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

Cannabinoids in current use such as nabilone activate both CB1 and CB2 receptors. Selective CB2 activation may provide some of the therapeutic effects of cannabinoids, such as their immuno-modulatory properties, without the psychoactive effects of CB1 activation. Therefore, cannabinoid CB2 receptors represent an attractive target for drug development. However, selective and potent CB2 agonists remain in development. CB1 and CB2 differ considerably in their amino acid sequence and tertiary structures. Therefore, clinical development of potent and selective CB2 agonists is probable. Mutational and ligand binding studies, functional mapping, and computer modelling have revealed key residues and domains in cannabinoid receptors that are involved in agonist and antagonist binding to CB1 and CB2. In addition, CB2 has undergone more rapid evolution, and results for ligand binding and efficacy cannot be automatically extrapolated from rat or mouse CB2 to human. Furthermore, loss of CB1 affinity is a crucial property for CB2-selective ligands, and although rat CB1 is 97% homologous with human CB1, critical differences do exist, with potential for further exploitation in drug design. In this paper we briefly review previous cannabinoid receptor models and mutation/binding studies. We also review binding affinity ratios with respect to CB1 and CB2. We then employ our own models to illustrate key cannabinoid receptor residues and binding subdomains that are involved in these differences in binding affinities and discuss how these might be exploited in the development of CB2 specific ligands. Published reports for species specific binding affinities for CB2 are scarce, and we argue that this needs to be corrected prior to the progression of CB2 agonists from pre-clinical to clinical research.

Topics: Animals; Arachidonic Acids; Benzoxazines; Cannabinoids; Cyclohexanols; Endocannabinoids; Humans; Ligands; Morpholines; Naphthalenes; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2

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

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

The endocannabinoid anandamide (AEA) causes vasorelaxation in animal studies. Although circulating AEA levels are increased in many pathologies, little is known about its vascular effects in humans. The aim of this work was to characterise the effects of AEA in human arteries. Ethical approval was granted to obtain mesenteric arteries from patients (n=31) undergoing bowel resection. Wire myography was used to probe the effects and mechanisms of action of AEA. RT-PCR was used to confirm the presence of receptor mRNA in human aortic endothelial cells (HAECs) and intracellular signalling proteins were measured using multiplex technology. AEA caused vasorelaxation of precontracted human mesenteric arteries with an R

Topics: Adult; Aged; Aged, 80 and over; Aorta; Arachidonic Acids; Cannabinoids; Cyclohexanols; Endocannabinoids; Endothelial Cells; Endothelium, Vascular; Female; Humans; Intracellular Signaling Peptides and Proteins; Male; Mesenteric Arteries; Middle Aged; Nitric Oxide; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; RNA, Messenger; Vasodilation

The endocannabinoid system (ECS) is involved in a considerable number of physiological processes in the Central Nervous System. Recently, a modulatory role of cannabinoid receptors (CBr) and CBr agonists on the reduction of the N-methyl-d-aspartate receptor (NMDAr) activation has been demonstrated. Quinolinic acid (QUIN), an endogenous analog of glutamate and excitotoxic metabolite produced in the kynurenine pathway (KP), selectively activates NMDAr and has been shown to participate in different neurodegenerative disorders. Since the early pattern of toxicity exerted by this metabolite is relevant to explain the extent of damage that it can produce in the brain, in this work we investigated the effects of the synthetic CBr agonist WIN 55,212-2 (WIN) and other agonists (anandamide or AEA, and CP 55,940 or CP) on early markers of QUIN-induced toxicity in rat striatal cultured cells and rat brain synaptosomes. WIN, AEA and CP exerted protective effects on the QUIN-induced loss of cell viability. WIN also preserved the immunofluorescent signals for neurons and CBr labeling that were decreased by QUIN. The QUIN-induced early mitochondrial dysfunction, lipid peroxidation and reactive oxygen species (ROS) formation were also partially or completely prevented by WIN pretreatment, but not when this CBr agonist was added simultaneously with QUIN to brain synaptosomes. These findings support a neuroprotective and modulatory role of cannabinoids in the early toxic events elicited by agents inducing excitotoxic processes.

Topics: Animals; Arachidonic Acids; Benzoxazines; Brain; Cannabinoid Receptor Agonists; Cell Survival; Cells, Cultured; Cyclohexanols; Endocannabinoids; Excitatory Amino Acid Agents; Lipid Peroxidation; Male; Mitochondria; Morpholines; Naphthalenes; Neurons; Oxidative Stress; Polyunsaturated Alkamides; Quinolinic Acid; Rats, Wistar; Reactive Oxygen Species; Receptors, Cannabinoid; Synaptosomes

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

Cannabinoid receptor (CBs) agonists affect the growth of tumor cells via activation of deadly cascades. The spectrum of action of these agents and the precise role of the endocannabinoid system (ECS) on oncogenic processes remain elusive. Herein we compared the effects of synthetic (CP 55-940 and WIN 55,212-2) and endogenous (anandamide or AEA) CBs agonists (10-20 μM) on morphological changes, cell viability, and induction of apoptosis in primary astrocytes and in two glioblastoma cell lines (C6 and U373 cells) in order to characterize their possible differential actions on brain tumor cells. None of the CBs agonist tested induced changes in cell viability or morphology in primary astrocytes. In contrast, CP 55-940 significantly decreased cell viability in C6 and U373 cells at 5 days of treatment, whereas AEA and WIN 55,212-2 moderately decreased cell viability in both cell lines. Treatment of U373 and C6 for 3 and 5 days with AEA or WIN 55,212-2 produced discrete morphological changes in cell bodies, whereas the exposure to CP 55-940 induced soma degradation. CP 55-940 also induced apoptosis in both C6 and U373 cell lines. Our results support a more effective action of CP 55-940 to produce cell death of both cell lines through apoptotic mechanisms. Comparative aspects between cannabinoids with different profiles are necessary for the design of potential treatments against glial tumors.

Topics: Animals; Apoptosis; Arachidonic Acids; Astrocytes; Benzoxazines; Cannabinoid Receptor Agonists; Cannabinoids; Cell Line, Tumor; Cell Survival; Cells, Cultured; Cyclohexanols; DNA; Endocannabinoids; Humans; Morpholines; Naphthalenes; Polyunsaturated Alkamides; Rats; Rats, Wistar

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

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

Modulation of type 1 cannabinoid receptor (CB1) activity has been touted as a potential means of treating addiction, anxiety, depression, and neurodegeneration. Different agonists of CB1 are known to evoke varied responses in vivo. Functional selectivity is the ligand-specific activation of certain signal transduction pathways at a receptor that can signal through multiple pathways. To understand cannabinoid-specific functional selectivity, different groups have examined the effect of individual cannabinoids on various signaling pathways in heterologous expression systems. In the current study, we compared the functional selectivity of six cannabinoids, including two endocannabinoids (2-arachidonyl glycerol (2-AG) and anandamide (AEA)), two synthetic cannabinoids (WIN55,212-2 and CP55,940), and two phytocannabinoids (cannabidiol (CBD) and Δ(9)-tetrahydrocannabinol (THC)) on arrestin2-, Gα(i/o)-, Gβγ-, Gα(s)-, and Gα(q)-mediated intracellular signaling in the mouse STHdh(Q7/Q7) cell culture model of striatal medium spiny projection neurons that endogenously express CB1. In this system, 2-AG, THC, and CP55,940 were more potent mediators of arrestin2 recruitment than other cannabinoids tested. 2-AG, AEA, and WIN55,212-2, enhanced Gα(i/o) and Gβγ signaling, with 2-AG and AEA treatment leading to increased total CB1 levels. 2-AG, AEA, THC, and WIN55,212-2 also activated Gα(q)-dependent pathways. CP55,940 and CBD both signaled through Gα(s). CP55,940, but not CBD, activated downstream Gα(s) pathways via CB1 targets. THC and CP55,940 promoted CB1 internalization and decreased CB1 protein levels over an 18-h period. These data demonstrate that individual cannabinoids display functional selectivity at CB1 leading to activation of distinct signaling pathways. To effectively match cannabinoids with therapeutic goals, these compounds must be screened for their signaling bias.

Topics: Animals; Arachidonic Acids; Arrestin; Benzoxazines; Blotting, Western; Cannabinoid Receptor Agonists; Cannabinoids; Cells, Cultured; Corpus Striatum; Cyclohexanols; Dendritic Spines; Dronabinol; Endocannabinoids; Fluorescence Resonance Energy Transfer; Glycerides; GTP-Binding Proteins; Ligands; Luminescent Proteins; Mice; Models, Biological; Morpholines; Naphthalenes; Neurons; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB1; Signal Transduction

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

The major constituent of the cannabis plant, Δ(9)-tetrahydrocannabinol, has stimulatory and depressant effects on cardiovascular functions. There is evidence from an in vivo study on the urethane-anaesthetized rat that part of the stimulatory effects is related to a tyramine-like activity. In the present study, we examined whether Δ(9)-tetrahydrocannabinol induces carrier-mediated noradrenaline release in vitro. The study was extended to another phytocannabinoid, cannabidiol, to the synthetic cannabinoids CP 55,940 and WIN 55,212-2 and to the endocannabinoids anandamide and 2-arachidonoyl glycerol. Tissue pieces of the renal cortex from the mouse and the rat were preincubated with (3)H-noradrenaline and superfused. The effect of the cannabinoids on basal (3)H-noradrenaline release was studied. Tyramine served as a positive control. In the mouse kidney, basal (3)H-noradrenaline release was increased by tyramine 0.1, 1 and 10 μM by 39, 91 and 212 %, respectively, and, in the rat kidney, (3)H-noradrenaline release was increased by tyramine 10 μM by 158 %. All effects were abolished by desipramine 1 μM, an inhibitor of the neuronal noradrenaline transporter. The cannabinoids at 0.1, 1 and 10 μM (CP 55,940 at 0.1, 1 and 3.2 μM) did not affect (3)H-noradrenaline release in the mouse kidney. The highest concentration of the cannabinoids (10 μM and in the case of CP 55,940 3.2 μM) also failed to affect (3)H-noradrenaline release in the rat kidney. In conclusion, the cannabinoids Δ(9)-tetrahydrocannabinol, cannabidiol, CP 55,940, WIN 55,212-2, anandamide and 2-arachidonoyl glycerol do not possess a tyramine-like effect on noradrenaline release.

Topics: Adrenergic Uptake Inhibitors; Animals; Arachidonic Acids; Benzoxazines; Cannabidiol; Cannabinoids; Cyclohexanols; Dose-Response Relationship, Drug; Dronabinol; Endocannabinoids; Glycerides; Kidney Cortex; Male; Mice; Mice, Inbred C57BL; Morpholines; Naphthalenes; Norepinephrine; Norepinephrine Plasma Membrane Transport Proteins; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Time Factors; Tyramine

Hyperactivation of the amygdala following chronic stress is believed to be one of the primary mechanisms underlying the increased propensity for anxiety-like behaviors and pathological states; however, the mechanisms by which chronic stress modulates amygdalar function are not well characterized. The aim of the current study was to determine the extent to which the endocannabinoid (eCB) system, which is known to regulate emotional behavior and neuroplasticity, contributes to changes in amygdalar structure and function following chronic stress. To examine the hypothesis, we have exposed C57/Bl6 mice to chronic restraint stress, which results in an increase in fatty acid amide hydrolase (FAAH) activity and a reduction in the concentration of the eCB N-arachidonylethanolamine (AEA) within the amygdala. Chronic restraint stress also increased dendritic arborization, complexity and spine density of pyramidal neurons in the basolateral nucleus of the amygdala (BLA) and increased anxiety-like behavior in wild-type mice. All of the stress-induced changes in amygdalar structure and function were absent in mice deficient in FAAH. Further, the anti-anxiety effect of FAAH deletion was recapitulated in rats treated orally with a novel pharmacological inhibitor of FAAH, JNJ5003 (50 mg per kg per day), during exposure to chronic stress. These studies suggest that FAAH is required for chronic stress to induce hyperactivity and structural remodeling of the amygdala. Collectively, these studies indicate that FAAH-mediated decreases in AEA occur following chronic stress and that this loss of AEA signaling is functionally relevant to the effects of chronic stress. These data support the hypothesis that inhibition of FAAH has therapeutic potential in the treatment of anxiety disorders, possibly by maintaining normal amygdalar function in the face of chronic stress.

Topics: Amidohydrolases; Amygdala; Animals; Anxiety; Arachidonic Acids; Chronic Disease; Cyclohexanols; Dendrites; Drug Evaluation, Preclinical; Endocannabinoids; Exploratory Behavior; Male; Maze Learning; Mice; Mice, Inbred C57BL; Mice, Knockout; Polyunsaturated Alkamides; Pyramidal Cells; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Restraint, Physical; Stress, Psychological

Inhibitors of fatty acid amide hydrolase (FAAH) and anandamide (AEA) uptake, which limit the degradation of endogenous cannabinoids, have received interest as potential therapeutics for pain. There is also evidence that endogenous cannabinoids mediate the antinociceptive effects of opioids. Assays of pain-elicited and pain-suppressed behavior have been used to differentiate the effects of drugs that specifically alter nociception from drugs that alter nociception caused by nonspecific effects such as catalepsy or a general suppression of activity. Using such procedures, this study examines the effects of the direct cannabinoid type 1 (CB1) agonist (-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol (CP55940), the FAAH inhibitor cyclohexylcarbamic acid 3'-carbamoylbiphenyl-3-yl ester (URB597), and the AEA uptake inhibitor N-(4-hydroxyphenyl) arachidonylamide (AM404). Additional experiments examined these compounds in combination with morphine. CP55940 produced antinociception in assays of pain-elicited, but not pain-suppressed, behavior and disrupted responding in an assay of schedule-controlled behavior. URB597 and AM404 produced antinociception in assays of pain-elicited and pain-suppressed behavior in which acetic acid was the noxious stimulus, but had no effect on the hotplate and schedule-controlled responding. CP55940 in combination with morphine resulted in effects greater than those of morphine alone in assays of pain-elicited and scheduled-controlled behavior but not pain-suppressed behavior. URB597 in combination with morphine resulted in enhanced morphine effects in assays of pain-elicited and pain-suppressed behavior in which diluted acetic acid was the noxious stimulus, but did not alter morphine's effects on the hotplate or schedule-controlled responding. These studies suggest that, compared with direct CB1 agonists, manipulations of endogenous cannabinoid signaling have enhanced clinical potential; however, their effects depend on the type of noxious stimulus.

Topics: Amidohydrolases; Analgesics; Animals; Arachidonic Acids; Benzamides; Cannabinoid Receptor Modulators; Carbamates; Cyclohexanols; Endocannabinoids; Male; Mice; Mice, Inbred C57BL; Morphine; Nociception; Pain; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB1

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

Comparison of human, rat and mouse cannabinoid CB(2) receptor primary sequences has shown significant divergence at the mRNA and protein sequence level, raising the possibility of species specific pharmacological properties. Additionally, given the importance of the dog as a non-rodent species for predicting human safety during the drug development process, we cloned the dog CB(2) receptor gene and characterized its in-vitro pharmacological properties in a recombinant expression system. A 1.1 kb dog peripheral cannabinoid receptor (dCB(2)) fragment encoding a 360 amino acid protein was cloned from dog spleen cDNA. Analysis of the cloned dCB(2) polypeptide sequence revealed that it shares between 76 and 82% homology with rat, mouse, human and predicted chimpanzee cannabinoid CB(2) receptors. The dog CB(2) receptor expressed in CHO cells displayed similar binding affinities for various synthetic and endogenous cannabinoids as compared to those measured for the human and rat cannabinoid CB(2) receptors. However, these ligands exhibited altered functional potencies and efficacies for the dog cannabinoid CB(2) receptor, which was also found to be negatively coupled to adenylate cyclase activity. These complex pharmacological differences observed across species for the cannabinoid CB(2) receptor suggest that caution should be exerted when analyzing the outcome of animal efficacy and safety studies, notably those involving cannabinoid CB(2) receptor targeting molecules tested in the dog.

Topics: Amino Acid Sequence; Animals; Arachidonic Acids; Base Sequence; Benzoxazines; Binding, Competitive; Camphanes; CHO Cells; Cloning, Molecular; Colforsin; Cricetinae; Cricetulus; Cyclic AMP; Cyclohexanols; Dogs; Dronabinol; Endocannabinoids; Humans; Mice; Molecular Sequence Data; Morpholines; Naphthalenes; Pan troglodytes; Polyunsaturated Alkamides; Pyrazoles; Rats; Receptor, Cannabinoid, CB2; Sequence Alignment

Cannabinoid effects on intestinal transit are commonly evaluated in rats. We characterized the cannabinoid receptors mediating the inhibitory effect of 5-(1,1-dimethylheptyl)-2-[5-hydroxy-2-(3-hydroxypropyl)-cyclohexyl]-phenol (CP 55,940), (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate (WIN 55,212-2), arachidonylethanolamide (AEA) and Delta(9)-tetrahydrocannabinol (Delta(9)-THC) on contractions of the rat ileum myenteric plexus-longitudinal muscle (MPLM) preparation.. The interaction of each agonist was examined with the CB(1) and CB(2) receptor antagonist rimonabant and SR 144,528 respectively, on contractions elicited by electrical field stimulation (EFS) or exogenous ACh. The interaction of AEA with capsazepine, a TRPV(1) receptor antagonist, was also investigated.. EFS with single and trains of pulses evoked neurogenic ACh-mediated twitch and rebound contractions respectively. The rank order of potency for inhibition was CP 55,940 = WIN 55,212-2 > AEA > Delta(9)-THC and AEA > WIN 55,212-2 =Delta(9)-THC = CP 55,940 respectively. The stereoisomer WIN 55,212-3 was without effect. Rimonabant antagonized the inhibition of the twitches with pK(B) values of around 8.60, but only antagonized rebound contractions induced by WIN 55,212-2, AEA and Delta(9)-THC, with pA(2) values of around 6.80. Rimonabant increased the twitches but inhibited the rebound contractions. Contractions to exogenous ACh were not altered. These observations extended to the guinea pig ileum MPLM.. The rat MPLM contains CB(1) receptors and at least two non-CB(1)-non-CB(2)-non-TRPV(1) receptors attenuating EFS-evoked ACh-mediated contractions in an EFS frequency-dependent pre-synaptic and stereo-specific manner. Augmentation of the twitches by rimonabant may be through antagonism of an endocannabinoid tone or inverse agonism, whereas inhibition of the rebound contractions involved partial agonism.

Topics: Animals; Arachidonic Acids; Benzoxazines; Cyclohexanols; Dronabinol; Electric Stimulation; Endocannabinoids; Female; Ileum; In Vitro Techniques; Male; Morpholines; Myenteric Plexus; Naphthalenes; Polyunsaturated Alkamides; Rats; Rats, Wistar; Receptors, Cannabinoid

Endocannabinoids are lipid signaling molecules that act via G-coupled receptors, CB(1) and CB(2). The endocannabinoid system is capable of activation of distinct signaling pathways on demand in response to pathogenic events or stimuli, hereby enhancing cell survival and promoting tissue repair. However, the role of endocannabinoids in hematopoietic stem and progenitor cells (HSPCs) and their interaction with hematopoietic stem cells (HSC) niches is not known. HSPCs are maintained in the quiescent state in bone marrow (BM) niches by intrinsic and extrinsic signaling. We report that HSPCs express the CB(1) receptors and that BM stromal cells secrete endocannabinoids, anandamide (AEA) (35 pg/10(7) cells), and 2-AG (75.2 ng/10(7) cells). In response to the endotoxin lipopolysaccharide (LPS), elevated levels of AEA (75.6 pg/10(7) cells) and 2-AG (98.8 ng/10(7) cells) were secreted from BM stromal cells, resulting in migration and trafficking of HSPCs from the BM niches to the peripheral blood. Furthermore, administration of exogenous cannabinoid CB(1) agonists in vivo induced chemotaxis, migration, and mobilization of human and murine HSPCs. Cannabinoid receptor knock-out mice Cnr1(-/-) showed a decrease in side population (SP) cells, whereas fatty acid amide hydrolase (FAAH)(-/-) mice, which have elevated levels of AEA, yielded increased colony formation as compared with WT mice. In addition, G-CSF-induced mobilization in vivo was modulated by endocannabinoids and was inhibited by specific cannabinoid antagonists as well as impaired in cannabinoid receptor knock-out mice Cnr1(-/-), as compared with WT mice. Thus, we propose a novel function of the endocannabinoid system, as a regulator of HSPC interactions with their BM niches, where endocannabinoids are expressed in HSC niches and under stress conditions, endocannabinoid expression levels are enhanced to induce HSPC migration for proper hematopoiesis.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Blotting, Western; Bone Marrow Cells; Cannabinoid Receptor Modulators; Cell Communication; Cell Movement; Cells, Cultured; Cyclohexanols; Endocannabinoids; Female; Flow Cytometry; Glycerides; Hematopoietic Stem Cell Mobilization; Hematopoietic Stem Cells; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Reverse Transcriptase Polymerase Chain Reaction; Side-Population Cells; Stem Cell Niche; Stromal Cells

Regulation of Ca(2+) homeostasis plays a critical role in oligodendrocyte function and survival. Cannabinoid CB(1) and CB(2) receptors have been shown to regulate Ca(2+) levels and/or K(+) currents in a variety of cell types. In this study we investigated the effect of cannabinoid compounds on the Ca(2+) influx elicited in cultured oligodendrocytes by transient membrane depolarization with an elevated extracellular K(+) concentration (50 mM). The CB(1) receptor agonist arachidonoyl-chloro-ethanolamide (ACEA) elicited a concentration-dependent inhibition of depolarization-evoked Ca(2+) transients in oligodendroglial somata with a maximal effect (94+/-3)% and an EC(50) of 1.3+/-0.03 microM. This activity was mimicked by the CB(1)/CB(2) agonist CP55,940, as well as by the endocannabinoids N-arachidonoyl-ethanolamine (anandamide, AEA) and 2-arachidonoylglycerol (2-AG), whereas the CB(2) receptor selective agonist JWH133 was ineffective. The CB(1) receptor antagonist AM251 (1 microM) also reduced the Ca(2+) response evoked by high extracellular K(+) and did not prevent the inhibition elicited by ACEA (3 microM). Nevertheless, the ability of ACEA and AEA to reduce depolarization-evoked Ca(2+) transients was significantly reduced in oligodendrocytes from CB(1) receptor knockout mice, as well as by pretreatment with pertussis toxin. Bath application of the inwardly rectifying K(+) channels (Kir channels) blockers BaCl(2) (300 microM) and CsCl(2) (1 mM) reduced the size of voltage-induced Ca(2+) influx and partially prevented the inhibitory effect of ACEA. Our results indicate that cannabinoids inhibit depolarization-evoked Ca(2+) transients in oligodendrocytes via CB(1) receptor-independent and -dependent mechanisms that involve the activation of PTX-sensitive G(i/o) proteins and the blockade of Kir channels.

Topics: Animals; Animals, Newborn; Arachidonic Acids; Calcium; Calcium Channels; Cannabinoid Receptor Modulators; Cannabinoids; Cyclohexanols; Dose-Response Relationship, Drug; Endocannabinoids; GTP-Binding Proteins; Immunosuppressive Agents; Mice; Mice, Knockout; Oligodendroglia; Optic Nerve; Polyunsaturated Alkamides; Potassium Channel Blockers; Potassium Channels; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; RNA, Messenger; Time Factors

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

In our previous studies, CB(1) cannabinoid receptor agonists stimulated production of cyclic GMP and translocation of nitric oxide (NO)-sensitive guanylyl cyclase in neuronal cells (Jones et al., Neuropharmacology 54:23-30, 2008). The purpose of these studies was to elucidate the signal transduction of cannabinoid-mediated neuronal nitric oxide synthase (nNOS) activation in neuronal cells. Cannabinoid agonists CP55940 (2-[(1S,2R,5S)-5-hydroxy-2-(3-hydroxypropyl) cyclohexyl]-5-(2-methyloctan-2-yl)phenol), WIN55212-2 (R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone mesylate), and the metabolically stable analog of anandamide, (R)-(+)-methanandamide stimulated NO production in N18TG2 cells over a 20-min period. Rimonabant (N-(piperidin-lyl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-H-pyrazole-3-carboxamide), a CB(1) receptor antagonist, partially or completely curtailed cannabinoid-mediated NO production. Inhibition of NOS activity (N ( G )-nitro-L: -arginine) or signaling via Gi/o protein (pertussis toxin) significantly limited NO production by cannabinoid agonists. Ca(2+) mobilization was not detected in N18TG2 cells after cannabinoid treatment using Fluo-4 AM fluorescence. Cannabinoid-mediated NO production was attributed to nNOS activation since endothelial NOS and inducible NOS protein and mRNA were not detected in N18TG2 cells. Bands of 160 and 155 kDa were detected on Western blot analysis of cytosolic and membrane fractions of N18TG2 cells, using a nNOS antibody. Chronic treatment of N18TG2 cells with cannabinoid agonists downregulated nNOS protein and mRNA as detected using Western blot analysis and real-time polymerase chain reaction, respectively. Cannabinoid agonists stimulated NO production via signaling through CB(1) receptors, leading to activation of Gi/o protein and enhanced nNOS activity. The findings of these studies provide information related to cannabinoid-mediated NO signal transduction in neuronal cells, which has important implications in the ongoing elucidation of the endocannabinoid system in the nervous system.

Topics: Arachidonic Acids; Benzoxazines; Blotting, Western; Calcium; Cannabinoid Receptor Modulators; Cannabinoids; Cell Line; Cyclic GMP; Cyclohexanols; Endocannabinoids; Enzyme Activation; Enzyme Inhibitors; Guanylate Cyclase; Humans; Morpholines; Naphthalenes; Neurons; Nitric Oxide Synthase Type I; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB1; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction

It has been suggested that disturbances in endocannabinoid signaling contribute to the development of depressive illness; however, at present there is insufficient evidence to allow for a full understanding of this role. To further this understanding, we performed an analysis of the endocannabinoid system in an animal model of depression. Male rats exposed to chronic, unpredictable stress (CUS) for 21 days exhibited a reduction in sexual motivation, consistent with the hypothesis that CUS in rats induces depression-like symptoms. We determined the effects of CUS, with or without concurrent treatment with the antidepressant imipramine (10 mg/kg), on CP55940 binding to the cannabinoid CB(1) receptor; whole tissue endocannabinoid content; and fatty acid amide hydrolase (FAAH) activity in the prefrontal cortex, hippocampus, hypothalamus, amygdala, midbrain and ventral striatum. Exposure to CUS resulted in a significant increase in CB(1) receptor binding site density in the prefrontal cortex and a decrease in CB(1) receptor binding site density in the hippocampus, hypothalamus and ventral striatum. Except in the hippocampus, these CUS-induced alterations in CB(1) receptor binding site density were attenuated by concurrent antidepressant treatment. CUS alone produced a significant reduction in N-arachidonylethanolamine (anandamide) content in every brain region examined, which was not reversed by antidepressant treatment. These data suggest that the endocannabinoid system in cortical and subcortical structures is differentially altered in an animal model of depression and that the effects of CUS on CB(1) receptor binding site density are attenuated by antidepressant treatment while those on endocannabinoid content are not.

Topics: Analgesics; Animals; Antidepressive Agents; Antidepressive Agents, Tricyclic; Arachidonic Acids; Brain; Cannabinoid Receptor Modulators; Cyclohexanols; Depressive Disorder, Major; Disease Models, Animal; Endocannabinoids; Imipramine; Male; Motivation; Polyunsaturated Alkamides; Rats; Rats, Long-Evans; Receptor, Cannabinoid, CB1; Sexual Behavior, Animal; Stress, Psychological

Experimental studies indicate a bidirectional, functional relationship between glucocorticoids and the endocannabinoid system; however, the effects of repeated glucocorticoid treatment on the endocannabinoid system have not been examined. In this study, we treated male rats with either a single dose or a 21-day course of treatment with corticosterone (20 mg/kg) and measured hippocampal cannabinoid CB(1) receptor expression and endocannabinoid content. The 21-day, but not the single, administration of corticosterone significantly reduced both the binding site density and amount of protein of the hippocampal cannabinoid CB(1) receptor without affecting affinity for the CB(1) receptor agonist, [(3)H]CP55940. With regard to hippocampal endocannabinoid content, acute corticosterone treatment resulted in a significant reduction in anandamide but did not affect 2-arachidonylglycerol, while repeated corticosterone treatment did not alter content of either anandamide or 2-arachidonylglycerol. These data support the hypothesis that the cannabinoid CB(1) receptor is under negative regulation by glucocorticoids in the hippocampus, and suggest that hippocampal cannabinoid CB(1) receptor signaling could be reduced under conditions associated with hypersecretion of glucocorticoids, such as chronic stress.

Topics: Animals; Anti-Inflammatory Agents; Arachidonic Acids; Corticosterone; Cyclohexanols; Dose-Response Relationship, Drug; Endocannabinoids; Glycerides; Hippocampus; Immunosuppressive Agents; Male; Polyunsaturated Alkamides; Rats; Rats, Long-Evans; Receptor, Cannabinoid, CB1; Tritium

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

The endocannabinoid system functions through two well characterized receptor systems, the CB1 and CB2 receptors. Work by a number of groups in recent years has provided evidence that the system is more complicated and additional receptor types should exist to explain ligand activity in a number of physiological processes.. Cells transfected with the human cDNA for GPR55 were tested for their ability to bind and to mediate GTPgammaS binding by cannabinoid ligands. Using an antibody and peptide blocking approach, the nature of the G-protein coupling was determined and further demonstrated by measuring activity of downstream signalling pathways.. We demonstrate that GPR55 binds to and is activated by the cannabinoid ligand CP55940. In addition endocannabinoids including anandamide and virodhamine activate GTPgammaS binding via GPR55 with nM potencies. Ligands such as cannabidiol and abnormal cannabidiol which exhibit no CB1 or CB2 activity and are believed to function at a novel cannabinoid receptor, also showed activity at GPR55. GPR55 couples to Galpha13 and can mediate activation of rhoA, cdc42 and rac1.. These data suggest that GPR55 is a novel cannabinoid receptor, and its ligand profile with respect to CB1 and CB2 described here will permit delineation of its physiological function(s).

Topics: Amino Acid Sequence; Animals; Arachidonic Acids; Binding Sites; Binding, Competitive; Cannabidiol; Cannabinoids; Cell Line; Cloning, Molecular; Cyclohexanols; Down-Regulation; Endocannabinoids; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Ligands; Mice; Molecular Sequence Data; Organ Specificity; Polymerase Chain Reaction; Polyunsaturated Alkamides; Rats; Receptors, Cannabinoid; Receptors, G-Protein-Coupled; RNA, Messenger; Signal Transduction; Structure-Activity Relationship

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

Certain fatty acid N-alkyl amides from the medicinal plant Echinacea activate cannabinoid type-2 (CB2) receptors. In this study we show that the CB2-binding Echinacea constituents dodeca-2E,4E-dienoic acid isobutylamide (1) and dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide (2) form micelles in aqueous medium. In contrast, micelle formation is not observed for undeca-2E-ene-8,10-diynoic acid isobutylamide (3), which does not bind to CB2, or structurally related endogenous cannabinoids, such as arachidonoyl ethanolamine (anandamide). The critical micelle concentration (CMC) range of 1 and 2 was determined by fluorescence spectroscopy as 200-300 and 7400-10000 nM, respectively. The size of premicelle aggregates, micelles, and supermicelles was studied by dynamic light scattering. Microscopy images show that compound 1, but not 2, forms globular and rod-like supermicelles with radii of approximately 75 nm. The self-assembling N-alkyl amides partition between themselves and the CB2 receptor, and aggregation of N-alkyl amides thus determines their in vitro pharmacological effects. Molecular mechanics by Monte Carlo simulations of the aggregation process support the experimental data, suggesting that both 1 and 2 can readily aggregate into premicelles, but only 1 spontaneously assembles into larger aggregates. These findings have important implications for biological studies with this class of compounds.

Topics: Amides; Arachidonic Acids; Cyclohexanols; Echinacea; Endocannabinoids; Humans; Models, Biological; Molecular Structure; Plants, Medicinal; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB2

Noradrenaline and ATP are sympathetic co-transmitters. In the rat perfused mesenteric bed cannabinoids have been shown to modify the overall response to sympathetic nerve stimulation. This study has assessed whether cannabinoid receptor activation modulates differentially the noradrenergic and purinergic components of sympathetic vasoconstriction.. Rat mesenteric beds were perfused with physiological salt solution and the effects of cannabinoids on responses to nerve stimulation, or exogenous noradrenaline or alpha,beta-methylene ATP (alpha,beta-meATP; P2X receptor agonist) were determined after raising tone with U46619. The effects of cannabinoids on the noradrenaline and ATP components of sympathetic neurotransmission were assessed using the alpha 1-adrenoceptor antagonist, prazosin, or after P2X receptor desensitization with alpha,beta-meATP.. Anandamide, WIN 55,212-2 and CP55,940 attenuated sympathetic neurogenic vasoconstrictor responses. The inhibitory actions of anandamide and WIN 55,212-2 were blocked by LY320135, a CB1 receptor antagonist, but not by SR144528, a CB2 receptor antagonist. The inhibitory actions of CP55,940 were unaffected by LY320135 and SR144528. WIN 55,212-3, the inactive S(-) enantiomer of WIN 55,212-2, had no effect on sympathetic neurogenic responses. None of the cannabinoids affected contractile responses to exogenous noradrenaline or alpha,beta-meATP. Anandamide and WIN 55,212-2 inhibited both the noradrenaline and ATP components of the sympathetic neurogenic contractile responses, with effects on the ATP component being most marked.. These results indicate that prejunctional CB1-like receptors mediate the sympathoinhibitory action of anandamide and WIN 55,212-2, but not CP55,940, in the rat mesenteric bed. Cannabinoids inhibit both the noradrenergic and purinergic components of sympathetic neurotransmission.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Triphosphate; Adrenergic Fibers; Animals; Arachidonic Acids; Benzofurans; Benzoxazines; Camphanes; Cannabinoids; Cyclohexanols; Dose-Response Relationship, Drug; Electric Stimulation; Endocannabinoids; In Vitro Techniques; Male; Mesenteric Arteries; Morpholines; Muscle Tonus; Muscle, Smooth, Vascular; Naphthalenes; Norepinephrine; Perfusion; Polyunsaturated Alkamides; Prazosin; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Receptors, Purinergic P2

Cannabis is widely abused by women at reproductive age and during pregnancy. Animal studies showed a particular vulnerability of the developing brain to prenatal chronic cannabinoid administration. We determined whether prenatal exposure to WIN 55,212-2, a potent cannabinoid receptor agonist, affected (1) density, affinity and/or function of cannabinoid CB(1) receptors, (2) endogenous levels of the endocannabinoid anandamide, (3) activities of the major anandamide synthesising and hydrolysing enzymes, N-acyl-phosphatidylethanolamine-specific phospholipase D (NAPE-PLD) and fatty acid amide hydrolase (FAAH), respectively, in brain areas of adult male offspring rats. Furthermore, the effect of prenatal WIN 55,212-2 on spontaneous motility was analyzed. Pregnant rats were treated daily with WIN 55,212-2 (0.5 mg/kg, gestation day 5-20) or vehicle. [(3)H]CP 55,940 and WIN 55,212-2-stimulated [(35)S] GTPgammaS binding were carried out in cerebellum, cerebral cortex, hippocampus, striatum and limbic areas of male adult offspring. Levels of anandamide, FAAH and NAPE-PLD activity were also determined. EC(50) values for WIN 55,212-2-stimulated [(35)S]GTPgammaS binding were significantly different in hippocampus (-26%) and striatum (+27%) in WIN 55,212-2-treated rats. Cannabinoid CB(1) receptor density and affinity were not affected in any analyzed region. In the striatum, increased anandamide levels were associated with reduced FAAH and enhanced NAPE-PLD activities. Opposite changes in anandamide levels and enzymatic activities were detected in limbic areas of WIN 55,212-2-treated rats. Ambulatory activity between WIN 55,212-2- and vehicle-treated adult offspring did not vary. Our results show that prenatal exposure to cannabinoid agonist induces a long-term alteration of endocannabinoid system in brain areas involved in learning-memory, motor activity and emotional behavior.

Topics: Analgesics; Animals; Arachidonic Acids; Behavior, Animal; Benzoxazines; Binding, Competitive; Brain; Cannabinoid Receptor Agonists; Cannabinoid Receptor Modulators; Cyclohexanols; Dose-Response Relationship, Drug; Endocannabinoids; Female; Guanosine 5'-O-(3-Thiotriphosphate); Litter Size; Male; Morpholines; Motor Activity; Naphthalenes; Phospholipase D; Polyunsaturated Alkamides; Pregnancy; Prenatal Exposure Delayed Effects; Radioligand Assay; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Receptors, Cannabinoid; Sulfur Radioisotopes; Weight Gain

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

Male Wistar rats were trained to discriminate (-)-nicotine (0.4 mg/kg) from saline under a two-lever, fixed-ratio 10 schedule of water reinforcement. During test sessions the following drugs were coadministered with saline (substitution studies) or nicotine (0.025-0.4 mg/kg; combination studies): the alpha4beta2 nicotinic acetylcholine receptor subtype antagonist dihydro-beta-erythroidine (DHbetaE), the non-selective nicotinic acetylcholine receptor subtype antagonist mecamylamine, the alpha7 nicotinic acetylcholine receptor subtype antagonist methyllycaconitine (MLA), the alpha4beta2 nicotinic acetylcholine receptor subtype agonist 5-iodo-3-(2(S)-azetidinylmethoxy)pyridine (5-IA), the cannabinoid CB1 receptor antagonist/partial agonist rimonabant, the cannabinoid CB2 receptor antagonist N-[(1S)-endo-1,3,3-trimethylbicyclo-[2.2.1]heptan-2-yl]5-(4-chloro-3-methyl-phenyl)-1-(4-methybenzyl)pyrazole-3-carboxamide (SR 144528), the cannabinoid CB1/2 receptor agonists (-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)-phenyl]-trans-4-(3-hydroxy-propyl)cyclohexanol (CP 55,940) or R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]-pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-(1-naphthalenyl)-methanone mesylate (WIN 55,212-2), the endogenous cannabinoid agonist and non-competitive alpha7 nicotinic acetylcholine receptor subtype antagonist anandamide, the anandamide uptake and fatty acid amide hydrolase inhibitor N-(4-hydroxyphenyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (AM-404), the fatty acid amide hydrolase inhibitor cyclohexylcarbamic acid 3'-carbamoyl-biphenyl-3-yl ester (URB 597), AM-404+anandamide or URB 597+anandamide. 5-IA (0.01 mg/kg) fully substituted for nicotine, while other drugs were inactive. In combination studies, DHbetaE and mecamylamine dose-dependently attenuated the discriminative stimulus effects of nicotine and the full substitution of 5-IA, while MLA, rimonabant, SR 144528, CP 55,940, WIN 55,212-2, and URB 597 did not alter the nicotine cue. Pretreatment with AM-404+anandamide or URB 597+anandamide weakly enhanced nicotine-lever responding. Our pharmacological analyses demonstrates that the expression of nicotine discrimination is under the control of nicotinic acetylcholine receptor subtypes composed of alpha4beta2 (but not of alpha7) subunits. Furthermore, we excluded the involvement of either cannabinoid CB1 and CB2 receptors or increases in the endocannabinoid tone in the nicotine discrimination.

Topics: Aconitine; Animals; Arachidonic Acids; Azetidines; Benzamides; Benzoxazines; Camphanes; Cannabinoid Receptor Antagonists; Carbamates; Cyclohexanols; Dihydro-beta-Erythroidine; Discrimination Learning; Dose-Response Relationship, Drug; Drug Synergism; Endocannabinoids; Male; Mecamylamine; Morpholines; Naphthalenes; Nicotine; Nicotinic Agonists; Nicotinic Antagonists; Polyunsaturated Alkamides; Pyrazoles; Pyridines; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Receptors, Cannabinoid; Receptors, Nicotinic

The present study investigated the effect of ethanol (EtOH) exposure and its withdrawal on the central endocannabinoid system utilizing an EtOH vapor inhalation model, which is known to produce functional tolerance and dependence to EtOH. Swiss Webster mice (n=24) were exposed to EtOH vapors for 72h. Mice were sacrificed after 72h following EtOH exposure (n=12) and 24h after its withdrawal (n=12). Radioligand binding assays were performed to measure the density of CB(1) receptor and CB(1) receptor agonist-stimulated [(35)S]GTPgammaS binding in crude synaptic membranes isolated from the cortex, hippocampus, striatum and cerebellum. The density of CB(1) receptor was significantly decreased (31-39%) in all the brain regions when compared to the control group. The CB(1) receptor-stimulated G(i/o) protein activation was also found to be decreased (29-40%) in these brain regions of EtOH exposed mice. Recovery of the CB(1) receptor density, in addition to, the CB(1) receptor-mediated G-protein activation was observed after 24h withdrawal from EtOH. The levels of cortical anandamide, which was significantly increased (147%) by EtOH exposure, returned to basal levels after 24h of withdrawal from EtOH exposure. A significant reduction (21%) in the activity of fatty acid amide hydrolase was found in the cortex of EtOH administered mice. Taken together, the neuroadaptation in the EC system may have a potential role in development of tolerance and dependence to EtOH.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Cannabinoid Receptor Modulators; Central Nervous System Depressants; Chromatography, Liquid; Cyclohexanols; Endocannabinoids; Ethanol; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Male; Mass Spectrometry; Mice; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB1; Substance Withdrawal Syndrome; Sulfur Radioisotopes

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

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

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

Immunomodulatory effects of endogenous and exogenous cannabinoids have been investigated in numerous studies, mostly performed with isolated cells or transformed cell lines, but only sparse data exist on human polymorphonuclear neutrophils (PMNs). We therefore investigated the respiratory burst reaction of human whole-blood PMNs under the influence of cannabinoids using flow cytometry. In their natural whole-blood milieu, a CB(2) receptor-dependent stimulation of the PMN respiratory burst was found at nanomolar concentrations of CP55 940 [(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol] and methanandamide after a 3-h incubation period, whereas the short-living and rapidly hydrolyzed endogenous ligand anandamide did not alter the burst reaction of whole-blood PMNs under the same experimental conditions. The stimulatory cannabinoid effect was totally absent in isolated PMNs but could be transferred onto isolated PMNs by adding the cell-free low-molecular mass plasma fraction (<5000 Da) of cannabinoid-incubated blood, indicating an indirect mechanism depending on humoral products or mediators. Results of our further experiments suggest that products of the arachidonic acid metabolism are mediators of the cannabinoid-induced enhancement of the respiratory burst reaction of whole-blood PMNs.

Topics: Arachidonic Acids; Cannabinoids; Cyclohexanols; Cyclooxygenase Inhibitors; Endocannabinoids; Humans; In Vitro Techniques; Lipoxygenase Inhibitors; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Polyunsaturated Alkamides; Reactive Oxygen Species; Receptor, Cannabinoid, CB2; Respiratory Burst; Stimulation, Chemical

As persistent behavioural changes, such as increased anxiety-related behaviours, can be predicted based on the phenomenon of psychostimulant-induced neuronal plasticity, the time course (3-, 5- and 10-day time points) of the effects of both a single and repeated (daily for 7 days) i.p. administrations of cocaine (COC) and methamphetamine (MA) on anxiety-related behavioural symptoms in the elevated plus-maze test were examined in mice. Furthermore, based on the reported interactions between brain dopamine versus cannabinoid (CB) receptors and the contribution of CB receptors to the occurrence of persistent anxiety-related behavioural symptoms, the interactions of the agonist CP 55940 (CP) and the endogenous ligands anandamide (arachidonylethanolamide: AEA), 2-arachidonylglycerol (ARA), N-arachidonyldopamine (NADA), noladin ether (NL), and virodhamine (VA) with the COC- or MA-induced anxiety-related behaviours were also studied. In both an acute experiment using a single COC (30 mg/kg) or MA (4 mg/kg) dose and a chronic experiment using repeated COC (15 mg/kg) or MA (2 mg/kg) doses, anxiety-related behavioural symptoms were observed similarly at 3- and 5-day time points, but disappeared at the 10-day time point. Among the CB ligands, the agonists CP, AEA, ARA, NADA, and NL provided strong protective effects against each parameter at 3- and 5-day time points. Therefore, it was concluded that both COC and MA caused persistent anxiety-related behavioural symptoms following both a single and repeated treatments. Since these anxiogenic effects were attenuated by the endogenous CB agonists, the involvement of brain CB receptors was suspected.

Topics: Analysis of Variance; Animals; Anxiety; Arachidonic Acids; Behavior, Animal; Cannabinoids; Cocaine; Cyclohexanols; Dopamine; Dose-Response Relationship, Drug; Drug Interactions; Endocannabinoids; Glycerides; Injections, Intraperitoneal; Male; Maze Learning; Methamphetamine; Mice; Mice, Inbred ICR; Polyunsaturated Alkamides; Receptor, Cannabinoid, CB1; Time Factors

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

Compelling evidence indicates that endocannabinoids are implicated in drug addiction. In the present study, we have addressed the interaction between cocaine and endocannabinoid system by means of neurochemical and neurophysiological experiments in rat brain slices. Using gas chromatography-electron impact mass spectrometry, we have found that cocaine increased the levels of the endocannabinoid anandamide in the striatum, a brain area primarily involved in the compulsive drug-seeking and drug-taking behaviors typical of addiction. This effect was attenuated by pharmacological inhibition of D2-like receptors but not D1-like receptors, and it was mimicked by D2-like but not D1-like receptor stimulation. The cocaine-induced increase in anandamide concentrations was attributable to both stimulation of its synthesis and inhibition of its degradation, as suggested by the ability of cocaine and quinpirole, a D2-like receptor agonist, to enhance the activity of NAPE-phospholipase D and to inhibit fatty acid amide hydrolase. By means of electrophysiological recordings from single striatal neurons, we have then observed that the ability of cocaine to inhibit, via D2-like receptors, GABA transmission was partially prevented following blockade of cannabinoid receptors, suggesting that endocannabinoids may act as downstream effectors in the action of cocaine in the striatum. Understanding the molecular and physiological effects of drugs of abuse in the brain is essential for the development of effective strategies against addiction.

Topics: 2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine; Amidohydrolases; Animals; Arachidonic Acids; Cerebral Cortex; Cocaine; Cyclohexanols; Dopamine Agonists; Dose-Response Relationship, Drug; Dronabinol; Electrophysiology; Endocannabinoids; Excitatory Postsynaptic Potentials; gamma-Aminobutyric Acid; In Vitro Techniques; Male; Neostriatum; Patch-Clamp Techniques; Polyunsaturated Alkamides; Quinpirole; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Receptors, Dopamine D2; Synaptic Transmission; Synaptosomes

2-Arachidonoylglycerol is an endogenous ligand for the cannabinoid receptors (CB1 and CB2). Previously, we provided evidence that 2-arachidonoylglycerol, but not anandamide (N-arachidonoylethanolamine), is the true natural ligand for the cannabinoid receptors. In the present study, we examined in detail the effects of 2-arachidonoylglycerol on the production of chemokines in human promyelocytic leukemia HL-60 cells. We found that 2-arachidonoylglycerol induced a marked acceleration in the production of interleukin 8. The effect of 2-arachidonoylglycerol was blocked by treatment of the cells with SR144528, a cannabinoid CB2 receptor antagonist, indicating that the effect of 2-arachidonoylglycerol is mediated through the CB2 receptor. Augmented production of interleukin 8 was also observed with CP55940, a synthetic cannabinoid, and an ether-linked analog of 2-arachidonoylglycerol. On the other hand, neither anandamide nor the free arachidonic acid induced the enhanced production of interleukin 8. A similar effect of 2-arachidonoylglycerol was observed in the case of the production of macrophage-chemotactic protein-1. The accelerated production of interleukin 8 by 2-arachidonoylglycerol was observed not only in undifferentiated HL-60 cells, but also in HL-60 cells differentiated into macrophage-like cells. Noticeably, 2-arachidonoylglycerol and lipopolysaccharide acted synergistically to induce the dramatically augmented production of interleukin 8. These results strongly suggest that the CB2 receptor and its physiological ligand, i.e., 2-arachidonoylglycerol, play important regulatory roles such as stimulation of the production of chemokines in inflammatory cells and immune-competent cells. Detailed studies on the cannabinoid receptor system are thus essential to gain a better understanding of the precise regulatory mechanisms of inflammatory reactions and immune responses.

Topics: Arachidonic Acid; Arachidonic Acids; Blotting, Northern; Calcitriol; Camphanes; Cell Differentiation; Chemokine CCL2; Chemokines; Cyclohexanols; Dose-Response Relationship, Drug; Endocannabinoids; Enzyme-Linked Immunosorbent Assay; Gene Expression; Glycerides; HL-60 Cells; Humans; Interleukin-8; Lipopolysaccharides; Polyunsaturated Alkamides; Pyrazoles; Receptor, Cannabinoid, CB2; RNA, Messenger; 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

Cannabinoids have been shown to affect various immune functions. To date, almost no data exist on PMN, which provide the first line antimicrobial defense. The objective of the present study was to investigate the effects of the synthetic dibenzopyrane ligand CP55 940, the endogenous cannabinoid anandamide and methanandamide on the "respiratory burst" of isolated human PMN in vitro. After preincubation with high micromolar concentrations of CP55 940, fMLP-stimulated PMN showed a reduction in superoxide production, whereas the spontaneous burst activity of resting PMN remained unaffected. This inhibitory effect of CP55 940 was not CB-receptor-mediated. In contrast, anandamide and methanandamide did not alter the oxidative microbicidal PMN function.

Topics: Adult; Arachidonic Acids; Cyclohexanols; Dronabinol; Endocannabinoids; Female; Humans; Male; Neutrophils; Polyunsaturated Alkamides; Receptors, Cannabinoid; Superoxides

Cannabinoids (CB) can act as retrograde synaptic mediators of depolarization-induced suppression of inhibition or excitation in hippocampus. This mechanism may underlie the impairment of some cognitive processes produced by these compounds, including short-term memory formation in the hippocampus. In this study, we investigated several compounds known to interact with CB receptors, evaluating their effects on K(+)-evoked release of [3H]D-aspartate ([3H]D-ASP) and [3H]GABA from superfused synaptosomes isolated from the rat hippocampus. [3H]D-ASP and [3H]GABA release were inhibited to different degrees by the synthetic cannabinoids WIN 55,212-2; CP 55,940, and arachidonyl-2'-chloroethylamide/N-(2-chloroethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (ACEA), as well as by the endocannabinoids, anandamide (AEA), and 2-arachidonoylglycerol (2-AG). Both types of release were also inhibited by capsaicin. The inhibition produced by each of the cannabinoid compounds and capsaicin was unaffected by capsazepine or by the CB1-receptor antagonists AM-251 and SR141716A. The mechanism underlying AEA- and synthetic CB-induced inhibition of the release of [3H]GABA and [3H]D-ASP from rat hippocampal synaptosomes might not involve activation of presynaptic CB1 receptors.

Topics: Animals; Arachidonic Acids; Aspartic Acid; Calcium; Cannabinoid Receptor Agonists; Cannabinoids; Capsaicin; Cyclohexanols; Endocannabinoids; gamma-Aminobutyric Acid; Hippocampus; Kinetics; Male; Polyunsaturated Alkamides; Potassium; Rats; Rats, Wistar; Synaptosomes

Pharmacological effects of cannabinoid ligands are thought to be mediated through cannabinoid CB1 and CB2 receptor subtypes. Sequence analysis revealed that rat and human cannabinoid CB2 receptors are divergent and share 81% amino acid homology. Pharmacological analysis of the possible species differences between rat and human cannabinoid CB2 receptors was performed using radioligand binding and functional assays. Pronounced species selectivity at the rat cannabinoid CB2 receptor (50- to 140-fold) was observed with AM-1710 (3-(1,1-Dimethyl-heptyl)-1-hydroxy-9-methoxy-benzo[c]chromen-6-one) and AM-1714 (3-(1,1-Dimethyl-heptyl)-1-9-dihydroxy-benzo[c]chromen-6-one). In contrast, JWH-015 ((2-Methyl-1-propyl-1H-indol-3-yl)-napthalen-1-yl-methanone) was 3- to 10-fold selective at the human cannabinoid CB2 receptor. Endocannabinoid ligands were more human receptor selective. Cannabinoid CB2 receptor antagonist, AM-630 ((6-Iodo-2-methyl-1-(2-morpholin-4-yl-ethyl)-1H-indol-3-yl)-(4-methoxy-phenyl)-methanone) was more potent at the rat receptor in radioligand binding and functional assays than that of the human receptor. The findings of the pharmacological differences between the human and rat cannabinoid CB2 receptors in this study provide critical information for characterizing cannabinoid ligands in in vivo rodent models for drug discovery purpose.

Topics: Animals; Arachidonic Acids; Benzoxazines; Binding, Competitive; Calcium; Cell Line; Chromones; Colforsin; Cyclic AMP; Cyclohexanols; DNA, Complementary; Dose-Response Relationship, Drug; Endocannabinoids; Glycerides; Humans; Indoles; Morpholines; Naphthalenes; Polyunsaturated Alkamides; Radioligand Assay; Rats; Receptor, Cannabinoid, CB2; Species Specificity; Transfection; Tritium

Mammalian tissues contain two types of cannabinoid receptors CB1 and CB2. The aim of our study was an evaluation of the influence of a single i.p. injection of a stable analogue of an endogenous cannabinoid anandamide--R-(+)-methanandamide (2.5 mg/kg) and CP 55,940 (0.25 mg/kg), which is an exogenous agonist of CB1 receptors, on the immunoreactivity of regulatory peptides, produced in rat thyroid C cells: calcitonin, CGRP, somatostatin and synaptophysin. This study indicates that a single injection of cannabinoids: R-(+)-methanandamide and CP 55,940 alters the immunoreactivity of regulatory peptides in thyroid parafollicular cells.

Topics: Animals; Arachidonic Acids; Calcitonin Gene-Related Peptide; Cannabinoids; Cyclohexanols; Endocannabinoids; Male; Polyunsaturated Alkamides; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Somatostatin; Synaptophysin; Thyroid Gland

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

Cannabinoid receptors and their endogenous ligands are potent inhibitors of neurotransmitter release in the brain. Here, we show that in a rat model of Parkinson's disease induced by unilateral nigral lesion with 6-hydroxydopamine (6-OHDA), the striatal levels of the endocannabinoid anandamide (AEA) were increased, while the activity of its membrane transporter and hydrolase (fatty-acid amide hydrolase, FAAH) were decreased. These changes were not observed in the cerebellum of the same animals. Moreover, the frequency and amplitude of glutamate-mediated spontaneous excitatory post-synaptic currents were augmented in striatal spiny neurones recorded from parkinsonian rats. Remarkably, the anomalies in the endocannabinoid system, as well as those in glutamatergic activity, were completely reversed by chronic treatment of parkinsonian rats with levodopa, and the pharmacological inhibition of FAAH restored a normal glutamatergic activity in 6-OHDA-lesioned animals. Thus, the increased striatal levels of AEA may reflect a compensatory mechanism trying to counteract the abnormal corticostriatal glutamatergic drive in parkinsonian rats. However, this mechanism seems to be unsuccessful, since spontaneous excitatory activity is still higher in these animals. Taken together, these data show that anomalies in the endocannabinoid system induced by experimental parkinsonism are restricted to the striatum and can be reversed by chronic levodopa treatment, and suggest that inhibition of FAAH might represent a possible target to decrease the abnormal cortical glutamatergic drive in Parkinson's disease.

Topics: Amidohydrolases; Animals; Antiparkinson Agents; Arachidonic Acids; Binding, Competitive; Cannabinoid Receptor Modulators; Cerebellum; Corpus Striatum; Cyclohexanols; Disease Models, Animal; Endocannabinoids; Excitatory Postsynaptic Potentials; Fatty Acids, Unsaturated; Glutamic Acid; Glycerides; In Vitro Techniques; Levodopa; Oxidopamine; Parkinsonian Disorders; Patch-Clamp Techniques; Phospholipase D; Polyunsaturated Alkamides; Rats; Rats, Wistar; Receptors, Cannabinoid; Receptors, Drug

Cyclohexyl hexadecanoate, hexadecyl propionate, and N-(3-hydroxypropionyl)pentadecanamide, respectively ester, retroester, and retroamide derivatives of N-palmitoylethanolamine, represent the first selective inhibitors of "N-palmitoylethanolamine hydrolase" described so far. These compounds are devoid of affinity for CB(1) and CB(2) receptors and characterized by high percentages of inhibition of N-palmitoylethanolamine-selective acid amidase (84.0, 70.5, and 76.7% inhibition at 100 microM, respectively) with much lower inhibitory effect on either fatty acid amide hydrolase or the uptake of anandamide.

Topics: Amides; Amidohydrolases; Animals; Arachidonic Acids; Benzoxazines; Binding, Competitive; Brain; Calcium Channel Blockers; CHO Cells; Cricetinae; Cyclohexanols; Endocannabinoids; Enzyme Inhibitors; Esters; Humans; Indicators and Reagents; Lung; Morpholines; Naphthalenes; Palmitates; Polyunsaturated Alkamides; Rats; Receptor, Cannabinoid, CB2; Receptors, Cannabinoid; Receptors, Drug

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

This study was aimed at finding structural requirements for the interaction of the acyl chain of endocannabinoids with cannabinoid receptors, membrane transporter protein, and fatty acid amide hydrolase (FAAH). To this end, the flexibility of the acyl chain was restricted by introduction of an 1-hydroxy-2Z,4E-pentadiene system in anandamide (N-arachidonoylethanolamine, AEA) and 2-arachidonoylglycerol (2-AG) at various positions using different lipoxygenases. This brought about selectivity and attenuated the binding potency of AEA and 2-AG. Although the displacement constants were modest, 15(S)-hydroxy-eicosa-5Z,8Z,11Z,13E-tetraenoyl-N-(2-hydroxyethyl)amine was found to bind selectively to the CB(1) receptor, whereas its 1-arachidonoyl-sn-glycerol analogue and 13(S)-hydroxy-octadeca-9Z,11E-dienoyl-N-(2-hydroxyethyl)amine could selectively bind to the CB(2) receptor. 11(S)-Hydroxy-eicosa-5Z,8Z,12E,14Z-tetraenoyl-N-(2-hydroxyethyl)amine did not bind to either receptor, whereas 12(S)-hydroxy-eicosa-5Z,8Z,10E,14Z-tetraenoyl-N-(2-hydroxyethyl)amine did bind to both CB receptors with an affinity similar to that of AEA. All oxygenated anandamide derivatives were good inhibitors of FAAH (low micromolar K(i)) but were ineffective on the AEA transporter. 2-AG rapidly isomerizes into 1(3)-arachidonoyl-sn-glycerol. Both 1- and 3-arachidonoyl-sn-glycerol did not bind to either CB receptor and did not interfere with AEA transport. Thus, after it is isomerized, 2-AG is inactivated, thereby decreasing effective concentrations of 2-AG. Analysis of (1)H NMR spectra revealed that chloroform did not induce notably different conformations in the acyl chain of 15(S)-hydroxy-eicosa-5Z,8Z,11Z,13E-tetraenoic acid as compared with water. Molecular dynamics (MD) simulations of AEA and its analogues in the presence of explicit water molecules revealed that a tightly folded conformation of the acyl chain is not the only requirement for CB(1) binding. Structural details of the C(2)-C(15) loop, such as an sp(2) carbon at position 11, are necessary for receptor binding. The MD simulations may suggest that the average orientations of the pentyl tail of AEA and 12(S)-hydroxy-eicosa-5Z,8Z,10E,14Z-tetraenoyl-N-(2-hydroxyethyl)amine are different from that of the low-affinity, inactive ligands.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Binding, Competitive; Biological Transport; Brain; Cannabinoid Receptor Modulators; Cannabinoids; Carrier Proteins; Chloroform; Cyclohexanols; Endocannabinoids; Glycerides; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Magnetic Resonance Spectroscopy; Male; Models, Molecular; Molecular Conformation; Polyunsaturated Alkamides; Rats; Rats, Wistar; Receptor, Cannabinoid, CB2; Receptors, Cannabinoid; Receptors, Drug; Tumor Cells, Cultured; Water

The aim of the present study was to classify release-inhibiting receptors on rat pheochromocytoma PC12 cells. Veratridine-evoked [3H]noradrenaline release from PC12 cells was inhibited by micromolar concentrations of the imidazoline and guanidine derivatives cirazoline, clonidine, aganodine, 1,3-di(2-tolyl)guanidine, BDF6143 and agmatine, and of the cannabinoid receptor agonist WIN55,212-2 (R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo-[1,2,3-de]-1,4-benzoxazin-yl](1-naphthalenyl)methanone mesylate), but not by noradrenaline. The inhibitory effect of clonidine was antagonized by micromolar concentrations of rauwolscine and SR141716A (N-[piperidin-1-yl]-5-[4-chlorophenyl]-1-[2,4-dichlorophenyl]-4-methyl-1H-pyrazole-3-carboxamide). The potencies of the agonists and antagonists were compatible with an action at previously characterized presynaptic imidazoline receptors. 1-Oleoyl-lysophosphatidic acid, but not sphingosine-1-phosphate, produced an inhibition of release that was antagonized by 30 microM rauwolscine, 1 microM SR141716A and 10 microM LY320135 as well as by pretreatment of the cells with 100 microM clonidine for 72 h. Polymerase chain reaction (PCR) experiments on cDNA from PC12 mRNA suggest mRNA expression of lysophospholipid receptors encoded by the genes edg2, edg3, edg5 and edg7, but not of receptors encoded by edg1, edg4, edg6 and edg8, and not of alpha(2A(-))nd CB(1) receptors. In conclusion, PC12 cells are not endowed with alpha(2)-adrenoceptors and CB(1) cannabinoid receptors, but with an inhibitory receptor recognizing imidazolines, guanidines and WIN55,212-2 similar to that on sympathetic nerves. The PCR results and the ability of 1-oleoyl-LPA to mimic these drugs (also with respect to their susceptibility to antagonists) suggest that the release-inhibiting receptor may be an edg-encoded lysophospholipid receptor.

Topics: Animals; Arachidonic Acids; Benzoxazines; Cannabinoids; Clonidine; Cyclohexanols; Endocannabinoids; Heterotrimeric GTP-Binding Proteins; Imidazoles; Imidazoline Receptors; Kinetics; Morpholines; Naphthalenes; Norepinephrine; PC12 Cells; Polyunsaturated Alkamides; Rats; Receptors, Adrenergic, alpha-2; Receptors, Cannabinoid; Receptors, Cell Surface; Receptors, Drug; Receptors, G-Protein-Coupled; Receptors, Lysophospholipid; Tritium; Veratridine

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

Interactions between cannabinoid CB(1) and GABA receptors and ligands were investigated in the myenteric plexus-longitudinal muscle of the guinea pig ileum. Electrically evoked contractions of the myenteric plexus-longitudinal muscle were inhibited by the cannabinoid receptor agonist CP55,940 ((-)-cis-3-[2-Hydroxy-4-(1,1-dimethylheptyl) phenyl]-trans-4-(3-hydroxypropyl) cyclohexanol), the GABA(B) receptor agonist, baclofen (4-amino-3-(chlorophenyl) butanoic acid), or exogenous GABA. Electrically evoked contractions of the myenteric plexus-longitudinal muscle were also inhibited by the addition of the GABA releasing agent ethylenediamine. CP55,940 (1 nM) or the endogenous cannabinoid anandamide (arachidonyl ethanolamide, 1 microM) reduced the inhibition produced by ethylenediamine, while in contrast, anandamide (10 microM) significantly increased the inhibition produced by ethylenediamine. The results suggest that while there is no interaction between cannabinoid CB(1) and GABA(B) receptors in the myenteric plexus-longitudinal muscle of the guinea pig, cannabinoid CB(1) receptor stimulation reduces the ethylenediamine-evoked GABA release. In addition, anandamide at higher concentrations also potentiates the inhibitory effect of ethylenediamine at least partly by stimulating vanilloid receptors.

Topics: Animals; Arachidonic Acids; Cannabinoids; Cyclohexanols; Electric Stimulation; Endocannabinoids; gamma-Aminobutyric Acid; Guinea Pigs; Ileum; In Vitro Techniques; Muscle Contraction; Myenteric Plexus; Polyunsaturated Alkamides; Receptors, Cannabinoid; Receptors, Drug; Receptors, GABA-B

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

Anandamide and the metabolically stabler analogs, (R)-1'-methyl-2'-hydroxy-ethyl-arachidonamide (Met-AEA) and N-(3-methoxy-4-hydroxy-benzyl)-arachidonamide (arvanil), are CB(1) cannabinoid and VR(1) vanilloid receptors agonists. We synthesized 1',1'-dimethylheptyl-arvanil (O-1839) and six other AEA analogs obtained by addition of either a hydroxy, cyano, or bromo group on the C-20 atom of 1,1'-dimethylpentyl-Met-AEA (O-1811, O-1812 and O-1860, respectively) or 1,1'-dimethylpentyl-arvanil (O-1856, O-1895 and O-1861, respectively). The compounds were tested for their (i) affinity for CB(1) and CB(2) receptors, (ii) capability to activate VR1 receptors, (iii) inhibitory effect on the anandamide hydrolysis and on the anandamide membrane transporter, and (iv) cannabimimetic activity in the mouse 'tetrad' of in vivo assays. O-1812 is the first ligand ever proven to be highly (500- to 1000-fold) selective for CB(1) vs both VR(1) and CB(2) receptors, while O-1861 is the first true "hybrid" agonist of CB(1)/VR(1) receptors and a compound with potential therapeutic importance. The activities of the seven compounds in vivo did not correlate with their activities at either CB(1) or VR(1) receptors, thus suggesting the existence of other brain sites of action mediating some of their neurobehavioral actions in mice.

Topics: Animals; Arachidonic Acids; Benzoxazines; Binding, Competitive; Calcium; Capsaicin; Cell Line; Cyclohexanols; Cytosol; Dose-Response Relationship, Drug; Endocannabinoids; Humans; Ligands; Membranes; Morpholines; Naphthalenes; Polyunsaturated Alkamides; Rats; Receptor, Cannabinoid, CB2; Receptors, Cannabinoid; Receptors, Drug; Structure-Activity Relationship; Tritium; TRPV Cation Channels; Tumor Cells, Cultured

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

Lipopolysaccharide (LPS) increases the levels of the endogenous cannabinoid anandamide (N-arachidonoylethanolamine, AEA) in rat macrophages, but the mechanism responsible for this effect has not been elucidated. Here we demonstrate that LPS enhances the levels of AEA (fourfold over controls) also in human lymphocytes. We show that in these cells LPS inhibits the activity of the AEA-degrading enzyme fatty acid amide hydrolase (FAAH), by downregulating the gene expression at transcriptional level. Lymphocytes have also a specific AEA transporter and a functional CB1 cannabinoid receptor, which were not modulated by LPS. The effect of this endotoxin on FAAH was not mediated by AEA-induced activation of cannabinoid receptors. Conversely, the stimulatory action of LPS on AEA levels might be due to inhibition of FAAH, as suggested by the observation that an increase of AEA amounts was also induced by an irreversible FAAH inhibitor. These results suggest that lymphocytes take part in regulating the peripheral endocannabinoid system and endocannabinoid homeostasis.

Topics: Amidohydrolases; Arachidonic Acids; Binding Sites; Biological Transport; Cannabinoid Receptor Modulators; Cyclohexanols; Down-Regulation; Endocannabinoids; Gene Expression Regulation, Enzymologic; Humans; In Vitro Techniques; Lipopolysaccharides; Lymphocytes; Polyunsaturated Alkamides; Tritium

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

Based on the previously reported co-localization and relationship between cannabinoid and dopamine receptors, the effects of cannabinoid receptor agonists against cocaine-induced toxic behavioural symptoms, including convulsive seizures, were examined in mice. The anticonvulsant effect of several cannabimimetics against seizures induced by other convulsants was also compared. The cannabinoid receptor agonists CP 55940 ((-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)-cyclohexanol) and WIN 55212-2 ((R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone), and the endogenous cannabinoid anandamide were co-administered intraperitoneally with cocaine (75 mg kg(-1)) or other convulsants such as bicuculline, methyl 6,7-dimethoxy-4-ethyl-beta-carboline-carboxylate (DMCM), L-glutamic acid and N-methyl-D-aspartate (NMDA). CP 55940 (2.5 mg kg(-1)) and anandamide (15 mg kg(-1)) significantly antagonized cocaine-induced lethality, and CP 55940 and WIN 55212-2 (2.5 mg kg(-1)) significantly attenuated the severity of cocaine-induced convulsive seizures. Furthermore, ataxic hyperactivity, which was observed only in the cocaine-treated group of mice and could be evaluated by their activity counts, was also depressed in the groups of mice co-treated with each of the three cannabinoid agonists. However, none of these agonists protected against bicuculline- or DMCM-induced lethality or convulsive seizures. In contrast, all of the cannabinoid agonists, most notably anandamide, antagonized both L-glutamic acid (2 g kg(-1))- and NMDA (200 mg kg(-1))-induced convulsive seizures. These data support the previously reported close correlation between dopamine and cannabinoid receptors, and between cannabinoid agonists, especially anandamide, and glutamate (NMDA) receptors. Furthermore, these results suggest a potential therapeutic role for cannabinoid agonists against cocaine- and other-convulsant-induced toxicities.

Topics: Animals; Arachidonic Acids; Behavior, Animal; Benzoxazines; Cocaine; Convulsants; Cyclohexanols; Dopamine Uptake Inhibitors; Drug Interactions; Endocannabinoids; Male; Mice; Mice, Inbred ICR; Morpholines; Naphthalenes; Polyunsaturated Alkamides; Receptors, Cannabinoid; Receptors, Drug; Seizures; Survival Rate

The effects of the endogenous cannabinoid (CB) anandamide (AEA) and its analogs on cocaine (COCA)-induced toxic symptoms such as lethality, convulsive seizures and hyperactivity were examined in mice. In addition to AEA, the effects of the AEA analogs arachidonyl-2-chloroethylamide (ACEA), arachidonylcyclopropylamide (ACPA) and R-(+)-methanandamide (METH) were compared to the selective and strong CB1 agonist CP 55940 (CP). Intraperitoneal (i.p.) coadministrations of these drugs with COCA (75 mg/kg) demonstrated that AEA (10 and 15 mg/kg), ACEA (5 mg/kg), ACPA (5 mg/kg), METH (5, 10 and 15 mg/kg) and CP (2.5 and 5 mg/kg) all antagonized the COCA-induced lethality, and that ACEA (5 and 10 mg/kg), ACPA (5 and 10 mg/kg), METH (5, 10 and 15 mg/kg) and CP (1, 2.5 and 5 mg/kg) antagonized the COCA-induced convulsive seizures. When alterations in the COCA-induced toxic behaviors were also evaluated by an activity counting instrument, antidotal effects against the COCA-induced hyperactivity were also observed using the above doses. The effects against hyperactivity were stronger in the groups of mice cotreated with CP or ACEA than in the groups of mice cotreated with AEA or METH. However, the antidotal effects against the lethality and convulsive seizures were stronger in the METH-treated group than in the AEA-, ACEA- or ACPA-treated groups, although the selectivity of METH for brain CB1 receptors was lower than for ACEA or ACPA. The correlation with other brain receptors and/or peripheral CB receptors seemed to contribute to the strong antidotal effects of METH, which were not exceeded even by CP.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Cocaine; Cyclohexanols; Drug Interactions; Drug Therapy, Combination; Endocannabinoids; Hyperkinesis; Ligands; Mice; Mice, Inbred ICR; Polyunsaturated Alkamides; Receptors, Cannabinoid; Receptors, Drug; Seizures

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 endogenous opioid dynorphin B was evaluated for its role in cannabinoid-induced antinociception. Previous work in our laboratory has shown that the synthetic, bicyclic cannabinoid, CP55,940, induces the release of dynorphin B whilst the naturally occurring cannabinoid, Delta(9)-tetrahydrocannabinol (Delta(9)-THC), releases dynorphin A. The dynorphins contribute in part to the antinociceptive effects of both cannabinoids at the level of the spinal cord. The present study compares dynorphin B released from perfused rat spinal cord in response to acute administration of anandamide (AEA), Delta(9)-THC and CP55,940 at two time points, 10 min and 30 min post administration, and attempts to correlate such release with antinociceptive effects of the drugs. Dynorphin B was collected from spinal perfusates of rats pretreated with Delta(9)-THC, CP55,940 or AEA. The supernatant was lyophilized and the concentrations of dynorphin B were measured via radioimmunoassay. At a peak time of antinociception (10 min), CP55,940 and Delta(9)-THC induced significant two-fold increases in the release of dynorphin B. AEA did not significantly release dynorphin B. Upon a 30-min pretreatment with the drugs, no significant dynorphin B release was observed, although antinociceptive effects persisted for CP55,940 and Delta(9)-THC. Previous work indicates that Delta(9)-THC releases dynorphin A while AEA releases no dynorphin A. This study confirms that although all three test drugs produced significant antinociception at 10 min, the endocannabinoid, AEA, does not induce antinociception via dynorphin release. Thus, our data indicate a distinct mechanism which underlies AEA-induced antinociception.

Topics: Analgesia; Analgesics; Analgesics, Non-Narcotic; Animals; Arachidonic Acids; Calcium Channel Blockers; Cannabinoid Receptor Modulators; Cannabinoids; Cyclohexanols; Dronabinol; Dynorphins; Endocannabinoids; Endorphins; Male; Pain; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Spinal Cord

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

N-Arachidonoylethanolamine (AEA) is a proposed endogenous ligand of the central cannabinoid receptor (CB1). Previous studies indicate that AEA is translocated across membranes via a process that has the characteristics of carrier-mediated facilitated diffusion. To date, studies of this mechanism have relied on [(3)H]AEA as a substrate for the carrier. We have synthesized an analog of AEA, SKM 4-45-1, that is nonfluorescent in the extracellular environment. When SKM 4-45-1 is exposed to intracellular esterases, it is de-esterified and becomes fluorescent. We have carried out studies to demonstrate that SKM 4-45-1 accumulation in cells occurs via the AEA carrier. SKM 4-45-1 is accumulated by both cerebellar granule cells and C6 glioma cells. Uptake of SKM 4-45-1 into C6 glioma is inhibited by AEA (IC(50)=53.8 +/- 1.8 microM), arachidonoyl-3-aminopyridine amide (IC(50)=10.1 +/- 1.4 microM), and arachidonoyl-4-hydroxyanilineamide (IC(50)=6.1 +/- 1.3 microM), all of which also inhibit [(3)H]AEA accumulation. Conversely, [(3)H]AEA accumulation by cerebellar granule cells is inhibited by SKM 4-45-1 with an IC(50) of 7.8 +/- 1. 3 microM. SKM 4-45-1 is neither a substrate nor inhibitor of fatty acid amide hydrolase, an enzyme that catabolizes AEA. SKM 4-45-1 does not bind the CB1 cannabinoid receptor at concentrations <10 microM. In summary, the cellular accumulation of SKM 4-45-1 occurs via the same pathway as AEA uptake and provides an alternative substrate for the study of this important cellular process.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Brain Neoplasms; Cannabinoids; Carrier Proteins; Cell Membrane; Cerebellum; Cyclohexanols; Endocannabinoids; Esterases; Fluorescent Dyes; Glioma; Humans; Lactones; Microscopy, Fluorescence; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Tumor Cells, Cultured

The endocannabinoids anandamide and 2-arachidonyl glycerol (2-AG) bind to G protein-coupled central and peripheral cannabinoid receptors CB1 and CB2, respectively. Due to the relatively high expression of the CB2 isotype on peripheral immune cells, it has been hypothesized that this receptor mediates the immunosuppressive effects of cannabinoids. Unfortunately, there was a dearth of pharmacological studies with the endocannabinoids and human CB2 (hCB2). These studies compare and contrast the potency and efficacy of anandamide, 2-AG, and the synthetic cannabinoid HU210 at hCB2. Using [(35)S]guanosine-5'-O-(3-thio)triphosphate (GTPgammaS) and radioligand bindings in insect Sf9-hCB2 membranes, we showed that both endocannabinoids bound hCB2 with similar affinity and that the cannabinoids acted as full agonists in stimulating [(35)S]GTPgammaS exchange, although 2-AG was 3-fold more potent than anandamide (EC(50) = 38.9 +/- 3.1 and 121 +/- 29 nM, respectively). In a mammalian expression system (Chinese hamster ovary-hCB2 cells), HU210 and 2-AG maximally inhibited forskolin-stimulated cAMP synthesis (IC(50) = 1.61 +/- 0.42 nM and 1.30 +/- 0.37 microM, respectively) although anandamide was ineffective. In Chinese hamster ovary-hCB2 membranes, HU210 and 2-AG were also full agonists in stimulating [(35)S]GTPgammaS binding (EC(50) = 1.96 +/- 0.35 and 122 +/- 17 nM, respectively), but anandamide was a weak partial agonist (EC(50) = 261 +/- 91 nM; 34 +/- 4% of maximum). Due to its low intrinsic activity, coincubation with anandamide effectively attenuated the functional activity of 2-AG at hCB2. Collectively, the data showed that both endocannabinoids bound hCB2 with similar affinity, but only 2-AG functioned as a full agonist. Moreover, the agonistic activity of 2-AG was attenuated by anandamide.

Topics: Animals; Arachidonic Acids; Binding, Competitive; Calcium Channel Blockers; Cannabinoid Receptor Modulators; Cannabinoids; Cell Membrane; CHO Cells; Colforsin; Cricetinae; Cyclic AMP; Cyclohexanols; Drug Antagonism; Endocannabinoids; Glycerides; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Immunosuppressive Agents; Insecta; Polyunsaturated Alkamides; Receptors, Cannabinoid; Receptors, Drug; Sulfur Radioisotopes; Transfection

Two putative endocannabinoids, N-arachidonylethanolamine (AEA) and 2-arachidonylglycerol, are inactivated by removal from the extracellular environment by a process that has the features of protein-mediated facilitated diffusion. We have synthesized and studied 22 N-linked analogues of arachidonylamide for the purpose of increasing our understanding of the structural requirements for the binding of ligands to the AEA transporter. We have also determined the affinities of these analogues for both the CB(1) cannabinoid receptor and fatty acid amide hydrolase (FAAH). We have identified several structural features that enhance binding to the AEA transporter in cerebellar granule cells. We have confirmed the findings of others that replacing the ethanolamine head group with 4-hydroxybenzyl results in a high-affinity ligand for the transporter. However, we find that the same molecule is also a competitive inhibitor of FAAH. Similarly, replacement of the ethanolamine of AEA with 3-pyridinyl also results in a high-affinity inhibitor of both the transporter and FAAH. We conclude that the structural requirements for ligand binding to the CB(1) receptor and binding to the transporter are very different; however, the transporter and FAAH share most, but not all, structural requirements.

Topics: Adjuvants, Immunologic; Amidohydrolases; Animals; Arachidonic Acids; Binding, Competitive; Biological Transport; Cannabinoid Receptor Modulators; Cannabinoids; Carrier Proteins; Cells, Cultured; Cerebellum; Cyclohexanols; Endocannabinoids; Glycerides; Immunosuppressive Agents; Ligands; Neurons; Polyunsaturated Alkamides; Prosencephalon; Rats; Receptors, Cannabinoid; Receptors, Drug; Structure-Activity Relationship; Tritium

We investigated the ability of the cannabinoid agonists CP55,940 (CB(1)/CB(2)) and anandamide (endogenous cannabinoid) to modulate electrical field stimulation (EFS)-induced acetylcholine (ACh) release from parasympathetic nerve terminals innervating guinea-pig trachea. We assessed whether modulation of transmitter release translated to an impact on functional responses by investigating the effect of these agents on contractile responses evoked by EFS and ACh. Furthermore, we evaluated the ability of these compounds to elicit bronchodilation in pre-contracted guinea-pig tracheal strips. CP55,940 and anandamide significantly inhibited EFS-evoked ACh release (maximal inhibition of 35.1+/-2.9% and 33.4+/-6.4% at 1 microM, P<0.05, respectively). The CB(1) receptor antagonist SR 141716A (1 microM), had no effect on ACh release and failed to reverse the inhibitory effect of CP55,940 (1 microM). Paradoxically, CP55,940 had no significant effect on EFS-evoked cholinergic contractile responses. Furthermore, CP55,940 did not relax pre-contracted tracheal strips or affect contractile responses to exogenous ACh. This lack of activity on smooth muscle tone is consistent with the fact that no detectable specific binding of [(3)H] CP55,940 was found in tracheal homogenates. These data suggest that cannabinoid agonists inhibit ACh release from cholinergic nerve terminals via activation of CB(2) receptors but that this inhibitory action does not impact on functional responses such as cholinergic contraction.

Topics: Acetylcholine; Analgesics; Analgesics, Non-Narcotic; Animals; Arachidonic Acids; Binding, Competitive; Cannabinoids; Carbachol; Cyclohexanols; Electric Stimulation; Endocannabinoids; Guinea Pigs; In Vitro Techniques; Male; Muscle Contraction; Muscle, Smooth; Parasympathetic Nervous System; Parasympatholytics; Polyunsaturated Alkamides; Trachea; Tritium

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 vitro receptor binding and quantitative autoradiography were used to assess the pre- and postsynaptic distribution of cannabinoid receptors in the cervical dorsal horn of the rat spinal cord. An extensive unilateral dorsal rhizotomy was performed across seven or eight successive spinal segments from C3 to T1 or T2. The densities of cannabinoid and mu opioid receptors in the central (C6) spinal segment were assessed 2, 4, 8, and 16 days post rhizotomy and compared with those of untreated rats. Rhizotomy induced approximately a 50% ipsilateral loss in the [3H]CP55,940 binding to spinal cannabinoid receptors that was maximal at 8 days post-rhizotomy. By comparison, the binding of [3H][d-Ala2-MePhe4, Gly-ol5]enkephalin (DAMGO) to mu receptors was depleted approximately 60% in near-adjacent sections. By contrast, changes in [3H]CP55,940 binding contralateral to the deafferentation were largely absent at all post-lesion delays. These data suggest that under conditions in which a spinal segment is completely deafferented, approximately 50% of cannabinoid receptors in the cervical (C6) dorsal horn reside presynaptically on central terminals of primary afferents. The present data provide anatomical evidence for presynaptic as well as postsynaptic localization of cannabinoid receptors in the spinal dorsal horn.

Topics: Analgesics; Analgesics, Opioid; Animals; Arachidonic Acids; Autoradiography; Calcium Channel Blockers; Cyclohexanols; Endocannabinoids; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Functional Laterality; Male; Neurons, Afferent; Polyunsaturated Alkamides; Presynaptic Terminals; Rats; Rats, Sprague-Dawley; Receptors, Cannabinoid; Receptors, Drug; Receptors, Opioid, mu; Rhizotomy; Spinal Cord; Tritium

Segments of human right atrial appendages preincubated with [3H]noradrenaline and superfused with physiological salt solution containing desipramine and corticosterone were used to examine whether the cardiac sympathetic nerves are endowed with cannabinoid receptors and to further study pharmacological properties of presynaptic imidazoline receptors. The cannabinoid CB1 receptor agonists CP55,940, HU210 and anandamide inhibited evoked [3H]noradrenaline release. The inhibition by CP55,940 and anandamide was abolished by the CB1 receptor antagonists SR141716A (1 microM) and LY320135 (1 microM). Rauwolscine at the imidazoline receptor-blocking concentration of 30 microM abolished the inhibitory effect of CP55,940 and anandamide. After blockade of alpha2-adrenoceptors with 1 microM rauwolscine, the imidazoline binding site ligand S23230, which is the (-)-enantiomer of the racemic oxazoline derivative S22687, exhibited low potency in inhibiting electrically evoked [3H]noradrenaline release (pIC30%=4.96), whereas the (+)-enantiomer S23229 and the racemate S22687 were ineffective. In the presence of 30 microM rauwolscine, S23230 did not significantly inhibit evoked release. The imidazoline receptor-mediated inhibitory effect of BDF 6143 and aganodine on evoked [3H]noradrenaline release was abolished by 1 microM SR141716A and by 1 microM LY320135. The inhibitory effect of moxonidine on evoked [3H]noradrenaline release, which is exclusively mediated via activation of alpha2-autoreceptors, was not antagonized by 1 microM SR141716A. In conclusion, inhibitory cannabinoid CB1 receptors are present on the sympathetic axon terminals of human atrial appendages. Presynaptic imidazoline receptors share the property of other receptors in that they can be stereoselectively activated. The cross-antagonism of imidazoline receptor agonists/antagonists with CB1 receptor antagonists/agonists suggests that these receptors may have certain binding domains in common or that they interact with each other in an unknown manner.

Topics: Adrenergic Uptake Inhibitors; Arachidonic Acids; Atrial Function; Corticosterone; Cyclohexanols; Desipramine; Dose-Response Relationship, Drug; Dronabinol; Drug Interactions; Electrophysiology; Endocannabinoids; Excitatory Amino Acid Agonists; Female; Guanidine; Humans; Imidazoles; Imidazoline Receptors; Ligands; Male; Norepinephrine; Polyunsaturated Alkamides; Receptors, Cannabinoid; Receptors, Drug; Receptors, Presynaptic; Sympathetic Nervous System; Sympathomimetics

In this study, we focused on the pharmacological characterization of cannabinoid receptor coupling to G protein-gated inwardly rectifying potassium (GIRK) channels. Cannabinoids were tested on Xenopus laevis oocytes coexpressing the CB(1) receptor and GIRK1 and GIRK4 channels (CB(1)/GIRK1/4) or the CB(2) receptor and GIRK1/4 channels (CB(2)/GIRK1/4). WIN 55,212-2 enhanced currents carried by GIRK channels in the CB(1)/GIRK1/4 and CB(2)/GIRK1/4 system; however, the CB(2) receptor did not couple efficiently to GIRK1/4 channels. In the CB(1)/GIRK1/4 system, WIN 55,212-2 was the most efficacious compound tested. CP 55,940 and anandamide acted as partial agonists. The rank order of potency was CP 55,940 > WIN 55,212-2 = anandamide. The CB(1)-selective antagonist SR141716A alone acted as a inverse agonist by inhibiting GIRK currents in oocytes expressing CB(1)/GIRK1/4, suggesting the CB(1) receptor is constitutively activated. A conserved aspartate residue, which was previously shown to be critical for G protein coupling in cannabinoid receptors, was mutated (to asparagine, D163N) and analyzed. Oocytes coexpressing CB(1)/GIRK1/4 or D163N/GIRK1/4 were compared. The potency of WIN 55, 212-2 at the mutant receptor was similar to wild type, but its efficacy was substantially reduced. CP 55,940 did not elicit currents in oocytes expressing D163N/GIRK1/4. In summary, it appears the CB(1) and CB(2) receptors couple differently to GIRK1/4 channels. In the CB(1)/GIRK1/4 system, cannabinoids evaluated demonstrated the ability to enhance or inhibit GIRK currents. Furthermore, a conserved aspartate residue in the CB(1) receptor is required for normal communication with GIRK channels in oocytes demonstrating the interaction between receptor and channels is G protein dependent.

Topics: Analgesics; Animals; Arachidonic Acids; Asparagine; Benzoxazines; Calcium Channel Blockers; Cyclohexanols; Dose-Response Relationship, Drug; Endocannabinoids; GTP-Binding Proteins; Morpholines; Mutation; Naphthalenes; Oocytes; Polyunsaturated Alkamides; Potassium Channels; Receptors, Cannabinoid; Receptors, Drug; RNA, Complementary; Time Factors; Xenopus

The electrically-evoked release of [3H]acetylcholine from hippocampal brain slices is inhibited by cannabinoid receptor agonists. The effect of palmitylsulphonyl fluoride (AM 374), a recently developed inhibitor of fatty acid amide hydrolase, in influencing the potency of exogenously added anandamide in this preparation was examined. Anandamide alone had relatively little effect on [3H]acetylcholine release. By contrast, in the presence of AM 374 (0.1 microM), anandamide produced a significant inhibition of [3H]acetylcholine release at all concentrations tested (0.1-10 microM). In addition to experiments with AM 374 the effects of N-(4-hydroxyphenyl)arachidonamide (AM 404), a putative anandamide uptake inhibitor, was also examined. However, AM 404 at concentrations up to 10 microM, was not found to significantly enhance the effect of anandamide on electrically-evoked [3H]acetylcholine release. These results indicate that AM 374 potently inhibits endogenous amidase activity and thus facilitates access of exogenous anandamide to cannabinoid receptors in the hippocampal tissue.

Topics: Acetylcholine; Amidohydrolases; Animals; Arachidonic Acids; Benzoxazines; Cannabinoids; Cyclohexanols; Drug Synergism; Electric Stimulation; Endocannabinoids; Enzyme Inhibitors; Hippocampus; In Vitro Techniques; Male; Morpholines; Naphthalenes; Palmitates; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Receptors, Cannabinoid; Receptors, Drug

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

We have detected the presence of a specific [3H] CP 55,940 binder in the cytosol of rat cerebral cortex. Competition studies showed that only cold CP 55,940 and to a lesser extent delta9THC was able to compete with [3H] CP 55,940; little competition was observed with either delta8THC or anandamide. Scatchard analysis of the data indicate the presence of two distinct binding components having affinity constants (Kd) of 0.97 +/- 0.03 nM, 5.83 +/- 0.08 nM, and Bmax of 3.31 +/- 0.06 pmol/mg protein, 22.2 +/- 1.2 pmol/mg protein respectively. The cytosolic CP 55,940 binder was heat stable up to 30 degrees C. Besides the brain cytosol, lesser amounts of binding were also detected in the spleen, and testis. Liver, kidney and muscle cytosol preparations were found to be devoid of this binder. Unlike the previously characterized brain membrane cannabinoid receptor, this binder was found to be salt, sulfhydryl blocking reagents and nucleotide resistant. Interestingly, dithiothreitol (DTT), a protein-disulfide group reducing agent, inhibited the binding of [3H] CP-55,940 to the receptor and approximately 80% binding inhibition was obtained at a 5 mM concentration. Western blot analysis using anti-receptor antibody reveal the presence of a 95-110, 50 and 38 kDa band in the brain, spleen and testis cytosolic preparations. In conclusion, we have identified the presence of a novel CP 55,940 binder in rat cerebral cortex cytosol possessing biochemical properties distinct from those previously observed using rat cerebral cortex membrane cannabinoid receptor.

Topics: Animals; Arachidonic Acids; Binding, Competitive; Cannabinoids; Cerebral Cortex; Cyclohexanols; Cytosol; Dithiothreitol; Dronabinol; Endocannabinoids; Kinetics; Male; Nucleotides; Organ Specificity; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Receptors, Cannabinoid; Receptors, Cytoplasmic and Nuclear; Receptors, Drug; Salts; Sulfhydryl Reagents; Temperature

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

Cannabinoids have been widely reported to produce antinociception in models such as tail flick and hot plate. However, their role in modulating thermal hyperalgesia is unknown. The potency of some drugs, such as the opioids, increases during hyperalgesia. Thus, we evaluated whether there is a change in the effectiveness of intrathecal cannabinoids with hyperalgesia. Additionally, we evaluated whether cannabinoids could inhibit capsaicin-evoked neurosecretion from isolated rat spinal cord. Our results indicate that 1 fmol anandamide (i.t.) completely blocked carrageenan-induced thermal hyperalgesia. However, anandamide at doses as high as 100 pmol had no effect on thermal latencies in normal animals. Additionally, anandamide inhibited K+- as well as capsaicin-evoked immunoreactive calcitonin gene-related peptide release. Finally, cannabinoid receptors were identified in sensory neurons. Collectively, these results indicate that there is an increased effectiveness of modulation of thermal nociceptive thresholds by spinal cannabinoids during hyperalgesia. This antihyperalgesic effect may be the result of cannabinoid-induced inhibition of neurosecretion from certain primary afferent fibers.

Topics: Animals; Arachidonic Acids; Calcitonin Gene-Related Peptide; Cannabinoids; Capsaicin; Carrageenan; Cyclohexanols; Endocannabinoids; Injections, Spinal; Male; Mice; Pain Threshold; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Spinal Cord

1. The actions of a number of cannabinoid receptor ligands were investigated using the myograph-mounted rat isolated mesenteric artery. Anandamide, CP 55,940, HU-210, palmitoylethanolamide and WIN 55,212-2 all caused concentration-dependent relaxations of methoxamine-precontracted vessels which were not affected by removal of the endothelium. 2. Precontracting vessels with 60 mM KCl instead of methoxamine greatly reduced the vasorelaxant effects of anandamide and palmitoylethanolamide. High K+ solution caused a modest decrease in the relaxant potency of CP 55,940 and HU-210, and had no effect on relaxations induced by WIN 55,212-2. 3. Relaxations of methoxamine-induced tone by anandamide, CP 55,940 and HU-210, but not palmitoylethanolamide and WIN 55,212-2, were attenuated by the cannabinoid receptor antagonist, SR 141716A. Relaxation of vessels contracted with 60 mM KCl by CP 55,940 was also sensitive to SR 141716A. 4. Anandamide and CP 55,940 caused small but concentration-dependent contractions in resting vessels in the absence of extracellular calcium. These were not sensitive to SR 141716A. Palmitoylethanolamide and WIN 55,212-2 produced smaller contractions only at higher concentrations. 5. Anandamide and CP 55,940, but not palmitoylethanolamide and WIN 55,212-2, caused concentration-dependent inhibition of the phasic contractions induced by methoxamine in calcium-free conditions, but only anandamide caused inhibition of contractions to caffeine under such conditions. These inhibitory effects were not antagonised by SR 141716A. 6. The present study provides the first detailed investigation of the actions of cannabinoid agonists on vascular smooth muscle. Our results show that these compounds exert both receptor-dependent and -independent effects on agonist-induced calcium mobilization in the rat isolated mesenteric artery.

Topics: Amides; Animals; Arachidonic Acids; Benzoxazines; Cannabinoids; Cyclohexanols; Dronabinol; Endocannabinoids; Endothelium, Vascular; Ethanolamines; In Vitro Techniques; Ligands; Male; Mesenteric Arteries; Methoxamine; Morpholines; Muscle Contraction; Muscle, Smooth, Vascular; Naphthalenes; Palmitic Acids; Polyunsaturated Alkamides; Rats; Rats, Wistar

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

Cannabinoid receptors couple to both Gs and Gi proteins and can consequently stimulate or inhibit the formation of cAMP. To test whether there is specificity among cannabinoid receptor agonists in activating Gs- or Gi-coupled pathways, the potency and intrinsic activity of various cannabinoid receptor ligands in stimulating or inhibiting cAMP accumulation were quantified. The rank order of potencies of cannabinoid receptor agonists in increasing or inhibiting forskolin-stimulated cAMP accumulation, in CHO cells expressing hCB1 receptors, was identical (HU-210 > CP-55,940 > THC > WIN-55212-2 > anandamide). However, the activities of these agonists were different in the two assays with anandamide and CP-55,940 being markedly less efficacious in stimulating the accumulation of cAMP than in inhibiting its formation. Studies examining the effects of forskolin on cannabinoid receptor mediated stimulation of adenyly cyclase also revealed differences among agonists in as much as forskolin enhanced the potency of HU-210 and CP-55,940 by approximately 100-fold but, by contrast, had no effect on the potency of WIN-55212-2 or anandamide. Taken together these findings demonstrate marked differences among cannabinoid receptor agonists in their activation of intracellular transduction pathways. This provides support for the emerging concept of agonist-specific trafficking of cellular responses and further suggests strategies for developing receptor agonists with increased therapeutic utility.

Topics: Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Animals; Arachidonic Acids; Benzoxazines; CHO Cells; Colforsin; Cricetinae; Cyclic AMP; Cyclohexanols; Dronabinol; Endocannabinoids; Enzyme Activation; Humans; Morpholines; Naphthalenes; Polyunsaturated Alkamides; Receptors, Cannabinoid; Receptors, Drug; Signal Transduction; Transfection

The effects of several nonclassical cannabinoids and the endogenous cannabinoid ligand, anandamide on the lipid ordering of rat brain synaptic plasma membranes (SPM) were examined and compared to delta 9-tetrahydrocannabinol (delta 9-THC). SPM order was determined using fluorescence polarization. All compounds tested affected membrane ordering. delta 9-THC, CP-55,940, CP-55,244 and WIN-55212 decreased lipid ordering in SPM. Some stereospecificity was observed with delta 9-THC and WIN-55212, but not other compounds. Anandamide also decreased lipid order as did its putative precursor, arachidonic acid. In contrast to these compounds, levonantradol increased SPM lipid order. Although all pharmacologically active cannabinoids affect SPM lipid order, potency on this measure does not correlate well with their pharmacological potency. The results of this study suggest that membrane perturbation (either increases or decreases in lipid order) may be a necessary characteristic for cannabinoid pharmacological activity, but it is not a primary or sufficient determinate of action for this class of drugs.

Topics: Analgesics; Animals; Arachidonic Acid; Arachidonic Acids; Benzoxazines; Brain; Cannabinoids; Cyclohexanols; Dronabinol; Endocannabinoids; Male; Membrane Lipids; Morpholines; Naphthalenes; Phenanthridines; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Structure-Activity Relationship; Synaptic Membranes

The endogenous ligand for the cannabinoid receptor, arachidonylethanolamide (anandamide), has been shown to produce antinociception using the tail-flick test following intrathecal administration. Anandamide was administered i.p. (40 mg kg) to mice four times per day for 3 days. Tolerance developed to anandamide: the ED50 for anandamide (i.t.) was shifted from 40 (26-61) to 139 (79-248) micrograms/mouse. Anandamide-tolerant mice were cross-tolerant to delta 9-THC and CP55,940, but not cross-tolerant to mu-, delta- or kappa- opioids, including dynorphins. Conversely, delta 9-THC-tolerant mice are cross-tolerant to anandamide, CP55,940 and kappa agonists. Our data indicate that anandamide and delta 9-THC differ in the mechanisms by which they induce tolerance, in particular the interaction with endogenous dynorphinergic systems.

Topics: Analgesics; Animals; Arachidonic Acids; Cannabinoids; Cyclohexanols; Dose-Response Relationship, Drug; Dronabinol; Drug Tolerance; Dynorphins; Endocannabinoids; Injections, Intraperitoneal; Injections, Spinal; Mice; Naltrexone; Narcotic Antagonists; Pain Measurement; Polyunsaturated Alkamides; Receptors, Cannabinoid; Receptors, Drug

Cannabinoids and their analogues have been found to inhibit N- and P/Q-type Ca2+ currents in cell lines and sympathetic neurons transfected with cannabinoid CB1 receptor. However, the effects of cannabinoids on Ca2+ currents in the CNS are largely unexplored. In this study we investigated whether these compounds inhibit Ca2+ channels in cultured rat hippocampal neurons. With the use of antibodies directed against the amino-terminus of the CB1 receptor, we found that in 5-day cultures pyramidally shaped neurons expressed somatic CB1 receptors, whereas in 4-wk cultures the receptor was predominately located on neurites. In early cultures, the cannabimimetic WIN 55,212-2 reversibly inhibited whole cell Ba2+ current in a concentration-dependent (K(1/2) = 21 nM) and pertussis-toxin-sensitive fashion. Inhibition was reduced by the CB1 antagonist SR141716. The current was unaffected by the nonpsychoactive enantiomer WIN 55,212-3. Maximal inhibition by the nonclassical cannabinoid agonist CP 55,940 and by an endogenous cannabinoid, anandamide, were similar to that seen with maximal concentrations of WIN 55,212-2. The Ba2+ current modulated by cannabinoids was carried by N-type (omega-conotoxin-GVIA-sensitive) and P/Q-type (omega-conotoxin-MVIIC-sensitive) channels. These results demonstrate cannabinoid-receptor-mediated inhibition of distinct Ca2+ channels in central neurons. Because the channels that underlie these currents are chiefly located presynaptically, and are required for evoked neurotransmitter release, our results suggest a major role for cannabinoids (endogenous and exogenous) in the modulation of synaptic transmission at CNS synapses.

Topics: Animals; Arachidonic Acids; Benzoxazines; Calcium Channel Blockers; Cannabinoids; Cells, Cultured; Cyclohexanols; Endocannabinoids; Hippocampus; Membrane Potentials; Morpholines; Naphthalenes; Neurons; Picrotoxin; Polyunsaturated Alkamides; Rats

Fourteen male rats were trained to discriminate between injections of 2 mg/kg delta-9-tetrahydrocannabinol (delta 9-THC) and vehicle in a 2-lever operant drug-discrimination paradigm. Following training, substitution tests using a cumulative dosing procedure revealed that anandamide (0.5-16 mg/kg ip), the putative endogenous camabinoid receptor ligand, failed to generalize to the discriminative stimulus properties of the training dose of delta 9-THC. However, dose-dependent generalization to the delta 9-THC cue was observed following administration of both CP-55,940 (0.05-0.8 mg/kg ip), a synthetic cannabinoid, and (R)-methanandamide (0.5-8 mg/kg ip), a metabolically stable analog of anandamide. Collectively, these results demonstrate a cannabinoid-specific in vivo effect of an anandamide compound and suggest that the naturally occurring form of anandamide may be metabolized too rapidly to produce a cannabimimetic intercceptive state when administered peripherally.

Topics: Animals; Arachidonic Acids; Cannabinoids; Cyclohexanols; Discrimination Learning; Discrimination, Psychological; Dose-Response Relationship, Drug; Dronabinol; Endocannabinoids; Generalization, Stimulus; Hallucinogens; Male; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley

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. Aminoalkylindoles, typified by WIN 55212-2, bind to G protein-coupled cannabinoid receptors in brain. Although cannabinoids inhibit adenylyl cyclase in NG108-15 neuroblastoma x glioma hybrid cells, cannabinoid receptor binding in these cells has not been described previously. This study compares pharmacological characteristics of [3H]WIN 55212-2 binding sites in rat cerebellar membranes and in NG108-15 membranes. 2. Although the KD of specified [3H]WIN 55212-2 binding was similar in brain and NG108-15 membranes, the Bmax was 10 times lower in NG108-15 than in cerebellar membranes. In both brain and NG108-15 membranes, aminoalkylindole analogues were relatively potent in displacing [3H]WIN 55212-2 binding. However, IC50 values for more traditional cannabinoids were significantly higher in NG108-15 membranes than in brain, e.g., the Ki values for CP55,940 were 1.2 nM in brain and > 5000nM in NG108-15 membranes. Moreover, sodium and GTP-gamma-S decreased [3H]WIN 55212-2 binding in brain but not in NG108-15 membranes. 3. These data suggest that WIN 55212-2 does not label traditional cannabinoid receptors in NG108-15 cells and that these novel aminoalkylindole binding sites are not coupled to G proteins.

Topics: Analgesics; Animals; Arachidonic Acids; Benzoxazines; Binding, Competitive; Calcium Channel Blockers; Cannabinoids; Cerebellum; Cyclohexanols; Endocannabinoids; Glioma; Guanosine 5'-O-(3-Thiotriphosphate); Hybrid Cells; Male; Membrane Proteins; Morpholines; Naphthalenes; Neuroblastoma; Polyunsaturated Alkamides; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptors, Cannabinoid; Receptors, Drug; Sensitivity and Specificity; Tritium

As yet, the physiological significance of hydroxylation of anandamide and linoleoyl amides is unknown. Therefore, we investigated whether hydroxylation of ODNHEtOH and ODNH2 influences their binding abilities to the CB-1 receptor and whether it alters their reactivity towards a fatty acid amide hydrolase (FAAH) from rat brain. Neither the fatty acid amides nor their hydroxylated derivatives were able to displace the potent cannabinoid [3H]CP 55.940 from the CB-1 receptor (Ki > 1 microM). Hydroxylation of ODNHEtOH resulted in a strong reduction of the maximum rate of hydrolysis by a FAAH, but the affinity of FAAH for the substrate remained of the same order of magnitude. Hydroxylation of ODNH2 led to a decrease in the affinity of FAAH for the substrate, but its maximum rate of conversion was unaffected. Furthermore, hydroxylation of ODNHEtOH enhanced its capacity to inhibit competitively the hydrolysis of anandamide. The resulting prolonged lifetime of anandamide and other fatty acid amide derivatives may have a considerable impact on cellular signal transduction.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Binding, Competitive; Brain; Cannabinoids; Cyclohexanols; Endocannabinoids; Enzyme Inhibitors; Hydroxylation; Kinetics; Linoleic Acids; Male; Polyunsaturated Alkamides; Rats; Rats, Wistar; Receptors, Cannabinoid; Receptors, Drug; Substrate Specificity

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

Topics: Animals; Arachidonic Acids; Binding, Competitive; Cannabinoids; Cyclohexanols; Endocannabinoids; Eukaryota; Ligands; Molecular Structure; Polyunsaturated Alkamides; Porifera; Receptors, Cannabinoid; Receptors, Drug; Seawater

Two cannabinoid receptors have been identified to date; one is located predominantly in the central nervous system (CB1), whereas the other is located exclusively in the periphery (CB2). The purposes of this study were to explore further the binding requirements of the CB2 receptor and to search for compounds displaying distinct affinities for either cannabinoid receptor. The binding affinities of a series of cannabinoids tested previously at the CB1 receptor were determined at cloned human CB1 and CB2 receptors using a filtration assay. In addition, possible allosteric regulation of the CB2 receptor was examined. Sodium and a GTP analog elicited a concentration-dependent decrease in specific binding to the CB2 receptor. The affinity of cannabinol for CB2 receptors (Ki = 96.3 +/- 14 nM) was confirmed to be in approximately the same range as that of delta 9-THC (Ki = 36.4 +/- 10 nM). Affinities at cloned CB1 and CB2 receptors were compared with affinities determined in the brain. Although most of the chosen compounds did not discriminate between CB1 and CB2, several ligands were identified that showed selectivity. Affinity ratios demonstrated that two 2'-fluoro analogs of anandamide were over 23-fold selective for the CB1 receptor and confirmed the CB1 selectivity of SR141716A {N- (piperidin-1-yl)-5-(4-chlorophenyl)-1-(2, 4-dichlorophenyl)-4- methyl-1H-pyrazole-3-carboxamidehydrochloride}. In addition, WIN-55, 212-2 {(R)-(+)-[2, 3-dihydro-5-methyl-3-[(4-morpholinyl) methyl] pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthalenyl) methanone} and a closely related propyl indole analog were shown to be 6.75- and 27.5- fold selective, respectively, for the CB2 receptor. These ligands can now serve as a basis for the design of compounds with even greater selectivity.

Topics: Animals; Binding, Competitive; Brain; Cannabinoids; Cell Membrane; Cells, Cultured; CHO Cells; Cricetinae; Cyclohexanols; Guanine Nucleotides; Humans; Ligands; Rats; Receptor, Cannabinoid, CB2; Receptors, Cannabinoid; Receptors, Drug; Recombinant Proteins; Sodium; Structure-Activity Relationship; Transfection

Several analogs of an endogenous cannabimimetic, arachidonylethanolamide (anandamide), were synthesized to study the structural requirements of the ethanolamide head group. CB1 receptor affinities of the analogs were evaluated by a standard receptor binding assay using tritiated CP-55,940 as the radioligand and compared to anandamide which was shown to have a Ki of 78 nM. Replacement of the amide carbonyl oxygen by a sulfur atom had a detrimental effect on the CB1 affinity. The thio analogs of both anandamide and (R)-methanandamide showed very weak affinity for CB1. The secondary nature of the amidic nitrogen was also shown to be important for affinity, indicating a possible hydrogen-bonding interaction between the amide NH and the receptor. Introduction of a phenolic moiety in the head group resulted in the loss of receptor affinity except when a methylene spacer was introduced between the amidic nitrogen and the phenol. A select group of analogs were also tested for their affinity for the CB2 receptor using a mouse spleen preparation and were found to possess low affinities for the CB2 sites. Notably, anandamide and (R)-methanandamide demonstrated high selectivity for the CB1 receptor. Overall, the data presented here show that structural requirements of the head group of anandamide are rather stringent.

Topics: Adjuvants, Immunologic; Analgesics; Animals; Arachidonic Acids; Brain; Calcium Channel Blockers; Cyclohexanols; Endocannabinoids; Kinetics; Mice; Polyunsaturated Alkamides; Rats; Receptor, Cannabinoid, CB2; Receptors, Cannabinoid; Receptors, Drug; Structure-Activity Relationship

Modulation of neuronal ion channels by the cloned rat brain CB1 cannabinoid receptor was investigated with the use of a heterologous neuronal expression system. Transient expression of the rat brain CB1 cannabinoid receptor was accomplished through microinjection of in vitro transcribed cRNA into the cytoplasm of enzymatically dissociated adult rat superior cervical ganglion neurons. The cannabimimetic aminoalkylindole WIN 55,212-2 inhibited whole-cell Ca2+ currents in neurons injected 16-25 hr previously with rat brain CB1 cannabinoid receptor cRNA. Inhibition of the Ca2+ current was voltage and concentration dependent, with a maximal inhibition of 73% and an IC50 value of 47 nM. The synthetic cannabinoid analogue CP55,940 also inhibited Ca2+ currents, with a maximal inhibition of 38% and an IC50 value of 7 nM. Ca2+ current inhibition was blocked by inclusion of guanosine-5'-O-(2-thiodiphosphate) in the intracellular patch pipette solution or by pretreatment with pertussis toxin. Pretreatment with the N-type Ca2+ channel antagonist omega-conotoxin GVIA reduced the inhibition by 100 nM WIN 55,212-2 from 44% to 6%, indicating that N-type Ca2+ channels are a target of cannabinoid action. On washout of WIN 55,212-2, the Ca2+ current amplitude "overrecovered" in 47% of the neurons tested. Anandamide, the endogenous cannabimimetic compound, had an inconsistent effect on the voltage-dependent Ca2+ currents in the majority of neurons microinjected with cannabinoid receptor cRNA. Ca2+ channels were a specific effector target of the cannabinoid receptor, as two different K+ currents, the M current and the A current, were not modulated by the cannabimimetic WIN 55,212-2.

Topics: Animals; Arachidonic Acids; Benzoxazines; Brain; Calcium Channels; Cyclohexanols; Dose-Response Relationship, Drug; Endocannabinoids; Morpholines; Naphthalenes; omega-Conotoxin GVIA; Peptides; Pertussis Toxin; Polyunsaturated Alkamides; Rats; Rats, Wistar; Receptors, Cannabinoid; Receptors, Drug; Superior Cervical Ganglion; Virulence Factors, Bordetella

The present study demonstrates that stereoselective binding sites for anandamide, a naturally occurring cannabinoid substance, can be found in invertebrate immunocytes and microglia. The anandamide-binding site is monophasic and of high affinity, exhibiting a Kd of 34.3 nM with a Bmax of 441 fmol/mg protein. These sites are highly selective, as demonstrated by the inability of other types of signaling molecules to displace [3H]anandamide. Furthermore, this binding site is coupled to nitric oxide release in the invertebrate tissues examined as well as in human monocytes. Interestingly, the cannabinoid-stimulated release of nitric oxide initiates cell rounding. Thus, these cannabinoid actions resemble those of opiate alkaloids. In this regard, we demonstrate that these signaling systems use the same effector system, i.e. nitric oxide release, but separate receptors. Last, the presence of a cannabinoid receptor in selected evolutionary diverse organisms indicates that this signaling system has been conserved for more than 500 million years.

Topics: Animals; Arachidonic Acids; Binding Sites; Cannabinoids; Cyclohexanols; Endocannabinoids; Humans; Microglia; Mollusca; Monocytes; Nitric Oxide; Polyunsaturated Alkamides; Protein Sorting Signals; Receptors, Cannabinoid; Receptors, Drug

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

The effects of anadamide, 2-arachidonoylglycerol and related compounds on the specific binding of a radiolabeled cannabinoid receptor ligand,[3H]CP55940, to synaptosomal membranes were examined. Anandamide, an endogenous cannabinoid receptor ligand, reduced the specific binding of [3H]CP55940 to synaptosomal membranes in a dose-dependent manner: the Ki value was 89 nM. 2-Arachidonoylglycerol was also shown to bind appreciably to the cannabinoid receptor in competitive inhibition experiments. The apparent binding affinity was markedly increased when the binding assay was carried out in the presence of the esterase inhibitor DFP or at 0 degrees C. Free arachidonic acid and N-palmitoylethanolamine were almost inactive in terms of binding to the cannabinoid receptor in synaptosomal membranes. 2-Arachidonoylglycerol may be an endogenous cannabinoid receptor ligand in the brain.

Topics: Animals; Arachidonic Acids; Binding, Competitive; Brain; Cannabinoids; Cyclohexanols; Dose-Response Relationship, Drug; Endocannabinoids; Glycerides; Lipase; Male; Polyunsaturated Alkamides; Rats; Rats, Wistar; Receptors, Cannabinoid; Receptors, Drug; Synaptic Membranes; Tritium

Anandamide, an endogenous arachidonic acid derivative that is released from neurons and activates cannabinoid receptors, may act as a transcellular cannabimimetic messenger in the central nervous system. The biological actions of anandamide and the identity of its target cells are, however, still poorly documented. Here we show that anandamide is a potent inhibitor of gap-junction conductance and dye permeability in striatal astrocytes. This inhibitory effect is specific for anandamide as compared to co-released congeners or structural analogues, is sensitive to pertussis toxin and to protein-alkylating agents, and is neither mimicked by cannabinoid-receptor agonists nor prevented by a cannabinoid-receptor antagonist. Glutamate released from neurons evokes calcium waves in astrocytes that propagate via gap junctions, and may, in turn, activate neurons distant from their initiation sites in astrocytes. We find that anandamide blocks the propagation of astrocyte calcium waves generated by either mechanical stimulation or local glutamate application. Thus, by regulating gap-junction permeability, anandamide may control intercellular communication in astrocytes and therefore neuron-glial interactions.

Topics: Animals; Arachidonic Acids; Astrocytes; Benzoxazines; Calcium; Cannabinoids; Cell Membrane Permeability; Cells, Cultured; Corpus Striatum; Cyclohexanols; Endocannabinoids; Gap Junctions; Glutamic Acid; Isoquinolines; Mice; Morpholines; Naphthalenes; Polyunsaturated Alkamides; Rats; Signal Transduction

The recently discovered endogenous agonist for the cannabinoid receptor, anandamide (arachidonylethanolamide), can be formed enzymatically by the condensation of arachidonic acid with ethanolamine. 5Z,8Z,11Z-Eicosatrienoic acid (mead acid) has been found to substitute for arachidonic acid in the sn-2 position of phospholipids and accumulate during periods of dietary fatty acid deprivation in rats. In the present study, the chemically synthesized ethanolamide of mead acid was evaluated as a potential agonist at the two known subtypes of cannabinoid receptor: CB1 (central) and CB2 (peripheral). This compound was equipotent to anandamide in competing with [3H]CP55,940 binding to plasma membranes prepared from L cells expressing the human CB1 receptor and from ATt-20 cells expressing the human CB2 receptor. Mead ethanolamide was also equipotent to anandamide in inhibiting forskolin-stimulated cAMP accumulation in cells expressing the CB1 receptor. It inhibited N-type calcium currents with a lower potency than anandamide. Mead and arachidonic acid were equally efficacious as substrates for the enzymatic synthesis of their respective ethanolamides in rat and adult human hippocampal P2 membranes. Palmitic acid was not an effective substrate for the enzymatic synthesis of palmitoyl ethanolamide. Mead ethanolamide exhibits several characteristics of a novel agonist to CB1 and CB2 receptors and may represent another candidate endogenous ligand for the CB1 receptor. Due to the anticonvulsant properties of GABA and the positional similarity of L-serine to ethanolamine in membrane phospholipids, these compounds were synthetically coupled to arachidonic acid, and their resulting arachidonamides were tested as potential cannabinoid agonists. The arachidonamides of GABA and L-serine were inactive in both binding and functional assays at the CB1 receptor.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Cannabinoids; CHO Cells; Cricetinae; Cyclohexanols; Endocannabinoids; Humans; L Cells; Mice; Polyunsaturated Alkamides; Rats; Receptor, Cannabinoid, CB2; Receptors, Cannabinoid; Receptors, Drug

A brain constituent, the N-amide derivative of arachidonic acid, termed anandamide, has been recently proposed as a possible endogenous ligand for the cannabinoid receptor. The present study has been designed to examine whether the acute or chronic exposure to anandamide affected the binding of cannabinoid receptors in specific brain areas as occurred with the exogenous cannabinoid agonist, delta 9-tetrahydrocannabinol (THC). To this end, we measured the maximum binding capacity (Bmax) and the affinity (Kd) of cannabinoid receptors, by using [3H]CP-55,940 binding assays, in membranes obtained from several brain areas of male rats acutely or chronically treated with anandamide or THC. Results were as follows. The acute administration of either anandamide or THC increased the Bmax of cannabinoid receptors in the cerebellum and, particularly, in the hippocampus. This effect was also observed after 5 days of a daily exposure to either anandamide or THC. However, whereas the increase in the Bmax after the acute treatment seems to be caused by changes in the receptor affinity (high Kd), the increase after the chronic exposure may be attributed to an increase in the density of receptors. On the contrary, the [3H]CP-55,940 binding to cannabinoid receptors in the striatum, the limbic forebrain, the mesencephalon, and the medial basal hypothalamus was not altered after the acute exposure to anandamide or THC. However, the chronic exposure to THC significantly decreased the Bmax of these receptors in the striatum and nonsignificantly in the mesencephalon. This effect was not elicited after the chronic exposure to anandamide and was not accompanied by changes in the Kd.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Analgesics; Animals; Arachidonic Acids; Brain Chemistry; Calcium Channel Blockers; Cannabinoids; Cannabis; Cyclohexanols; Dronabinol; Endocannabinoids; In Vitro Techniques; Kinetics; Male; Membranes; Polyunsaturated Alkamides; Rats; Rats, Wistar; Receptors, Cannabinoid; Receptors, Drug

The endogenous cannabinoid receptor ligand, anandamide, produced a concentration related inhibition of electrically evoked contractions of the guinea-pig myenteric plexus preparation. Its potency was markedly enhanced by phenylmethylsulphonyl fluoride (2.0-200 microM) which presumably acts by inhibiting the hydrolysis of anandamide in this preparation. The degree of this potentiation increased with the concentration of phenylmethylsulphonyl fluoride used. The methyl analogue of anandamide, R-(+)-arachidonyl-1'-hydroxy-2'-propylamide, also inhibited contractions of the guinea-pig myenteric plexus preparation. The potency of this compound was much less affected by phenylmethylsulphonyl fluoride than was the potency of anandamide, confirming its greater resistance to hydrolysis. Phenylmethylsulphonyl fluoride did not alter the inhibitory potency of the cannabinoid, CP 55,940 ((-)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4- [3-hydroxypropyl]cyclohexan-1-ol), which is not an amidase substrate. Nor did phenylmethylsulphonyl fluoride affect the ability of anandamide to inhibit electrically evoked contractions of the mouse vas deferens, suggesting that anandamide does not undergo hydrolysis in this tissue.

Topics: Analgesics; Animals; Arachidonic Acids; Cannabinoids; Cannabis; Cyclohexanols; Electric Stimulation; Endocannabinoids; Guinea Pigs; Hydrolysis; In Vitro Techniques; Ligands; Male; Muscle Contraction; Muscle, Smooth; Myenteric Plexus; Phenylmethylsulfonyl Fluoride; Polyunsaturated Alkamides; Receptors, Cannabinoid; Receptors, Drug; Stereoisomerism; Vas Deferens

1. Effects of cannabinoid agonists on the serotonin (5-HT)3 receptor-mediated current were investigated in rat nodose ganglion neurons. Anandamide, Win 55212-2, and CP55940 inhibited the 5-HT-induced current in a concentration dependent manner. IC50 values were 190, 310, and 94 nM for anandamide, Win 55212-2, and CP55940, respectively, and 1.6 microM for the nonpsychoactive enantiomer CP56667. This inhibition was slowly developing, noncompetitive, not dependent on membrane potential, and not affected by adenosine 3',5'-cyclic monophosphate (cAMP) analogues, guanosine-5'-O-(2-thiodiphosphate) (GDP-beta-S), and opioid receptor antagonist naltrexone. These data suggest that 5-HT3 receptor ion-channel is a site acted upon by cannabinoid agonists in the nervous system, and the action of cannabinoid agonists on 5-HT3 receptors may be a possible mechanism for some of the behavioral effects of cannabinoids, such as antiemesis and analgesia.

Topics: Animals; Arachidonic Acids; Benzoxazines; Cannabinoids; Cyclohexanols; Endocannabinoids; Morpholines; Naphthalenes; Neurons; Nodose Ganglion; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Receptors, Serotonin

AM630 (iodopravadoline), a novel aminoalkylindole, has been found to attenuate the ability of a number of cannabinoids to inhibit electrically-evoked twitches of the mouse isolated vas deferens. It did not block the inhibitory effects of morphine or clonidine on the twitch response. AM630 behaved as a competitive antagonist of CP 55,940, WIN 55,212-2, anandamide and (R)-(+)-arachidonyl-1'-hydroxy-2'-propylamide (AM356), producing rightward shifts in the log concentration response curves of these cannabinoid receptor agonists that were concentration-dependent, essentially parallel and not accompanied by any decrease in the size of maximal response. AM630 also produced concentration-dependent, parallel rightward shifts in the log concentration-response curve of delta 9-THC. However, these shifts were accompanied by a decrease in the maximal response. AM630 was markedly more potent as an antagonist of delta 9-THC and CP 55,940 (Kd = 14.0 and 17.3 nM respectively) than as an antagonist of WIN 55,212-2, AM356 or anandamide (Kd = 36.5, 85.9 and 278.8 nM respectively). These differences in dissociation constant imply that the mouse vas deferens may contain more than one type of cannabinoid receptor. The data also indicate that the receptors for which AM630 has the highest affinity may not be CB1 cannabinoid receptors as the CB1 selective antagonist, SR141716A, is known to be equally potent in attenuating the inhibitory effects of CP 55,940 and anandamide on the twitch response of the mouse vas deferens.

Topics: Animals; Arachidonic Acids; Benzoxazines; Binding, Competitive; Cannabinoids; Cyclohexanols; Endocannabinoids; In Vitro Techniques; Indoles; Male; Mice; Morpholines; Naphthalenes; Polyunsaturated Alkamides; Receptors, Cannabinoid; Receptors, Drug

Anandamide (arachidonylethanolamide), isolated from porcine brain, has been shown to bind to the cannabinoid receptor and also to produce cannabimimetic activity in pharmacological assays. This study examined structure-activity relationships in alkylated anandamide analogs. The analogs were evaluated for their ability to displace [3H]CP-55,940 in a filtration binding assay using rat brain membranes in the presence and absence of the enzyme inhibitor phenylmethylsulfonyl fluoride (PMSF). Behavioral activity was assessed by the ability of the analogs to produce hypomotility and antinociception. Methylations at carbons 2 and 1 produced compounds stable in the absence of PMSF with similar affinities and behavioral activity as anandamide. Addition of larger alkyl groups at these positions or nitrogen methylation reduced receptor affinity and behavioral potency. These results indicate that methylations at specific carbons of anandamide confer stability in vitro.

Topics: Alkylation; Analgesics; Animals; Arachidonic Acids; Binding, Competitive; Cannabinoids; Cyclohexanols; Endocannabinoids; Male; Motor Activity; Polyunsaturated Alkamides; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptors, Cannabinoid; Receptors, Drug; Structure-Activity Relationship; Swine

Anandamide (arachidonylethanolamide), a putative endogenous ligand for the cannabinoid receptor, produces a tetrad of behavioral effects in mice characteristic of psychoactive cannabinoids including catalepsy, antinociception, hypothermia, and hypomobility. The present study examined the discriminative stimulus effects of anandamide in rats trained to discriminate delta 9-tetrahydrocannabinol or the potent cannabinoid receptor ligand CP 55,940 [(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)-phenyl]-trans-4-(3- hydroxypropyl)cyclohexanol)] from vehicle. Intraperitoneal injections of anandamide substituted for delta 9-tetrahydrocannabinol and for CP 55,940; however, unlike substitution dose-effect curves with the training drugs, anandamide substitution occurred at a single dose (30 or 45 mg/kg) and was accompanied by severe decreases in response rates. The results of the present study suggest that, although systemic anandamide administration may have cannabimimetic effects similar to those of delta 9-tetrahydrocannabinol and CP 55,940, some differences in the behavioral effects of anandamide and other psychoactive cannabinoids also are apparent.

Topics: Animals; Arachidonic Acids; Cannabinoids; Cyclohexanols; Discrimination Learning; Dose-Response Relationship, Drug; Dronabinol; Endocannabinoids; Male; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley

Recent evidence implicates anandamide as the endogenous ligand for the cannabinoid receptor. One purpose of this study was to determine the structural requirements for anandamide's receptor interaction and the influence of phenylmethylsulfonyl fluoride (PMSF), an enzyme inhibitor, on receptor affinity. A second objective was evaluation of the correlation between affinities of the analogs and in vivo pharmacological activities. The ability of anandamide and analogs to displace [3H]CP-55,940 ([3](-)-3-[2-hydroxyl-4-(1,1-dimethylheptyl)phenyl]-4-[3- hydroxylpropyl]cyclohexan-1-ol) was determined by a filtration assay. Displacement curves for anandamide in the presence of PMSF produced a Ki of 89 +/- 10 nM; without PMSF the Ki increased to 5400 +/- 1600 nM. Anandamide analogs were evaluated for their ability to produce antinociception and hypomotility. The levels of saturation of the anandamide structure were critical to receptor affinity and in vivo potency, with complete saturation and hydroxyl substitution with a fluorine moiety resulting in a compound with increased potency in the spontaneous activity and antinociception assays. Substitution of the hydroxyl with a fluorine atom increased affinity only in the presence of PMSF and reduced potency in the antinociception assay. Ethanolamide substitution with bromobenzenesulfonamide produced an inactive compound in all assays. Increasing the length of the N-substituent by one or two carbons decreased receptor binding affinity and potency in the tail-flick assay only. Certain structural modifications, such as methylations, allowed the analogs to retain affinity without the addition of PMSF. Linear correlation between the behavioral and binding assays were performed, and the greatest correlation was obtained with compounds that were either very potent or inactive.

Topics: Animals; Arachidonic Acids; Behavior, Animal; Cannabinoids; Cyclohexanols; Endocannabinoids; Kinetics; Male; Mice; Mice, Inbred ICR; Models, Molecular; Phenylmethylsulfonyl Fluoride; Polyunsaturated Alkamides; Protein Binding; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptors, Cannabinoid; Receptors, Drug; Structure-Activity Relationship

The antinociceptive effects of the putative endogenous cannabinoid ligand anandamide (ANA) and its fluorinated analog, fluoroanandamide (FA), were determined as measured by the tail-flick and p-phenylquinone (PPQ) stretch tests. The ED50 values (confidence limits) for ANA and FA were 77 (52-13) and 7 (2-21) micrograms/mouse, respectively, for the tail-flick test and 30 (23-41) and 0.5 (0.1-2) micrograms/mouse, respectively, for the PPQ test after intrathecal (i.t.) administration. ANA was not significantly less potent than delta 9-tetrahydrocannabinol (THC) in the tail-flick test, but it was less potent in the PPQ test. FA was more potent than either ANA or THC in tail-flick test. The antinociceptive effects of all drugs (administered i.t.) were blocked significantly or nearly abolished by the pretreatment of the mice with pertussis toxin (i.t.). Pretreatment of the mice with 5 and 25 micrograms forskolin per mouse or 10 micrograms 8-(4-chlorophenyl-thio)-adenosine-3',5'-monophosphate cyclic monosodium salt per mouse (both i.t.) significantly attenuated the antinociception produced by THC but not by ANA or FA. Various calcium modulators were tested in combination with THC, ANA, and FA, but they failed to alter the antinociceptive effects of the drugs. Various potassium channel blockers were tested in combination with the drugs. Apamin, a blocker of small (low)-conductance calcium-gated potassium channels that attenuates THC-induced antinociception, failed to alter ANA- or FA-induced antinociception. In contrast to THC, which is blocked by the kappa antagonist nor-binaltorphimine, ANA- and FA-induced antinociception was not altered by classic opioid antagonists. Also in contrast to THC, which enhances mu and delta opioid-induced antinociceptive effects, ANA failed to significantly alter opioid antinociception. ANA significantly shifted the THC dose-effect curve to the right. Thus, ED50 for DMSO/THC in the tail-flick test was shifted from 14 (7-29) to 54 (38-77) micrograms/mouse and was shifted in the hot-plate test from 22 (12-42) to 63 (43-92) micrograms/mouse. The magnitude of the shift in the ED50 was 3.8-fold in the tail-flick test and 2.9-fold in the hot-plate test. The shifts were parallel and significant. The Ki for the displacement of 3H-CP 55,940 binding by ANA and FA was 214 nM (+/- 45 S.E.M.) and 72 nM (+/- 5 S.E.M.), respectively, in pure spinal cord synaptosomes from the rat. ANA and FA were significantly cross-tolerant to THC. Although simila

Topics: Analgesics; Animals; Arachidonic Acids; Binding Sites; Calcium Channel Blockers; Cyclohexanols; Dronabinol; Drug Tolerance; Endocannabinoids; Injections, Spinal; Mice; Narcotic Antagonists; Pertussis Toxin; Polyunsaturated Alkamides; Potassium Channel Blockers; Rats; Spinal Cord; Synaptosomes; Virulence Factors, Bordetella

Ligand binding to the cannabinoid receptor of brain membranes has been characterized using [3H]CP 55,940 and the Multiscreen Filtration System. Binding of [3H]CP 55,940 is saturable and reaches equilibrium by 45 min at room temperature. At a concentration of 10 micrograms of membrane protein/well, the KD for [3H]CP 55,940 is 461 pM and the Bmax is 860 fmol/mg of protein. The apparent KD of [3H]CP 55,940 is dependent upon tissue protein concentration, increasing to 2,450 pM at 100 micrograms of membrane protein. Binding of [3H]CP 55,940 is dependent upon the concentration of bovine serum albumin in the buffer; the highest ratio of specific to nonspecific binding occurs between 0.5 and 1.0 mg/ml. The Ki of anandamide, a putative endogenous ligand of the cannabinoid receptor, is 1.3 microM in buffer alone and 143 nM in the presence of 0.15 mM phenylmethylsulfonyl fluoride. When [14C]anandamide is incubated with rat forebrain membranes at room temperature, it is degraded to arachidonic acid; the hydrolysis is inhibited by 0.15 mM phenylmethylsulfonyl fluoride. These results support the hypothesis that anandamide is a high-affinity ligand of the cannabinoid receptor and that it is rapidly degraded by membrane fractions.

Topics: Animals; Arachidonic Acids; Brain; Cannabinoids; Cyclohexanols; Endocannabinoids; Filtration; Ligands; Male; Membranes; Neurochemistry; Phenylmethylsulfonyl Fluoride; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Receptors, Cannabinoid; Receptors, Drug; Time Factors

The arachidonic acid derivative anandamide (arachidonylethanolamide) has been isolated from porcine brain and has been shown to bind competitively to the cannabinoid receptor. Although the pharmacological activity of this compound has not yet been fully determined, preliminary data suggest that it produces several effects similar ot the cannabinoids. In the present experiments anandamide produced effects similar to those of delta 9-tetrahydrocannabinol, including antinociception (as determined in a latency to tail-flick evaluation), hypothermia, hypomotility and catalepsy in mice after i.v., i.t. and i.p. administration. In general, the effects of anandamide occurred with a rapid onset, but with a rather short duration of action. Prominent antinociceptive effects (> 80% maximal possible effect) were measured immediately after i.v. and i.t. administration. Anandamide produced significant decreases in rectal temperature (2-4 degrees C) after either i.v. or i.t. injection. Maximal effects on motor activity (approximately 85% inhibition) were observed immediately after i.v. and i.p. administration and 10 min after i.t. administration. Maximum immobility observed after i.v. administration was over 80%, yet that produced after i.p. and i.t. administration was too small (< or = 20%) to be considered pharmacologically relevant. Anandamide was less potent (1.3 to 18 times) than delta 9-tetrahydrocannabinol in all behavioral assays. Pretreatment with nor-binaltorphimine, a kappa opioid antagonist which blocks i.t. delta 9-tetrahydrocannabinol-induced antinociception, failed to alter antinociception after i.t. anandamide administration. Binding studies demonstrating that anandamide displaces [3H]CP-55,940 from rat whole brain P2 membrane preparations with a KD of 101 +/- 15 nM. These findings demonstrate that anandamide produces effects in a tetrad of tests used to predict cannabimimetic activity and supports the contention of its role as an endogenous cannabinoid ligand. However, there appear to be distinct differences between anandamide and the cannabinoids with regard to their antinociceptive properties, and other properties vary as a function of route of administration.

Topics: Analgesics; Animals; Arachidonic Acids; Binding, Competitive; Brain; Calcium Channel Blockers; Cyclohexanols; Dronabinol; Drug Administration Routes; Drug Interactions; Endocannabinoids; Male; Membranes; Mice; Mice, Inbred ICR; Naltrexone; Polyunsaturated Alkamides; Receptors, Purinergic P2; Tritium

Arachidonoyl ethanolamide (anandamide) is a naturally occurring brain constituent that binds to a specific brain cannabinoid receptor (CBR1). An amidase activity (anandamide amidase) in membrane fractions of brain and in cultured neuroblastoma cells rapidly degrades anandamide to arachidonic acid (Deutsch, D. G., and Chin, S. (1993) Biochem. Pharmacol. 46, 791-796). In the current study, analogs of anandamide representing three classes of putative transition-state inhibitor (trifluoromethyl ketones, alpha-keto esters, and alpha-keto amides) were synthesized and tested as inhibitors of anandamide hydrolysis in vitro and as ligands for CBR1. The trifluoromethyl ketones and alpha-keto esters showed nearly 100% inhibition of anandamide hydrolysis in vitro at 7.5 microM inhibitor and 27.7 microM anandamide. Arachidonyl trifluoromethyl ketone was the only synthetic compound in the series of fatty acid derivatives able to displace [3H]CP-55940 binding to CBR1 with a Ki of 0.65 microM. It was also the most effective inhibitor in intact neuroblastoma cells, leading to a 12-fold increase of cellular anandamide levels at 12 microM. From the action of these inhibitors on this hydrolytic enzyme, it seems likely that anandamide is cleaved by a mechanism that involves an active-site serine hydroxyl group. These inhibitors may serve as useful tools to elucidate the role anandamide plays in vivo.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Binding, Competitive; Cannabinoids; Cyclohexanols; Endocannabinoids; Esters; Fatty Acids; Hydrolysis; Ketones; Polyunsaturated Alkamides; Rats; Tumor Cells, Cultured

1. Mice pretreated intraperitoneally for 2 days with delta-9-tetrahydrocannabinol (delta-9-THC) at a dose of 20 mg kg-1 day-1 and then challenged intravenously with this drug, 24 h after the second pretreatment, showed a 6 fold tolerance to the hypothermic effect of delta-9-THC. This pretreatment also induced tolerance to the hypothermic effects of the cannabimimetic agents, CP 55,940 (4.6 fold) and WIN 55,212-2 (4.9 fold), but not to the hypothermic effect of the putative endogenous cannabinoid, anandamide. 2. Vasa deferentia removed from mice pretreated intraperitoneally with delta-9-THC twice at a dose of 20 mg kg-1 day-1 were less sensitive to its inhibitory effect on electrically-evoked contractions than vasa deferentia obtained from control animals. The cannabinoid pretreatment induced a 30 fold parallel rightward shift in the lower part of the concentration-response curve of delta-9-THC and a marked reduction in the maximal inhibitory effect of the drug. It also induced tolerance to the inhibitory effects on the twitch response of CP 55,940 (8.7 fold), WIN 55,212-2 (9.6 fold) and anandamide (12.3 fold). 3. The results confirm that cannabinoid tolerance can be rapid in onset and support the hypothesis that it is mainly pharmacodynamic in nature. The finding that in vivo pretreatment with delta-9-THC can produce tolerance not only to its own inhibitory effect on the vas deferens but also to that of three other cannabimimetic agents, suggests that this tissue would be suitable as an experimental model for investigating the mechanisms responsible for cannabinoid tolerance. 4. Further experiments are required to establish why tolerance to anandamide-induced hypothermia was not produced by a pretreatment with delta-9-THC that did induce tolerance to the hypothermic effects of delta-9-THC, CP 55,940 and WIN 55,212-2 and to the inhibitory effects of delta-9-THC,CP 55,940, WIN 55,212-2 and anandamide on the twitch response of the vas deferens.

Topics: Amides; Animals; Arachidonic Acids; Benzoxazines; Body Temperature; Cannabinoids; Cyclohexanols; Dronabinol; Drug Tolerance; Electric Stimulation; Endocannabinoids; Fatty Acids, Unsaturated; Male; Mice; Morpholines; Naphthalenes; Polyunsaturated Alkamides; Vas Deferens

Arachidonylethanolamide (anandamide), a candidate endogenous cannabinoid ligand, has recently been isolated from porcine brain and displayed cannabinoid-like binding activity to synaptosomal membrane preparations and mimicked cannabinoid-induced inhibition of the twitch response in isolated murine vas deferens. In this study, anandamide and several congeners were evaluated as cannabinoid agonists by examining their ability to bind to the cloned cannabinoid receptor, inhibit forskolin-stimulated cAMP accumulation, inhibit N-type calcium channels, and stimulate one or more functional second messenger responses. Synthetic anandamide, and all but one congener, competed for [3H]CP55,940 binding to plasma membranes prepared from L cells expressing the rat cannabinoid receptor. The ability of anandamide to activate receptor-mediated signal transduction was evaluated in Chinese hamster ovary (CHO) cells expressing the human cannabinoid receptor (HCR, termed CHO-HCR cells) and compared to control CHO cells expressing the muscarinic m5 receptor (CHOm5 cells). Anandamide inhibited forskolin-stimulated cAMP accumulation in CHO-HCR cells, but not in CHOm5 cells, and this response was blocked with pertussis toxin. N-type calcium channels were inhibited by anandamide and several active congeners in N18 neuroblastoma cells. Anandamide stimulated arachidonic acid and intracellular calcium release in both CHOm5 and CHO-HCR cells and had no effect on the release of inositol phosphates or phosphatidylethanol, generated after activation of phospholipase C and D, respectively. Anandamide appears to exhibit the essential criteria required to be classified as a cannabinoid/anandamide receptor agonist and shares similar nonreceptor effects on arachidonic acid and intracellular calcium release as other cannabinoid agonists.

Topics: Amides; Animals; Arachidonic Acids; Binding, Competitive; Calcium Channels; Cannabinoids; Cell Membrane; CHO Cells; Cloning, Molecular; Colforsin; Cricetinae; Cyclic AMP; Cyclohexanols; Endocannabinoids; Fatty Acids, Unsaturated; Humans; Kinetics; Pertussis Toxin; Polyunsaturated Alkamides; Radioligand Assay; Receptors, Cannabinoid; Receptors, Drug; Signal Transduction; Structure-Activity Relationship; Transfection; Tritium; Virulence Factors, Bordetella