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: 2154736
• 负责人: EVELYN TIFFANY-CASTIGLIONI
• 金额: $ 21.98万
• 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/2154736
• 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.

铅的神经毒性研究的两个目标是确定分子和 与低血糖相关的行为缺陷的细胞改变 水平暴露，并确定铅的吸收和耐受机制， 积累铅的细胞。细胞培养是一种实用的工具， 追求这些目标，提供细胞外环境， 精确操作和直接观察。神经元和神经胶质 是脑铅损伤的可能部位。星形胶质细胞，一种 神经胶质细胞被认为是脑中铅的储存库或过滤器。在 培养这些细胞从周围介质中积累铅并储存它 细胞内。作为一种非生理金属，铅必须进入人体， 细胞，如星形胶质细胞，通过颠覆机制，存在的 其他分子的运输。有两种运输机制， 确定：红细胞血影和钙通道中的阴离子交换剂， 肾上腺嗜铬细胞上述Pb进入的机制都没有 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Ca 2+通道。鉴于有能力 星形胶质细胞从培养基中吸收铅并储存铅 在细胞内，再加上它们对明显的铅毒性的抗性， 将是合理的假设机制，铅的耐受性，在这些 细胞大量关于低水平铅暴露的实验结果 支持两个概念：第一，铅诱导的损伤是一种 从离散的分子位点开始的连续的累积过程， 除非受到细胞内防御机制的控制，否则会发生进展;以及 第二，铅渗透通常使用的代谢途径， 金属.在拟议的研究中，将测试三个假设，第一个 其中铅通过多种机制进入星形胶质细胞，包括 Ca 2+通道和阴离子交换剂。这一假设将由以下人员进行检验： 荧光分光光度法监测Pb 2+进入培养的星形胶质细胞 存在离子通道激动剂和阻断剂以及阴离子转运 阻断剂通过使用Ca 2+荧光团Fura-2，其结合到Pb 2+。 表征Pb迁移动力学的Km和Vmax值 将确定吸收的能量需求，以及 将评估竞争金属和细胞外蛋白。 第二 假设谷胱甘肽是一种防御机制， 细胞内积累的Pb。将通过测量细胞溶质 交互式激光细胞仪测定培养星形胶质细胞谷胱甘肽含量 作为铅暴露的函数以及与谷胱甘肽相关的细胞损伤 程度.此外，还将对Pb诱导的细胞损伤进行定量， 谷胱甘肽耗尽和谷胱甘肽富集的细胞。最后一个假设 是铅渗入细胞内钙储存，从而破坏钙 循环和代谢过程依赖于Ca 2+信号传导。这 将通过表征细胞内二价 暴露于Pb的星形胶质细胞中的阳离子池，其被细胞外 用离子霉素刺激，鉴定模拟 离子霉素效应，并定量立即生理 二价阳离子迁移的后果，包括瞬时闭合 蛋白激酶C的激活和肽酶的激活。