TRANSPORT AND ACTIONS OF METAL IONS

金属离子的传输和作用

• 批准号: 3254189
• 负责人: PATRICIA M. HINKLE
• 金额: $ 13.73万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-01-01 至 1995-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3254189
• 关键词: G protein; animal tissue; biological signal transduction; cadmium; dialysis; digital imaging; endocrine gland /system; fluorescence; fluorescence spectrometry; hormone biosynthesis; hormone receptor; human tissue; ion transport; lead; membrane channels; membrane permeability; neoplastic cell culture for noncancer research; protein tyrosine kinase; radioimmunoassay; reagent /indicator; receptor mediated endocytosis; secretion; tissue /cell culture; toxicant interaction; transcription factor; voltage gated channel

The overall goal of this research project is to elucidate the molecular mechanisms involved in the transport and toxic actions of heavy metals such as cadmium and lead. This proposal addresses three questions: how do these metals get into cells, where do they go, and how do they act? We previously discovered that Cd2+ enters pituitary cells via voltage sensitive calcium channels and that calcium channel blockers afford protection from cadmium toxicity. Pb2+ has also been shown to enter some cells through voltage-gated calcium channels. We will capitalize on our finding that Cd2+ and Pb2+ increase fluorescence of intracellularly trapped Fura 2 and that Fura 2 and Quin 2 can be used to measure the concentration of intracellular free CD2+ and Pb2+ directly. We will also define the effects of Cd2+ and Pb2+ on calcium homeostasis, distinguishing between effects that are due to metal ion-induced changes in calcium concentration and effects that are due to metal ion substitution in calcium-mediated processes. Initial studies will be performed in endocrine systems where calcium effects have been carefully delineated. Other models, such as neuronal cells with and without active voltage-sensitive calcium channels, will be used later to establish whether the results can be extrapolated to other cell types. Specific aims are as follows. First, the mechanisms of Cd2+ and Pb2+ uptake will be determined. Fluorescence methods will be used to measure free intracellular metal ions. The importance of voltage-sensitive calcium channels in the transport and toxicity of Cd2+ and Pb2+ will be quantified. Other routes of uptake of both free and protein-bound metal ions will be characterized. Second, intracellular free Cd2+ and Pb2+ will be visualized by digital fluorescence imaging techniques. Imaging techniques will be applied to study the localization of metal ions as they enter cells and the localization of free Cd2+ and Pb2+ in chronically exposed cells. The last objective is to determine systematically the effects of Cd2+ and Pb2+ on the function of endocrine cells. The effects of Cd2+ and Pb2+ to disrupt hormone synthesis and secretion stimulated by different signal transduction pathways. The effects of the metal ions on receptors that act via g-proteins (stimulatory and inhibitory adenylate cyclase pathways and phosphoinositide pathways), and on receptors with tyrosine kinase activity will be determined. Finally, the effects of Cd2+ and Pb2+ on hormone synthesis and on nuclear hormone receptors that function as transcription factors will be measured.

这项研究项目的总体目标是阐明分子 重金属迁移和毒性作用的机制 比如镉和铅。这项提议解决了三个问题：如何 这些金属进入细胞了吗，它们去了哪里，它们是如何作用的？ 我们先前发现，CD2+通过电压进入脑垂体细胞 敏感的钙通道和钙通道阻滞剂 防止镉中毒。Pb2+也被显示进入 一些细胞通过电压门控钙通道。我们将利用这一点 关于Cd~(2+)和Pb~(2+)对细胞内荧光的增强作用 细胞内捕获的Fura 2以及Fura 2和Quin 2可用于 直接测定细胞内游离Cd~(2+)、Pb2+浓度。 我们还将确定Cd~(2+)和Pb~(2+)对钙稳态的影响， 区分由金属离子引起的变化引起的影响 钙离子浓度和金属离子的影响 钙介导的过程中的替代。初步研究将是 在内分泌系统中进行，在内分泌系统中钙的影响已经被仔细地 勾勒出来的。其他模型，如有和没有的神经细胞 激活的电压敏感钙通道，稍后将用于 确定结果是否可以外推到其他类型的单元格。 具体目标如下。第一，Cd~(2+)和Pb~(2+)的作用机制 摄取量将被确定。将使用荧光方法来测量 胞内游离金属离子。电压敏感型的重要性 钙通道在Cd~(2+)和Pb~(2+)的转运和毒性中的作用 量化的。其他吸收游离金属和蛋白质结合金属的途径 将对离子进行表征。第二，细胞内游离的CD2+和Pb2+ 将通过数字荧光成像技术可视化。成象 将应用技术来研究金属离子的局域化 它们进入细胞和游离Cd~(2+)、Pb~(2+)在细胞内的定位 长期暴露的细胞。最后一个目标是确定 Cd~(2+)、Pb~(2+)对内分泌功能的系统影响 细胞。Cd~(2+)和Pb~(2+)对激素合成的干扰作用 不同的信号转导途径刺激分泌。这个 金属离子对通过G蛋白作用的受体的影响 (刺激性和抑制性腺苷环化酶途径和 磷脂酰肌醇途径)，以及酪氨酸激酶受体 活动将被确定。最后，Cd~(2+)和Pb~(2+)对玉米生长的影响 激素合成和核激素受体的功能 转录因子将被测量。