) and muscarinic (by atropine 0.5 microM) acetylcholine receptors was not depressed by WIN55212-2. WIN55212-3 (10 microM) had no effect on adrenaline release. 4. No detectable specific CB1 receptor binding and mRNA expression were found in rabbit adrenal glands with autoradiography and in situ hybridization. 5. The results show that cannabinoids inhibit adrenaline secretion in rabbit isolated adrenal glands; the likely mechanism is a presynaptic CB1 receptor-mediated inhibition of acetylcholine release from preganglionic sympathetic neurons. The inhibition of adrenaline secretion in adrenal glands most probably accounts for the decrease in the plasma adrenaline concentration observed after cannabinoid administration in pithed rabbits.

Topics: Adrenal Glands; Adrenal Medulla; Analgesics; Animals; Benzoxazines; Blood Pressure; Cannabinoids; Chromaffin Cells; Cyclohexanols; Decerebrate State; Dose-Response Relationship, Drug; Electric Stimulation; Epinephrine; Heart Rate; In Vitro Techniques; Injections, Intravenous; Morpholines; Naphthalenes; Piperidines; Pyrazoles; Rabbits; Receptors, Cannabinoid; Receptors, Drug; Rimonabant

Activated brain microglial cells release inflammatory mediators such as nitric oxide (NO) that may play important roles in central nervous system antibacterial, antiviral, and antitumor activities. However, excessive release of these factors has been postulated to elicit immune-mediated neurodegenerative inflammatory processes and to cause brain injury. Recent studies using the rat animal model indicate that select cannabinoids may modulate production of these inflammatory factors. Treatment of neonatal rat brain cortical microglial cells with the cannabinoid paired enantiomers CP55940 and CP56667 resulted in a stereoselective differential effect on inducible NO production. The analog CP55940 exerted a dose-dependent inhibition of interferon gamma (IFNy)/bacterial lipopolysaccharide (LPS)-inducible NO production which was significantly greater than that exerted by CP56667. Pretreatment of microglial cells with the CB1 cannabinoid receptor-selective antagonist SR141716A reversed this CP55940-mediated inhibition. MRT-PCR demonstrated the presence of CB1 receptor mRNA within microglial cells consistent with the presence of CB1 receptors. Collectively, these results indicate that the cannabinoid analog CP55940 selectively inhibits inducible NO production by microglial cells and that this inhibition is effected, at least in part, through the CB1 receptor.

Topics: Animals; Cannabinoids; Cyclohexanols; Gene Expression; Microglia; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Piperidines; Pyrazoles; Rats; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; RNA, Messenger

Elevated intraocular pressure is the primary risk factor for glaucoma. Cannabinoids interact with molecular targets in the eye and lower intraocular pressure by an unknown mechanism. The purpose of the present study was to examine eye tissues for functional cannabinoid receptors of the neuronal, CB(1) class, and an endogenous ligand, anandamide. The trabecular meshwork and ciliary processes are the primary structures of the eye that contribute to intraocular pressure and thus were our focus. Total RNA, frozen sections, cellular membranes and primary cultures of cells were prepared from both bovine and cadaveric human tissues. Using cannabinoid CB(1) receptor-specific oligodeoxynucleotide primers, cannabinoid CB(1) receptor antiserum, and cannabinoid-specific compounds (CP-55,940, WIN55,212-2 and SR-141716A), the presence of cannabinoid CB(1) receptors in ciliary processes and trabecular meshwork was determined. Using reverse transcription-polymerase chain reaction, we identified mRNA encoding cannabinoid CB(1) receptor protein in ciliary process and trabecular meshwork cells. Specific binding of anti-CB(1) immunoglobulin-G in tissue sections localized cannabinoid CB(1) receptor protein to the non-pigmented epithelial cells of the ciliary process and cells of the trabecular meshwork. While CP-55,940 and WIN55,212-2 failed to stimulate [(35)S]GTP gamma S binding in membrane preparations from trabecular meshwork and ciliary process, CP-55,940 significantly stimulated whole cell [(35)S]GTP gamma S binding by 51% over basal in ciliary process epithelial cells and 69% over basal in trabecular meshwork cells permeabilized with 5 microM digitonin (p<0.001). Specificity of agonist stimulation was verified by complete blockade with the specific cannabinoid CB(1) receptor antagonist, SR-141716A. Moreover, activation of cannabinoid CB(1) receptors by CP-55,940 resulted in a 2.3+/-0.3 and 1.7+/-0.3-fold stimulation of cAMP accumulation in trabecular meshwork and ciliary process cells, respectively (p<0.01). Lastly, anandamide was detected in human trabecular meshwork (3.08 pmol/g), ciliary process (49.42 pmol/g) and neurosensory retinal (4.48 pmol/g) tissues. These data, for the first time, demonstrate in a single study the presence of both CB(1) mRNA and protein in trabecular meshwork and ciliary processes from two different species. Activation of heterotrimeric G-proteins and stimulation of cAMP accumulation by cannabinoids in vitro suggest that their intraocular pr

Topics: Animals; Arachidonic Acids; Cannabinoids; Cattle; Cell Separation; Ciliary Body; Cyclic AMP; Cyclohexanols; Endocannabinoids; Fluorescent Antibody Technique; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Intraocular Pressure; Ligands; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Reverse Transcriptase Polymerase Chain Reaction; Rimonabant; RNA, Messenger; Trabecular Meshwork

Lewis rats were trained to self-stimulate the medial forebrain bundle (MFB) using a rate-frequency paradigm. They were then tested for the effects of the cannabinoid receptor agonist CP 55,940, the selective cannabinoid receptor antagonist SR 141716 and the dopamine D1 receptor antagonist SCH 23390. CP 55,940 (0, 10, 25 and 50 microg/kg i.p.) had no effect on MFB self-stimulation behaviour as assessed by the M50, the stimulation frequency at which half-maximal response rates were obtained. With SR 141716, only a very high dose (20 mg/kg i.p.) caused a significant inhibition of the rewarding efficacy of the stimulation. This was seen as an increase in the M50. All other doses of SR 141716 (0, 1, 3, 10 mg/kg i.p.) were ineffective in modulating the M50. By comparison, a relatively low dose (0.06 mg/kg i.p.) of SCH 23390 caused a large increase in M50. These results indicate a relatively modest influence, if any at all, of exogenous or endogenous cannabinoids on reward-relevant neurotransmission.

Topics: Animals; Benzazepines; Brain; Cannabinoids; Cyclohexanols; Dopamine Antagonists; Dose-Response Relationship, Drug; Electric Stimulation; Piperidines; Pyrazoles; Rats; Rats, Inbred Lew; Receptors, Cannabinoid; Receptors, Drug; Reward; Rimonabant; Self Stimulation

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.

Topics: Amides; Arachidonic Acids; Calcium Signaling; Camphanes; Cannabinoids; Cyclohexanols; Cyclooxygenase Inhibitors; Drug Interactions; Endocannabinoids; Ethanolamines; Glycerides; HL-60 Cells; Humans; Ligands; Lipoxygenase Inhibitors; Molecular Mimicry; Palmitic Acids; Pertussis Toxin; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; RNA, Messenger; Structure-Activity Relationship; Virulence Factors, Bordetella

The effect of cannabinoid drugs on peristalsis in the guinea-pig ileum was studied. Peristalsis was induced by delivering fluid into the oral end of an isolated intestinal segment. Longitudinal muscle reflex contraction, threshold pressure and threshold volume to trigger peristalsis, compliance of the intestinal wall during the preparatory phase (a reflection of the resistance of the wall to distension) and maximal ejection pressure during the emptying phase of peristalsis were measured. The cannabinoid agonists WIN 55,212-2 (0.3 - 300 nM) and CP55,940 (0.3 - 300 nM) significantly decreased longitudinal muscle reflex contraction, compliance and maximal ejection pressure, while increased threshold pressure and volume to elicit peristalsis. These effects were not modified by the opioid antagonist naloxone (1 microM) and by the alpha-adrenoceptor antagonist phentolamine (1 microM). The inhibitory effect of both WIN 55,212-2 and CP55,940 on intestinal peristalsis was antagonized by the cannabinoid CB(1) receptor antagonist SR141716A (0.1 microM), but not by the cannabinoid CB(2) receptor antagonist SR144528 (0.1 microM). In absence of other drugs, the CB(1) receptor antagonists SR141716A (0.01 - 1 microM) and AM281 (0.01 - 1 microM) slightly (approximatively 20%) but significantly increased maximal ejection pressure during the empty phase of peristalsis without modifying longitudinal muscle reflex contraction, threshold pressure, threshold volume to trigger peristalsis and compliance. It is concluded that activation of CB(1) receptors reduces peristalsis efficiency in the isolated guinea-pig, and that the emptying phase of peristalsis could be tonically inhibited by the endogenous cannabinoid system.

Topics: Animals; Benzoxazines; Camphanes; Cannabinoids; Compliance; Cyclohexanols; Guinea Pigs; Ileum; In Vitro Techniques; Ligands; Male; Morpholines; Muscle Contraction; Naphthalenes; Peristalsis; Piperidines; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant

The role of cannabinoid CB(1) receptors in sympathetic neurotransmission was characterised in nerve-mediated responses of isolated right atria, vasa deferentia and small mesenteric resistance arteries using the cannabinoid CB(1) receptor agonists Delta(9)-tetrahydrocannabinol, CP 55,940 and anandamide and the cannabinoid CB(1)-selective antagonist SR 141716A. In the mouse vas deferens, the twitch response was completely inhibited by each of the putative cannabinoid receptor agonists with pIC(50) values of CP 55,940, 9.2+/-0.1; Delta(9)-tetrahydrocannabinol, 8.4+/-0.1; anandamide, 7.1+/-0.1. SR 141716A 10-100 nM was a competitive antagonist of all three agonists with a pK(B) value of 8.4-8.6, consistent with an interaction at the cannabinoid CB(1) receptor. In the rat vas deferens CP 55,940 (0.01-10 microM) inhibited the contractions to a significant extent (88.5+/-0.5% at 10 microM; pIC(50) of 7.1+/-0.1) while Delta(9)-tetrahydrocannabinol and anandamide (both up to 10 microM) were inactive. CP 55,940 exhibited low potency in rat compared with mouse vas deferens and the rat concentration-response curve was not competitively antagonised by SR 141716A (100 nM) or SR 144528 (10 nM-10 microM), suggesting an interaction at a receptor(s) distinct from cannabinoid CB(1) or CB(2). Sympathetic nerve-induced tachycardia in rat and mouse atria, and rat mesenteric artery smooth muscle contractile responses to perivascular nerve stimulation, were not inhibited by Delta(9)-tetrahydrocannabinol, CP 55,940 or anandamide up to 1 microM. These data indicate that cannabinoid CB(1) receptor activation inhibits sympathetic neurotransmission only in the mouse vas deferens and thus point to species and regional differences in cannabinoid CB(1) receptor involvement in pre-synaptic inhibition of sympathetic neurotransmission and CP 55,940 may have inhibitory actions in rat vas deferens unrelated to cannabinoid receptor activity.

Topics: Animals; Arachidonic Acids; Camphanes; Cannabinoids; Cyclohexanols; Dronabinol; Endocannabinoids; Heart Atria; In Vitro Techniques; Male; Mesenteric Artery, Inferior; Mice; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB2; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Sympathetic Nervous System; Synaptic Transmission; Vas Deferens; Vascular Resistance

The psychoactive component of marijuana, delta9-tetrahydrocannabinol (THC) suppresses different functions of immunocytes, including the antimicrobicidal activity of macrophages. The triggering of cannabinoid receptors of CB1 and CB2 subtypes present on leukocytes may account for these effects. We investigated the influence of specific CB1 or CB2 receptor antagonists (SR141716A and SR144528, respectively) and nonselective CB1/CB2 cannabinoid receptor agonists (CP55,940 or WIN 55212-2) on macrophage infection by Brucella suis, an intracellular gram-negative bacteria. None of the compounds tested affected bacterial phagocytosis. By contrast, the intracellular multiplication of Brucella was dose-dependently inhibited in cells treated with 10-500 nM SR141716A and 1 microM SR141716A-induced cells exerted a potent microbicidal effect against the bacteria. SR144528, CP55,940, or WIN 55212-2 did not affect (or slightly potentiated) the growth of phagocytized bacteria. However, CP55,940 or WIN 55212-2 reversed the SR141716A-mediated effect, which strongly suggested an involvement of macrophage CB1 receptors in the phenomenon. SR141716A was able to pre-activate macrophages and to trigger an activation signal that inhibited Brucella development. The participation of endogenous cannabinoid ligand(s) in Brucella infection was discussed. Finally, our data show that SR141716A up-regulates the antimicrobial properties of macrophages in vitro and might be a pharmaceutical compound useful for counteracting the development of intramacrophagic gram-negative bacteria.

Topics: Animals; Anti-Bacterial Agents; Antigens, CD; Benzoxazines; Brucella; Calcitriol; Camphanes; Cell Differentiation; Cell Line; Colforsin; Cyclic AMP; Cyclohexanols; Dose-Response Relationship, Drug; Humans; Intercellular Adhesion Molecule-1; Macrophage Activation; Macrophages; Mice; Monocytes; Morpholines; Naphthalenes; Phagocytosis; Piperidines; Pyrazoles; Reactive Oxygen Species; Receptors, Cannabinoid; Receptors, Drug; Rimonabant

Cannabinoids which impair rat working memory appear to inhibit hippocampal extracellular acetylcholine (Ach) release and reduce choline uptake through an interaction with CB1 cannabinoid receptors. Here we report that CP 55,940, a potent bicyclic synthetic cannabinoid analog, dose-dependently impaired rat performance, when given i.p. 20 min before an eight-arm radial maze test. The selective CB1 cannabinoid receptor antagonist SR 141716A, given i.p. 20 min earlier, significantly reduced the memory deficit Pretreatment with eptastigmine, a second generation cholinesterase inhibitor, given orally 100 min before the cannabinoid agonist, relieved the memory impairment without affecting CP 55,940-induced behavioural alterations such as reduced spontaneous motor activity, analgesia and hind limb splaying. These data suggest that cannabinoid-induced working memory impairment is mediated through a central cholinergic blockade.

Topics: Animals; Behavior, Animal; Cannabinoids; Cholinesterase Inhibitors; Cyclohexanols; Male; Maze Learning; Memory; Memory Disorders; Motor Activity; Physostigmine; Piperidines; Pyrazoles; Rats; Rats, Wistar; Rimonabant

Systemically administered cannabinoids elicit marked cardiovascular effects, and the role of the central and the peripheral nervous system in these effects is not clarified. The aim of this study was to characterize the actions of cannabinoids on cardiovascular regulatory centers in conscious rabbits. A catheter for administration of drugs into the cisterna cerebellomedullaris and an electrode for recording renal sympathetic nerve activity were implanted under halothane anesthesia. Experiments were carried out later in conscious animals. Two cannabinoid receptor agonists were injected intracisternally: the aminoalkylindole WIN55212-2 (0.1, 1, and 10 microg kg(-1)) and the bicyclic Delta(9)-tetrahydrocannabinol analog CP55940 (0.1, 1, and 10 microg kg(-1)). WIN55212-2 and CP55940 dose dependently increased renal sympathetic nerve activity and the plasma noradrenaline concentration and also lowered the heart rate. The highest doses of WIN55212-2 and CP55940 increased blood pressure. In contrast, intracisternal injection of WIN55212-3 (0.1, 1, and 10 microg kg(-1)), an enantiomer of WIN55212-2 with very low affinity for cannabinoid binding sites, had no effects. The CB(1) cannabinoid receptor antagonist SR141716A (0.5 mg kg(-1), i.v. ) attenuated the effects of intracisternally administered WIN55212-2 (0.1, 1, and 10 microg kg(-1)). The results indicate that cannabinoids, acting directly on cardiovascular regulatory centers, elicit sympathoactivation and bradycardia. These effects were likely mediated by CB(1) cannabinoid receptors, because they were elicited by two cannabinoid agonists belonging to different chemical classes (WIN55212-2 and CP55940), but not by the inactive enantiomer WIN55212-3, and because they were attenuated by the CB(1) cannabinoid receptor antagonist SR141716A.

Topics: Animals; Benzoxazines; Blood Pressure; Bradycardia; Cannabinoids; Cyclohexanols; Drug Interactions; Female; Heart Rate; Injections, Intraventricular; Kidney; Male; Morpholines; Naphthalenes; Norepinephrine; Piperidines; Pyrazoles; Rabbits; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Stereoisomerism; Sympathetic Nervous System

Cannabinoids have been shown to affect immune responses, acting on different populations of immune cells. In the present paper we analyze the ability of in vivo and in vitro treatment with the potent synthetic cannabinoid CP55,940 to interfere with an important function of rat peritoneal macrophages, i.e. spontaneous migration and formyl-metionyl-leucine-phenylalanine (fMLP)-induced chemotaxis, that were assessed by the use of a Boyden-modified microchemotaxis chamber. When added in vitro, CP55,940 induced a significant and dose-dependent inhibition of both spontaneous migration and fMLP-induced chemotaxis. Both the Cannabinoid Receptor 1 (CB1) and the Cannabinoid Receptor 2 (CB2) antagonists were able to block the CP55,940-induced inhibition of spontaneous migration, although the CB2 antagonist was more potent and only the CB2 antagonist was able to reverse the effect of CP55,940 on fMLP-induced chemotaxis. Similarly, in the in vivo experiments, 1 h after the acute subcutaneous administration of 0.4 mg/kg of CP55,940, both spontaneous motility and chemotaxis were reduced. The pretreatment with the CB2 antagonist, but not with the CB1 antagonist, was able to prevent this effect. Our data confirm that cannabinoids can affect some macrophage functions, mainly throughout CB2 receptors, and suggest that the development of specific CB2 ligands may lead to an interesting new class of anti-inflammatory drugs.

Topics: Animals; Camphanes; Cannabinoids; Cell Movement; Cells, Cultured; Cyclohexanols; Dose-Response Relationship, Drug; Immunosuppressive Agents; In Vitro Techniques; Macrophages, Peritoneal; Male; Piperidines; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB2; Receptors, Cannabinoid; Receptors, Drug; Rimonabant

Using a new antibody developed against the C-terminus of the cannabinoid receptor (CB1), the immunostaining in the hippocampus revealed additional axon terminals relative to the pattern reported previously with an N-terminus antibody. Due to a greater sensitivity of this antibody, a large proportion of boutons in the dendritic layers displaying symmetrical (GABAergic) synapses were also strongly immunoreactive for CB1 receptors, as were axon terminals of perisomatic inhibitory cells containing cholecystokinin. Asymmetrical (glutamatergic) synapses, however, were always negative for CB1. To investigate the effect of presynaptic CB1 receptor activation on hippocampal inhibition, we recorded inhibitory postsynaptic currents (IPSCs) from principal cells. Bath application of CB1 receptor agonists (WIN55,212-2 and CP55,940) suppressed IPSCs evoked by local electrical stimulation, which could be prevented or reversed by the CB1 receptor antagonist SR141716A. Action potential-driven IPSCs, evoked by pharmacological stimulation of a subset of interneurons, were also decreased by CB1 receptor activation. We also examined the effects of CB1 receptor agonists on Ca2+-independent miniature IPSCs (mIPSC). Both agonists were without significant effect on the frequency or amplitude of mIPSCs. Synchronous gamma oscillations induced by kainic acid in the CA3 region of hippocampal slices were reversibly reduced in amplitude by the CB1 receptor agonist CP 55,940, which is consistent with an action on IPSCs. We used CB1-/- knock-out mice to confirm the specificity of the antibody and of the agonist (WIN55,212-2) action. We conclude that activation of presynaptic CB1 receptors decreases Ca2+-dependent GABA release, and thereby reduces the power of hippocampal network oscillations.

Topics: Action Potentials; Analgesics; Animals; Antibodies; Benzoxazines; Cannabinoids; Cyclohexanols; Electrophysiology; Epitopes; gamma-Aminobutyric Acid; Hippocampus; Interneurons; Male; Mice; Mice, Inbred Strains; Mice, Knockout; Microscopy, Electron; Morpholines; Naphthalenes; Neural Inhibition; Neural Pathways; Periodicity; Piperidines; Presynaptic Terminals; Pyramidal Cells; Pyrazoles; Rats; Rats, Wistar; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Synaptic Transmission

Chronic exposure to CP55,940 produced a significant down-regulation of cannabinoid receptors in the striatum, cortex, hippocampus, and cerebellum of rat brain. At 24 h after SR141716-precipitated withdrawal, we observed a tendency to return to basal levels in the striatum and cortex, whereas the specific binding remained lower in the hippocampus and cerebellum. When we surveyed cannabinoid receptor-activated G proteins, in chronic CP55,940-treated rats the guanosine 5'-O:-(3-[(35)S]thiotriphosphate) ([(35)S]GTPgammaS) binding assay revealed a decrease of activated G proteins in the striatum, cortex, and hippocampus, whereas no significant changes were seen in the cerebellum. At 24 h after the SR141716-precipitated withdrawal, [(35)S]GTPgammaS binding increased compared with that of rats chronically exposed to CP55,940, attaining the control level except for cerebellum, where we observed a trend to overcome the control amounts. Concerning the cyclic AMP (cAMP) cascade, which represents the major intracellular signaling pathway activated by cannabinoid receptors, in the cerebral areas from rats chronically exposed to CP55,940 we found alteration in neither cAMP levels nor protein kinase A activity. In the brain regions taken from CP55, 940-withdrawn rats, we only observed a significant up-regulation in the cerebellum. Our findings suggest that receptor desensitization and down-regulation are strictly involved in the development of cannabinoid tolerance, whereas alterations in the cAMP cascade in the cerebellum could be relevant in the mediation of the motor component of cannabinoid abstinence.

Topics: Animals; Behavior, Animal; Brain; Cannabinoids; Cerebellum; Cerebral Cortex; Corpus Striatum; Cyclic AMP; Cyclohexanols; Disease Models, Animal; Down-Regulation; Drug Administration Schedule; Drug Tolerance; GTP-Binding Proteins; Hippocampus; Injections, Intraperitoneal; Male; Marijuana Abuse; Piperidines; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Signal Transduction

The effect of CP 55,940, on electroencephalographic (EEG) spectral power decrease and hyperlocomotion induced by transient global ischemia in gerbils, was investigated. Animals were treated with CP 55,940 (4 mg/kg intraperitoneal (i.p.)) 5 min after bilateral carotid occlusion or SR 141716A (3 mg/kg i.p.) 5 min before or with both. Mean total and relative spectral power was evaluated for 1 h before (basal) and 1 and 24 h, 3 and 7 days after ischemia. Spontaneous locomotor activity was evaluated at the same times. CP 55,940 antagonized the reduction in mean total spectral power and the hyperlocomotion induced by ischemia, in comparison with vehicle group, starting from 24 h and lasting 7 days (P<0.001). Pretreatment with SR 141716A completely blocked the neuroprotective effect of CP 55,940. These findings suggest a potential therapeutic role of cannabinoids in cerebral ischemia.

Topics: Analgesics; Animals; Brain; Brain Ischemia; Cyclohexanols; Electroencephalography; Gerbillinae; Hyperkinesis; Male; Neuroprotective Agents; Piperidines; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant

An endogenous cannabimimetic molecule, 2-arachidonoylglycerol, induces a rapid, transient increase in intracellular free Ca2+ concentrations in NG108-15 cells through a cannabinoid CB1 receptor-dependent mechanism. We examined the activities of 24 relevant compounds (2-arachidonoylglycerol, its structural analogues, and several synthetic cannabinoids). We found that 2-arachidonoylglycerol is the most potent compound examined so far: its activity was detectable from as low as 0.3 nM, and the maximal response induced by 2-arachidonoylglycerol exceeded the responses induced by others. Activities of HU-210 and CP55940, potent cannabinoid receptor agonists, were also detectable from as low as 0.3 nM, whereas the maximal responses induced by these compounds were low compared with 2-arachidonoylglycerol. Anandamide was also found to act as a partial agonist in this assay system. We confirmed that free arachidonic acid failed to elicit a response. Furthermore, we found that a metabolically stable ether-linked analogue of 2-arachidonoylglycerol possesses appreciable agonistic activity, although its activity was apparently lower than that of 2-arachidonoylglycerol. We also confirmed that pretreating cells with various cannabinoid receptor agonists nullified the response induced by 2-arachidonoylglycerol, whereas pretreating cells with other neurotransmitters or neuromodulators did not affect the response. These results strongly suggested that the cannabinoid CB1 receptor is originally a 2-arachidonoylglycerol receptor, and 2-arachidonoylglycerol is the intrinsic physiological ligand for the cannabinoid CB1 receptor.

Topics: Animals; Arachidonic Acids; Benzoxazines; Cannabinoids; Cell Line; Cyclohexanols; Dronabinol; Endocannabinoids; Models, Chemical; Morpholines; Naphthalenes; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Structure-Activity Relationship

In our previous study, we demonstrated that chronic ethanol (EtOH) exposure down-regulated the cannabinoid receptors (CB1) in mouse brain synaptic plasma membrane (SPM) (Basavarajappa et al., Brain Res. 793 (1998) 212-218). In the present study, we investigated the effect of chronic EtOH (4-day inhalation) on the CB1 agonist stimulated guanosine-5'-O-(3-[35S]thio)-triphosphate ([35S]GTP gamma S) binding in SPM from mouse. Our results indicate that the net CP55,940 stimulated [35S]GTP gamma S binding was increased with increasing concentrations of CP55,940 and GDP. This net CP55,940 (1.5 microM) stimulated [35S]GTP gamma S binding was reduced significantly (-25%) in SPM from chronic EtOH group (175 +/- 5.25%, control; 150 +/- 8.14%, EtOH; P < 0.05). This effect occurs without any significant changes on basal [35S]GTP gamma S binding (152.1 +/- 10.7 for control, 147.4 +/- 5.0 fmol/mg protein for chronic EtOH group, P > 0.05). Non-linear regression analysis of net CP55,940 stimulated [35S]GTP gamma S binding in SPM showed that the Bmax of cannabinoid stimulated binding was significantly reduced in chronic EtOH exposed mouse (Bmax = 7.58 +/- 0.22 for control; 6.42 +/- 0.20 pmol/mg protein for EtOH group; P < 0.05) without any significant changes in the G-protein affinity (Kd = 2.68 +/- 0.24 for control; 3.42 +/- 0.31 nM for EtOH group; P > 0.05). The pharmacological specificity of CP55,940 stimulated [35S]GTP gamma S binding in SPM was examined with CB1 receptor antagonist, SR141716A and these studies indicated that CP55,940 stimulated [35S]GTP gamma S binding was blocked by SR141716A with a decrease (P < 0.05) in the IC50 values in the SPM from chronic EtOH group. These results suggest that the observed down-regulation of CB1 receptors by chronic EtOH has a profound effect on desensitization of cannabinoid-activated signal transduction and possible involvement of CB1 receptors in EtOH tolerance and dependence.

Topics: Analgesics; Animals; Binding, Competitive; Brain Chemistry; Central Nervous System Depressants; Chronic Disease; Cyclohexanols; Dose-Response Relationship, Drug; Down-Regulation; Ethanol; Guanosine 5'-O-(3-Thiotriphosphate); Kinetics; Male; Mice; Piperidines; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Sulfur Radioisotopes; Synaptic Membranes

Upon activation, brain microglial cells release proinflammatory mediators, such as nitric oxide (NO), which may play an important role in the central nervous system antibacterial, antiviral, and antitumor activities. However, excessive release of NO has been postulated to elicit immune-mediated neurodegenerative inflammatory processes and to cause brain injury. In the present study, the effect of cannabinoids on the release of NO from endotoxin/cytokine-activated rat cortical microglial cells was evaluated. A drug dose-dependent (0.1 microM-8 microM) inhibition of NO release from rat microglial cells was exerted by the cannabinoid receptor high-affinity binding enantiomer (-)-CP55940. In contrast, a minimal inhibitory effect was exerted by the lower affinity binding paired enantiomer (+)-CP56667. Pretreatment of microglial cells with the Galphai/Galphao protein inactivator pertussis toxin, cyclic AMP reconstitution with the cell-permeable analog dibutyryl-cAMP, or treatment of cells with the Galphas activator cholera toxin, resulted in reversal of the (-)-CP55940-mediated inhibition of NO release. A similar reversal in (-)-CP55940-mediated inhibition of NO release was effected when microglial cells were pretreated with the central cannabinoid receptor (CB1) selective antagonist SR141716A. Mutagenic reverse transcription-polymerase chain reaction, Western immunoblot assay using a CB1 receptor amine terminal domain-specific antibody, and cellular colocalization of CB1 and the microglial marker Griffonia simplicifolia isolectin B4 confirmed the expression of the CB1 receptor in rat microglial cells. Collectively, these results indicate a functional linkage between the CB1 receptor and cannabinoid-mediated inhibition of NO production by rat microglial cells.

Topics: Animals; Animals, Newborn; Cannabinoids; Cells, Cultured; Cyclohexanols; Immunohistochemistry; Microglia; Nitric Oxide; Nitric Oxide Synthase; Piperidines; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptors, Cannabinoid; Receptors, Drug; Reverse Transcriptase Polymerase Chain Reaction; Rimonabant; RNA, Messenger

The effects of cannabinoid agonists on noxious heat-evoked firing of 62 spinal wide dynamic range (WDR) neurons were examined in urethan-anesthetized rats (1 cell/animal). Noxious thermal stimulation was applied with a Peltier device to the receptive fields in the ipsilateral hindpaw of isolated WDR neurons. To assess the site of action, cannabinoids were administered systemically in intact and spinally transected rats and intraventricularly. Both the aminoalkylindole cannabinoid WIN55,212-2 (125 microg/kg iv) and the bicyclic cannabinoid CP55,940 (125 microg/kg iv) suppressed noxious heat-evoked activity. Responses evoked by mild pressure in nonnociceptive neurons were not altered by CP55,940 (125 microg/kg iv), consistent with previous observations with another cannabinoid agonist, WIN55,212-2. The cannabinoid induced-suppression of noxious heat-evoked activity was blocked by pretreatment with SR141716A (1 mg/kg iv), a competitive antagonist for central cannabinoid CB1 receptors. By contrast, intravenous administration of either vehicle or the receptor-inactive enantiomer WIN55,212-3 (125 microg/kg) failed to alter noxious heat-evoked activity. The suppression of noxious heat-evoked activity induced by WIN55,212-2 in the lumbar dorsal horn of intact animals was markedly attenuated in spinal rats. Moreover, intraventricular administration of WIN55,212-2 suppressed noxious heat-evoked activity in spinal WDR neurons. By contrast, both vehicle and enantiomer were inactive. These findings suggest that cannabinoids selectively modulate the activity of nociceptive neurons in the spinal dorsal horn by actions at CB1 receptors. This modulation represents a suppression of pain neurotransmission because the inhibitory effects are selective for pain-sensitive neurons and are observed with different modalities of noxious stimulation. The data also provide converging lines of evidence for a role for descending antinociceptive mechanisms in cannabinoid modulation of spinal nociceptive processing.

Topics: Analgesics; Animals; Axotomy; Benzoxazines; Cannabinoids; Cyclohexanols; Hot Temperature; Injections, Intravenous; Injections, Intraventricular; Lumbosacral Region; Male; Mechanoreceptors; Microinjections; Morpholines; Naphthalenes; Neurons; Pain Measurement; Piperidines; Pyrazoles; Rats; Rats, Sprague-Dawley; Rimonabant; Spinal Cord

1. The aim of the present study was to analyse the cardiovascular actions of the synthetic CB1/CB2 cannabinoid receptor agonist WIN55212-2, and specifically to determine its sites of action on sympathetic cardiovascular regulation. 2. Pithed rabbits in which the sympathetic outflow was continuously stimulated electrically or which received a pressor infusion of noradrenaline were used to study peripheral prejunctional and direct vascular effects, respectively. For studying effects on brain stem cardiovascular regulatory centres, drugs were administered into the cisterna cerebellomedullaris in conscious rabbits. Overall cardiovascular effects of the cannabinoid were studied in conscious rabbits with intravenous drug administration. 3. In pithed rabbits in which the sympathetic outflow was continuously electrically stimulated, intravenous injection of WIN55212-2 (5, 50 and 500 microg kg(-1)) markedly reduced blood pressure, the spillover of noradrenaline into plasma and the plasma noradrenaline concentration, and these effects were antagonized by the CB1 cannabinoid receptor-selective antagonist SR141716A. The hypotensive and the sympathoinhibitory effect of WIN55212-2 was shared by CP55940, another mixed CB1/CB2 cannabinoid receptor agonist, but not by WIN55212-3, the enantiomer of WIN55212-2, which lacks affinity for cannabinoid binding sites. WIN55212-2 had no effect on vascular tone established by infusion of noradrenaline in pithed rabbits. 4. Intracisternal application of WIN55212-2 (0.1, 1 and 10 microg kg(-1)) in conscious rabbits increased blood pressure and the plasma noradrenaline concentration and elicited bradycardia; this latter effect was antagonized by atropine. 5. In conscious animals, intravenous injection of WIN55212-2 (5 and 50 microg kg(-1)) caused bradycardia, slight hypotension, no change in the plasma noradrenaline concentration, and an increase in renal sympathetic nerve firing. The highest dose of WIN55212-2 (500 microg kg(-1)) elicited hypotension and tachycardia, and sympathetic nerve activity and the plasma noradrenaline concentration declined. 6. The results obtained in pithed rabbits indicate that activation of CB1 cannabinoid receptors leads to marked peripheral prejunctional inhibition of noradrenaline release from postganglionic sympathetic axons. Intracisternal application of WIN55212-2 uncovered two effects on brain stem cardiovascular centres: sympathoexcitation and activation of cardiac vagal fibres. The highest dose of

Topics: Analgesics; Animals; Benzoxazines; Blood Pressure; Bradycardia; Cannabinoids; Cardiovascular Physiological Phenomena; Cisterna Magna; Consciousness; Cyclohexanols; Decerebrate State; Dose-Response Relationship, Drug; Electric Stimulation; Female; Heart Rate; Infusions, Intravenous; Injections; Injections, Intravenous; Kidney; Male; Morpholines; Naphthalenes; Norepinephrine; Piperidines; Pyrazoles; Rabbits; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Stereoisomerism; Sympathetic Nervous System; Sympathomimetics

The human cannabinoid receptor CB1 functionally couples primarily to Gi-, but also to Gs-mediated pathways to modulate intracellular cyclic AMP (cAMP) levels. To probe the features of the receptor that may be involved in promoting interactions with one G protein type over another, we generated the L341A/A342L mutant CB1 receptor. The double mutation involved the swap in position of two adjacent residues in the carboxyl-terminal segment of the third intracellular loop of CB1. This resulted in partial constitutive activation of the receptor and an agonist-independent enhancement in cAMP levels. Characterization following treatment with either pertussis or cholera toxin indicated that the constitutive activity is selective for a Gs- and not a Gi-mediated pathway. Treatment with the CB1-specific inverse agonist SR141716A inhibited the basal accumulation of cAMP in the presence of pertussis toxin, establishing that the effect is CB1 mediated. The binding of the agonist CP-55,940 to the L341A/A342L receptor was not markedly different from that for the wild-type receptor despite the constitutive Gs activity. This may reflect a preference of this ligand for an activated receptor state associated with the Gi coupling form and underscores the potential for developing therapeutics that selectively activate one pathway over another.

Topics: Animals; CHO Cells; Cholera Toxin; Cricetinae; Cyclic AMP; Cyclohexanols; GTP-Binding Proteins; Humans; Mutation; Peptide Fragments; Pertussis Toxin; Piperidines; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Virulence Factors, Bordetella

We previously showed that the cannabinoid receptor CB1 stably transfected in Chinese hamster ovary cells was constitutively active and could be inhibited by the inverse agonist SR 141716A. In the present study, we demonstrate that the cannabinoid agonist CP-55940 induced cytosol alkalinization of CHO-CB1 cells in a dose- and time-dependent manner via activation of the Na+/H+ exchanger NHE-1 isoform. By contrast, the inverse agonist SR 141716A induced acidification of the cell cytosol, suggesting that the Na+/H+ exchanger NHE-1 was constitutively activated by the CB1 receptor. CB1-mediated NHE1 activation was prevented by both pertussis toxin treatment and the specific MAP kinase inhibitor PD98059. NHE-1 and p42/p44 MAPK had a similar time course of activation in response to the addition of CP-55940 to CHO-CB1 cells. These results suggest that CB1 stimulates NHE-1 by G(i/o)-mediated activation of p42/p44 MAP kinase and highlight a cellular physiological process targeted by CB1.

Topics: Animals; Calcium-Calmodulin-Dependent Protein Kinases; Cannabinoids; CHO Cells; Cricetinae; Cyclohexanols; Hydrogen-Ion Concentration; Piperidines; Protein Isoforms; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Signal Transduction; Sodium-Hydrogen Exchangers

A series of experiments examined the effects of the cannabinoid CB1 receptor agonist CP 55,940 ((-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hyd roxypropyl)cyclohexanol) on the motivation to consume beer, near-beer (a beer-like beverage containing <0.5% ethanol) and sucrose solutions in rats. The experiments employed a 'lick-based progressive ratio paradigm' in which an ever increasing number of licks had to be emitted at a tube for each successive fixed unit of beverage delivered. Break point, the lick requirement at which responding ceased, was used as an index of motivation. In the first experiment, CP 55,940 (10, 30 or 50 microg/kg) caused a dose-dependent increase in break points for beer (containing 4.5% ethanol v/v) and for near-beer. The highest (50 microg/kg) dose of CP 55,940 also significantly decreased locomotor activity. In the second experiment, CP 55,940 (10 or 30 microg/kg) dose-dependently increased break points in rats licking for 'light' beer (containing 2.7% ethanol v/v) or for a sucrose solution (8.6% w/v) containing the same number of calories as the beer. In the third experiment, the facilitatory effects of CP 55,940 (30 microg/kg) on responding for beer and near-beer were reversed by both the cannabinoid CB1 receptor antagonist SR 141716 (N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-me thyl-1H-pyrazole-3-carboxamide hydrochloride) (1.5 mg/kg) and the opioid receptor antagonist naloxone (2.5 mg/kg). Naloxone had a proportionally greater effect on rats licking for beer compared to near-beer, consistent with previous reports of opioid receptor mediation of alcohol craving. These results show that cannabinoids modulate the motivation for beer via both cannabinoid CB1 receptors and opioid receptors. The similar effect of CP 55,940 on the motivation for beer, near-beer and sucrose suggests that the drug effect may reflect a general stimulatory effect on appetite for palatable beverages, although a more specific effect on the desire for alcohol cannot be ruled out.

Topics: Animals; Beer; Cannabinoids; Cyclohexanols; Dose-Response Relationship, Drug; Drinking Behavior; Ethanol; Male; Motor Activity; Naloxone; Piperidines; Pyrazoles; Rats; Rats, Wistar; Receptors, Cannabinoid; Receptors, Drug; Receptors, Opioid; Rimonabant; Self Administration; Sucrose

The hypothesis of these studies is that ligand efficacy at the neuronal CB1 receptor is dependent on the ratio of ligand affinities for the active and inactive states of the receptor. Agonist efficacy was determined in rat cerebellar membranes using agonist-induced guanosine 5'-O-(3-[35S]thiotriphosphate) binding; efficacy was variable among the CB1 agonists examined. Ligand affinities for the active and inactive state of the CB1 receptor were determined by competition with [3H]CP55940 and [3H]SR141716A in the presence of 5'-guanylylimidodiphosphate, respectively. All of the agonists investigated had a higher affinity for the active state than the inactive state. The fraction of CB1 receptors in the active state at a maximally effective concentration was calculated for each agonist and was found to correlate significantly with agonist efficacy. These studies demonstrate that the CB1 receptor of the cerebellum can assume an active conformation in the absence of agonist and that the variability in efficacy among CB1 receptor agonists can be explained by the relative affinities of these ligands for the CB1 receptor in the active and inactive states.

Topics: Animals; Binding, Competitive; Cannabinoids; Cell Membrane; Cerebellum; Cyclohexanols; Dronabinol; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Guanosine Triphosphate; Kinetics; Ligands; Male; Piperidines; Pyrazoles; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Tritium

We have used an ex vivo binding assay in the mouse to evaluate the brain penetration of cannabinoid receptor ligands. After intraperitoneal or oral administration, the pharmacological activity linked to the compound was assessed by using by [3H]WIN 55212-2 binding on cerebellar membranes. The brain penetration was high for compounds like methanandamide or delta9-tetrahydrocannabinol but poor for synthetic agonists such as (cis)-3-(2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-(trans)-4-(3-hydr oxypropyl)cyclohexanol (CP 55940) or, R-(+)-(2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrol[1,2,3-d e]-1,4-benzoxazin-6-yl)(1-napthalenyl)methanone monomethane-sulfonate (WIN 55212-2). After oral administration the duration of action of delta9-tetrahydrocannabinol, methanandamide and WIN 55212-2 is limited and decreased 4 h after administration. The cannabinoid CB1 receptor antagonist: N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-met hyl-1 H-pyrazole-3-carboxamide hydrochloride (SR141716A) exhibited a good brain penetration and a long duration of action.

Topics: Animals; Arachidonic Acids; Benzoxazines; Binding, Competitive; Brain; Cannabinoids; Cyclohexanols; Male; Mice; Morpholines; Naphthalenes; Perfusion; Piperidines; Pyrazoles; Radioligand Assay; Receptors, Drug; Rimonabant; Tritium

It has long been established that the cannabinoid CB1 receptor transduces signals through a pertussis toxin-sensitive Gi/Go inhibitory pathway. Although there have been reports that the cannabinoid CB1 receptor can also mediate an increase in cyclic AMP levels, in most cases the presence of an adenylyl cyclase costimulant or the use of very high amounts of agonist was necessary. Here, we present evidence for dual coupling of the cannabinoid CB receptor to the classical pathway and to a pertussis toxin-insensitive adenylyl cyclase stimulatory pathway initiated with low quantities of agonist in the absence of any costimulant. Treatment of Chinese hamster ovary (CHO) cells expressing the cannabinoid CB1 receptor with the cannabinoid CP 55,940, {(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hyd roxypropyl) cyclohexan-1-ol} resulted in cyclic AMP accumulation in a dose-response manner, an accumulation blocked by the cannabinoid CB1 receptor-specific antagonist SR 141716A, {N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-me thyl-1H-pyrazole-3-carboxamide hydrochloride}. In CHO cells coexpressing the cannabinoid CB1 receptor and a cyclic AMP response element (CRE)-luciferase reporter gene system, CP 55,940 induced luciferase expression by a pathway blocked by the protein kinase A inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide hydrochloride (H-89). Under the same conditions the peripheral cannabinoid CB2 receptor proved to be incapable of inducing cAMP accumulation or luciferase activity. This incapacity allowed us to study the luciferase activation mediated by CB /CB2 chimeric constructs, from which we determined that the first and second internal loop regions of the cannabinoid CB1 receptor were involved in transducing the pathway leading to luciferase gene expression.

Topics: Animals; Binding Sites; Cannabinoids; CHO Cells; Corticotropin-Releasing Hormone; Cricetinae; Cyclic AMP; Cyclic AMP Response Element-Binding Protein; Cyclohexanols; Dose-Response Relationship, Drug; Enzyme Induction; Enzyme Inhibitors; Gene Expression; Genes, Reporter; GTP-Binding Proteins; Humans; Isoquinolines; Luciferases; Piperidines; Protein Kinase Inhibitors; Pyrazoles; Receptors, Cannabinoid; Receptors, Corticotropin-Releasing Hormone; Receptors, Drug; Recombinant Fusion Proteins; Rimonabant; Signal Transduction; Sulfonamides

Tolerance develops rapidly to cannabis, cannabinoids, and related drugs acting at the CB1 cannabinoid receptor. However, little is known about what happens to the receptor as tolerance is developing. In this study, we have found that CB1 receptors are rapidly internalized following agonist binding and receptor activation. Efficacious cannabinoid agonists (WIN 55,212-2, CP 55,940, and HU 210) caused rapid internalization. Methanandamide (an analogue of an endogenous cannabinoid, anandamide) was less effective, causing internalization only at high concentration, whereas delta9-tetrahydrocannabinol caused little internalization, even at 3 microM. CB1 internalized via clathrin-coated pits as sequestration was inhibited by hypertonic sucrose. Internalization did not require activated G protein alpha(i), alpha(o), or alpha(s) subunits. A region of the extreme carboxy terminus of the receptor was necessary for internalization, as a mutant CB1 receptor lacking the last 14 residues did not internalize, whereas a mutant lacking the last 10 residues did. Steps involved in the recycling of sequestered receptor were also investigated. Recovery of CB1 to the cell surface after short (20 min) but not long (90 min) agonist treatment was independent of new protein synthesis. Recycling also required endosomal acidification and dephosphorylation. These results show that CB1 receptor trafficking is dynamically regulated by cannabimimetic drugs.

Topics: Acids; Ammonium Chloride; Animals; Arachidonic Acids; Benzoxazines; Calcium Channel Blockers; Cannabinoids; Cells, Cultured; Clathrin; Coated Vesicles; Cyclohexanols; Dronabinol; Drug Tolerance; Endocytosis; Endosomes; Excitatory Amino Acid Antagonists; GTP-Binding Proteins; Immunosuppressive Agents; Morpholines; Mutagenesis; Naphthalenes; Phosphorylation; Piperidines; Protein Binding; Protein Structure, Tertiary; Pyrazoles; Rats; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Transfection; Up-Regulation; Virulence Factors, Bordetella

Guinea-pig hippocampal slices preincubated with [3H]noradrenaline were superfused with medium containing desipramine and rauwolscine and rat striatal slices preincubated with [3H]dopamine were superfused with medium containing nomifensine; the effect of cannabinoid receptor ligands on tritium overflow stimulated by NMDA or kainate was examined. Furthermore, the affinity of the drugs for cannabinoid CB1 receptors was determined in rat brain cortex membranes using [3H]SR 141716. In guinea-pig hippocampal slices preincubated with [3H]noradrenaline, tritium overflow stimulated by NMDA 100 microM and 1000 microM and by kainate 1000 microM was inhibited by the cannabinoid receptor agonists CP-55,940 and/or WIN 55,212-2. The CB1 receptor antagonist SR 141716 increased the NMDA (1000 microM)-stimulated tritium overflow but did not affect tritium overflow stimulated by NMDA 100 microM or kainate 1000 microM. The inhibitory effect of WIN 55,212-2 on the NMDA (100 microM)- and kainate (1000 microM)-evoked tritium overflow was antagonized by SR 141716. In rat striatal slices preincubated with [3H]dopamine, WIN 55,212-2 inhibited the NMDA (1000 microM)-stimulated tritium overflow. SR 141716, which, by itself, did not affect tritium overflow, counteracted the inhibitory effect of WIN 55,212-2. [3H]SR 141716 binding to rat cortical membranes was inhibited by SR 141716, CP-55,940 and WIN 55,212-2 (pKi 8.53, 7.34 and 5.93, respectively) but not affected by desipramine, rauwolscine and nomifensine (pKi < 5). In conclusion, activation of CB1 receptors inhibits the NMDA- and kainate-stimulated noradrenaline release in guinea-pig hippocampus and the NMDA-stimulated dopamine release in rat striatum. The explanation for the facilitatory effect of SR 141716 might be that it acts as an inverse agonist at CB1 receptors or that these receptors are activated by endogenous cannabinoids.

Topics: Animals; Benzoxazines; Binding, Competitive; Brain Chemistry; Cannabinoids; Cerebral Cortex; Cyclohexanols; Dopamine; Excitatory Amino Acid Agonists; Guinea Pigs; Kainic Acid; Male; Membranes; Morpholines; N-Methylaspartate; Naphthalenes; Norepinephrine; Piperidines; Pyrazoles; Rats; Rats, Wistar; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Stimulation, Chemical

Intrathecal administration of anandamide, delta9-tetrahydrocannabinol (THC) and (-)-3-[2-hydroxy-4-(1,1-dimethyheptyl)ptyl)phenyl]-4-(3-hydr oxypropyl)-cicloexan-1-ol (CP55,940) induced spinal antinociception accompanied by differential kappa-opioid receptor involvement and dynorphin A peptide release. Antinociception using the tail-flick test was induced by the classical cannabinoid THC and was blocked totally by 17,17'-bis(cyclopropylmethyl)-6',6,7,7'-tetrahydro-4,5,4'5'-diepoxy++ +-6,6'-(imino)[7,7'-bimorphinan]-3,3',14,14'-tetrol (norbinaltorphimine) indicating a significant and critical kappa-opioid receptor component. The endogenous cannabinoid, anandamide and the non-classical bicyclic cannabinoid, CP55,940, induced non-nor-BNI-sensitive effects. The N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazo le-carboxamide (SR141716A)-mediated attenuation of spinal antinociception imparted by the various cannabinoids indicates cannabinoid CB1 receptor involvement. THC-induced an enhancement of immunoreactive dynorphin A release which coincided with the onset, but not duration antinociception. The release of dynorphin A was also attenuated by SR141716A suggesting it is cannabinoid CB1 receptor-mediated. These data indicate a critical role for dynorphin A release in the initiation of the antinociceptive effects of the cannabinoids at the spinal level.

Topics: Analgesics; Animals; Cannabinoids; Cyclohexanols; Dimethyl Sulfoxide; Dronabinol; Dynorphins; Injections, Spinal; Male; Naltrexone; Narcotic Antagonists; Nociceptors; Pain; Pain Measurement; Piperidines; Pyrazoles; Rats; Rats, Sprague-Dawley; Rimonabant

The presence of CB(2) receptors was reported in the rat basophilic cell line RBL-2H3 and N-palmitoylethanolamide was proposed as an endogenous, potent agonist of this receptor. We synthesized a series of 10 N-palmitoylethanolamide homologues and analogues, varying by the elongation of the fatty acid chain from caproyl to stearoyl and by the nature of the amide substituent, respectively, and evaluated the affinity of these compounds to cannabinoid receptors in the rat spleen, RBL-2H3 cells and CHO-CB(1) and CHO-CB(2) receptor-transfected cells. In rat spleen slices, CB(2) receptors were the predominant form of the cannabinoid receptors. No binding of [(3)H]SR141716A was observed. [(3)H]CP-55,940 binding was displaced by WIN 55,212-2 and anandamide. No displacement of [(3)H]CP-55,940 or [(3)H]WIN 55,212-2 by palmitoylethanolamide derivatives was observed in rat spleen slices. In RBL-2H3 cells, no binding of [(3)H]CP-55,940 or [(3)H]WIN 55,212-2 could be observed and conversely, no inhibitory activity of N-palmitoylethanolamide derivatives and analogues was measurable. These compounds do not recognize the human CB(1) and CB(2) receptors expressed in CHO cells. In conclusion, N-palmitoylethanolamide was, in our preparations, a weak ligand while its synthesized homologues or analogues were essentially inactive. Therefore, it seems unlikely that N-palmitoylethanolamide is an endogenous agonist of the CB(2) receptors but it may be a compound with potential therapeutic applications since it may act via other mechanisms than cannabinoid CB(1)-CB(2) receptor interactions.

Topics: Animals; Benzoxazines; Binding, Competitive; Cannabinoids; CHO Cells; Cricetinae; Cyclohexanols; Ethanolamines; Humans; Molecular Structure; Morpholines; Naphthalenes; Piperidines; Pyrazoles; Rats; Receptor, Cannabinoid, CB2; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Spleen; Transfection; Tumor Cells, Cultured

We have studied the effects of the cannabinoid receptor agonists (R)-(+)[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2, 3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (WIN 55,212-2, 0. 3-5 mg/kg, i.p.) and (-)-cis-3-[2-hydroxy-4-(1, 1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol) (CP 55,940, 0.03-1 mg/kg, i.p.), the cannabinoid CB(1) receptor antagonist (N-piperidin-1-yl)-5-(4-chlorophenyl)-1-2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716A, 0. 3-5 mg/kg, i.p.) and the cannabinoid CB(2) receptor antagonist N-[-(1S)-endo-1,3,3-trimethyl bicyclo [2.2.1] heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazo le- 3-carboxamide (SR144528, 1 mg/kg, i.p.) on intestinal motility, defaecation and castor-oil (1 ml/100 g rat, orally)-induced diarrhoea in the rat. SR141716A, but not SR144528, increased defaecation and upper gastrointestinal transit, while WIN 55,212-2 and CP 55,940 decreased upper gastrointestinal transit but not defaecation. WIN 55,212-3 (5 mg/kg), the less active enantiomer of WIN 55,212-2, was without effect. A per se non-effective dose of SR141716A (0.3 mg/kg), but not of SR144528 (1 mg/kg) or the opioid receptor antagonist, naloxone (2 mg/kg i.p.), counteracted the inhibitory effect of both WIN 55,212-2 (1 mg/kg) and CP 55,940 (0.1 mg/kg) on gastrointestinal motility. WIN 55,212-2 did not modify castor-oil-induced diarrhoea, while CP 55,940 produced a transient delay in castor-oil-induced diarrhoea at the highest dose tested (1 mg/kg), an effect counteracted by SR141715A (5 mg/kg). These results suggest that (i) intestinal motility and defaecation could be tonically inhibited by the endogenous cannabinoid system, (ii) exogenous activation of cannabinoid CB(1) receptors produces a reduction in intestinal motility in the upper gastrointestinal tract but not in defaecation, (iii) endogenous or exogenous activation of cannabinoid CB(2) receptors does not affect defaecation or intestinal motility and (iv) the cannabinoid receptor agonist, CP 55, 940, possesses a weak and transient antidiarrhoeal effect while the cannabinoid receptor agonist, WIN 55,212-2, does not possess antidiarrhoeal activity.

Topics: Analgesics; Animals; Antidiarrheals; Benzoxazines; Castor Oil; Cyclohexanols; Defecation; Diarrhea; Dose-Response Relationship, Drug; Gastrointestinal Motility; Gastrointestinal Transit; Loperamide; Male; Morpholines; Naphthalenes; Piperidines; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB2; Receptors, Cannabinoid; Receptors, Drug; Rimonabant

Cannabinoids, such as Delta9-THC, are capable of inhibiting nociception, i.e., pain transmission, at least in part, by interacting with spinal Gi/Go-coupled cannabinoid receptors. What is not known, however, is the antinociceptive role of endogenous spinal cannabinoids. If endogenous cannabinoids modulate basal nociceptive thresholds, then alterations in this system could be involved in the etiology of certain pain states. In this report we provide evidence for tonic modulation of basal thermal nociceptive thresholds by the spinal cannabinoid system. Administration of oligonucleotides directed against CB1 cannabinoid receptor mRNA significantly reduced spinal cannabinoid binding sites and produced significant hyperalgesia when compared with a randomer oligonucleotide control. A second method used to reduce activity of the spinal cannabinoid receptor was intrathecal administration of the cannabinoid receptor antagonist SR 141716A. SR 141716A evoked thermal hyperalgesia with an ED50 of 0.0012 fmol. The SR 141716A-induced hyperalgesia was dose-dependently blocked by the administration of D-AP-5 or MK-801, two antagonists to the NMDA receptor. These results indicate that there is tonic activation of the spinal cannabinoid system under normal conditions. Furthermore, hypoactivity of the spinal cannabinoid system results in an NMDA-dependent hyperalgesia and thus may participate in the etiology of certain chronic pain states.

Topics: 2-Amino-5-phosphonovalerate; Analgesics; Animals; Cannabinoids; Cyclohexanols; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Hot Temperature; Hyperalgesia; Male; Mice; Nociceptors; Pain Measurement; Piperidines; Pyrazoles; Receptors, N-Methyl-D-Aspartate; Rimonabant; Spinal Cord; Tritium

Anandamide is the newly discovered endogenous cannabinoid ligand that binds to brain cannabinoid receptors and shares most, but not all, of the pharmacological properties of delta 9-THC. Therefore, this study was undertaken to determine whether its interaction with the CB1 receptor in brain was identical to that of delta 9-THC. Anandamide depressed spontaneous activity and produced hypothermia, antinociception and immobility in mice after i.v. administration. However, none of these effects was blocked by pretreatment with the selective CB1 antagonist, SR 141716A. However, the metabolically stable analog 2-methyl-2'-fluoroethylanandamide produced reductions in motor activity and antinociception in mice, effects that were blocked by the antagonist. To determine whether anandamide's receptor binding mimicked that of other cannabinoids, an autoradiographic comparison of anandamide, SR 141716A and CP 55,940 competition for [3H]CP55,940 binding was conducted throughout rat brain. The receptor affinities for all three compounds did not change according to brain area. As expected, Bmax values differed dramatically among differ brain areas. However, the Bmax values for each brain area were similar regardless of the compound used for displacement. These data suggest that anandamide, SR 141716A and CP 55,940 compete for the same cannabinoid receptor throughout brain despite SR 141716A's failure to block anandamide's pharmacological effects. Although there is no question that anandamide binds to the cannabinoid receptor, failure of SR 141716A to block its pharmacological effects in mice poses a dilemma. The results presented herein raise the possibility that anandamide may not be producing all of its effects by a direct interaction with the CB1 receptor.

Topics: Animals; Arachidonic Acids; Autoradiography; Brain; Cannabinoids; Cyclohexanols; Endocannabinoids; Male; Mice; Mice, Inbred ICR; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptors, Cannabinoid; Receptors, Drug; Rimonabant

To further characterize neuronal cannabinoid receptors, we compared the ability of known and novel cannabinoid analogs to compete for receptor sites labeled with either [3H]SR141716A or [3H]CP-55,940. These efforts were also directed toward extending the structure-activity relationships for cannabinoid agonists and antagonists. A series of alternatively halogenated analogs of SR141716A were synthesized and tested in rat brain membrane binding assays along with the classical cannabinoids, Delta9-tetrahydrocannabinol, cannabinol, cannabidiol, the nonclassical cannabinoid CP-55,940, the aminoalkylindole WIN55212-2 and the endogenous fatty acid ethanolamide, anandamide. Saturation binding isotherms were performed with both radioligands, as were displacement studies, allowing an accurate comparison to be made between the binding of these various compounds. Competition studies demonstrated that all of the compounds were able to displace the binding of [3H]CP-55,940 with rank order potencies that agreed with previous studies. However, the rank order potencies of these compounds in competition studies with [3H]SR141716A differed significantly from those determined with [3H]CP-55,940. These results suggest that CP-55,940, WIN55212-2 and other agonists interact with cannabinoid binding sites within the brain which are distinguishable from the population of binding sites for SR141716A, its analogs and cannabidiol. Structural modification of SR141716A significantly altered the affinity of the compound and its relative ability to displace either [3H]CP-55,940 or [3H]SR141716A preferentially within the rat brain receptor membrane preparation.

Topics: Analgesics; Animals; Binding, Competitive; Cannabinoids; Cyclohexanols; Male; Models, Molecular; Piperidines; Pyrazoles; Rats; Rats, Inbred F344; Rimonabant

The effect of anandamide, an endogenous ligand for central (CB1) and peripheral (CB2) cannabinoid receptors, was investigated on the growth of the murine IL-6-dependent lymphoid cell line B9 and the murine IL-3-dependent myeloblastic cell line FDC-P1. In conditions of low serum level, anandamide potentiated the growth of both cytokine-dependent cell lines. Comparison with other fatty acid cannabinoid ligands such as (R)-methanandamide, a ligand with improved selectivity for the CB1 receptor, or palmitylethanolamide, an endogenous ligand for the CB2 receptor, showed a very similar effect, suggesting that cell growth enhancement by anandamide or its analogs could be mediated through either receptor subtype. However, several lines of evidence indicated that this growth-promoting effect was cannabinoid receptor-independent. First, the potent synthetic cannabinoid agonist CP 55940, which displays high affinity for both receptors, was inactive in this model. Second, SR 141716A and SR 144528, which are potent and specific antagonists of CB1 and CB2 receptors respectively, were unable, alone or in combination, to block the anandamide-induced effect. Third, inactivation of both receptors by pretreatment of cells with pertussis toxin did not affect the potentiation of cell growth by anandamide. These data demonstrated that neither CB1 nor CB2 receptors were involved in the anandamide-induced effect. Moreover, using CB2-transfected Chinese hamster ovary cells, we demonstrated that after complete blockade of the receptors by the specific antagonist SR 144528, anandamide was still able to strongly stimulate a mitogen-activated protein (MAP) kinase activity, clearly indicating that the endogenous cannabinoid can transduce a mitogenic signal in the absence of available receptors. Finally, arachidonic acid, a structurally related compound and an important lipid messenger without known affinity for cannabinoid receptors, was shown to trigger MAP kinase activity and cell growth enhancement similar to those observed with anandamide. These findings provide clear evidence for a functional role of anandamide in activating a signal transduction pathway leading to cell activation and proliferation via a non-cannabinoid receptor-mediated process.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Calcium-Calmodulin-Dependent Protein Kinases; Camphanes; Cannabinoids; Cell Division; CHO Cells; Cricetinae; Cyclohexanols; Endocannabinoids; Enzyme Activation; Gene Expression Regulation; Mice; Pertussis Toxin; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; RNA, Messenger; Signal Transduction; Transfection; Tumor Cells, Cultured; Virulence Factors, Bordetella

The subthalamic nucleus contains cannabinoid receptors and cannabinoid receptor mRNA. However, the role of cannabinoid receptors in this nucleus has not been examined. In order to investigate the functional role of cannabinoid receptors in the rat subthalamic nucleus, turning activity was observed following unilateral microinjection of the synthetic cannabinoid CP 55,940. CP 55,940 (10 microg) induced ipsilateral turning. This effect was blocked by coadministration of the cannabinoid receptor antagonist SR141716A (5 microg). These results suggest that cannabinoid receptors in the subthalamic nucleus mediate an inhibition of motor activity.

Topics: Animals; Behavior, Animal; Cannabinoids; Cyclohexanols; Functional Laterality; Globus Pallidus; Injections, Intraventricular; Male; Mesencephalon; Microinjections; Motor Activity; Piperidines; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Thalamic Nuclei

1. CB-1 cannabinoid receptors are strongly expressed in the molecular layer of the cerebellar cortex. We have analysed, in patch-clamped Purkinje cells (PCs) in rat cerebellar slices, the effect of the selective CB-1 agonists WIN55,212-2 and CP55,940 and of the selective CB-1 antagonist SR141716-A on excitatory synaptic transmission and synaptic plasticity. 2. Bath application of both agonists markedly depressed parallel fibre (PF) EPSCs. This effect was reversed by SR141716-A. In contrast, responses of PCs to ionophoretic application of glutamate were not affected by WIN55, 212-2. 3. The coefficient of variation and the paired-pulse facilitation of these PF-mediated EPSCs increased in the presence of WIN55,212-2. 4. WIN55,212-2 decreased the frequency of miniature EPSCs and of asynchronous synaptic events evoked in the presence of strontium in the bath, but did not affect their amplitude. 5. WIN55, 212-2 did not change the excitability of PFs. 6. WIN55,212-2 impaired long-term depression induced by pairing protocols in PCs. This effect was antagonized by SR141716-A. The same impairment of LTD was produced by 2-chloroadenosine, a compound that decreases the probability of release of glutamate at PF-PC synapses. 7. The present study demonstrates that cannabinoids inhibit synaptic transmission at PF-PC synapses by decreasing the probability of release of glutamate, and thereby impair LTD. These two effects might represent a plausible cellular mechanism underlying cerebellar dysfunction caused by cannabinoids.

Topics: Animals; Benzoxazines; Calcium Channel Blockers; Cannabinoids; Cerebellar Cortex; Cyclohexanols; Electric Stimulation; Electrophysiology; Excitatory Postsynaptic Potentials; In Vitro Techniques; Male; Membrane Potentials; Morpholines; Naphthalenes; Nerve Fibers; Neuronal Plasticity; Patch-Clamp Techniques; Piperidines; Purkinje Cells; Pyrazoles; Rats; Rats, Sprague-Dawley; Rimonabant; Synaptic Transmission

Delta9-tetrahydrocannabinol (delta9-THC), cannabinol and cannabidiol are three important natural cannabinoids from the Marijuana plant (Cannabis sativa). Using [35S]GTP-gamma-S binding on rat cerebellar homogenate as an index of cannabinoid receptor activation we show that: delta9-THC does not induce the maximal effect obtained by classical cannabinoid receptor agonists such as CP55940. Moreover at high concentration delta9-THC exhibits antagonist properties. Cannabinol is a weak agonist on rat cerebellar cannabinoid receptors and cannabidiol behaves as an antagonist acting in the micromolar range.

Topics: Analgesics; Animals; Binding, Competitive; Brain; Cannabidiol; Cell Membrane; Cerebellar Cortex; Cyclohexanols; Dronabinol; Guanosine Triphosphate; Male; Piperidines; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptors, Cannabinoid; Receptors, Drug; Rimonabant

Two subtypes of G-protein-coupled cannabinoid receptors have been identified to date: the CB1 central receptor subtype, which is mainly expressed in the brain, and the CB2 peripheral receptor subtype, which appears particularly abundant in the immune system. We investigated the expression of CB2 receptors in leukocytes using anti-CB2 receptor immunopurified polyclonal antibodies. We showed that peripheral blood and tonsillar B cells were the leukocyte subsets expressing the highest amount of CB2 receptor proteins. Dual-color confocal microscopy performed on tonsillar tissues showed a marked expression of CB2 receptors in mantle zones of secondary follicles, whereas germinal centers (GC) were weakly stained, suggesting a modulation of this receptor during the differentiation stages from virgin B lymphocytes to memory B cells. Indeed, we showed a clear downregulation of CB2 receptor expression during B-cell differentiation both at transcript and protein levels. The lowest expression was observed in GC proliferating centroblasts. Furthermore, we investigated the effect of the cannabinoid agonist CP55,940 on the CD40-mediated proliferation of both virgin and GC B-cell subsets. We found that CP55,940 enhanced the proliferation of both subsets and that this enhancement was blocked by the CB2 receptor antagonist SR 144528 but not by the CB1 receptor antagonist SR 141716. Finally, we observed that CB2 receptors were dramatically upregulated in both B-cell subsets during the first 24 hours of CD40-mediated activation. These data strongly support an involvement of CB2 receptors during B-cell differentiation.

Topics: Animals; B-Lymphocyte Subsets; Camphanes; CD40 Antigens; Cell Differentiation; CHO Cells; Cricetinae; Cricetulus; Cyclohexanols; Germinal Center; Humans; Microscopy, Confocal; Palatine Tonsil; Peptide Fragments; Piperidines; Pyrazoles; Rabbits; Receptors, Cannabinoid; Receptors, Drug; Recombinant Fusion Proteins; Rimonabant; Transfection

The present study attempts to indirectly determine if a neuronal cannabinoid (CB1) receptor mediates the intraocular pressure (IOP) reduction effects of arachidonoyl ethanolamide (AEA), its R-alpha-isopropyl analog, and the non-classical cannabinoid, CP-55,940. A series of these cannabinoids were dissolved in an aqueous 10-20% 2-hydroxypropyl-beta-cyclodextrin (2-HP-beta-CD) solution (containing 3% polyvinyl alcohol) and administered (25-62.5 microg) unilaterally to normotensive rabbit eyes. This was repeated on animals pre-treated with a subcutaneous injection (2.5 mg/kg) of the highly specific CB1 receptor antagonist, SR 141716A, dissolved in an aqueous 42% 2-HP-beta-CD solution. AEA, its R-alpha-isopropyl analog, and CP-55,940 reduced IOP upon topical application to a greater degree than was detected in the untreated eye. This reduction was eliminated for the latter two compounds by subcutaneous (s.c.) pretreatment of the rabbits with the CB1 receptor antagonist, but the IOP properties of AEA remained unchanged. SR 141716A administered alone (s.c.), elevated the IOP of both eyes. A CB1 receptor seems involved in the IOP reduction induced by either R-alpha-isopropyl anandamide or CP-55,940. However, AEA apparently functions through a different mechanism.

Topics: Animals; Arachidonic Acids; Cannabinoids; Cyclohexanols; Endocannabinoids; Female; Intraocular Pressure; Male; Ocular Hypotension; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rabbits; Receptors, Cannabinoid; Receptors, Drug; Rimonabant

We have investigated the nature of cannabinoid receptors in guinea-pig small intestine by establishing whether this tissue contains cannabinoid receptors with similar binding properties to those of brain CB1 receptors. The cannabinoids used were the CB1-selective antagonist SR141716A, the CB2-selective antagonist SR144528, the novel cannabinoid receptor ligand, 6'-azidohex-2'-yne-delta8-tetrahydrocannabinol (O-1184), and the agonists CP55940, which binds equally well to CB1 and CB2 receptors, and WIN55212-2, which shows marginal CB2 selectivity. [3H]-CP55940 (1 nM) underwent extensive specific binding both to forebrain membranes (76.3%) and to membranes obtained by sucrose density gradient fractionation of homogenates of myenteric plexus-longitudinal muscle of guinea-pig small intestine (65.2%). Its binding capacity (Bmax) was higher in forebrain (4281 fmol mg(-1)) than in intestinal membranes (2092 fmol mg(-1)). However, the corresponding KD values were not significantly different from each other (2.29 and 1.75 nM respectively). Nor did the Ki values for its displacement by CP55940, WIN55212-2, O-1184, SR141716A and SR144528 from forebrain membranes (0.87, 4.15, 2.85, 5.32 and 371.9 respectively) differ significantly from the corresponding Ki values determined in experiments with intestinal membranes (0.99, 5.03, 3.16, 4.95 and 361.5 nM respectively). The Bmax values of [3H]-CP55940 and [3H]-SR141716A in forebrain membranes did not differ significantly from each other (4281 and 5658 fmol mg(-1)) but were both greater than the Bmax of [3H]-WIN55212-2 (2032 fmol mg(-1)). O-1184 (10 or 100 nM) produced parallel dextral shifts in the log concentration-response curves of WIN55212-2 and CP55940 for inhibition of electrically-evoked contractions of the myenteric plexus-longitudinal muscle preparation, its KD values being 0.20 nM (against WIN55212-2) and 0.89 nM (against CP55940). We conclude that cannabinoid binding sites in guinea-pig small intestine closely resemble CB1 binding sites of guinea-pig brain and that 0-1184 behaves as a cannabinoid receptor antagonist in the guinea-pig myenteric plexus-longitudinal muscle preparation.

Topics: Analgesics; Animals; Benzoxazines; Binding Sites; Binding, Competitive; Camphanes; Cyclohexanols; Dronabinol; Guinea Pigs; Intestine, Small; Kinetics; Male; Morpholines; Muscle Contraction; Muscle, Smooth; Myenteric Plexus; Naphthalenes; Neuromuscular Junction; Piperidines; Prosencephalon; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Tritium

We have investigated the adaptive changes of the human central cannabinoid receptor (CB1) stably expressed in Chinese hamster ovary cells (CHO-CB1), after agonist (CP 55,940) or selective CB1 inverse agonist (SR 141716) treatment. CB1 receptor density and affinity constant as measured by binding assays with both tritiated ligands remained essentially unchanged after varying period exposure of CHO-CB1 cells (from 30 min to 72 hr) to saturating concentrations of CP 55,940 or SR 141716. However, using a C-myc-tagged version of the CB1 receptor, FACS analysis and confocal microscopy studies on CB1 expression indicated that the agonist promoted a disappearance of cell surface receptor although inverse agonist increased its cell surface density. Taken together these results suggest that 1) agonist induces internalization of the receptor into a cellular compartment that would be still accessible to both the hydrophobic ligands CP 55,940 or SR 141716; 2) inverse-agonist promotes externalization of the receptor from an intracellular preexisting pool to the cell surface. In parallel, we also investigated the associated effects of CP 55,940 and SR 141716 on CB1 receptor-coupled second messengers. We showed that preexposure of cells to CP 55,940 induced a rapid desensitization of the CB1 to the agonist response. The ability of CP 55,940 to inhibit the forskolin-stimulated adenylyl cyclase and to activate the mitogen-activated protein kinase activity was dramatically reduced. By striking contrast, SR 141716 pretreatment of CHO-CB1 cells not only had no significant effect on the potency of CP 55,940 to inhibit the forskolin-stimulated adenylyl cyclase but also induced a significant enhancement of the CP 55,940 ability to stimulate the mitogen-activated protein kinase activity. These results suggest that the modulation of the number of cell surface receptor could lead to functional desensitization or sensitization of the CB1 receptors.

Topics: Animals; Calcium-Calmodulin-Dependent Protein Kinases; Cannabinoids; Cell Membrane; CHO Cells; Cricetinae; Cyclic AMP; Cyclohexanols; Humans; Piperidines; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Signal Transduction; Transfection

Topics: Action Potentials; Analgesics; Animals; Basal Ganglia; Benzoxazines; Bicuculline; Cannabinoids; Cyclohexanols; Dopamine; Dopamine Agonists; Electrophysiology; GABA Antagonists; gamma-Aminobutyric Acid; Morpholines; Motor Neurons; Naphthalenes; Nerve Degeneration; Neurotransmitter Agents; Oxidopamine; Piperidines; Pyrazoles; Quinpirole; Rats; Rimonabant; Sympatholytics; Thalamic Nuclei

In the present study we examined the effect of the cannabinoid receptor agonist, [[1 a,2-(R)-5-(1,1-dimethylheptyl)-2-[5-hydroxy-2-(3-hydroxypropyl)cyc lohexyl]-phenol; CP 55,940] on [14C]acetylcholine and [3H]norepinephrine release from hippocampal slices and on [14C]acetylcholine release from striatal slices. CP 55,940 potently inhibited electrically evoked [14C]acetylcholine release from hippocampal slices, with an EC50 of 0.02 microM and a maximal inhibition of 61% at 1 microM. The inhibition of acetylcholine release by CP 55,940 was partially antagonized (60%) by the cannabinoid receptor antagonist, [[N-piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-me thyl-1H-pyrazole-3-carboxamide hydrochloride; SR 141716A]. Alone, SR 141716A significantly enhanced stimulated [14C]acetylcholine release. In contrast to the effects of CP 55,940 on [14C]acetylcholine release, electrically evoked [3H]norepinephrine release from hippocampal slices and [14C]acetylcholine release from striatal slices were both unaffected by this compound. Similarly, hippocampal [3H]norepinephrine release and striatal [14C]acetylcholine release were not affected by SR 141716A. In conclusion, the results of this study extend our previous data indicating that cannabinoid receptors modulate acetylcholine release in the hippocampus. The effects of cannabinoid receptor activation on [3H]acetylcholine release in the hippocampus does not appear to extend to [3H]norepinephrine release from this region or to acetylcholine release from the striatum.

Topics: Acetylcholine; Analgesics; Animals; Benzoxazines; Cannabinoids; Corpus Striatum; Cyclohexanols; Hippocampus; Male; Morpholines; Naphthalenes; Norepinephrine; Piperidines; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptors, Cannabinoid; Receptors, Drug; Rimonabant

Previous evidence suggests that the endogenous cannabinoid system could emerge and be operative early during brain development. In the present study, we have explored the distribution of specific binding for cannabinoid receptors in rat brain at gestational day 21 (GD21), postnatal days 5 (PND5) and 30 (PND30), and at adult age (> 70 days after birth) by using autoradiography with [3H]CP-55,940. Our results indicated that specific binding for cannabinoid receptors can be detected in the brain of rat fetuses at GD21 in the classic areas that contain these receptors in adulthood-in particular, in the cerebellum and the hippocampus and, to a lesser extent, in the basal ganglia, several limbic structures, and cerebral cortex. The density of cannabinoid receptors in all these structures increased progressively at all postnatal ages studied until reaching the classical adult values in 70-day-old animals. Interestingly, cannabinoid receptor binding can also be detected at GD21 in regions, in which they are scarcely distributed or not located in the adult brain and that have the particularity of all being enriched in neuronal fibers. Among these were the corpus callosum, anterior commissure, stria terminalis, fornix, white matter areas of brainstem, and others. This atypical location was quantitatively high at GD21, tended to wane at PND5, and practically disappeared at PND30 and in adulthood, with the only exception being the anterior commissure, which exhibited a moderate density for cannabinoid receptors. Moreover, the binding of [3H]CP-55,940 to cannabinoid receptors in the white matter regions at GD21 seems to be functional and involves a GTP-binding protein-mediated mechanism. Thus, the activation of these receptors with an agonist such as WIN-55,212-2 increased the binding of [35S]-guanylyl-5'-O-(gamma-thio)-triphosphate, measured by autoradiography, in the corpus callosum and white matter areas of brainstem of fetuses at GD21. This increase was reversed by coincubation of WIN-55,212-2 with SR141716, a cannabinoid receptor antagonist. As this antagonist is specific for the cerebral cannabinoid receptor subtype, called CB1, we can assert that the signal found for cannabinoid receptor binding in the fetal and early postnatal brain likely corresponds to this receptor subtype. Collectively, all these data suggest the existence of a transient period of the brain development in the rat, around the last days of the fetal period and the first days of postnatal lif

Topics: Animals; Animals, Newborn; Autoradiography; Benzoxazines; Brain; Brain Chemistry; Cannabinoids; Cyclohexanols; Female; Guanosine 5'-O-(3-Thiotriphosphate); Morpholines; Naphthalenes; Piperidines; Pregnancy; Pyrazoles; Rats; Rats, Wistar; Receptors, Cannabinoid; Receptors, Drug; Rimonabant

Two synthetic cannabinoids, WIN 55,212-2 {R-(+)-(2,3-dihydro-5-methyl-3-[{4-morpholinylmethyl]pyrol [1,2,3-de]-1,4-benzoxazin-6-yl)(1-naphthalenyl)methanone monomethanesulfonate} (5.0 and 10 mg/kg i.p.) and CP 55,940 {[1a,2-(R)-5-(1.1-dimethylheptyl)-2-[5-hydroxy-2-(3-hydroxypropyl) cyclohexyl]-phenol} {[1a,2-(R)-5-(1,1-dimethylheptyl)-2-[5-hydroxy-2-(3-hydroxypropyl) cyclohexyl]-phenol} (0.5 and 1.0 mg/kg i.p.), inhibited acetylcholine release in the rat hippocampus. The inhibition was prevented by the cannabinoid receptor antagonist, SR 141716A {N-(piperidin-1-yl)-5-(4- chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide} HCl, at the dose of 0.1 mg/kg i.p. Higher doses of SR 141716A (1.0 and 3.0 mg/kg i.p.) themselves increased hippocampal acetylcholine release, suggesting that acetylcholine output is tonically inhibited by endogenous cannabinoids. The results also suggest that the negative effects of marijuana on learning and memory may depend on cannabinoid receptor-mediated inhibition of acetylcholine release.

Topics: Acetylcholine; Animals; Benzoxazines; Cannabinoids; Cyclohexanols; Hippocampus; Male; Morpholines; Naphthalenes; Piperidines; Pyrazoles; Rats; Rats, Sprague-Dawley; Rimonabant

In the present study, we showed that Chinese hamster ovary (CHO) cells transfected with human central cannabinoid receptor (CB1) exhibit high constitutive activity at both levels of mitogen-activated protein kinase (MAPK) and adenylyl cyclase. These activities could be blocked by the CB1-selective ligand, SR 141716A, that functions as an inverse agonist. Moreover, binding studies showed that guanine nucleotides decreased the binding of the agonist CP-55,940, an effect usually observed with agonists, whereas it enhanced the binding of SR 141716A, a property of inverse agonists. Unexpectedly, we found that CB1-mediated effects of SR 141716A included inhibition of MAPK activation by pertussis toxin-sensitive receptor-tyrosine kinase such as insulin or insulin-like growth factor 1 receptors but not by pertussis toxin-insensitive receptor-tyrosine kinase such as the fibroblast growth factor receptor. We also observed similar results when cells were stimulated with Mas-7, a mastoparan analog, that directly activates the Gi protein. Furthermore, SR 141716A inhibited guanosine 5'-0-(thiotriphosphate) uptake induced by CP-55,940 or Mas-7 in CHO-CB1 cell membranes. This indicates that, in addition to the inhibition of autoactivated CB1, SR 141716A can deliver a biological signal that blocks the Gi protein and consequently abrogates most of the Gi-mediated responses. By contrast, SR 141716A had no effect on MAPK activation by insulin or IGF1 in CHO cells lacking CB1 receptors, ruling out the possibility of a direct interaction of SR 141716A with the Gi protein. This supports the notion that the Gi protein may act as a negative intracellular signaling cross-talk molecule. From these original results, which considerably enlarge the biological properties of the inverse agonist, we propose a novel model for receptor/ligand interactions.

Topics: Adenylate Cyclase Toxin; Animals; Calcium-Calmodulin-Dependent Protein Kinases; Cannabinoids; CHO Cells; Cricetinae; Cyclohexanols; Enzyme Activation; Humans; Insulin; Insulin-Like Growth Factor I; Pertussis Toxin; Piperidines; Pyrazoles; Receptor Protein-Tyrosine Kinases; Receptor, IGF Type 1; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Signal Transduction; Virulence Factors, Bordetella

Using the endogenous cannabinoid receptor agonist anandamide, the synthetic agonist CP 55940 [[1alpha,2beta(R)5alpha]-(-)-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-me thyl-1H-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 microM) and anandamide (10 microM) caused significant reductions in forskolin-stimulated cyclic AMP accumulation in rat striatal slices, which were reversed in the presence of SR 141716 (1 microM). CP 55940 (1 microM) 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-prelabelied 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.

Topics: Acetylcholine; Adenylyl Cyclases; Animals; Arachidonic Acids; Cannabinoids; Cerebral Cortex; Corpus Striatum; Cyclic AMP; Cyclohexanols; Dopamine; Electric Stimulation; Endocannabinoids; In Vitro Techniques; Kinetics; Male; Phosphatidylinositols; Piperidines; Polyunsaturated Alkamides; Potassium Chloride; Pyrazoles; Rats; Rats, Wistar; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Second Messenger Systems

1. The dose-related inhibition of the twitch responses of the myenteric plexus-longitudinal muscle preparation of the guinea-pig small intestine by cannabinoid (CB) agonists, (+)-WIN 55212 and CP 55940 during stimulation at 0.1 Hz with supramaximal voltage was confirmed. These agonists inhibited acetylcholine (ACh) release in the presence of physostigmine (7.7 microM) thus indicating a prejunctional site of action. 2. Inhibition of twitch responses and ACh release by CB agonists was reversed by the CB1-selective cannabinoid receptor antagonist, SR141716A. Dose-response curves to (+)-WIN 55212 and CP 55940 were shifted to the right, with no reduction of maximal response, by pretreatment with SR141716A (31.6-1000 nM), but not its vehicle, Tween 80 (1 microM). However, at very high concentrations (25-400 microM), Tween 80 itself caused a dose-related inhibition of the twitch response which was significantly reduced in the presence of SR141716A (1 microM). The opioid receptor antagonist, naloxone (1 microM) had no significant effect on the inhibition by CP 55940 of the twitch response. 3. (+)-WIN 55212, CP 55940 and Tween 80 (50 microM) had no effect on responses to exogenous ACh, confirming that their actions were prejunctional. SR141716A (1 microM) did not increase the sensitivity of the longitudinal muscle to either ACh or histamine, but inhibited the responses to high doses of ACh. 4. The (-)-enantiomer of WIN 55212, was approximately 300 times less active than the (+) enantiomer in inhibiting the twitch response, had no CB1 antagonist activity against the active isomer and did not inhibit the release of ACh in the presence of physostigmine. 5. The dissociation constant (KD) values for SR 141716A against the inhibitory effect of (+)-WIN 55212 and CP 55940 on the twitch response were 12.07 nM (95% confidence intervals 8.55 and 20.83) and 6.44 nM (95% confidence intervals 4.70 and 10.24), respectively. In experiments in which the release of ACh was inhibited by (+)-WIN 55212, the KD values were 9.21 nM and 10.53 nM at SR141716A concentrations of 31.6 nM and 100 nM, respectively. The KD values for the antagonism by naloxone of the inhibition of the twitch responses and the inhibition of ACh release by normorphine in this preparation were found to be 2.38 +/- 0.69 nM and 2.00 +/- 0.9 nM, respectively. 6. During maximal inhibition of ACh release by (+)-WIN 55212, the addition of normorphine (400 nM) caused a further significant decrease in ACh output. 7. SR141

Topics: Acetylcholine; Benzoxazines; Child, Preschool; Cyclohexanols; Histamine; Humans; In Vitro Techniques; Male; Morpholines; Myenteric Plexus; Naloxone; Naphthalenes; Norepinephrine; Piperidines; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Stereoisomerism

The binding of [123I]AM251 (a radioiodinated analog of the cannabinoid CB1 receptor antagonist SR141716A) was compared to that of [3H]CP 55,940 in mouse and rat brain preparations. Scatchard analysis of the binding of [123I]AM251 and [3H]CP 55,940 to membranes prepared from mouse cerebellum, striatum and hippocampus yielded similar Bmax values (15-41 pmol/g wet wt tissue). Kd values were lower for [123I]AM251 (0.23-0.62 nM) than for [3H]CP 55,940 (1.3-4 nM). CP 55,940 and SR141716A increased dissociation of [123I]AM251 from binding sites in mouse cerebellar homogenates to a similar extent. The structurally dissimilar cannabinoid receptor ligands THC, methanandamide, WIN 55, 212-2, CP 55,940 and SR141716A were each able to fully compete with binding of both [123I]AM251 and [3H]CP 55,940 in mouse cerebellum. In vitro autoradiography demonstrated that the distribution of binding sites for [123I]AM251 in rat brain was very similar to published distributions of binding sites for [3H]CP 55,940. Together, these observations suggest that AM251 binds to the same site (the cannabinoid CB1 receptor) in rodent brains as CP 55,940. However, the binding site domains which interact with AM251 and CP 55,940 may not be identical, since IC50 values for cannabinoid receptor ligands depended on whether [123I]AM251 or [3H]CP 55,940 was used as radioligand.

Topics: Animals; Autoradiography; Brain; Cyclohexanols; Iodine Radioisotopes; Male; Mice; Piperidines; Protein Binding; Pyrazoles; Radioligand Assay; Rats; Receptors, Cannabinoid; Receptors, Drug

We measured (-)-5-(1,1-dimethylheptyl)-2-[5-hydroxy-2-(3-hydroxypropyl)cyclohe xyl]-phenol (CP 55,940)-, (-)11-OH-delta8-tetrahydrocannabinol-dimethylheptyl (HU-210)-, anandamide- and delta9-tetrahydrocannabinol-stimulated G protein activation in mouse brain using the [35S]GTPgammaS functional assay. The Ki values for these drugs were determined by agonist competition binding with the cannabinoid CB1 receptor antagonist [3H]N-(piperidin-1-yl-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4- methyl-1H-pyrazole-3-carboxamidehydrochloride ([3H]SR141716A). This information was used to calculate the efficacy for drug stimulation of G protein activity. The rank order of efficacy was CP 55,940 > HU-210 > anandamide > delta9-tetrahydrocannabinol with the latter two drugs being partial agonists. Since efficacy values relate receptor occupancy to functional responses, we believe efficacy values are a better measure of drug-mediated functional responses compared with measurements of drug potency.

Topics: Animals; Arachidonic Acids; Brain; Cannabinoids; Cells, Cultured; Cyclohexanols; Dronabinol; Endocannabinoids; Enzyme Activation; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Male; Mice; Mice, Inbred ICR; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant

Changes in mitogen-induced splenocyte proliferation and NK activity were evaluated after acute (1 h) and chronic (6 d) in vivo treatment of rats with the synthetic cannabinoid compound CP-55,940. At a dose of 0.4 mg/kg i.p. it significantly inhibited the splenocyte proliferative response to PHA and NK activity but half this dose (0.2 mg/kg) had no effect on immune responses. Pretreatment of rats with the cannabinoid receptor CB1 antagonist SR141716A did not antagonize the CP-55,940-induced immunosuppression, excluding the activation of this receptor subtype in the mediation of this effect. When immune function studies were done on rats tolerant to CP-55,940-induced analgesia, full tolerance also developed for the inhibition of splenocyte proliferation and NK activity. The data provided indicate that CB1 cannabinoid receptors are not involved in mediating the acute and chronic effects of cannabinoids on the immune system and suggest a possible implication of CB2 receptor although other modalities of CP-55,940 action can not be ruled out.

Topics: Animals; Behavior, Animal; Cannabinoids; Cyclohexanols; Cytotoxicity, Immunologic; Drug Administration Schedule; Immunosuppressive Agents; Injections, Intraperitoneal; Killer Cells, Natural; Lymphocyte Activation; Male; Piperidines; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB2; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Spleen

The ability of cannabinoid receptor stimulation or blockade to alter catalepsy produced by dopamine D1 and D2 receptor antagonists was studied in rats. The cannabinoid receptor antagonist SR 141716A (N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-me thyl-1H- pyrazole-3-carboxamidehydrochloride) (0.5 and 2.5 mg/kg) reduced catalepsy elicited by the cannabinoid receptor agonist CP 55,940 (1 alpha,2-(R)-5-(1,1-dimethylheptyl)-2-[5-hydroxy-2-(3-hydroxypropyl ) cyclohexyl-phenol) (0.5 mg/kg). However, SR 141716A (0.5 and 2.5 mg/kg) did not decrease catalepsy produced by the dopamine D1 receptor antagonist SCH 23390 (R-(+)-7chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5- tetrahydro-1-H-3-benzazepine) (0.5 mg/kg) or the dopamine D2 receptor antagonist raclopride (S(-)-3,5-dichloro-N-(1-ethyl-2-pyrrolidinyl)-methyl-6-methoxysalicylami de) (2.5 mg/kg), suggesting that, under these conditions, endogenous cannabinoid ligands do not modulate the cataleptic effects of dopamine D1 or D2 receptor antagonists. In contrast, CP 55,940 (0.025 and 0.1 mg/kg), at doses which do not produce catalepsy when administered alone, enhanced catalepsy produced by SCH 23390 and raclopride. These results suggest that stimulation, but not blockade, of brain cannabinoid receptors modifies catalepsy behavior produced by selective dopamine D1 and D2 receptor blockade.

Topics: Animals; Benzazepines; Brain; Catalepsy; Cyclohexanols; Dopamine Antagonists; Dose-Response Relationship, Drug; Male; Piperidines; Pyrazoles; Raclopride; Rats; Rats, Sprague-Dawley; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Salicylamides

SR141716A (Sanofi Recherche), a pyrazole derivative with high affinity for rat and human CB1 cannabinoid receptors, has recently been reported to reverse biochemical, physiological and behavioral effects induced by cannabinoid agonists. The present experiments characterized the activity of SR141716A (SR) in behavioral procedures designed to assess its antagonistic and intrinsic effects on unconditioned behavior and on complex learned behaviors. Six adult male pigeons were trained to discriminate injections of 0.56 mg/kg delta 9-tetrahydrocannabinol (delta 9-THC) from vehicle under a two-key, fixed-ratio schedule of food reinforcement. SR (IM) produced a nearly complete blockade of THC-appropriate responding occasioned by the training dose without inducing significant changes in session response rates, but also produced partial substitution for delta 9-THC when administered alone. In another group of pigeons trained under a multiple schedule of signaled and unsignaled fixed consecutive number (FCN) responding, SR had little effect on accuracy, but delta 9-THC produced dose-related decreases in accuracy under both schedule components. SR was also evaluated in acoustic startle procedures in rats. SR produced little effect either on startle amplitude or prepulse inhibition of acoustic startle. In contrast, the potent cannabinomimetic CP-55, 940 produced large decreases in startle responses elicited by 120 dB [A] broad-band noise. These decreases were completely reversed by SR (10 mg/kg, IP). In concurrent measures, SR blocked the hypothermic effect CP-55,940. These results suggest that SR is an effective antagonist of the psychoactive effects of cannabinoids.

Topics: Animals; Columbidae; Cyclohexanols; Discrimination Learning; Dronabinol; Male; Piperidines; Pyrazoles; Rats; Rats, Sprague-Dawley; Rats, Wistar; Receptors, Cannabinoid; Receptors, Drug; Reflex, Startle; Rimonabant

Our results provide further evidence for the hypothesis that the mouse vas deferens contains cannabinoid CB1 receptors. Thus we found that in the presence of forskolin, the cannabinoid receptor agonist, CP 55,940 ((-)-3-[2-hydroxy-4-(1,1-dimethyl-heptyl)phenyl]-4-(3- hydroxypropyl)cyclohexan-1-ol) produced a concentration related inhibition of cyclic AMP production by the vas deferens (EC50 = 6.0 nM). At 100 nM, SR141716A (N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-me thyl-1H- pyrazole-3-carboxamide hydrochloride) attenuated this effect of CP 55,940, producing a parallel rightward shift in its log concentration-response curve (Kd = 4.3 nM). We also found that cyclic AMP production was inhibited by (-)-11-hydroxy-1',1'-dimethylheptyl-delta 8- tetrahydrocannabinol but not by the (+) enantiomer.

Topics: Animals; Cannabinoids; Colforsin; Cyclic AMP; Cyclohexanols; In Vitro Techniques; Male; Mice; Piperidines; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Stereoisomerism; Vas Deferens

1. CP 50,556, CP 55,940, nabilone, CP 56,667, delta 9 -tetrahydrocannabinol (THC) and cannabinol each inhibited electrically-evoked contractions of the myenteric plexus-longitudinal muscle preparation of guinea-pig small intestine in a concentration-related manner. The IC50 values of these cannabinoids, respectively 3.45, 3.46, 30.61, 162.94, 214.63, and 3913.5 nM, correlate well with previously obtained potency values for displacement of [3H]-CP 55,940 from cannabinoid binding sites. 2. Electrically-evoked contractions of the myenteric plexus-longitudinal muscle preparation were also inhibited by AM 630 (6-iodo-pravadoline) and by WIN 55,212-2 (IC50 = 1923.0 and 5.54 nM, respectively). The present finding that AM 630 is an agonist, contrasts with a previous observation that it behaves as a cannabinoid receptor antagonist in the mouse isolated vas deferens. 3. SR141716A produced dose-related parallel rightward shifts in the log concentration-response curves of CP 55,940, WIN 55,212-2, THC and AM 630 for inhibition of electrically-evoked contractions of the myenteric plexus-longitudinal muscle preparation. SR141716A (1 microM) did not reverse the inhibitory effects of normorphine and clonidine on electrically-evoked contractions or potentiate the contractile response to acetylcholine. 4. Doses of naloxone and yohimbine that reversed the inhibitory effects of normorphine or clonidine on electrically-evoked contractions of the myenteric plexus-longitudinal muscle preparation did not affect the inhibitory response to WIN 55,212-2. 5. Electrically-evoked release of acetylcholine from strips of myenteric plexus-longitudinal muscle was decreased by 200 nM CP 55,940 and this inhibitory effect was almost completely reversed by 1 microM SR141716A. Acetylcholine-induced contractions were not affected by 200 nM CP 55,940. 6. These results support the hypothesis that guinea-pig small intestine contains prejunctional cannabinoid CB1 receptors through which cannabinoids act to inhibit electrically-evoked contractions by reducing release of the contractile transmitter, acetylcholine. 7. THC was found to be more susceptible to antagonism by SR141716A than CP 55,940 or AM 630, raising the possibility that guinea-pig small intestine contains more than one type of cannabinoid receptor. 8. At concentrations of 10 nM and above, SR141716A produced small but significant increases in the amplitude of electrically-evoked contractions of the myenteric plexus-longitudinal muscle pre

Topics: Acetylcholine; Analgesics; Animals; Cyclohexanols; Dose-Response Relationship, Drug; Guinea Pigs; Intestine, Small; Male; Muscle Contraction; Myenteric Plexus; Piperidines; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant

The recent identification and cloning of guanine nucleotide regulatory protein-coupled brain-type and spleen-type cannabinoid receptors (CB1-R and CB2-R, respectively) provide evidence that many of the effects of cannabinoids are mediated via these receptors. Our recent observation of expression of both CB1-R and CB2-R genes in the preimplantation mouse embryo suggests that it could also be a target for cannabinoids. Indeed, cannabinoid agonists interfered with preimplantation embryo development in vitro. To examine whether cannabinoid effects on preimplantation embryos are mediated via CB1-R, we developed rabbit antipeptide antibodies against the N-terminal region of CB1-R and examined the receptor protein in the blastocyst by Western blotting and its spatiotemporal distribution in preimplantation mouse embryos by immunohistochemistry. Cannabinoid binding sites in the blastocyst were examined by Scatchard analysis, while the reversibility of cannabinoid-induced embryonic arrest in vitro was monitored using a specific antagonist to CB1-R, SR141716A. Western blot analysis detected a major band of approximately 59 kDa and a minor band of approximately 54 kDa in the blastocyst. Immunocytochemistry detected this receptor protein from the 2-cell through the blastocyst stages. Scatchard analysis using 3H-anandamide (an endogenous ligand) showed a single class of binding sites in Day 4 blastocysts with an apparent Kd of 1.0 nM and Bmax of 0.09 fmol/blastocyst. Considering the total number of cells (approximately 50) and total protein content (approximately 20 ng) of a blastocyst, it is apparent that the mouse blastocyst has many more high-affinity receptors than those in the mouse brain (Kd: 1.8 nM and Bmax: 18.8 pmol/mg membrane protein). Cannabinoid agonists and the CB1-R antagonist SR141716A effectively competed for anandamide binding in the blastocyst. To determine whether cannabinoid inhibition of embryonic development could be reversed by SR141716A, 2-cell embryos were cultured in the presence of cannabinoid agonists with or without SR141716A for 72 h. Most of the 2-cell embryos cultured in the absence of the agonists developed into blastocysts (approximately 90%). In contrast, the addition of cannabinoid agonists anandamide, Win 55212-2, or CP 55,940 in the culture medium severely compromised embryonic development: more than 60% of the 2-cell embryos failed to develop to blastocysts. A reduction in trophectoderm cell numbers was noted in those blastocysts

Topics: Analgesics; Animals; Arachidonic Acids; Benzoxazines; Binding, Competitive; Blastocyst; Blotting, Western; Cannabinoids; Cyclohexanols; Embryo, Mammalian; Embryonic and Fetal Development; Embryonic Development; Endocannabinoids; Female; Immunohistochemistry; Male; Mice; Morpholines; Naphthalenes; Piperidines; Polyunsaturated Alkamides; Pregnancy; Pyrazoles; Radioligand Assay; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Tritium

This study tested the hypothesis that cannabinoid agonists, applied locally into the pars reticulata of substantia nigra (SNpr), could modulate striatonigral transmission, without affecting the response of SNpr neurons to iontophoretically-applied GABA. Multibarreled glass capillary electrode assemblies were used for extracellular recording of the spontaneous electrical activity of single SNpr cells in anesthetized rats. Local pressure ejection of the cannabinoid agonists Win 55212-2 (WIN2) and CP 55940 increased SNpr spontaneous firing rate by 13-46%, similar to the effects of systemic injections. Neither WIN2 nor CP 55940 had an effect on the slowing of SNpr neuron activity in response to iontophoretic GABA. Local pressure application of Win 55212-3 (the much less active enantiomer of WIN2) produced an insignificant decrease in SNpr firing rate. Similarly, locally applied vehicle (45% 2-hydroxypropyl-beta-cyclodextrin) produced insignificant decreases in SNpr firing. A second application of cannabinoid agonist produced a much smaller effect, suggesting desensitization. Increasing the interval between CP 55940 applications to 45 min showed recovery of sensitivity to the agonist. Local application of the cannabinoid antagonist, SR 141716A, significantly decreased spontaneous cell firing by 34%. CP 55940, when given immediately following or concurrently with the antagonist application failed to produce the expected increase in discharge rate over baseline. A second application of CP 55940 45 min later produced a 26% increase in firing rate. Bicuculline methiodide (BMI) was applied locally causing a significant increase in SNpr cell firing. CP 55940, when locally administered concurrently with bicuculline methiodide, had no further effect on the firing rate of the cell. Based on the reported presynaptic localization of cannabinoid receptors in SNpr, these findings suggest that cannabinoids act within the SNpr to modulate striatonigral neurotransmission presynaptically. The effect of SR 141716A suggests that an endogenous cannabinoid may mediate striato-nigral transmission.

Topics: Action Potentials; Analgesics; Analysis of Variance; Animals; Benzoxazines; Bicuculline; Cannabinoids; Cyclohexanols; gamma-Aminobutyric Acid; Iontophoresis; Male; Morpholines; Naphthalenes; Neurons; Piperidines; Pressure; Pyrazoles; Rats; Rats, Sprague-Dawley; Rimonabant; Substantia Nigra

The present study investigated the effects of the cannabinoid receptor agonist CP 55,940 (1-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl) phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol) and the cannabinoid receptor antagonist SR 141716A (N-(piperidin-l-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-me thyl-1 H-pyrazole-3-carboxamide hydrochloride) on ultrasonic vocalizations, body temperature and activity in 11-13-day-old rat pups. Testing occurred in a 5-min session 30 min following drug administration. CP 55,940 produced a dose-dependent decrease in ultrasonic vocalizations, with a 1000-micrograms/kg dose causing an almost complete inhibition of calls. Doses of 100 and 1000 micrograms/kg of CP 55,940, but not 10 micrograms/kg, caused significant hypothermia in the pups and the 1000 micrograms/kg dose also inhibited activity. The cannabinoid receptor antagonist SR 141716A (20 mg/kg) reversed the effects of 1000 micrograms/kg CP 55,940 on ultrasonic vocalizations and body temperature, but the benzodiazepine receptor antagonist flumazenil (20 mg/kg), the dopamine D1 receptor antagonist SCH 23390 (0.5 mg/kg) and the opioid receptor antagonist naloxone (1 mg/kg) did not. When administered alone, SR 141716A (20 mg/kg) increased pup ultrasonic vocalizations without affecting body temperature or activity. These results indicate that cannabinoids modulate ultrasonic vocalization production in rat pups in a manner that is independent of hypothermia. The increase in ultrasonic vocalizations produced by SR 141716A is one of the first reported behavioural effects of this drug and suggests that the endogenous cannabinoid ligand anandamide may be involved in the regulation of ultrasonic vocalizations.

Topics: Analysis of Variance; Animals; Animals, Newborn; Behavior, Animal; Benzazepines; Body Temperature; Cannabinoids; Cyclohexanols; Dopamine Antagonists; Dose-Response Relationship, Drug; Flumazenil; GABA Modulators; Hypothermia; Motor Activity; Naloxone; Narcotic Antagonists; Piperidines; Pyrazoles; Rats; Rimonabant; Vocalization, Animal

This study examined the effect of cannabinoid ligands on human tonsillar B-cells activated either through cross-linking of surface immunoglobulins or ligation of the CD40 antigen. The two synthetic cannabinoids, CP55,940 and WIN55212-2, as well as delta 9-tetrahydrocannabinol (THC), the psychoactive component of marijuana, caused a dose-dependent increase of B-cell proliferation and displayed EC50 at low nanomolar concentrations. This cannabinoid-induced enhancing activity was inhibited by pertussis toxin which suggested a G-protein-coupled receptor process. In addition, the absence of antagonistic effect of SR141716A, a specific CB1 receptor antagonist, together with the demonstration that human B-cells displayed large amount of CB2 receptor mRNAs, led us to assume that the growth enhancing activity observed on B-cells at very low concentrations of cannabinoids could be mediated through the CB2 receptor.

Topics: Antigens, CD; Antigens, Differentiation, B-Lymphocyte; B-Lymphocytes; Base Sequence; Benzoxazines; Cannabinoids; CD40 Antigens; Cell Division; Cyclohexanols; DNA; Dronabinol; Humans; Ligands; Lymphocyte Activation; Molecular Sequence Data; Morpholines; Naphthalenes; Palatine Tonsil; Pertussis Toxin; Piperidines; Pyrazoles; Receptors, Antigen, B-Cell; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; RNA, Messenger; Virulence Factors, Bordetella

CP 55,940 is a potent synthetic bicyclic cannabinoid analog that has been used in a number of studies as a radioligand for the cannabinoid receptor. This compound shares behavioral and biochemical properties with naturally occurring cannabinoids such as delta 9-THC. The purpose of the present study was 3-fold: to establish the ability of CP 55,940 to serve as a discriminative stimulus, to determine whether this discriminative stimulus is identical to that of delta 9-THC, and to examine whether a newly developed cannabinoid antagonist, SR141716A, would antagonize the discriminative stimulus effects of CP 55,940. Rats were trained to discriminate 0.1 mg/kg CP 55,940 from vehicle in standard 2-lever operant conditioning chambers. CP 55,940 produced dose-dependent generalization from the training dose in dose-effect determinations conducted before and after testing with other drugs. The effects of the training dose of CP 55,940 were dose-dependently antagonized by co-administration of SR141716A. Results of substitution tests showed that delta 9-THC, WIN 55,212-2, and cannabinol substituted completely for CP 55,940 in a dose-dependent manner; however, CP 55,940 was approx 10-fold more potent than any of the other drugs in producing CP 55,940-like discriminative stimulus effects. Several drugs with CNS depressant properties (phencyclidine, haloperidol and diazepam) failed to produce reliable substitution for CP 55,940. These results demonstrate that CP 55,940 has discriminative stimulus effects and that it shares these effects with structurally dissimilar compounds that, like CP 55,940, bind to the cannabinoid receptor. Further, these effects are blocked by SR141716A, a cannabinoid receptor antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Analgesics; Animals; Behavior, Animal; Cannabinoids; Cyclohexanols; Discrimination, Psychological; Dose-Response Relationship, Drug; Dronabinol; Injections, Intraperitoneal; Male; Phencyclidine; Piperidines; Pyrazoles; Rats; Rats, Sprague-Dawley; Rimonabant

When injected unilaterally into the mouse striatum, cannabinoid agonists such as Win 55212-2 (1-100 ng/mouse), CP 55940 (0.1-50 ng/mouse), and anandamide (0.5-50 ng/mouse), the putative endogenous ligand of CB1 receptor, dose-dependently induced turning behavior. SR 141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-me thyl-1H- pyrazole-3-carboxamide hydrochloride], the selective antagonist of CB1 receptor, antagonized the three cannabinoid receptor agonists-induced turning with similar ED50s (0.13-0.15 mg/kg, IP). Spiroperidol (a D2 receptor blocker), (+)-SCH 23390 (a D1 receptor blocker), or prior 6-OHDA lesions of the striatum blocked Win 55212-2- and CP 55940-induced turning, thus suggesting the involvement of DA transmission in cannabinoid-induced turning. Taken together, these findings reinforce the notion of a cannabinoid receptor-mediated control of nigrostriatal function.

Topics: Analgesics; Animals; Benzoxazines; Cannabis; Corpus Striatum; Cyclohexanols; Dose-Response Relationship, Drug; Female; Injections; Mice; Morpholines; Naphthalenes; Neurons; Oxidopamine; Piperidines; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Receptors, Neurokinin-2; Rimonabant; Stereoisomerism; Stereotyped Behavior

The G-protein-coupled central cannabinoid receptor (CB1) has been shown to be functionally associated with several biological responses including inhibition of adenylate cyclase, modulation of ion channels and induction of the immediate-early gene Krox-24. Using stably transfected Chinese Hamster Ovary cells expressing human CB1 we show here that cannabinoid treatment induces both phosphorylation and activation of mitogen-activated protein (MAP) kinases, and that these effects are inhibited by SR 141716A, a selective CB1 antagonist. The two p42 and p44 kDa MAP kinases are activated in a time- and dose-dependent manner. The rank order of potency for the activation of MAP kinases with various cannabinoid agonists is CP-55940 > delta 9-tetrahydrocannabinol > WIN 55212.2, in agreement with the pharmacological profile of CB1. The activation of MAP kinases is blocked by pertussis toxin but not by treatment with hydrolysis-resistant cyclic AMP analogues. This suggests that the signal transduction pathway between CB1 and MAP kinases involves a pertussis-toxin-sensitive GTP-binding protein and is independent of cyclic AMP metabolism. This coupling of CB1 subtype and mitogenic signal pathway, also observed in the human astrocytoma cell line U373 MG, may explain the mechanism of action underlying cannabinoid-induced Krox-24 induction.

Topics: 1-Methyl-3-isobutylxanthine; 8-Bromo Cyclic Adenosine Monophosphate; Adenylate Cyclase Toxin; Analgesics; Animals; Benzoquinones; Bucladesine; Calcium-Calmodulin-Dependent Protein Kinases; Cannabinoids; Cell Line; CHO Cells; Cricetinae; Cyclohexanols; DNA-Binding Proteins; Early Growth Response Protein 1; Enzyme Activation; Enzyme Inhibitors; GTP-Binding Proteins; Humans; Immediate-Early Proteins; Kinetics; Lactams, Macrocyclic; Pertussis Toxin; Piperidines; Pyrazoles; Quinones; Receptors, Cannabinoid; Receptors, Drug; Recombinant Proteins; Rifabutin; Rimonabant; Tetradecanoylphorbol Acetate; Transcription Factors; Transfection; Virulence Factors, Bordetella

The novel compounds, 1-pentyl-2-methyl-3-(1-naphthoyl)indole, 1-pentyl-3-(1-naphthoyl)pyrrole and 1-heptyl-3-(1-naphthoy)indole, produced a dose-related inhibition of electrically evoked contractions of the mouse vas deferens, with IC50 values of 2.56 nM, 3.38 nM and 639 nM respectively. Kd values of the selective CB1 cannabinoid receptor antagonist, SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-me thyl-1 H-pyrazole-3-carboxamide hydrochloride], determined in the vas deferens from experiments with these compounds are 1.34 nM, 3.86 nM and 8.06 nM respectively, indicating their susceptibility to antagonism by SR141716A is similar to that of their parent compound, the CB1 cannabinoid receptor agonist WIN 55,212-2 ¿(R)-(+)-[2,3-dihydro-5-methyl-3-[4-methylino)methyl]pyrrolo-[1,2, 3-de]-1,4-benzoxazin-6-yl](1-naphthyl)methanone}. SR141716A (100 nM) had no effect on the actions of two non-cannabinoid receptor agonists, morphine and clonidine. These results provide strong support for the hypothesis that 1-pentyl-2-methyl-3-(1-naphthoyl)indole, 1-pentyl-3-(1-naphthoyl)pyrrole and 1-heptyl-3-(1-naphthoyl)indole are cannabinoid receptor agonists and confirm that the WIN 55,212-2 molecule can be modified considerably without detectable loss of cannabinoid activity.

Topics: Animals; Benzoxazines; Cannabinoids; Cyclohexanols; Dose-Response Relationship, Drug; In Vitro Techniques; Male; Mice; Morpholines; Muscle Contraction; Naphthalenes; Piperidines; Pyrazoles; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Vas Deferens

SR141716A is the first selective and orally active antagonist of the brain cannabinoid receptor. This compound displays nanomolar affinity for the central cannabinoid receptor but is not active on the peripheral cannabinoid receptor. In vitro, SR141716A antagonises the inhibitory effects of cannabinoid receptor agonists on both mouse vas deferens contractions and adenylyl cyclase activity in rat brain membranes. After intraperitoneal or oral administration SR141716A antagonises classical pharmacological and behavioural effects of cannabinoid receptor agonists. This compound should prove to be a powerful tool for investigating the in vivo functions of the anandamide/cannabinoid system.

Topics: Animals; Benzoxazines; Binding, Competitive; Biological Assay; Brain; Cannabinoids; Cell Membrane; Cyclohexanols; In Vitro Techniques; Mice; Morpholines; Naphthalenes; Piperidines; Pyrazoles; Rats; Receptors, Cannabinoid; Receptors, Drug; Rimonabant