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BIOSYNTHESIS AND ACTION OF NEUROSTEROIDS IN THE BRAIN

大脑中神经类固醇的生物合成和作用

基本信息

批准号：
08454265
负责人：
TSUTSUI Kazuyoshi
金额：
$ 4.86万
依托单位：
HIROSHIMA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

项目摘要

Steroid hormones supplied by peripheral steroidogenic glands regulate neuronal functions during development and in adulthood. Peripheral steroids cross the blood-brain barrier as a result of their lipid solubility and act on brain tissues through intracellular receptor-mediated mechanisms that regulate the transcription of specific genes. Therefore, the brain is considered to be a target site of peripheral steroids. Recently, we obtained new findings that the avian brain also possesses a steroidogenic enzyme, cytochrome P450scc, and produces pregnenolone and its sulfate ester, by biochemical and immunochemical approaches. In addition, our immunohistochemical studies with avian brain indicated that an intense immunoreaction with the polyclonal antibody directed against the purified bovine adrenal P450scc is present in soma and dendrites of the Purkinje cell, a typical cerebellar neuron. We further demonstrated that steroidogenic enzyme P450scc appears in the rat Purkinje cell immediatel … More y after its differentiation.To understand the mode of action of pregnenolone and its sulfate ester, we examined the effects of these neurosteroids on synaptic currents in cerebellar Purkinje neurons. Inhibitory postsynaptic currents (IPSCs) in Purkinje neurons were recorded in a cerebellar slice by the patch-clamp method. Pregnenolone sulfate increased, in a dose-related way, the frequency of IPSCs within 1 min of perfusion, indicating that this effect is unlikely to be induced via gene transcription. In contrast, pregnenolone had no effect on the frequency of IPSCs. The IPSCs recorded in the Purkinje neurons were completely blocked by bicuculine, a gamma-aminobutyric acid A (GABAA) receptor antagonist, suggesting that they are mediated by GABAA receptors. It is therefore possible that pregnenolone sufate, produced in Purkinje neurons, may modulate GABAergic transmission by paracrine acitons on GAGAergic neurons rather than by genomic mechanisms. Behavioral studies are now in progress to clarify the neurosteroidal function.More recently, we demonstrated that Purkinje neurons possess not only P450scc but also 3beta-HSD and produces progesterone by molecular and biochemical approaches. Interestingly, expression of the P450scc mRNA was relatively constant during neonatal life and in adulthood, whereas the 3beta-HSD mRNA expression was evident in early in early neonatal life. It is considered that progesterone, a product of 3beta-HSD activity, may play a role in the development of cerebellar nervous system structures in this period. Less

由外周类固醇生成腺提供的类固醇激素在发育和成年期调节神经元功能。外周类固醇因其脂溶性而穿过血脑屏障，并通过调节特定基因转录的细胞内受体介导机制作用于脑组织。因此，大脑被认为是外周类固醇的目标部位。最近，我们通过生化和免疫化学方法获得新发现，禽脑还具有类固醇生成酶细胞色素P450scc，并产生孕烯醇酮及其硫酸酯。此外，我们对禽脑的免疫组织化学研究表明，针对纯化的牛肾上腺 P450scc 的多克隆抗体存在强烈的免疫反应，存在于浦肯野细胞（典型的小脑神经元）的体细胞和树突中。我们进一步证明，类固醇生成酶 P450scc 在大鼠浦肯野细胞分化后立即出现。为了了解孕烯醇酮及其硫酸酯的作用模式，我们检查了这些神经类固醇对小脑浦肯野神经元突触电流的影响。通过膜片钳方法在小脑切片中记录浦肯野神经元的抑制性突触后电流（IPSC）。孕烯醇酮硫酸盐以剂量相关的方式增加了灌注后 1 分钟内 IPSC 的频率，表明这种效应不太可能是通过基因转录诱导的。相反，孕烯醇酮对 IPSC 的频率没有影响。浦肯野神经元中记录的 IPSC 被荷包牡丹碱（一种 γ-氨基丁酸 A (GABAA) 受体拮抗剂）完全阻断，表明它们是由 GABAA 受体介导的。因此，浦肯野神经元中产生的孕烯醇酮硫酸盐可能通过 GAGA 能神经元上的旁分泌激子而不是基因组机制来调节 GABA 能传递。目前正在进行行为研究以阐明神经甾体功能。最近，我们证明浦肯野神经元不仅具有 P450scc，还具有 3beta-HSD，并通过分子和生化方法产生黄体酮。有趣的是，P450scc mRNA 的表达在新生儿生命和成年期相对恒定，而 3β-HSD mRNA 表达在新生儿生命早期很明显。认为3β-HSD活性产物黄体酮可能在这一时期小脑神经系统结构的发育中发挥作用。较少的