LOW-LEVEL LEAD METABOLISM AND TOXICITY IN GLIAL CULTURES

胶质培养物中的低水平铅代谢和毒性

• 批准号: 3254214
• 负责人: EVELYN TIFFANY-CASTIGLIONI
• 金额: $ 21.14万
• 依托单位: TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-04-01 至 1996-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3254214
• 关键词: anions; biological signal transduction; brain metabolism; calcium channel; calcium channel blockers; cytotoxicity; glia; glutathione; ion transport; laboratory rat; lead poisoning; metal metabolism; microspectrophotometry; neurons; neuroprotectants; neurotoxins; tissue /cell culture

Two goals of Pb neurotoxicity research are to identify molecular-and cellular alterations that underlie behavioral deficits associated with low level exposure and to define mechanisms of Pb uptake and tolerance in cells that accumulate Pb. Cell cultures are practical tools with which to pursue these goals, offering an extracellular environment that can be precisely manipulated and direct observation. Both neurons and neuroglia are probable sites of Pb-induced damage in brain. Astroglia, a type of neuroglia, are proposed to serve as a Pb depot or filter in brain. In culture these cells accumulate Pb from the surrounding medium and store it intracellularly. As a non-physiological metal, Pb must gain entry into cells such as astroglia by subverting mechanisms that exist for the transport of other molecules. Two transport mechanisms have been identified: an anion exchanger in red cell ghosts and calcium channels in adrenal chromaffin cells. Neither of the above mechanisms for Pb entry has been studied in cultured astroglia, although both anion exchangers and Ca2+ channels are found in these cells. In view of the ability of astroglia to take up Pb from the culture medium and store it intracellularly, coupled with their resistance to overt Pb toxicity, it would be reasonable to postulate mechanisms of Pb tolerance in these cells. The bulk of experimental findings concerning low-level Pb exposure of cells supports two concepts: first, that Pb-induced damage is a continuous, cumulative process that begins at discrete molecular sites and progresses unless contained by defensive mechanisms within the cell; and second, that Pb infiltrates metabolic pathways normally used by essential metals. In the proposed study, three hypotheses will be tested, the first of which is that Pb enters astroglia via multiple mechanisms, including Ca2+ channels and anion exchangers. This hypothesis will be tested by spectrofluorometrically monitoring Pb2+ entry into cultured astroglia in the presence of ion channel agonists and blockers and anion transport blockers by the use of the Ca2+ fluorophore Fura-2, which binds-to Pb2+. Values for Km and Vmax that characterize the kinetics of Pb transport energy requirements for uptake will be determined, and the effects of competing metals and extracellular proteins will be assessed. The second hypothesis is that glutathione is a defense mechanism against intracellularly accumulated Pb. It will be tested by measuring cytosolic glutathione content in astroglia in culture by interactive laser cytometry as a function of Pb exposure and correlating cell injury to glutathione levels. In addition, Pb-induced cell injury will be quantified in glutathione-depleted and glutathione-enriched cells. The final hypothesis is that Pb infiltrates intracellular Ca stores, thereby disrupting Ca cycling and metabolic processes dependent on Ca2+ signaling. This hypothesis will be tested by characterizing an intracellular divalent cation pool in Pb-exposed astroglia that is mobilized by extracellular stimulation with ionomycin, identifying physiological agents that mimic the ionomycin effect, and quantitating immediate physiological consequences of divalent cation mobilization, including transient closing of gap junctions, activation of PKC, and activation of peptidases.

铅神经毒性研究的两个目标是确定分子和 与低水平相关的行为缺陷背后的细胞改变 水平暴露并定义铅吸收和耐受的机制 积累 Pb 的细胞。细胞培养是实用的工具，可以用来 追求这些目标，提供一个可以 精确操纵和直接观察。神经元和神经胶质细胞 是铅引起的大脑损伤的可能部位。星形胶质细胞，一种 神经胶质细胞被认为充当大脑中的铅库或过滤器。在 培养这些细胞从周围介质中积累铅并将其储存 细胞内。作为一种非生理金属，Pb 必须进入 通过颠覆星形胶质细胞等细胞存在的机制 其他分子的运输。两种运输机制 确定：红细胞鬼影中的阴离子交换剂和钙通道 肾上腺嗜铬细胞。上述两种 Pb 进入机制都没有 在培养的星形胶质细胞中进行了研究，尽管阴离子交换剂和 在这些细胞中发现了 Ca2+ 通道。鉴于能力 星形胶质细胞从培养基中吸收铅并储存 细胞内，再加上它们对明显的铅毒性的抵抗力，它 假设这些中的 Pb 耐受机制是合理的 细胞。大量关于低浓度铅暴露的实验结果 细胞支持两个概念：首先，Pb 引起的损伤是 从离散的分子位点开始的连续的、累积的过程 除非被细胞内的防御机制所抑制，否则就会进展；和 其次，铅渗透到必需物质通常使用的代谢途径中。 金属。在拟议的研究中，将测试三个假设，第一个 其中，Pb 通过多种机制进入星形胶质细胞，包括 Ca2+ 通道和阴离子交换剂。这个假设将被检验 荧光分光光度法监测 Pb2+ 进入培养的星形胶质细胞 离子通道激动剂和阻断剂以及阴离子转运的存在 通过使用 Ca2+ 荧光团 Fura-2 来阻断剂，该荧光团可与 Pb2+ 结合。 表征 Pb 传输动力学的 Km 和 Vmax 值 吸收的能量需求将被确定，并且影响 将评估竞争性金属和细胞外蛋白质。 第二个 假设谷胱甘肽是一种防御机制 细胞内积累的Pb。它将通过测量细胞溶质来测试 通过交互式激光细胞术测定培养物星形胶质细胞中的谷胱甘肽含量 作为铅暴露和与谷胱甘肽相关的细胞损伤的函数 水平。此外，Pb 诱导的细胞损伤将量化为 谷胱甘肽耗尽的细胞和谷胱甘肽富集的细胞。最终假设 是 Pb 渗透到细胞内的 Ca 储存，从而破坏 Ca 循环和代谢过程依赖于 Ca2+ 信号传导。这 假设将通过表征细胞内二价来检验 暴露于铅的星形胶质细胞中的阳离子池由细胞外动员 用离子霉素刺激，识别模仿的生理制剂 离子霉素效应，并定量立即生理 二价阳离子动员的后果，包括瞬时关闭 间隙连接、PKC 激活和肽酶激活。