Mechanisms of Hepatic Electrolyte Transport

描述（由申请方提供）：本提案中描述的研究阐述了负责快速调节肝细胞离子转运以满足不断变化的生理需求的细胞机制。 在肝细胞中，细胞代谢、细胞体积和离子转运之间的动态相互作用在调节对循环激素、底物可用性和代谢刺激的反应中起关键作用。 先前的研究表明，这种反应在很大程度上是由通道介导的Na+内流（其驱动细胞体积增加）与K+和Cl-外流（其导致细胞体积减小）之间的平衡介导的。 有趣的是，初步观察表明：i）离子通道蛋白的分布在质膜和细胞内膜（囊泡）地址之间受到仔细调节，和ii）含有通道的囊泡的不同池可以被快速和选择性地动员以形成质膜的导电性质。 事实上，10，000个或更多个囊泡的胞吐对于细胞对激素的反应是必不可少的，并且足以在几分钟内替换20-40%的质膜。 因此，特定目的旨在解决以下工作假设：含通道囊泡的不同池的调节插入和回收提供了一种快速调节肝细胞转运能力的机制，以响应不断变化的生理需求，并代表了激素和其他因子的重要作用位点，这些因子利用细胞体积作为调节细胞代谢和转运的中间信号。 具体目的集中于1）评价非选择性阳离子（NSC）通道作为容量调节Na +内流的主要机制，2）表征将容量敏感性信号传导与含通道囊泡的不同池的快速胞吐作用耦合的细胞信号，和3）评估编码环核苷酸门控（CNG）阳离子通道的cDNA作为肝细胞中NSC传导的分子候选物。 细胞代谢、离子通道和细胞体积之间的界面代表了肝脏转运、分泌和胆汁形成的生理调节的新的和强大的作用位点;以及开发调节肝细胞和器官功能的药理学方法的重要靶点。

DESCRIPTION (provided by applicant): The studies described in this proposal address the cellular mechanisms responsible for rapid modulation of hepatocyte ion transport to meet changing physiologic demands. In liver cells, dynamic interactions between cell metabolism, cell volume and ion transport play a key role in regulation of the response to circulating hormones, substrate availability, and metabolic stimuli. Previous studies indicate that this response is mediated in large part by a balance between channel-mediated Na+ influx, which drives cell volume increases, versus reciprocal K+ and Cl- efflux, which are responsible for cell volume decreases. Interestingly, preliminary observations indicate that; i) the distribution of ion channel proteins is carefully regulated between plasma membrane and intracellular membrane (vesicular) addresses, and ii) distinct pools of channel-containing vesicles can be mobilized rapidly and selectively to shape the conductive properties of the plasma membrane. Indeed, exocytosis of 10,000 or more vesicles is essential for the cellular response to hormones, and is sufficient to replace 20-40% of the plasma membrane within minutes. Accordingly, the Specific Aims are designed to address the following working hypothesis: regulated insertion and retrieval of distinct pools of channel-containing vesicles provides a mechanism for rapid modulation of the transport capacity of hepatocytes in response to changing physiologic demands, and represents an important site of action for hormones and other factors that utilize cell volume as an intermediary signal regulating cell metabolism and transport. The Specific Aims focus on 1) evaluation of non-selective cation (NSC) channels as the dominant mechanism for volume-regulatory Na + influx, 2) characterization of the cellular signals that couple volume-sensitive signaling to rapid exocytosis of distinct pools of channel-containing vesicles, and 3) assessment of a cDNA encoding cyclic nucleotide-gated (CNG) cation channels as a molecular candidate for the NSC conductance in liver cells. The interface between cell metabolism, ion channels, and cell volume represents a novel and powerful site of action for physiologic modulation of hepatic transport, secretion, and bile formation; and an important target for development of pharmacological approaches to modulation of liver cell and organ function.