Abstract:Cannabinoids receptors (CB) are being targeted therapeutically for the treatment of anxiety, obesity, movement disorders, glaucoma, and pain. More recently, cannabinoid agonists have displayed anti-proliferative activity against breast cancer and prostate cancer in animal models.
In order to study cannabinoid receptor ligands, we have developed a novel plate-based assay that measures internalization of CB1/CB2 receptors by determining the change in the intracellular levels of the radiolabeled agonists: [3H]-Wi… Show more
“…As demonstrated by the increased GFAP staining, astrocytes remained activated after pre-treatment with AM251 (Figure 2 B-D), suggesting that the endocannabinoid system is involved in MCS-induced analgesia by inhibiting astrocyte activity. Given that (1) MCS inhibits astrocyte and microglial activation; (2) the CB2 receptor, rather than CB1, is directly involved in astrocyte and microglial inhibition in neuropathic pain conditions [ 47 - 49 ]; and (3) AM251 can act as a CB2 inverse agonist [ 50 , 51 ], we next evaluated the CB2 labeling pattern in the spinal cord of MCS-responsive and MCS-refractory animals. Figure 2 Participation of the cannabinoid system in motor cortex stimulation (MCS)-induced analgesia.…”
BackgroundMotor cortex stimulation (MCS) is an effective treatment in neuropathic pain refractory to pharmacological management. However, analgesia is not satisfactorily obtained in one third of patients. Given the importance of understanding the mechanisms to overcome therapeutic limitations, we addressed the question: what mechanisms can explain both MCS effectiveness and refractoriness? Considering the crucial role of spinal neuroimmune activation in neuropathic pain pathophysiology, we hypothesized that modulation of spinal astrocyte and microglia activity is one of the mechanisms of action of MCS.MethodsRats with peripheral neuropathy (chronic nerve injury model) underwent MCS and were evaluated with a nociceptive test. Following the test, these animals were divided into two groups: MCS-responsive and MCS-refractory. We also evaluated a group of neuropathic rats not stimulated and a group of sham-operated rats. Some assays included rats with peripheral neuropathy that were treated with AM251 (a cannabinoid antagonist/inverse agonist) or saline before MCS. Finally, we performed immunohistochemical analyses of glial cells (microglia and astrocytes), cytokines (TNF-α and IL-1β), cannabinoid type 2 (CB2), μ-opioid (MOR), and purinergic P2X4 receptors in the dorsal horn of the spinal cord (DHSC).FindingsMCS reversed mechanical hyperalgesia, inhibited astrocyte and microglial activity, decreased proinflammatory cytokine staining, enhanced CB2 staining, and downregulated P2X4 receptors in the DHSC ipsilateral to sciatic injury. Spinal MOR staining was also inhibited upon MCS. Pre-treatment with AM251 blocked the effects of MCS, including the inhibitory mechanism on cells. Finally, MCS-refractory animals showed similar CB2, but higher P2X4 and MOR staining intensity in the DHSC in comparison to MCS-responsive rats.ConclusionsThese results indicate that MCS induces analgesia through a spinal anti-neuroinflammatory effect and the activation of the cannabinoid and opioid systems via descending inhibitory pathways. As a possible explanation for MCS refractoriness, we propose that CB2 activation is compromised, leading to cannabinoid resistance and consequently to the perpetuation of neuroinflammation and opioid inefficacy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-014-0216-1) contains supplementary material, which is available to authorized users.
“…As demonstrated by the increased GFAP staining, astrocytes remained activated after pre-treatment with AM251 (Figure 2 B-D), suggesting that the endocannabinoid system is involved in MCS-induced analgesia by inhibiting astrocyte activity. Given that (1) MCS inhibits astrocyte and microglial activation; (2) the CB2 receptor, rather than CB1, is directly involved in astrocyte and microglial inhibition in neuropathic pain conditions [ 47 - 49 ]; and (3) AM251 can act as a CB2 inverse agonist [ 50 , 51 ], we next evaluated the CB2 labeling pattern in the spinal cord of MCS-responsive and MCS-refractory animals. Figure 2 Participation of the cannabinoid system in motor cortex stimulation (MCS)-induced analgesia.…”
BackgroundMotor cortex stimulation (MCS) is an effective treatment in neuropathic pain refractory to pharmacological management. However, analgesia is not satisfactorily obtained in one third of patients. Given the importance of understanding the mechanisms to overcome therapeutic limitations, we addressed the question: what mechanisms can explain both MCS effectiveness and refractoriness? Considering the crucial role of spinal neuroimmune activation in neuropathic pain pathophysiology, we hypothesized that modulation of spinal astrocyte and microglia activity is one of the mechanisms of action of MCS.MethodsRats with peripheral neuropathy (chronic nerve injury model) underwent MCS and were evaluated with a nociceptive test. Following the test, these animals were divided into two groups: MCS-responsive and MCS-refractory. We also evaluated a group of neuropathic rats not stimulated and a group of sham-operated rats. Some assays included rats with peripheral neuropathy that were treated with AM251 (a cannabinoid antagonist/inverse agonist) or saline before MCS. Finally, we performed immunohistochemical analyses of glial cells (microglia and astrocytes), cytokines (TNF-α and IL-1β), cannabinoid type 2 (CB2), μ-opioid (MOR), and purinergic P2X4 receptors in the dorsal horn of the spinal cord (DHSC).FindingsMCS reversed mechanical hyperalgesia, inhibited astrocyte and microglial activity, decreased proinflammatory cytokine staining, enhanced CB2 staining, and downregulated P2X4 receptors in the DHSC ipsilateral to sciatic injury. Spinal MOR staining was also inhibited upon MCS. Pre-treatment with AM251 blocked the effects of MCS, including the inhibitory mechanism on cells. Finally, MCS-refractory animals showed similar CB2, but higher P2X4 and MOR staining intensity in the DHSC in comparison to MCS-responsive rats.ConclusionsThese results indicate that MCS induces analgesia through a spinal anti-neuroinflammatory effect and the activation of the cannabinoid and opioid systems via descending inhibitory pathways. As a possible explanation for MCS refractoriness, we propose that CB2 activation is compromised, leading to cannabinoid resistance and consequently to the perpetuation of neuroinflammation and opioid inefficacy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-014-0216-1) contains supplementary material, which is available to authorized users.
“…We have recently reported a plate-based assay that measures internalization of radiolabeled agonists for CB1 and CB2 receptors in intact CHO cells (Dossou et al 2013 ). Consistent with our hypothesis, MNF was found to selectively block internalization of the CB1 receptor agonist, Win55-212-2, with an IC 50 of 101 nmol/L (Fig.…”
Section: Resultsmentioning
confidence: 99%
“…The cells were routinely maintained in DMEM supplemented with l -glutamine, 1% penicillin/streptomycin solution, and 10% FBS. CHO cells stably expressing CB1 receptors or CB2 receptors (Dossou et al 2013 ) were maintained in DMEM:F12 (1:1) supplemented with l -glutamine, 2.44 g/L sodium bicarbonate, penicillin/streptomycin, and 10% FBS. All cell lines were cultured in a humidified CO 2 incubator at 37°C.…”
(R,R’)-4-methoxy-1-naphthylfenoterol (MNF) inhibits cancer cell proliferation in vitro through cell-type specific modulation of β2-adrenergic receptor and/or cannabinoid receptor function. Here, we report an investigation into antitumor activity of MNF in rat C6 glioma cells. The potent antiproliferative action of MNF in these cells (IC50 of ∼1 nmol/L) was refractory to pharmacological inhibition of β2-adrenergic receptor while a synthetic inverse agonist of cannabinoid receptor 1 significantly blocked MNF activity. The antitumor activity of MNF was then assessed in a C6 glioblastoma xenograft model in mice. Three days after subcutaneous implantation of C6 cells into the lower flank of nude mice, these animals were subjected to i.p. injections of saline or MNF (2 mg/kg) for 19 days and tumor volumes were measured over the course of the experiment. Gene expression analysis, quantitative RT-PCR and immunoblot assays were performed on the tumors after treatment. Significant reduction in mean tumor volumes was observed in mice receiving MNF when compared with the saline-treated group. We identified clusters in expression of genes involved in cellular proliferation, as well as molecular markers for glioblastoma that were significantly downregulated in tumors of MNF-treated mice as compared to saline-injected controls. The efficacy of MNF against C6 glioma cell proliferation in vivo and in vitro was accompanied by marked reduction in the expression of cell cycle regulator proteins. This study is the first demonstration of MNF-dependent chemoprevention of a glioblastoma xenograft model and may offer a potential mechanism for its anticancer action in vivo.
“…Our previous work has demonstrated that first-generation CB1 receptor blockers, such as AM251 and rimonabant, stimulated β-cell proliferation both in vitro and in vivo 11 26 . Recently, a novel CB1 receptor inverse agonist with low brain penetrance named JD-5037 has been characterized 27 28 29 . This compound was shown to protect β-cell function by reducing islet inflammation and to restore glycemic plasma levels to normal in ZDF rats 30 .…”
Therapeutics aimed at blocking the cannabinoid 1 (CB1) receptor for treatment of obesity resulted in significant improvements in liver function, glucose uptake and pancreatic β-cell function independent of weight loss or CB1 receptor blockade in the brain, suggesting that peripherally-acting only CB1 receptor blockers may be useful therapeutic agents. Neuropsychiatric side effects and lack of tissue specificity precluded clinical use of first-generation, centrally acting CB1 receptor blockers. In this study we specifically analyzed the potential relevance to diabetes of human CB1 receptor isoforms in extraneural tissues involved in glucose metabolism. We identified an isoform of the human CB1 receptor (CB1b) that is highly expressed in β-cells and hepatocytes but not in the brain. Importantly, CB1b shows stronger affinity for the inverse agonist JD-5037 than for rimonabant compared to CB1 full length. Most relevant to the field, CB1b is a potent regulator of adenylyl cyclase activity in peripheral metabolic tissues. CB1b blockade by JD-5037 results in stronger adenylyl cyclase activation compared to rimonabant and it is a better enhancer of insulin secretion in β-cells. We propose this isoform as a principal pharmacological target for the treatment of metabolic disorders involving glucose metabolism.
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