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

基本信息

批准号：
7966868
负责人：
Toni Shippenberg
金额：
$ 11.79万
依托单位：
NATIONAL INSTITUTE ON DRUG ABUSE
依托单位国家：
美国
项目类别：
财政年份：
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/7966868
关键词：
2-arachidonylglycerol Acetylcholine Affect Affinity Agonist Amides Amidohydrolases Arachidonic Acids Attenuated Basal Ganglia Binding Biochemical Biotinylation Brain Brain region Cannabinoids Cannabis Cell Line Cell membrane Cell surface Cells Confocal Microscopy Cytosol Dopamine Down-Regulation Drug Addiction Eating Emotions Endocannabinoids Equilibrium Esters Etiology Extracellular Space G-Protein-Coupled Receptors GTP-Binding Proteins Gated Ion Channel Gene Family Glycine Receptors Human Hypothalamic structure Imaging Techniques Integral Membrane Protein Ion Channel Learning Ligands Lipid Bilayers Lipids Measures Mediating Membrane Membrane Proteins Memory Metabolic Molecular Molecular Target Motor Activity Movement Neurotransmitters Nucleus Accumbens Parkinson Disease Pertussis Toxin Phenylmethylsulfonyl Fluoride Physiological Polyunsaturated Fatty Acids Process Property Protein Conformation Psychotropic Drugs Resolution Rewards Role Schizophrenia Serotonin Signal Transduction Spinal Spinal Cord Structure Synaptosomes System Techniques Testing Tetrahydrocannabinol Time Tissues anandamide cannabinoid receptor dopamine transporter extracellular inhibitor/antagonist member mesolimbic system neurotransmission novel receptor coupling response reward processing trafficking transmission process uptake voltage

2-arachidonylglycerol Acetylcholine Affect Affinity Agonist Amides Amidohydrolases Arachidonic Acids Attenuated Basal Ganglia Binding Biochemical Biotinylation Brain Brain region Cannabinoids Cannabis Cell Line Cell membrane Cell surface Cells Confocal Microscopy Cytosol Dopamine Down-Regulation Drug Addiction Eating Emotions Endocannabinoids Equilibrium Esters Etiology Extracellular Space G-Protein-Coupled Receptors GTP-Binding Proteins Gated Ion Channel Gene Family Glycine Receptors Human Hypothalamic structure Imaging Techniques Integral Membrane Protein Ion Channel Learning Ligands Lipid Bilayers Lipids Measures Mediating Membrane Membrane Proteins Memory Metabolic Molecular Molecular Target Motor Activity Movement Neurotransmitters Nucleus Accumbens Parkinson Disease Pertussis Toxin Phenylmethylsulfonyl Fluoride Physiological Polyunsaturated Fatty Acids Process Property Protein Conformation Psychotropic Drugs Resolution Rewards Role Schizophrenia Serotonin Signal Transduction Spinal Spinal Cord Structure Synaptosomes System Techniques Testing Tetrahydrocannabinol Time Tissues anandamide cannabinoid receptor dopamine transporter extracellular inhibitor/antagonist member mesolimbic system neurotransmission novel receptor coupling response reward processing trafficking transmission process uptake voltage

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项目摘要

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 anandomide addition. Increased DAT function was long-lasting. A significant inhihibition 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 anandomide. 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.

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。添加肛门后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，ASP+积累的显着不hi肽仍然显而易见。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Anandomide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。