MERCURY IMPAIRMENT OF CELL VOLUME REGISTRATION IN SKATE HEPATOCYTES

总的目标是确定分子机制和目标， 汞对鳐肝脏质膜转运系统的损害， 特别是参与细胞体积调节的蛋白质和牛磺酸 流出 我们最近证明，从肝细胞中分离的肝细胞， 小鳐（Raja erinacea）为研究其生物学行为提供了一个强有力模式系统 汞对细胞体积调节机制的影响。 我们 研究表明，质膜离子和溶质的变化 渗透率是敏感的和接近的事件，在表达 汞引起的细胞损伤，并提供直接支持， 假设细胞膜是目标细胞器。 此外，委员会认为， 三十年来，汞暴露导致细胞肿胀，我们的结果是， 表明汞也会阻止正常的调节体积， 牛磺酸渗透转运后观察到RVD降低， Na+渗透性，这两种机制可能有助于受损的 RVD. 拟议研究的重点是检查分子 汞与体积激活的牛磺酸转运蛋白的相互作用： 1. 定义驱动力、底物特异性和 汞与牛磺酸外排转运蛋白，使用分离的 基底侧质膜小泡。 2. 因为C1通道 认为调节体积激活的牛磺酸转运和RVD在其他 细胞类型，我们计划研究HgC 12对C1通道的影响， 应用膜片钳技术研究鳐肝细胞的活性和牛磺酸转运 技术. 3. 测试汞是否干扰膜 循环（胞吐）在滑冰肝细胞，以及是否这是相关的 抑制RVD; 4. 一个长期目标是 鳐牛磺酸外排转运系统cDNA的克隆 肝细胞，并确定潜在的汞结合位点 (eg半胱氨酸残基）。 这些研究应该提供重要的洞察机制的体积 调节和牛磺酸体内平衡，以及汞如何破坏这些 基本的细胞过程 需要数量监管流程 通过所有动物细胞来平衡跨膜粘附梯度， 但对细胞或生物体的存活特别重要 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.