BIOCHEMICAL CORRELATES OF 5-HT NEURONAL ACTIVITY

5-HT 神经元活动的生化相关性

• 批准号: 3395393
• 负责人: MARGARET C BOADLE-BIBER
• 金额: $ 12.41万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-12-01 至 1990-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3395393
• 关键词: brain metabolism; brain stem; calmodulin; caudate nucleus; cofactor; dorsal raphe nucleus; electrostimulus; enzyme induction /repression; enzyme mechanism; enzyme substrate; experimental brain lesion; gamma aminobutyrate; gas chromatography mass spectrometry; high performance liquid chromatography; hydroxyindoleacetate; laboratory rat; local anesthetics; morphine; neural information processing; neural inhibition; neural initiation; neural transmission; neurochemistry; neuropeptides; neuropharmacology; neurotransmitter biosynthesis; noise biological effect; norepinephrine; oxidoreductase inhibitor; phosphorylation; protein kinase C; pteridines; serotonin; stereotaxic techniques; synaptosomes; tryptophan 5 monooxygenase

This continuation proposal focusses on the regulation of tryptophan hydroxylase (Trp H) activity in the serotonergic neurons of the rat brain. Since this enzyme is rate limiting in the synthesis of the transmitter, serotonin, (5-HT) its activity is central for determining the availability of this important substance, which is now implicated as a mediator in a wide variety of functions including mood or affect, anxiety, antinociception, sleep, temperature regulation and central control of blood pressure. Synthesis of 5-HT is enhanced in response to increased 5-HT neuronal impulse flow by a mechanism that may involve activation of Trp H. Thus enzyme prepared from stimulated cortical 5-HT projections shows an activation that is frequency dependent, and reversed by alkaline phosphatase. Inhibition of 5-HT firing on the other hand decreases enzyme activity, again by an alkaline phosphatase reversible process. Experiments are now planned 1) to further characterize the changes in kinetic behaviour of supernatant preparations of the enzyme in response to different levels of 5- HT neuronal firing in chloral hydrate anaesthetized male Sprague Dawley rats in different regions of CNS, with the natural cofactor, 6R-L-erythro-5,6,7,8-tetrahydrobiopterin; 2) to examine the role of candidate transmitter substances, microinfused stereotaxically into the dorsal raphe nucleus on the regulation of enzyme activity in ascending 5-HT projections supplied by this nucleus in awake rats; 3) to examine the effects of various transmitters or putative transmitters on tryptophan hydroxylase activity after incubation with slice preparations; 4) to establish whether in vitro changes in activity and kinetic properties of supernatant enzyme in response to these manipulations are expressed in vivo; 5) to initiate studies aimed at determining whether the activation of enzyme in response to physiological stimuli results from a direct phosphorylation of the enzyme. These latter studies will require an antibody to tryptophan hydroxylase. 5-HT metabolites and 5-HTP, generated in the Trp H assay, are isolated and quantitated by HPLC with electrochemical detection. Important insights should be gained from this work regarding cellular and molecular mechanisms for maintenance of 5-HT levels and hence 5-HT mediated function.

这项延续建议的重点是规管 色氨酸羟化酶（Trp H）活性 老鼠大脑的神经元。 由于这种酶是限速的， 合成的递质，血清素，（5-羟色胺），其活动是 关键是要确定这一重要的 物质，现在被广泛认为是一种媒介 各种功能，包括情绪或情感，焦虑， 抗伤害、睡眠、体温调节和中枢神经系统 控制血压。 5-羟色胺的合成增强 反应增加5-HT神经冲动流由一个 可能涉及Trp H激活的机制。 因此酶 从刺激的皮质5-HT投射制备的显示， 激活是频率依赖性的，并通过碱性逆转 磷酸酶。 另一方面，抑制5-HT激发 降低酶的活性，同样是通过碱性磷酸酶 可逆过程 实验现在计划1）进一步 表征上清液动力学行为的变化 酶制剂响应于不同水平的5- 水合氯醛麻醉雄性SD大鼠HT神经元放电 道利大鼠中枢神经系统的不同区域，与自然 辅因子，6 R-L-胆甾-5，6，7，8-四氢生物蝶呤; 2） 检查候选递质物质的作用， 立体定向注入中缝背核， 上升5-HT投射中酶活性的调节 在清醒的大鼠中由该核供应; 3）检查影响 各种各样的递质或假定的递质对色氨酸 与切片制备物孵育后的羟化酶活性; 4） 以确定是否在体外改变活性和动力学 响应于这些的上清液酶的性质 操作在体内表达; 5）启动研究， 在确定是否响应于 生理刺激是由蛋白质的直接磷酸化引起的。 酵素 这些后面的研究将需要一种抗体， 色氨酸羟化酶 生成的5-HT代谢物和5-HTP 在Trp H测定中，分离并通过HPLC定量， 电化学检测 应该获得重要的见解 从这项关于细胞和分子机制的工作中， 维持5-HT水平并因此维持5-HT介导的功能。