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，多巴胺可调节控制运动，情感和奖励的大脑区域的兴奋性和抑制性神经传递。 DA传播的失调与多种病理状况的病因有关，包括帕金森氏病，吸毒和精神分裂症。多巴胺转运蛋白（DAT）是一种整体膜蛋白，是Na+ - 和Cl-依赖性共转运蛋白基因家族的成员。它通过清除释放到细胞外空间中的多巴胺来终止多巴胺传播方面起着关键作用。内源性大麻素（anandamide）的全身性给药增加了伏伏核中细胞外多巴胺浓度，这是介导各种精神活性药物的滥用责任的大脑区域。响应合成大麻素激动剂的响应也观察到了多巴胺浓度的增加。尽管这些作用归因于释放的改变，但证据表明anandamide和其他大麻素抑制了天然组织中转运蛋白介导的多巴胺摄取，并且已经提出了异源表达系统。这些发现值得注意的是，它们表明内源性大麻素可以通过调节DAT功能来调节多巴胺的传播。介导anandamide与DAT的相互作用的细胞机制尚不清楚。尽管有证据表明它可以通过CB1独立机制来调节DAT功能，但anandamide的作用是否是通过在大脑中表达的其他大麻素受体（例如CB2和GPR55）的激活介导的。另外，尽管细胞或突触体的长时间孵育可降低多巴胺的摄取，但尚未评估这种作用的时间过程和细胞机制。我们先前证明，使用荧光，高亲和力DAT底物，ASP+ 4-（4-（dimethylamino）styrl）-n-甲基吡啶丹）与共聚焦显微镜相结合，可以实时，实时，空间分辨出异源性表达系统中传输函数的空间分析。 ASP+积累的分析不仅允许在同一细胞中分辨底物结合和摄取，还可以量化dat功能的快速变化（例如1分钟）变化（请参阅：Bolan等，2007； Zapata等，2007）。使用此技术，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，该细胞系未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运输贩运在介导Anandamide诱发的DAT功能变化中的作用。在瞬时转染以表达荧光标记的HDAT的EM4细胞中添加anandamide产生了浓度依赖于ASP+积累的抑制作用。添加anandamide后1分钟内，这种效果迅速发生。提高DAT功能是长期的。添加Anandamide后10分钟，对ASP+积累的显着抑制作用仍然明显。 用百日咳毒素温育细胞并没有减弱蜘蛛酰胺的作用，这表明与GI/GO偶联受体无关。淀粉酶抑制剂，苯基磺酰基氟化物未能改变anandamide的作用。响应于花生四烯酸，没有观察到ASP+积累的变化，这表明anandamide的作用不是由其代谢产物介导的。 DAT功能的下调与使用共聚焦显微镜和生物素化技术测量的HDAT显着重新分布到细胞质。这些结果表明，anandamide通过大麻素受体独立的机制调节DAT功能。此外，他们认为内源性大麻素可以部分通过增加DAT内在化来增加细胞外多巴胺。 羟色胺三型（5-HT3）受体是配体门控离子通道家族的成员，介导了中心和外周神经系统中5-HT的快速和瞬时去极化效应。据报道，5-HT3受体参与疼痛传播，情绪障碍和药物滥用。此外，5-HT3受体拮抗剂是用于治疗化学疗法引起的恶心和呕吐的有效治疗剂。较早的研究表明，5-HT3受体拮抗剂和大麻素会产生类似的药理作用，例如非阿片类受体介导的镇痛和抗抑制作用。实际上，美国食品和药物管理局批准了合成的δ-四氢大麻醇（THC），dronabinol和THC类似物（例如nabilone），用于化学疗法引起的恶心和呕吐对常规抗疾病治疗的难治性。 THC的治疗实用性和上述化学类似物的局限性是，它们可以通过大麻素1受体（CB1）大麻二酚（CBD）产生精神活性作用，是抗氧化剂，抗抗激素抗炎和抗生素的抗氧化剂sativa最丰富的大麻素之一。它的耐受性良好，并且在长期施用对人类时没有副作用。此外，由于对CB1和CB2受体的亲和力低，CBD没有精神活性特性。因此，近年来，该化合物的药物兴趣已大大增加。 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受体功能尚不清楚。通过评估CBD对使用两电极电压夹技术在异爪蟾卵母细胞中表达的5-HT3A受体功能的影响来解决这个问题。 CBD可逆地以浓度依赖性方式（IC50 =0.6μm）抑制5-HT（1μM）诱发的电流。 CBD没有改变5-HT3A拮抗剂3HGR65630的特定结合。在存在CBD的情况下，还抑制了最大5-HT诱导的电流。在不存在和存在CBD的情况下，EC50值为1.2μm和1.4μm，表明CBD充当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均未改变CBD诱发的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受体电流密度成反比。抑制蛋白转录的放线菌素D进行预处理，降低了平均5-HT3受体电流密度并增加了CBD抑制的幅度。这些数据表明，CBD是在异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，通过CBD对5-HT3受体的变构抑制可能有助于其在伤害受伤和发育的调节中的生理作用。

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