DISCOVERY OF NOVEL MOLECULAR TARGETS FOR ENDOGENOUS AND SYNTHETIC CANNABINOIDS

内源性和合成大麻素的新分子靶标的发现

• 批准号: 8336464
• 负责人: Toni Shippenberg
• 金额: $ 19.57万
• 依托单位: NATIONAL INSTITUTE ON DRUG ABUSE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8336464
• 关键词: 2-arachidonylglycerol; Absence of pain sensation; Acetylcholine; Address; Adverse effects; Affect; Affinity; Agonist; Amides; Amidohydrolases; Anti-Inflammatory Agents; Anti-inflammatory; Antiemetic Effect; Antiemetics; Antioxidants; Arachidonic Acids; Attenuated; Basal Ganglia; Binding; Biochemical; Biotinylation; Brain; Brain region; CNR2 gene; Cannabidiol; Cannabinoids; Cannabis; Cannabis sativa plant; Cell Line; Cell membrane; Cell surface; Cells; Chemicals; Confocal Microscopy; Cytosol; Dactinomycin; Data; Dopamine; Down-Regulation; Drug Addiction; Drug abuse; Eating; Electrodes; Emotions; Endocannabinoids; Equilibrium; Esters; Etiology; Extracellular Space; Family; G-Protein-Coupled Receptors; GTP-Binding Proteins; Gated Ion Channel; Gene Family; Genetic Transcription; Glycine Receptors; Human; Hypothalamic structure; Imaging Techniques; In Vitro; Inhibitory Concentration 50; Injection of therapeutic agent; Integral Membrane Protein; Ion Channel; Learning; Ligands; Lipid Bilayers; Lipids; Measures; Mediating; Membrane; Membrane Proteins; Memory; Metabolic; Molecular; Molecular Target; Mood Disorders; Motor Activity; Movement; Nabilone; Nausea and Vomiting; Neurotransmitters; Nociception; Nucleus Accumbens; Pain; Parkinson Disease; Peripheral Nervous System; Pertussis Toxin; Pharmacologic Substance; Phenylmethylsulfonyl Fluoride; Physiological; Polyunsaturated Fatty Acids; Process; Property; Protein Conformation; Proteins; Psychotropic Drugs; Refractory; Reporting; Resolution; Rewards; Role; Schizophrenia; Serotonin; Serotonin Receptors 5-HT-3; Signal Transduction; Spinal; Spinal Cord; Structure; Synaptosomes; System; Techniques; Testing; Tetrahydrocannabinol; Therapeutic; Therapeutic Agents; Time; Tissues; United States Food and Drug Administration; Voltage-Clamp Technics; Vomiting; Xenopus oocyte; analog; anandamide; cannabinoid receptor; chemotherapy; density; dopamine transporter; extracellular; inhibitor/antagonist; interest; member; mesolimbic system; neurotransmission; novel; receptor; receptor coupling; receptor function; response; reward processing; trafficking; transmission process; uptake; voltage

The neurotransmitter, dopamine, modulates excitatory and inhibitory neurotransmission in brain regions that control movement, emotion, and reward. Dysregulation of DA transmission is implicated in the etiology of several pathological conditions including Parkinsons disease, drug addiction, and schizophrenia. The dopamine transporter (DAT) is an integral membrane protein that is a member of the Na+- and Cl-- dependent cotransporter gene family. It serves a key role in terminating dopamine transmission by clearing dopamine released into the extracellular space. Systemic administration of the endocannabinoid, anandamide, increases extracellular dopamine concentrations in the nucleus accumbens, a brain region implicated in mediating the abuse liability of various psychoactive drugs. Increased dopamine concentrations are also observed in response to synthetic cannabinoid agonists. Although these effects have been attributed to alterations in release, evidence that anandamide and other cannabinoids inhibit transporter-mediated dopamine uptake in native tissue and heterologous expression systems has been presented. Such findings are noteworthy in that they suggest that endocannabinoids may modulate dopamine transmission by regulating DAT function. The cellular mechanisms mediating the interaction of anandamide with DAT are unclear. Although there is evidence that it may modulate DAT function by a CB1 independent mechanism, whether the effect of anandamide is mediated by the activation of other cannabinoid receptors such as CB2 and GPR55 that are expressed in the brain is unclear. In addition, although prolonged incubation of cells or synaptosomes decreases dopamine uptake, the time course and the cellular mechanisms of this effect have not been assessed. We previously demonstrated that use of the fluorescent, high affinity DAT substrate, ASP+ 4-(4-(dimethylamino)styrl)-N-methylpyridinium))in combination with confocal microscopy enables real-time, spatially resolved analysis of transporter function in heterologous expression systems. Analysis of ASP+ accumulation not only permits resolution of substrate binding and uptake in the same cell but enables quantification of rapid (e.g. 1 min) changes in DAT function (see: Bolan et al., 2007, Zapata et al 2007). Using this technique we have investigated the effect of anandamide on the function of human DAT (hDAT) expressed in the EM4 HEK 293 cell line which does not endogenously express known cannabinoid receptors. In parallel studies, biochemical and confocal imaging techniques were employed to assess the role of transporter trafficking in mediating anandamide-evoked alterations in DAT function. Addition of anandamide to EM4 cells transiently transfected to express fluorescently tagged hDAT produced a concentration-dependent inhibition of ASP+ accumulation. This effect was rapid occurring within 1 min after anandamide addition. Increased DAT function was long-lasting. A significant inhibition of ASP+ accumulation was still apparent 10 min after anandamide addition. Incubation of cells with pertussis toxin did not attenuate the effects of anandamide suggesting a mechanism independent of Gi/Go coupled receptors. The amidohydrolase inhibitor, phenylmethylsulfonyl fluoride, failed to alter the effects of anandamide. No change in ASP+ accumulation was observed in response to arachidonic acid suggesting that the effects of anandamide are not mediated by its metabolic products. The downregulation of DAT function was associated with a significant redistribution of hDAT from the membrane to the cytosol as measured using both confocal microscopy and biotinylation techniques. These results demonstrate that anandamide modulates DAT function via a cannabinoid receptor-independent mechanism. Furthermore, they suggest that endocannabinoids may increase extracellular dopamine, in part, by increasing DAT internalization. The serotonin type three (5-HT3) receptor, a member of the ligand-gated ion channel family, mediates rapid and transient membrane depolarizing effect of 5-HT in the central and peripheral nervous system. An involvement of 5-HT3 receptors in pain transmission, mood disorders, and drug abuse has been reported. Furthermore, 5-HT3 receptor antagonists are effective therapeutic agents for the treatment of chemotherapy-induced nausea and vomiting. Earlier studies showed that 5-HT3-receptor antagonists and cannabinoids produce similar pharmacological effects such as non-opioid receptor-mediated analgesia and antiemesis. In fact, synthetic Δ-tetrahydrocannabinol (THC), dronabinol, and THC analogs such as nabilone are approved by the US Food and Drug Administration for use in chemotherapy-induced nausea and vomiting refractory to conventional antiemetic therapy. The limitation of the therapeutic utility of THC and the above-mentioned chemical analogs is that they can produce psychoactive effects through the cannabinoid 1 receptor (CB1) Cannabidiol (CBD) is one of the most abundant cannabinoids of Cannabis sativa with reported antioxidant, anti-inflammatory, and antiemetic effects. It is well tolerated and is without side effects when chronically administered to humans. Furthermore, CBD is devoid of psychoactive properties due to a low affinity for the CB1 and CB2 receptors. Thus, pharmaceutical interest in this compound has increased significantly in recent years. Although the effects of THC, synthetic cannabinoid receptor agonists (e.g., WIN55,212- 2, CP55,940, and JWH-015), and the endocannabinoid, anandamide, on the functional properties of 5-HT3-receptors were demonstrated in in-vitro studies, whether CBD, a nonpsychotropic cannabinoid affects 5-HT3A-receptor function is unknown. This issue was addressed by assessing the effects of CBD on the function of 5-HT3A receptors expressed in Xenopus oocytes using two-electrode voltage clamp techniques. CBD reversibly inhibited 5-HT (1 μM)-evoked currents in a concentration-dependent manner (IC50=0.6μM). CBD did not alter specific binding of the 5-HT3A antagonist 3HGR65630. In the presence of CBD, the maximum 5-HT-induced currents were also inhibited. The EC50 values were 1.2 μM and 1.4 μM, in the absence and presence of CBD, indicating that CBD acts as a noncompetitive antagonist of 5-HT3 receptors. Neither intracellular BAPTA injection nor pertussis toxin pretreatment altered the CBD-evoked inhibition of 5-HT-induced currents. CBD inhibition was inversely correlated with 5-HT3A expression levels and mean 5-HT3 receptor current density. Pretreatment with actinomycin D, which inhibits protein transcription, decreased the mean 5-HT3 receptor current density and increased the magnitude of CBD inhibition. These data demonstrate that CBD is an allosteric inhibitor of 5-HT3 receptors expressed in Xenopus oocytes. They further suggest that allosteric inhibition of 5-HT3 receptors by CBD may contribute to its physiological role in the modulation of nociception and emesis.

神经递质多巴胺调节控制运动、情绪和奖励的大脑区域的兴奋性和抑制性神经传递。 DA 传输失调与多种病理状况的病因有关，包括帕金森病、药物成瘾和精神分裂症。多巴胺转运蛋白 (DAT) 是一种完整的膜蛋白，属于 Na+- 和 Cl-- 依赖性协同转运蛋白基因家族的成员。它通过清除释放到细胞外空间的多巴胺来终止多巴胺传输。全身施用内源性大麻素 anandamide 会增加伏隔核中细胞外多巴胺的浓度，伏隔核是一个与介导各种精神活性药物滥用倾向有关的大脑区域。还观察到合成大麻素激动剂引起的多巴胺浓度增加。尽管这些效应归因于释放的改变，但已有证据表明大麻素和其他大麻素抑制天然组织和异源表达系统中转运蛋白介导的多巴胺摄取。这些发现值得注意，因为它们表明内源性大麻素可能通过调节 DAT 功能来调节多巴胺的传递。介导 anandamide 与 DAT 相互作用的细胞机制尚不清楚。尽管有证据表明它可能通过 CB1 独立机制调节 DAT 功能，但 anandamide 的作用是否是通过激活大脑中表达的 CB2 和 GPR55 等其他大麻素受体介导的。此外，虽然细胞或突触体的长时间孵育会减少多巴胺的摄取，但这种效应的时间过程和细胞机制尚未得到评估。我们之前证明，使用荧光高亲和力 DAT 底物 ASP+ 4-(4-(二甲氨基)苯乙烯)-N-甲基吡啶鎓))与共焦显微镜相结合，可以对异源表达中的转运蛋白功能进行实时、空间分辨分析系统。 ASP+ 积累的分析不仅可以解析同一细胞中的底物结合和摄取，还可以量化 DAT 功能的快速（例如 1 分钟）变化（参见：Bolan 等人，2007 年；Zapata 等人，2007 年）。使用这项技术，我们研究了 anandamide 对 EM4 HEK 293 细胞系中表达的人 DAT (hDAT) 功能的影响，该细胞系不内源表达已知的大麻素受体。在平行研究中，采用生化和共聚焦成像技术来评估转运蛋白运输在介导 anandamide 引起的 DAT 功能改变中的作用。向瞬时转染表达荧光标记 hDAT 的 EM4 细胞中添加 anandamide 会产生浓度依赖性的 ASP+ 积累抑制。添加 anandamide 后 1 分钟内，这种效果迅速发生。 DAT 功能的增强是持久的。添加 anandamide 10 分钟后，对 ASP+ 积累的显着抑制仍然很明显。 细胞与百日咳毒素一起孵育并没有减弱 anandamide 的作用，表明其机制不依赖于 Gi/Go 偶联受体。酰胺水解酶抑制剂苯甲基磺酰氟未能改变 anandamide 的作用。没有观察到花生四烯酸对 ASP+ 积累的影响，这表明花生四烯酸的作用不是由其代谢产物介导的。使用共聚焦显微镜和生物素化技术测量，DAT 功能的下调与 hDAT 从膜到细胞质的显着重新分布相关。这些结果表明 anandamide 通过大麻素受体独立机制调节 DAT 功能。此外，他们认为内源性大麻素可能部分通过增加 DAT 内化来增加细胞外多巴胺。 血清素三型 (5-HT3) 受体是配体门控离子通道家族的成员，介导中枢和周围神经系统中 5-HT 的快速且短暂的膜去极化作用。据报道，5-HT3 受体参与疼痛传递、情绪障碍和药物滥用。此外，5-HT3受体拮抗剂是治疗化疗引起的恶心和呕吐的有效治疗剂。早期研究表明5-HT3受体拮抗剂和大麻素产生相似的药理作用，例如非阿片受体介导的镇痛和止吐。事实上，合成的 Δ-四氢大麻酚 (THC)、屈大麻酚和 THC 类似物（例如大麻龙）已被美国食品和药物管理局批准用于治疗传统止吐疗法难以治疗的化疗引起的恶心和呕吐。 THC 和上述化学类似物的治疗用途的局限性在于，它们可以通过大麻素 1 受体 (CB1) 产生精神作用。 大麻二酚 (CBD) 是大麻中最丰富的大麻素之一，据报道具有抗氧化、抗具有抗炎、止吐作用。人类长期服用它具有良好的耐受性并且没有副作用。此外，由于 CBD 与 CB1 和 CB2 受体的亲和力较低，因此不具有精神活性。因此，近年来，制药界对该化合物的兴趣显着增加。尽管 THC、合成大麻素受体激动剂（例如 WIN55,212-2、CP55,940 和 JWH-015）以及内源性大麻素、大麻素对 5-HT3 受体功能特性的影响已在体外研究表明，CBD（一种非精神类大麻素）是否影响 5-HT3A 受体功能尚不清楚。通过使用两电极电压钳技术评估 CBD 对爪蟾卵母细胞中表达的 5-HT3A 受体功能的影响解决了这个问题。 CBD 以浓度依赖性方式可逆地抑制 5-HT (1 μM) 诱发电流 (IC50=0.6μM)。 CBD 不会改变 5-HT3A 拮抗剂 3HGR65630 的特异性结合。在 CBD 存在的情况下，最大 5-HT 诱导电流也受到抑制。在不存在和存在 CBD 的情况下，EC50 值分别为 1.2 μM 和 1.4 μM，表明 CBD 作为 5-HT3 受体的非竞争性拮抗剂。细胞内 BAPTA 注射和百日咳毒素预处理都没有改变 CBD 对 5-HT 诱导电流的抑制作用。 CBD 抑制与 5-HT3A 表达水平和平均 5-HT3 受体电流密度呈负相关。用抑制蛋白质转录的放线菌素 D 进行预处理，降低了平均 5-HT3 受体电流密度，并增加了 CBD 抑制的程度。这些数据表明 CBD 是非洲爪蟾卵母细胞中表达的 5-HT3 受体的变构抑制剂。他们进一步表明，CBD 对 5-HT3 受体的变构抑制可能有助于其在调节伤害感受和呕吐中的生理作用。

项目成果

The endogenous cannabinoid, anandamide, inhibits dopamine transporter function by a receptor-independent mechanism.

• DOI: 10.1111/j.1471-4159.2009.06557.x
• 发表时间: 2010-03
• 期刊: Journal of neurochemistry
• 影响因子: 4.7
• 作者: Oz M;Jaligam V;Galadari S;Petroianu G;Shuba YM;Shippenberg TS
• 通讯作者: Shippenberg TS

The nonpsychoactive cannabinoid cannabidiol inhibits 5-hydroxytryptamine3A receptor-mediated currents in Xenopus laevis oocytes.

非精神活性大麻素大麻二酚可抑制非洲爪蟾卵母细胞中 5-羟色胺3A 受体介导的电流。

• DOI: 10.1124/jpet.109.162594
• 发表时间: 2010
• 期刊: The Journal of pharmacology and experimental therapeutics
• 作者: Yang,Keun-Hang;Galadari,Sehamuddin;Isaev,Dmytro;Petroianu,Georg;Shippenberg,ToniS;Oz,Murat
• 通讯作者: Oz,Murat