Pharmacology of Dopamine Release by Amphetamine

安非他明释放多巴胺的药理学

• 批准号: 8635996
• 负责人: MARGARET E GNEGY
• 金额: $ 41.98万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-01 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8635996
• 关键词: Accident and Emergency department; Adderall; Affect; Affinity; Agonist; Amphetamine Abuse; Amphetamines; Attention deficit hyperactivity disorder; Autoreceptors; Behavior; Behavioral; Cells; Complex; Data; Development; Dopamine; Drug Addiction; Effectiveness; Electric Stimulation; Exocytosis; Exocytosis Inhibition; Extracellular Fluid; Food; Goals; Investigation; Kinetics; Knowledge; Lead; Learning; Mediating; Mental disorders; Modality; Molecular; Motivation; N-terminal; Neuroblastoma; Neurodegenerative Disorders; Neurons; Opiates; Parkinson Disease; Pharmaceutical Preparations; Pharmacology; Phosphorylation; Procedures; Property; Protein Kinase C; Protein Kinase C Inhibitor; Psychological reinforcement; Regulation; Rewards; Role; Schizophrenia; Self Administration; Self-Administered; Serine; Signal Transduction; Site; Specificity; Surface; Synapses; System; Testing; Translating; Visit; base; design; dopamine transporter; drug seeking behavior; extracellular; in vivo; meetings; mutant; nonmedical use; novel therapeutics; reinforced behavior; remifentanil; response; reuptake; therapeutic target; trafficking; uptake

DESCRIPTION (provided by applicant): This proposal will fill a gap in our understanding of normal and amphetamine (AMPH)-induced regulation of the dopamine transporter (DAT) which may lead to new therapeutic modalities. Reinforcing properties of AMPHs depend on the level of extracellular dopamine (DA), which is regulated by DA release, DAT and DA autoreceptors (D2S). We find that PKC? regulates the functions of DAT and D2S and their interaction. Inhibition or deletion of PKC? reduces AMPH-stimulated DA efflux and AMPH-stimulated locomotor and rewarded behaviors, and enhances direct D2S inhibition of exocytosis. Both DAT and D2S are PKC? substrates but it is unknown how phosphorylation by PKC? will regulate these activities. We propose that inhibition of PKC? reduces AMPH-stimulated increases in extracellular DA and thus the reinforcing effects of AMPH, suggesting a potential therapeutic target for AMPH abuse. Our objectives are to: a. examine molecular mechanisms by which PKC? regulates AMPH action and D2S-DAT functional interactions, providing significant new information on regulation of this crucial system; b. integrate the principles learned in mechanisti studies to test if PKC? inhibition will reduce exocytotic and AMPH-evoked DA release and drug-taking behaviors in vivo. To meet these objectives, the following hypotheses will be tested: 1. PKC? activation enhances AMPH-stimulated DA efflux by phosphorylating DAT N-terminal serines. PKC?-phosphorylated serines will be determined and relevant non-PKC?-phosphorylatable DAT mutants will be synthesized and tested for AMPH-stimulated DA efflux, DA uptake, and Michaelis-Menton kinetics of inward and outward transport in neuroblastoma N2A cells. 2. PKC?-stimulated phosphorylation of DAT or D2S or both is required for D2S agonists to increase surface DAT. Non-PKC?-phosphorylatable DAT and D2S mutants will be synthesized and tested for D2S-stimulation of DAT function, D2S trafficking and D2S effects on DA release. 3. Inhibition of PKC? will reduce electrical- and AMPH-evoked levels of extracellular DA thereby lessening the reinforcing effects of AMPH. We predict: a. that PKC? inhibition will blunt extracellular DA in response to electrical stimulation and AMPH because of enhanced D2S inhibition of DA exocytosis and reduced outward transport through DAT with no reduction in DA reuptake, and b. the reduction in extracellular DA will lead to reduced drug-taking and drug- seeking behavior for AMPH in a self-administration procedure. The functional consequences of PKC? inhibition on extracellular DA following electrically-stimulated DA release will be examined using cyclic voltammetry, giving simultaneous assessment of DA release and reuptake parameters. To examine if PKC? is a potential therapeutic target for AMPH abuse, the effect of PKC? inhibition on drug-taking behavior, drug-primed reinstatement, and motivation to self-administer AMPH will be evaluated. A greater mechanistic understanding of factors regulating synaptic DA will be attained, advancing us toward the unmet need of designing an effective, non-reinforcing treatment for AMPH abuse.

描述（由申请人提供）：该提案将填补我们对多巴胺转运蛋白（DAT）的正常和苯丙胺（AMPH）诱导调节的理解中的空白，这可能导致新的治疗方式。AMPH的增强特性取决于细胞外多巴胺（DA）的水平，其由DA释放、DAT和DA自身受体（D2 S）调节。找到蛋白激酶C了吗？调节DAT和D2 S的功能及其相互作用。抑制或删除PKC？减少AMPH刺激的DA流出和AMPH刺激的运动和奖励行为，并增强胞吐的直接D2 S抑制。DAT和D2 S都是PKC吗？底物，但它是未知的如何磷酸化的PKC？将规范这些活动。我们建议，抑制PKC？减少AMPH刺激的细胞外DA增加，从而增强AMPH的作用，提示AMPH滥用的潜在治疗靶点。我们的目标是：a。研究PKC？调节AMPH作用和D2 S-DAT功能性相互作用，提供关于该关键系统的调节的重要新信息; B.整合mechanisti研究中学到的原则，以测试PKC？抑制将减少胞吐和AMPH诱发的DA释放和体内药物摄取行为。为了实现这些目标，将测试以下假设：1。PKC？激活通过磷酸化DAT N-末端丝氨酸增强AMPH刺激的DA流出。蛋白激酶C磷酸化丝氨酸将被确定，相关的非PKC？将合成可磷酸化的DAT突变体，并测试成神经细胞瘤N2 A细胞中AMPH刺激的DA流出、DA摄取和向内和向外转运的Michaelis-Menton动力学。2.蛋白激酶C D2 S激动剂需要DAT或D2 S或两者的刺激磷酸化来增加表面DAT。将合成可磷酸化的DAT和D2 S突变体，并测试DAT功能的D2 S刺激、D2 S运输和D2 S对DA释放的影响。3.抑制PKC？将降低细胞外DA的电和AMPH诱发水平，从而减轻AMPH的增强作用。我们预测：a.这个PKC？抑制将钝化细胞外DA对电刺激和AMPH的响应，因为增强的D2 S对DA胞吐的抑制和减少的通过DAT的向外转运，而不减少DA再摄取，和B.细胞外DA的减少将导致在自我给药过程中AMPH的药物摄取和药物寻求行为减少。PKC的功能后果？将使用循环伏安法检查电刺激DA释放后对细胞外DA的抑制，同时评估DA释放和再摄取参数。检查是否有PKC？是AMPH滥用的潜在治疗靶点，PKC的作用？将评价对药物服用行为的抑制、药物引发的恢复和自我施用AMPH的动机。将获得对调节突触DA的因素的更大的机械理解，推进我们对设计有效的未满足的需求，非强化治疗AMPH滥用。