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ARGININE SYNTHESIS AND UTILIZATION IN BRAIN

精氨酸在脑中的合成和利用

基本信息

批准号：
2332974
负责人：
Marian Jackson
金额：
$ 10.38万
依托单位：
UNIVERSITY OF MARYLAND BALTIMORE
依托单位国家：
美国
项目类别：
财政年份：
1993
资助国家：
美国
起止时间：
1993-02-01 至 1999-01-31
项目状态：
已结题

项目摘要

Argininosuccinate synthetase (AS) and argininosuccinate lyase (AL) are enzymes of the urea cycle whose heritable deficiencies result in severe disease including mental retardation. The combined actions of these two enzymes leads to synthesis of arginine from citrulline + aspartate. While the complete urea cycle is not present in the brain, AS and AL are expressed in brain tissue. The role(s) of arginine in brain metabolism and neurological function are not clear. Nitric oxide synthase (NOS), catalyzes the conversion of arginine to nitric oxide + citrulline. Recent evidence indicates that nitric oxide (NO) is a novel second messenger molecule which is important in signal transduction processes in vascular endothelium and neural tissue. Regulation of nitric oxide production in vascular endothelium is pertinent to understanding cerebrovascular diseases such as stroke and migraine. Glutamate receptor stimulation is coupled to NO production. The synthesis of NO in response to stimulation of N-methyl-D-aspartate receptors may play a role in synaptic plasticity and long term potentiation. It Is our hypothesis that neural AS and AL play critical roles In nitric oxide-dependent processes by converting citrulline back to arginine for further nitric oxide synthesis. The long term goals of this research are to understand the role(s) of arginine metabolism in the brain and the mechanisms by which it is regulated during development and maturation of the nervous system. In the proposed studies we will determine the distribution of AS, AL and NOS in different regions and cell types of mouse brain using a combination of enzyme assays and immunocytochemical localization techniques. These studies will provide correlations of arginine metabolism with brain functions and will indicate whether the three enzymes are present in distinct compartments. AL expression is detected earlier in fetal brain development than is AS. In contrast, AS and AL are coordinately induced in developing liver and kidney. We will use molecular genetic approaches to characterize the molecular mechanisms which lead to discrete localization of AS and AL in different cell types at different times during development. These studies will compare and contrast the chromatin structure for the AS and AL genes in brain, liver and kidney. We will also determine whether AL is transcribed from an alternative, brain-specific promoter in the central nervous system. Glucocorticoids, cAMP and insulin are known to modulate the transcription and expression of AS and AL in liver and kidney. We will determine the effects of these effectors on AL expression in cultured brain cells. These studies will correlate enzyme activity with steady state mRNA levels and will determine whether regulation is exerted at the level of transcription. Ultimately, we will use recombinant minigenes transfected into cultured brain cells to characterize the sequences required for establishing the temporal and spatial patterns of AL expression observed in the brain.

精氨基琥珀酸合成酶（AS）和精氨基琥珀酸裂解酶（AL）是尿素循环的酶，其遗传缺陷导致严重的包括智力迟钝在内的疾病。这两个人的联合行动酶导致从瓜氨酸+天冬氨酸合成精氨酸。虽然完整的尿素循环不存在于大脑中，但AS和AL存在于大脑中。在脑组织中表达。精氨酸在脑代谢中的作用和神经功能尚不清楚。一氧化氮合酶（NOS），催化精氨酸转化为一氧化氮+瓜氨酸。最近的证据表明，一氧化氮（NO）是一种新的第二，信使分子是重要的信号转导过程中，血管内皮和神经组织。一氧化氮的调节血管内皮细胞的生产与理解脑血管疾病，如中风和偏头痛。谷氨酸受体刺激与NO的产生相结合。一氧化氮的合成 N-甲基-D-天冬氨酸受体的刺激可能在突触可塑性和长时程增强。这是我们的假设神经AS和AL在一氧化氮依赖性中发挥关键作用通过将瓜氨酸转化为精氨酸，氧化物合成这项研究的长期目标是了解精氨酸代谢在脑中的作用以及它在神经系统的发育和成熟过程中受到调节系统在拟议的研究中，我们将确定用免疫组织化学方法观察了小鼠不同脑区和不同细胞类型的AS、AL和NOS的变化。酶测定和免疫细胞化学定位的组合技术. 这些研究将提供精氨酸代谢与大脑功能，并将表明是否三个酶存在于不同的区室中。检测到AL表达早于AS的胎儿大脑发育。相反，AS和AL 在肝、肾的发育中协调诱导。我们将使用分子遗传学方法来表征分子机制这导致AS和AL在不同细胞类型中的离散定位在发育的不同时期。这些研究将比较和对比脑、肝、肝中AS和AL基因的染色质结构，和肾脏。我们还将确定AL是否转录自中枢神经系统中的脑特异性启动子。已知糖皮质激素、cAMP和胰岛素调节转录 AS和AL在肝、肾组织中的表达。康贝特人将以这些效应物对培养的脑细胞中AL表达的影响。这些研究将酶活性与稳态mRNA 并将决定是否在一级实施监管，转录。最终，我们将使用重组小基因转染植入培养的脑细胞中，建立观察到的AL表达的时空模式在大脑中。