MERCURY IMPAIRMENT OF CELL VOLUME REGISTRATION IN SKATE HEPATOCYTES

总体目标是确定分子机制和靶点 汞对滑冰肝脏质膜转运系统的损害 特别是与细胞体积调节和牛磺酸有关的蛋白质 外流。我们最近证明了从肝细胞中分离出的肝细胞 小溜鱼(Raja Erinacea)为研究提供了一个强大的模型系统 汞对细胞体积调节机制的影响。我们的 研究表明，质膜离子和溶质的变化 渗透性是表达的敏感和接近的事件 汞诱导的细胞损伤，并为 假设细胞膜是目标细胞器。此外， 三十年来，汞暴露导致细胞肿胀，我们的结果 证明汞也阻止了正常的调节量 在渗透牛磺酸转运后观察到的降低(RVD)，并增加 NA通透性，两种可能对受损的人起作用的机制 房租和房租。拟议的研究的重点是检查分子 汞与容量激活的牛磺酸转运体的相互作用： 1.确定驱动力、底物专一性和相互作用 汞与牛磺酸的外排转运蛋白，使用分离 基侧质膜小泡。2.因为c1通道是 认为调节容量激活的牛磺酸转运和RVD 细胞类型，我们计划研究HgC12对C1-通道的影响 膜片钳技术在冰刀肝细胞活性和牛磺酸转运中的应用 技巧。3.测试汞是否干扰膜 滑冰动物肝细胞的循环(胞吐作用)，以及这是否与 对RVD的抑制，以及4。长期目标是分子 滑冰动物牛磺酸外排转运系统基因的克隆 肝细胞和潜在汞结合部位的鉴定 (例如，半胱氨酸残基)。 这些研究应该会对体积的机制提供重要的见解 调节和牛磺酸动态平衡，以及汞是如何破坏这些的 基本的细胞过程。需要进行批量监管流程 被所有动物细胞用来抵消跨膜肿瘤梯度， 但对细胞或有机体的生存尤其重要 这必须适应不同的细胞外渗透压。

The overall objective is to identify molecular mechanisms and targets for mercury's impairment of plasma membrane transport systems in skate liver, specifically the proteins involved in cell volume regulation and taurine efflux. We recently demonstrated that hepatocytes isolated from the little skate (Raja erinacea) provide a powerful model system for studying the effects of mercury on mechanisms of cell volume regulation. Our studies indicate that changes in plasma membrane ion and solute permeability are sensitive and proximate events in the expression of mercury-induced cell injury, and provide direct support for the hypothesis that cell membrane is the target organelle. Furthermore, three decades that mercury exposure leads to cell swelling, our results demonstrated that mercury also prevents the normal regulatory volume decrease (RVD) observed after osmotic taurine transport, and increased Na+ permeability, two mechanisms that probably contribute to the impaired RVD. The focus of the proposed studies is to examine the molecular interactions of mercury with the volume-activated taurine transporter: 1. Define the driving forces, substrate specificity, and interaction of mercury with the taurine efflux transport proteins, using isolated basolateral plasma membrane vesicles. 2. Because C1-channels are thought to modulate volume-activated taurine transport and RVD in other cell types, we plan to examine the effects of HgC12 on C1- channel activity and taurine transport in skate hepatocytes using patch clamp techniques. 3. Test whether mercury is interfering with membrane recycling (exocytosis) in skate hepatocytes, and whether this is related to the inhibition of RVD, and 4. A long-term goal is the molecular cloning of the cDNA for the taurine efflux transport system in skate hepatocytes, and the identification of potential mercury binding sites (eg, cysteine residues) in the gene product. These studies should provide important insights into mechanisms of volume regulation and taurine homeostasis, and how mercury disrupts these fundamental cellular processes. Volume regulatory processes are required by all animal cells to counterbalance transmembrane oncotic gradients, but are particularly important for the survival of cells or organisms that must adapt to varying extracellular osmolarities.