TRANSPORT AND ACTIONS OF METAL IONS

本研究项目的总体目标是阐明 重金属的迁移和毒性作用机制 例如镉和铅。 这一建议涉及三个问题： 这些金属会进入细胞吗？它们会去哪里？它们又是如何起作用的？ 我们以前发现Cd 2+通过电压进入垂体细胞， 敏感的钙通道和钙通道阻滞剂提供 防止镉中毒。 Pb 2+也被证明可以进入 一些细胞通过电压门控钙通道。 我们将利用 在我们的发现，Cd 2+和Pb 2+增加荧光的 胞内捕获Fura 2，Fura 2和Quin 2可用于 直接测定细胞内游离CD 2+和Pb 2+浓度。 我们还将确定Cd 2+和Pb 2+对钙稳态的影响， 区分由于金属离子引起的变化 在钙浓度和由于金属离子 在钙介导的过程中的替代。 初步研究将 在内分泌系统中进行，其中钙的作用已经被仔细地 描绘的。 其他模型，如神经元细胞， 活性电压敏感钙通道，稍后将用于 确定结果是否可以外推到其他细胞类型。 具体目标如下。 第一，Cd 2+和Pb 2+的作用机制 将确定摄取量。 将使用荧光方法测量 游离的细胞内金属离子。 电压敏感的重要性 钙通道在Cd 2+和Pb 2+的转运和毒性中的作用 量化。 游离金属和蛋白结合金属的其他摄取途径 离子将被表征。 第二，细胞内游离Cd 2+和Pb 2 + 将通过数字荧光成像技术可视化。 成像 技术将被应用于研究金属离子的定位， 它们进入细胞，游离Cd ~（2+）和Pb ~（2+）在 长期暴露的细胞。 最后一个目标是确定 系统地研究了Cd ~（2+）、Pb ~（2+）对大鼠内分泌功能的影响， 细胞 Cd ~（2+）和Pb ~（2+）对激素合成的影响 不同的信号转导途径刺激分泌。 的 金属离子对通过G蛋白起作用的受体的影响 （刺激性和抑制性腺苷酸环化酶途径， 磷脂酰肌醇途径），并与酪氨酸激酶受体 活动将被确定。 最后，研究了Cd ~（2+）和Pb ~（2+）对 激素合成和对核激素受体的作用， 将测量转录因子。

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.