Evidence for cannabinoid receptor-dependent and -independent mechanisms of action in leukocytes.
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Cannabinoids exhibit immunosuppressive actions that include inhibition of interleukin-2 production in response to a variety of T cell activation stimuli. Traditionally, the effects of these compounds have been attributed to cannabinoid receptors CB1 and CB2, both of which are expressed in mouse splenocytes. Therefore, N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorphenyl)-4-methyl-H-pyrazole-3 carboxyamidehydrochloride (SR141716A), a CB1 antagonist, and N-[(1S)-endo-1,3,3,-trimethyl-bicyclo[2,2,1]heptan-2-yl]-5-(4-chloro-3-methylphen yl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR144528), a CB2 antagonist, were used to investigate the role of cannabinoid receptors in the cannabinoid-induced inhibition of phorbol ester plus calcium ionophore (PMA/Io)-stimulated interleukin-2 production by mouse splenocytes. PMA/Io-stimulated interleukin-2 production was inhibited by cannabinol, cannabidiol, and both WIN 55212-2 stereoisomers with a rank order potency of R-(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxaz inyl]-(1-napthanlenyl) methanone mesylate (WIN 55212-2) approximately cannabidiol > S-(-)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxaz inyl]-(1-napthanlenyl) methanone mesylate (WIN 55212-3) approximately cannabinol. Cannabinoid-induced inhibition of PMA/Io-stimulated interleukin-2 was not attenuated by the presence of both SR144528 and SR141716A. Using pertussis toxin to address the role of G protein-coupled receptors in this response, it was determined that pertussis toxin treatment did not attenuate cannabinol-induced inhibition of PMA/Io-stimulated interleukin-2. With the demonstration that cannabinoid-induced inhibition of PMA/Io-stimulated interleukin-2 was not mediated via CB1 or CB2, alternative targets of cannabinoids in T cells were examined. Specifically, it was demonstrated that cannabinoids elevated intracellular calcium concentration in resting splenocytes and that the cannabinol-induced elevation in intracellular calcium concentration was attenuated by treatment with both SR144528 and SR141716A. Interestingly, pretreatment of splenocytes with agents that elevate intracellular calcium concentration inhibited PMA/Io-stimulated interleukin-2 production, suggesting that an elevation in intracellular calcium concentration might be involved in the mechanism of interleukin-2 inhibition. These studies suggest that immune modulation produced by cannabinoids involves multiple mechanisms, which might be both cannabinoid receptor-dependent and -independent. (+info)
Effects of delta9-tetrahydrocannabinol and cannabidiol on a Mg2+-ATPase of synaptic vesicles prepared from rat cerebral cortex.
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1. delta9-Tetrahydrocannabinol and cannabidiol both exhibited a concentration-related inhibition of Mg2+-ATPase of vesicles prepared from synaptosomes isolated from rat cerebral cortex. Cannabidiol was about 3 times more potent than tetrahydrocannabinol. 2. These results were similar to those obtained previously using drugs with well established anticonvulsant activity. 3. Tetrahydrocannabinol at a sub-inhibitory concentration (1 micronM) increased the activity of the Mg2+-ATPase relative to values obtained with vehicle controls. (+info)
Neuroprotective effect of (-)Delta9-tetrahydrocannabinol and cannabidiol in N-methyl-D-aspartate-induced retinal neurotoxicity: involvement of peroxynitrite.
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In glaucoma, the increased release of glutamate is the major cause of retinal ganglion cell death. Cannabinoids have been demonstrated to protect neuron cultures from glutamate-induced death. In this study, we test the hypothesis that glutamate causes apoptosis of retinal neurons via the excessive formation of peroxynitrite, and that the neuroprotective effect of the psychotropic Delta9-tetrahydroxycannabinol (THC) or nonpsychotropic cannabidiol (CBD) is via the attenuation of this formation. Excitotoxicity of the retina was induced by intravitreal injection of N-methyl-D-aspartate (NMDA) in rats, which also received 4-hydroxy-2,2,6,6-tetramethylpiperidine-n-oxyl (TEMPOL,a superoxide dismutase-mimetic), N-omega-nitro-L-arginine methyl ester (L-NAME, a nitric oxide synthase inhibitor), THC, or CBD. Retinal neuron loss was determined by TDT-mediated dUTP nick-end labeling assay, inner retinal thickness, and quantification of the mRNAs of ganglion cell markers. NMDA induced a dose- and time-dependent accumulation of nitrite/nitrate, lipid peroxidation, and nitrotyrosine (foot print of peroxynitrite), and a dose-dependent apoptosis and loss of inner retinal neurons. Treatment with L-NAME or TEMPOL protected retinal neurons and confirmed the involvement of peroxynitrite in retinal neurotoxicity. The neuroprotection by THC and CBD was because of attenuation of peroxynitrite. The effect of THC was in part mediated by the cannabinoid receptor CB1. These results suggest the potential use of CBD as a novel topical therapy for the treatment of glaucoma. (+info)
Effects of cannabidiol (CBD) on regional cerebral blood flow.
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Animal and human studies have suggested that cannabidiol (CBD) may possess anxiolytic properties, but how these effects are mediated centrally is unknown. The aim of the present study was to investigate this using functional neuroimaging. Regional cerebral blood flow (rCBF) was measured at rest using (99m)Tc-ECD SPECT in 10 healthy male volunteers, randomly divided into two groups of five subjects. Each subject was studied on two occasions, 1 week apart. In the first session, subjects were given an oral dose of CBD (400 mg) or placebo, in a double-blind procedure. SPECT images were acquired 90 min after drug ingestion. The Visual Analogue Mood Scale was applied to assess subjective states. In the second session, the same procedure was performed using the drug that had not been administered in the previous session. Within-subject between-condition rCBF comparisons were performed using statistical parametric mapping (SPM). CBD significantly decreased subjective anxiety and increased mental sedation, while placebo did not induce significant changes. Assessment of brain regions where anxiolytic effects of CBD were predicted a priori revealed two voxel clusters of significantly decreased ECD uptake in the CBD relative to the placebo condition (p<0.001, uncorrected for multiple comparisons). These included a medial temporal cluster encompassing the left amygdala-hippocampal complex, extending into the hypothalamus, and a second cluster in the left posterior cingulate gyrus. There was also a cluster of greater activity with CBD than placebo in the left parahippocampal gyrus (p<0.001). These results suggest that CBD has anxiolytic properties, and that these effects are mediated by an action on limbic and paralimbic brain areas. (+info)
Antitumor effects of cannabidiol, a nonpsychoactive cannabinoid, on human glioma cell lines.
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Recently, cannabinoids (CBs) have been shown to possess antitumor properties. Because the psychoactivity of cannabinoid compounds limits their medicinal usage, we undertook the present study to evaluate the in vitro antiproliferative ability of cannabidiol (CBD), a nonpsychoactive cannabinoid compound, on U87 and U373 human glioma cell lines. The addition of CBD to the culture medium led to a dramatic drop of mitochondrial oxidative metabolism [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide test] and viability in glioma cells, in a concentration-dependent manner that was already evident 24 h after CBD exposure, with an apparent IC(50) of 25 microM. The antiproliferative effect of CBD was partially prevented by the CB2 receptor 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; SR2) and alpha-tocopherol. By contrast, the CB1 cannabinoid receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole -3-carboximide hydrochloride (SR141716; SR1), capsazepine (vanilloid receptor antagonist), the inhibitors of ceramide generation, or pertussis toxin did not counteract CBD effects. We also show, for the first time, that the antiproliferative effect of CBD was correlated to induction of apoptosis, as determined by cytofluorimetric analysis and single-strand DNA staining, which was not reverted by cannabinoid antagonists. Finally, CBD, administered s.c. to nude mice at the dose of 0.5 mg/mouse, significantly inhibited the growth of subcutaneously implanted U87 human glioma cells. In conclusion, the nonpsychoactive CBD was able to produce a significant antitumor activity both in vitro and in vivo, thus suggesting a possible application of CBD as an antineoplastic agent. (+info)
A novel synthetic, nonpsychoactive cannabinoid acid (HU-320) with antiinflammatory properties in murine collagen-induced arthritis.
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OBJECTIVE: To explore the antiarthritic potential of a novel synthetic cannabinoid acid, Hebrew University-320 (HU-320), in the DBA/1 mouse model of arthritis, and to investigate in vitro antiinflammatory and immunosuppressive effects of HU-320 on macrophages and lymphocytes. METHODS: DBA/1 mice were immunized with bovine type II collagen (CII) to induce arthritis and then injected intraperitoneally daily with HU-320. The effects of treatment on arthritic changes in hind feet were assessed clinically and histologically, and draining lymph node responses to CII were assayed. Murine splenic and human blood lymphocytes were cultured to study the effect of HU-320 on polyclonal mitogenic stimulation. Macrophage cultures were set up to evaluate in vitro effects of HU-320 on production of tumor necrosis factor alpha (TNF alpha) and reactive oxygen intermediates (ROIs). The effect of HU-320 administration on lipopolysaccharide-induced serum TNF levels was assayed using C57BL/6 mice. Bioactive TNF production was measured using BALB/c clone 7 target cells. Evaluation of HU-320 psychoactivity was performed using established laboratory tests on Sabra mice. RESULTS: Systemic daily administration of 1 and 2 mg/kg HU-320 ameliorated established CII-induced arthritis. Hind foot joints of treated mice were protected from pathologic damage. CII-specific and polyclonal responses of murine and human lymphocytes were down-modulated. HU-320 inhibited production of TNF from mouse macrophages and of ROIs from RAW 264.7 cells and suppressed the rise in serum TNF level following endotoxin challenge. HU-320 administration yielded no adverse psychotropic effects in mice. CONCLUSION: Our studies show that the novel synthetic cannabinoid acid HU-320 has strong antiinflammatory and immunosuppressive properties while demonstrating no psychoactive effects. The profound suppressive effects on cellular immune responses and on the production of proinflammatory mediators all indicate its usefulness as a novel nonpsychoactive, synthetic antiinflammatory product. (+info)
Vanilloid TRPV1 receptor mediates the antihyperalgesic effect of the nonpsychoactive cannabinoid, cannabidiol, in a rat model of acute inflammation.
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Cannabidiol (CBD), a nonpsychoactive marijuana constituent, was recently shown as an oral antihyperalgesic compound in a rat model of acute inflammation. We examined whether the CBD antihyperalgesic effect could be mediated by cannabinoid receptor type 1 (CB1) or cannabinoid receptor type 2 (CB2) and/or by transient receptor potential vanilloid type 1 (TRPV1). Rats received CBD (10 mg kg(-1)) and the selective antagonists: SR141716 (N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazol e-3-carboxamide) for CB1, SR144528 (N-[(1S)-endo-1,3,3-trimethylbicyclo[2.2.1]heptan-2-yl]-5-(4-chloro-3-methylpheny l)-1-(4-methylbenzyl)pyrazole-3 carboxamide) for CB2 and capsazepine (CPZ) for TRPV1 receptors. The intraplantar injection of carrageenan in rats induced a time-dependent thermal hyperalgesia, which peaked at 3 h and decreased at the following times. CBD, administered 2 h after carrageenan, abolished the hyperalgesia to the thermal stimulus evaluated by plantar test. Neither SR141716 (0.5 mg kg(-1)) nor SR144528 (3 and 10 mg kg(-1)) modified the CBD-induced antihyperalgesia; CPZ partially at the lowest dose (2 mg kg(-1)) and fully at the highest dose (10 mg kg(-1)) reversed this effect. These results demonstrate that TRPV1 receptor could be a molecular target of the CBD antihyperalgesic action. (+info)
Cannabidiol inhibits human glioma cell migration through a cannabinoid receptor-independent mechanism.
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We evaluated the ability of cannabidiol (CBD) to impair the migration of tumor cells stimulated by conditioned medium. CBD caused concentration-dependent inhibition of the migration of U87 glioma cells, quantified in a Boyden chamber. Since these cells express both cannabinoid CB1 and CB2 receptors in the membrane, we also evaluated their engagement in the antimigratory effect of CBD. The inhibition of cell was not antagonized either by the selective cannabinoid receptor antagonists SR141716 (CB1) and SR144528 (CB2) or by pretreatment with pertussis toxin, indicating no involvement of classical cannabinoid receptors and/or receptors coupled to Gi/o proteins. These results reinforce the evidence of antitumoral properties of CBD, demonstrating its ability to limit tumor invasion, although the mechanism of its pharmacological effects remains to be clarified. (+info)