TETRAHYDROBIOPTERIN BIOSYNTHESIS BY DOPAMINE NEURONS

有明确的证据表明，人类遗传缺陷， 中枢生物单胺神经传递， 四氢生物蝶呤（BH 4）基因缺陷的直接结果 新陈代谢. 这一证据支持了 单胺能神经元内的异常功能， 精神分裂症和情感障碍的生物学假说 病 我们对BH4代谢的理解， 酪氨酸和色氨酸羟化酶必需辅因子 单胺能神经元内的活性，受到 使用完整动物进行生化研究的困难。 的 本提案概述了一项旨在研究BH4的研究计划 在两个解剖学定义的完整的 多巴胺（DA）神经元来源于胚胎 小鼠下丘脑和中脑，并保持在组织中 文化 这些研究将联合收割机免疫组化、高 高效液相色谱和放射性核素示踪 技术与完整的和无细胞的制备论文 培养，以研究细胞定位和代谢 BH4。 此外，提出了一系列实验， 旨在确定慢性BH4减少的影响 DA水平和合成的内容。 已知的BH4抑制剂 将测试合成物对BH4的急性抑制能力 合成. 然后将与抑制剂长期孵育 并监测BH4含量。 的功能影响 DA水平上细胞内BH4含量降低， 生物合成将被检查，因为将外源的能力， 将BH4添加到孵育培养基中以恢复DA功能。 它 希望这些研究将有助于阐明可能的差异， DA神经元之间的BH4代谢，并将 也导致了完整的细胞系统的发展， 多巴胺生物合成与BH_4相互作用的研究 DA神经元的代谢。

There is unequivocal evidence of inherited deficiencies in human central biogenic monoamine neurotransmission which are the direct result of genetic defects in tetrahydrobiopterin (BH4) metabolism. This evidence lends support to the theory of abnormal function within monoaminergic neurons as expressed in current hypotheses of the biology of schizophrenia and affective illness. Our understanding of the metabolism of BH4, the essential cofactor for tyrosine and tryptophan hydroxylase activity within monoaminergic neurons, is limited by the difficulty of biochemical studies using intact animals. The present proposal outlines a research plan designed to study BH4 metabolism within two anatomically defined populations of intact dopamine (DA) containing neurons derived from the embryonic mouse hypothalamus and mesencephalon and maintained in tissue culture. These studies will combine immunohistochemical, high performance liquid chromatographic and radionuclide tracer techniques with intact and cell-free preparations of theses cultures to investigate the cellular localization and metabolism of BH4. In addition, a series of experiments are proposed which are intended to determine the effect of chronically decreased BH4 content on DA levels and synthesis. Known inhibitors of BH4 synthesis will be tested for their capacity to acutely inhibit BH4 synthesis. Long term incubation with inhibitor(s) will then be undertaken, and BH4 content monitored. The functional impact of decreased intracellular BH4 content on DA levels and biosynthesis will be examined, as will the capacity of exogenous BH4 added to the incubation medium to reinstate DA function. It is hoped that these studies will help elucidate possible differences between DA neurons with respect to BH4 metabolism, and will also lead to the development of an intact cell system for the study of the interaction between DA biosynthesis and BH4 metabolism in DA neurons.