IONIC SIGNALING MECHANISMS IN HEPATOCYTES

本提案侧重于两类不同的 在肝细胞中起关键作用的离子通道 生理应激时释放的激素引起的反应， 如与肝再生有关的生长因子。 这些渠道， 主要研究者最近发现的有：（a） 钙渗透性阳离子选择性通道和（B）钾通道。 本实验室和其他实验室的观察结果支持以下观点 假设：阳离子选择性通道和钾通道是 协调调节，使得钾流出的激素激活 通过钾通道提供有利的电驱动力 通过阳离子选择性促进钙内流的激活 渠道 拟议实验的总体目标是 这是检验这一假设的第一步。 具体目标 （1）确定肝细胞阳离子- 选择性通道和钾通道被激活，（2）发展 药理学工具，以进一步阐明的生理作用， 肝细胞中的阳离子选择性通道和钾通道，和（3） 分离编码肝细胞阳离子选择性 钾离子通道和钾离子通道 本实验室的初步研究 支持实现这些具体目标的可行性。 一是这些 研究已经开始揭示通道调节的新机制， 钙和/或环AMP依赖性蛋白激酶。 其次，他们有 确定了几个候选通道阻滞剂。 第三， 证实了阳离子选择性和钾通道的激活， 钙或环AMP介导的信号通路在非洲爪蟾卵母细胞， 表达肝脏mRNA。 此外，它们还导致了肝脏的分离， 在电激发的细胞中显示出与离子通道同源性的cDNA 组织，并可能编码肝细胞阳离子选择性通道， 钾离子通道 拟议的实验将采用细胞 目前用于此的生理和分子克隆技术 实验室 这些技术方法包括测量单个 使用膜片钳记录技术的通道和全细胞电流， 用荧光法测量细胞内阳离子浓度 染料，基于与已知离子通道cDNA的同源性进行克隆，以及离子 通道在非洲爪蟾卵母细胞中的表达。 条例的定义和 肝细胞阳离子选择性通道和钾离子的药理学 通道及其cDNA的分离有望为 控制肝细胞中钙进入的新见解，钙调节 肝细胞过程，并通过扩展，肝脏的激素调节 功能 就像其他细胞类型中离子通道的特征一样 在高血压等多种疾病上取得了临床突破 和糖尿病表征的阳离子选择性通道和钾 肝细胞中的通道可能最终允许开发新的 治疗伴随肝病的代谢紊乱。

This proposal focuses on the characterization of two distinct classes of ion channels that are positioned to play pivotal roles in hepatocellular responses evoked by hormones released during physiological stress as well as by growth factors linked to liver regeneration. These channels, which have been recently identified by the principal investigator, are: (a) calcium-permeable cation-selective channels and (b) potassium channels. Observations in this laboratory and by others support the following hypothesis: Cation-selective channels and potassium channels are coordinately regulated such that hormonal activation of potassium efflux through potassium channels provides favorable electrical driving forces that facilitate activation of calcium influx through cation-selective channels. The overall objectives of the proposed experiments represent a necessary first step toward testing this hypothesis. The specific aims are: (1) to determine the mechanisms by which hepatocellular cation- selective channels and potassium channels are activated, (2) to develop pharmacological tools to further elucidate the physiological roles of cation-selective channels and potassium channels in hepatocytes, and (3) to isolate functional cDNAs encoding hepatocellular cation-selective channels and potassium channels. Preliminary studies in this laboratory support the feasibility of achieving these specific aims. First, these studies have begun to reveal novel mechanisms of channel regulation by calcium and/or cyclic AMP-dependent protein kinase. Second they have identified several candidate channel blockers. Third, they have demonstrated activation of cation-selective and potassium channels by calcium or cyclic AMP-mediated signaling pathways in Xenopus oocytes that express liver mRNA. Moreover, they have led to the isolation of liver cDNAs that exhibit homology to ion channels in electrically excitable tissues and may encode hepatocellular cation-selective channels and potassium channels. The proposed experiments will employ cell physiological and molecular cloning techniques currently used in this laboratory. These technical approaches include measurements of single channel and whole cell currents using patch clamp recording techniques, measurements of intracellular cation concentrations using fluorescent dyes, cloning based on homology to known ion channel cDNAs, and ion channel expression in Xenopus oocytes. Definition of the regulation and pharmacology of hepatocellular cation-selective channels and potassium channels and isolation of their cDNAs are expected to provide a basis for new insights into control calcium entry in hepatocytes, calcium-regulated hepatocellular processes, and by extension, hormonal regulation of liver function. Just as characterization of ion channels in other cell types has led to clinical breakthroughs in diseases as diverse as hypertension and diabetes characterization of cation-selective channels and potassium channels in hepatocytes may ultimately permit the development of novel therapies for the metabolic derangements that attend liver disease.