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: 7593315
• 负责人: Toni Shippenberg
• 金额: $ 6.69万
• 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/7593315
• 关键词: 2-arachidonylglycerol; Acetylcholine; Affect; Amides; Basal Ganglia; Behavior; Binding Proteins; Brain; Cannabinoids; Cannabis; Cardiac; Cell Line; Cell membrane; Cell surface; Cells; Cholinergic Receptors; Class; Coupling; Cytosol; Data; Dopamine; Eating; Endocannabinoids; Equilibrium; Esters; Feedback; G-Protein-Coupled Receptors; GABA Receptor; GTP-Binding Proteins; Gated Ion Channel; Glucose; Glycine; Glycine Receptors; Hypothalamic structure; Imagery; Imaging Techniques; Insulin; Integral Membrane Protein; Ion Channel; Learning; Life; Ligands; Link; Lipid Bilayers; Lipids; Mammalian Cell; Mediating; Membrane; Membrane Proteins; Memory; Metabolic; Modeling; Molecular; Molecular Target; Motor Activity; Mus; Neurons; Neurotransmitters; Nociception; Nociceptive Stimulus; Opioid Receptor; Pain; Periodicity; Pertussis; Physiological; Polyunsaturated Fatty Acids; Potassium; Potassium Channel; Process; Property; Protein Conformation; Receptor Inhibition; Research; Role; Sensory Ganglia; Serotonin; Serotonin Receptors 5-HT-3; Signal Transduction; Slice; Spinal; Spinal Cord; Spinal Ganglia; Structure; Synaptic Transmission; Synaptosomes; System; Testing; Tetrahydrocannabinol; Thalamic structure; Time; Work; Xenopus oocyte; anandamide; cannabinoid receptor; cellular imaging; dopamine transporter; extracellular; gamma-Aminobutyric Acid; insulin secretion; ligand gated channel; mesolimbic system; neurotransmission; novel; oocyte maturation; receptor; receptor coupling; reward processing; trafficking; vasoconstriction; voltage

We have been studying the effects of endocannabinoids such as anandamide and 2-AG on ligand-gated ion channels, potassium channels, and G-protein couple receptors. Our work has focused on three receptor coupled ligand-gated channels, the alpha-7 and alpha-4-beta-2 subtypes of nicotinic ACh receptors, 5-HT3 receptors and GABA receptors. In other studies we have examined ATP-sensitive potassium (KATP) channel. The KATP channels form an important link between metabolic state and cell excitability. They are implicated in the control of insulin secretion, vasoconstriction, cardiac rhythmicity, and oocyte maturation. We have found that anandamide inhibits the function of KATP channels in Xenopus oocytes and suppress the maturation process. Similar results were obtained in mammalian cell lines expressing KATP channels. KATP channels mediate glucose induced insulin release. In on-going collaborative studies, we are studying the effects of endocannabinoids on these channels and insulin secretion processes. In our earlier studies, using the Xenopus oocyte expression system, we were the first to show that alpha-7 nicotinic ACh, 5-HT3, and Glycine receptors are directly modulated by endocannabinoids. Recently, we have directed our research to mammalian cell lines and synaptosomes from the thalamic region of mice brain. Our work has shown that the endocannabinoid, anandamide, potently inhibits the function of the 4-beta-2 nACh receptor subtype expressed in SH-EP1 cells. Furthermore, similar effects of anandamide were observed on Rb efflux mediated by the activation of alpha-4-beta-2 nACh receptors in thalamic synaptosomes. Currently, we are extending our studies to investigate the role of direct cannabinoid modulation of alpha-7 and alpha-4-beta-2 nicotinic ACh receptors, 5-HT3 receptors and various GABA receptor subtypes on synaptic transmission and circuit behaviours of brain slices. We have initiated studies examining endocannabinoid modulation of the dopamine transporter (DAT); a membrane bound protein that regulates extracellular concentrations of dopamine in the CNS. Earlier studies indicated that the effects of cannabinoids on the function of DAT are not altered by cannabinoid receptor antagonists. Using a novel live cell imaging technique that enables visualization of DAT function in real-time, we have obtained evidence that anandamide inhibits DAT function. This effect is pertussis txoin independent indicating that it occurs independently of Gi/Go coupled receptors. Data regarding the influence of anandamide on trafficking of the transporter between the membrane and cytosol suggest that this effect is mediated, at least in part, via an increase in transporter internalization. Collaborative studies with Drs. Sanders-Bush and Zhang are examining endocannabionoid modulation of serotonin type 2C and mu-opioid receptor coupling to GIRK channels. We have found that anandamide inhibits the coupling of these channels to GIRK channels and other effectors. Since the activation of serotonin type 2C induces endocannabinoid synthesis, it is likely that anandamide inhibition of receptor-effector coupling constitutes a novel inhibitory feedback mechanism for these G-protein coupled receptors. The antinociceptive effects of cannabinoids are well established. However, in many pain models tested, the effects of cannabinoids are not reversed by cannabinoid receptor antagonists. Interestingly, receptors (e.g., glycine and GABA) and several Ca2+ channel subtypes that are implicated in nociceptive processing are potently modulated by anandamide and THC in a cannabinoid-receptor independent manner. Thus, we are investigating the role of cannabinoid modulation of these ion channels in cannabinoid-induced antinociception. We have developed a nociceptive dorsal root ganglion sensory neuronal line that enables us to examine endocannabinoid modulation of these ion channel functions in processing of nociceptive stimuli. Currently, we are testing the effects of endocannabinoids on the functional properties of ion channels expressed in these cell lines.

我们一直在研究内源性大麻素（例如anandamide和2AG）对配体门控离子通道，钾通道和G蛋白夫妇受体的影响。 我们的工作集中于三个受体耦合配体门控通道，即烟碱ACH受体，5-HT3受体和GABA受体的α-7和α-4-β-2亚型。在其他研究中，我们研究了ATP敏感钾（KATP）通道。 KATP通道形成了代谢状态和细胞兴奋性之间的重要联系。 它们与控制胰岛素分泌，血管收缩，心律节奏和卵母细胞成熟有关。 我们发现，anandamide抑制了Xenopus卵母细胞中KATP通道的功能并抑制成熟过程。 在表达KATP通道的哺乳动物细胞系中获得了相似的结果。 KATP通道介导葡萄糖诱导的胰岛素释放。 在正在进行的合作研究中，我们正在研究内源性大麻素对这些渠道和胰岛素分泌过程的影响。 在我们较早的研究中，使用爪蟾卵母细胞表达系统，我们是第一个表明α-7烟碱ACH，5-HT3和甘氨酸受体由内源性大麻素直接调节的。 最近，我们将研究指向了小鼠脑丘脑区域的哺乳动物细胞系和突触体。 我们的工作表明，内源性大麻素（anandamide）有效抑制在SH-EP1细胞中表达的4-β-2 NACH受体亚型的功能。 此外，观察到anandamide对丘脑突触体中α-4-β-2 NACH受体的激活介导的RB外排的相似作用。 目前，我们正在扩展研究以研究α-7和α-4-β-2烟碱ACH受体，5-HT3受体以及各种GABA受体亚型在突触传播和脑切片的电路行为上的直接大麻素调节。 我们已经开始研究多巴胺转运蛋白（DAT）的内源性大麻素调节。一种调节CNS中多巴胺的细胞外浓度的膜结合蛋白。较早的研究表明，大麻素受体拮抗剂不会改变大麻素对DAT功能的影响。 使用一种新型的活细胞成像技术，该技术可以实时可视化DAT功能，我们获得了Anandamide抑制DAT功能的证据。这种作用是百日咳TXOIN独立的，表明它与GI/GO耦合受体无关。有关仙化胺对膜和胞质溶胶之间运输蛋白运输的影响的数据表明，这种作用至少部分是通过转运蛋白内在化的增加而介导的。 与博士的合作研究。 Sanders-Bush和Zhang正在检查5-羟色胺2C型和Mu-Apioid受体偶联到GIRK通道的内源性调节。 我们发现，Anandamide抑制了这些通道与Girk通道和其他效应子的耦合。 由于5-羟色胺2C的激活诱导内源性大麻素的合成，因此对这些G蛋白偶联受体的Anandamide抑制受体抑制受体抑制是一种新型的抑制反馈机制。 大麻素的抗伤害感受作用已良好。但是，在许多测试的疼痛模型中，大麻素受体拮抗剂不会逆转大麻素的作用。 有趣的是，与伤害性加工有关的受体（例如甘氨酸和GABA）和几种与伤害性处理有关的Ca2+通道亚型通过大麻素受体独立的方式有效地调节了Anandamide和THC。 因此，我们正在研究这些离子通道在大麻素诱导的抗伤害感受中的大麻素调节作用。 我们已经开发了一种伤害性背根神经节感官神经元线，使我们能够检查这些离子通道功能在伤害性刺激的处理中的内源性大麻素调节。 目前，我们正在测试内源性大麻素对这些细胞系中表达的离子通道功能特性的影响。