LOW-LEVEL LEAD METABOLISM AND TOXICITY IN GLIAL CULTURES

铅神经毒性研究的两个目标是确定分子和 与低血压相关的行为缺陷的细胞变化 以明确铅摄取和耐受的机制 积累铅的细胞。细胞培养是一种实用的工具 追求这些目标，提供一个可以 精确操作和直观的观察。神经元和神经胶质细胞 是铅致脑损伤的可能部位。星形胶质细胞，一种 神经胶质细胞被认为是脑内铅的储存库或过滤器。在……里面 培养这些细胞会从周围的培养液中积累铅并储存它 在细胞内。作为一种非生理性金属，铅必须进入 通过颠覆现有的机制，如星形胶质细胞 其他分子的运输。已经有两种传输机制 鉴定：红细胞膜和钙通道中的一种阴离子交换器 肾上腺嗜铬细胞。上述两种铅进入机制都没有 在培养的星形胶质细胞中进行了研究，尽管阴离子交换器和 在这些细胞中发现了钙通道。鉴于……的能力 星形胶质细胞从培养液中摄取铅并储存 在细胞内，再加上它们对公开的铅毒性的抵抗力，它 可以合理地推测这些动物的铅耐受机制 细胞。关于低水平铅暴露的大部分实验结果 支持两个概念：第一，铅引起的损伤是一种 连续、累积的过程，开始于离散的分子位置和 进步，除非被细胞内的防御机制所遏制；以及 第二，铅渗透到人体必需的代谢途径中。 金属。在拟议的研究中，将检验三个假设，第一个 其中之一是铅通过多种机制进入星形胶质细胞，包括 钙离子通道和阴离子交换器。这一假设将通过以下方式检验 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滞剂的存在与阴离子转运 通过使用与Pb2+结合的钙荧光载体Fura-2来阻滞剂。 表征铅迁移动力学的Km值和Vmax 将确定摄取的能量需求，并确定 相互竞争的金属和胞外蛋白将被评估。第二 假设谷胱甘肽是一种防御机制 细胞内铅的积累。它将通过测量胞质来进行测试 交互式激光细胞术测定培养星形胶质细胞内谷胱甘肽含量 作为铅暴露的函数以及谷胱甘肽对细胞损伤的相关性 级别。此外，铅诱导的细胞损伤将在 谷胱甘肽缺乏和谷胱甘肽丰富的细胞。最后一个假设 是铅渗入细胞内的钙储存，从而扰乱钙 循环和代谢过程依赖于钙信号。这 假说将通过表征细胞内的二价体来检验 铅暴露星形胶质细胞的阳离子池被细胞外动员 用离子霉素刺激，识别模拟的生理剂 离子霉素效应，以及即时生理定量 二价阳离子动员的后果，包括瞬时关闭 缝隙连接、PKC的激活和多肽酶的激活。

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.