Pharmacology of Dopamine Release by Amphetamine

描述（由申请人提供）：该提案将填补我们对正常和安非他明（AMPH）诱导的多巴胺转运体（DAT）调节的理解空白，这可能会导致新的治疗方式。AMPHs的增强特性取决于细胞外多巴胺（DA）的水平，而细胞外多巴胺（DA）的水平受DA释放、DAT和DA自受体（D2S）的调节。我们发现PKCß调节DAT和D2S的功能及其相互作用。PKCß的抑制或缺失减少了amph刺激的DA外排和amph刺激的运动和奖励行为，增强了D2S对胞吐的直接抑制。DAT和D2S都是PKCß的底物，但PKCß的磷酸化如何调节这些活性尚不清楚。我们提出，抑制PKCß可减少AMPH刺激的细胞外DA增加，从而增强AMPH的作用，提示滥用AMPH的潜在治疗靶点。我们的目标是：a.研究PKCß调节AMPH作用和D2S-DAT功能相互作用的分子机制，为这一关键系统的调节提供重要的新信息；b.整合在机制研究中所学到的原理来测试PKCß抑制是否会减少胞外分泌和amph诱发的DA释放和体内用药行为。为了实现这些目标，将测试以下假设：PKCß激活通过磷酸化DAT n端丝氨酸增强amph刺激的DA外排。将测定pkc ß-磷酸化丝氨酸，并合成相关的非pkc ß-磷酸化DAT突变体，并测试amph刺激的DA外排、DA摄取和神经母细胞瘤N2A细胞内向和外向运输的Michaelis-Menton动力学。2. D2S激动剂增加表面DAT需要pkc ß刺激的DAT或D2S或两者的磷酸化。将合成非pkc ß-磷酸化的DAT和D2S突变体，并测试D2S刺激DAT功能、D2S转运和D2S对DA释放的影响。3. 抑制PKCß会降低电和AMPH诱发的细胞外DA水平，从而减弱AMPH的强化作用。我们预测：a. PKCß抑制会减弱细胞外DA对电刺激和AMPH的响应，因为D2S增强了DA胞吐的抑制作用，减少了通过DAT向外运输，但没有减少DA的再摄取；b.细胞外DA的减少将导致在自我给药过程中减少AMPH的药物服用和药物寻求行为。电刺激DA释放后PKCß抑制细胞外DA的功能后果将使用循环伏安法进行检查，同时评估DA释放和再摄取参数。为了研究PKCß是否是AMPH滥用的潜在治疗靶点，我们将评估PKCß抑制对吸毒行为、药物启动恢复和自我给药动机的影响。对调节突触DA的因素有了更深入的机制理解，这将推动我们设计一种有效的、非强化的AMPH滥用治疗方法。

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 PKCß regulates the functions of DAT and D2S and their interaction. Inhibition or deletion of PKCß reduces AMPH-stimulated DA efflux and AMPH-stimulated locomotor and rewarded behaviors, and enhances direct D2S inhibition of exocytosis. Both DAT and D2S are PKCß substrates but it is unknown how phosphorylation by PKCß will regulate these activities. We propose that inhibition of PKCß 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 PKCß 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 PKCß 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. PKCß activation enhances AMPH-stimulated DA efflux by phosphorylating DAT N-terminal serines. PKCß-phosphorylated serines will be determined and relevant non-PKCß-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. PKCß-stimulated phosphorylation of DAT or D2S or both is required for D2S agonists to increase surface DAT. Non-PKCß-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 PKCß will reduce electrical- and AMPH-evoked levels of extracellular DA thereby lessening the reinforcing effects of AMPH. We predict: a. that PKCß 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 PKCß 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 PKCß is a potential therapeutic target for AMPH abuse, the effect of PKCß 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.