ARGININE SYNTHESIS AND UTILIZATION IN BRAIN

精氨酸在脑中的合成和利用

• 批准号: 3478526
• 负责人: Marian Jackson
• 金额: $ 9.16万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-02-01 至 1998-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3478526
• 关键词: DNA methylation; amine oxidoreductase; aminoacid biosynthesis; arginine; astrocytes; brain; brain metabolism; chromatin; complementary DNA; cyclic AMP; gene expression; genetic promoter element; genetic regulatory element; genetic transcription; glucocorticoids; growth /development; immunocytochemistry; insulin; kidney; laboratory mouse; laboratory rat; liver; messenger RNA; molecular cloning; neurogenesis; neurons; nitric oxide; nuclear runoff assay; nucleic acid sequence; recombinant DNA; tissue /cell culture; transfection

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 尿素循环酶，其遗传性缺陷会导致严重的 疾病包括精神发育迟滞。 这两者的联合行动 酶导致瓜氨酸+天冬氨酸合成精氨酸。 虽然大脑中不存在完整的尿素循环，但 AS 和 AL 却存在 在脑组织中表达。 精氨酸在脑代谢中的作用 及神经功能尚不明确。 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，在信号转导过程中起重要作用 血管内皮和神经组织。 一氧化氮的调节 血管内皮的产生与理解有关 脑血管疾病，如中风和偏头痛。 谷氨酸受体 刺激与一氧化氮的产生相结合。 反应中 NO 的合成 刺激 N-甲基-D-天冬氨酸受体可能在 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 通过将瓜氨酸转化回精氨酸以进一步产生硝酸的过程 氧化物合成。 这项研究的长期目标是了解 精氨酸代谢在大脑中的作用及其机制 它在神经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 小鼠大脑不同区域和细胞类型的 AS、AL 和 NOS 酶测定和免疫细胞化学定位的结合 技术。 这些研究将提供精氨酸的相关性 新陈代谢与大脑功能的关系，并将表明这三者是否 酶存在于不同的区室中。 检测到 AL 表达 胎儿大脑发育比 AS 早。 相比之下，AS 和 AL 协调诱导肝脏和肾脏的发育。 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 在开发过程中的不同时期。 这些研究将比较并 对比大脑、肝脏中 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 中枢神经系统中的替代大脑特异性启动子。 已知糖皮质激素、cAMP 和胰岛素可调节转录 肝、肾中AS、AL的表达。 我们将确定 这些效应器对培养脑细胞中 AL 表达的影响。 这些研究将酶活性与稳态 mRNA 关联起来 水平，并将决定是否在以下水平实施监管 转录。 最终，我们将使用转染的重组小基因 进入培养的脑细胞以表征所需的序列 建立观察到的 AL 表达的时间和空间模式 在大脑中。