NEUROBIOLOGY OF BRAIN ARGININE DECARBOXYLASE

1994年，我们从牛脑中分离出精氨酸（agmatine），它是精氨酸脱羧酶（ADC）从精氨酸合成的一种胺，能与α 2肾上腺素能受体和咪唑啉受体结合。这一发现令人惊讶，因为在细菌、植物和寄生虫中普遍存在的agmatine和ADC都没有在哺乳动物中检测到。在我们最初的发现之后，其他一些研究证实了agmatine和ADC在哺乳动物组织中的存在。虽然ADC已经从细菌和植物中得到了广泛的表征、纯化和克隆，但从未在哺乳动物系统中进行过研究。哺乳动物（大鼠脑）ADC的已知性质清楚地将其与其他亚型和哺乳动物鸟氨酸脱羧酶（一种密切相关的酶）区分开来。进一步研究ADC在哺乳动物脑内的分子特性、定位和调控，对理解胍丁氨酸的神经生物学具有重要意义。该提案将验证哺乳动物ADC是一种具有独特初级结构的新型酶的假设，该酶定位于选择性神经细胞并调节胍丁氨酸的产生，胍丁氨酸是一种假定的神经递质/神经调节剂。研究1将描述大鼠肝/脑ADC与大鼠肝脏ODC在亚细胞定位、反应特性、抑制谱和调控方面的平行特征。证明哺乳动物ADC是一种新的，迄今为止未被认识的酶，将通过分子克隆建立其初级结构。我们将通过两种方法克隆哺乳动物ADC。由于已经克隆了几种非哺乳动物ADC的同种异构体，因此将对哺乳动物ADC进行PCR同源性筛选，获得可能的部分克隆，并利用它们筛选cDNA文库，获得全长cDNA克隆。作为一种替代方法，我们将从大鼠肝脏中纯化ADC，获得肽序列，并基于这些序列设计寡聚探针，用于基于PCR或cDNA文库的筛选。研究2将验证ADC可能是一种胶质酶的假设，其产物agmatine被释放到细胞外空间，被吸收并储存在神经元中。我们将通过生化和免疫细胞化学方法研究ADC和agmatine在培养的神经细胞和全脑中的定位，以验证这一假设。研究3将验证ADC活性受胶质细胞炎症刺激调节的假设。我们将研究细菌毒素、LPS和细胞因子对培养的星形胶质细胞和巨噬细胞中ADC表达的调节。比较ADC与NOS-2的调节，我们将能够理解精氨酸代谢是如何与胍丁氨酸或一氧化氮合成协同调节的。在本项目完成后，我们将能够证明哺乳动物ADC是一种具有独特序列、定位和调控特性的新型酶，从而为进一步了解agmatine在哺乳动物大脑中的生物学作用提供有价值的信息和工具。

In 1994, we isolated agmatine, an amine synthesized from arginine by arginine decarboxylase (ADC), which bound to alpha2 adrenergic and imidazoline receptors of all subclasses, from cow brain. The discovery was a surprise, as neither agmatine nor ADC, prevalent in bacteria, plants and parasites, had been detected in mammals. Following our initial discovery, a number of other studies confirmed the presence of agmatine and ADC in mammalian tissues. Although ADC had been extensively characterized, purified and cloned from bacteria and plants, it was never studied in mammalian systems. The known properties of mammalian (rat brain) ADC clearly distinguishes it from other isoforms and mammalian ornithine decarboxylase, a closely related enzyme. Further studies on the molecular properties, localization and regulation of ADC in mammalian brain is critical in understanding the neurobiology of agmatine. This Proposal will test the hypothesis that mammalian ADC is a novel enzyme with unique primary structure, localized in selective neuronal cells and regulating the production of agmatine, a putative neurotransmitter/neuromodulator. Study 1 will characterize rat liver/brain ADC in parallel with rat liver ODC with respect to subcellular localization, reaction properties, inhibition profiles, and regulation. The proof that mammalian ADC is a novel, and heretofore unrecognized, enzyme will be to establish its primary structure by molecular cloning. We will clone mammalian ADC by two approaches. As several isoforms of non- mammalian ADC have been cloned, mammalian ADC will be cloned by homology screening by PCR to obtain possible partial clones and to use them to screen cDNA libraries to obtain the full length cDNA clone. As an alternate approach, we will purify ADC from rat liver, obtain peptide sequences, and design oligoprobes based on these sequences to be used in PCR based, or cDNA library, screening. Study 2 Will test the hypothesis that ADC may be a glial enzyme, and that its product, agmatine, is released into the extracellular space and taken up into, and stored in, neurons. We will investigate the localization of ADC and agmatine in cultured neuronal cells and whole rat brain by biochemical and immunocytochemical methods to verify this hypothesis. Study 3 will test the hypothesis that activity of ADC is regulated by inflammatory stimuli in glial cells. We will investigate the regulation of the expression of ADC in cultured astrocytes and macrophages by bacterial toxin, LPS, and cytokines. Comparing the regulation of ADC with NOS-2, we will be able to understand how arginine metabolism is regulated in concert with agmatine or nitric oxide synthesis. At the completion of this project, we will be able to demonstrate that mammalian ADC is a novel enzyme with unique sequence, and localization and regulatory properties, thereby providing valuable information and tools to further understand the biological role of agmatine in mammalian brain.