MECHANISMS OF HEPATIC ELECTROLYTE TRANSPORT

本提案中描述的研究将调查两个相关的 关于膜离子渗透性的激素调节的假说， 和表征最近描述的Na+/HCO 3- 肝细胞共转运。 这些代表了基本的，但很差的 直接和广泛影响许多人的理解机制 肝脏的特殊功能，包括肝脏再生、胆汁分泌 形成和膜溶质运输。 拟议的研究将 使用多种方法，但强调直接测量整体 膜片钳记录细胞和单通道电流 其在这些或其它方面的应用很少 肝细胞功能 第一个假设的长期目标 是为了确定连接受体-受体相互作用的机制 调节膜K+和其他离子通道活性的变化， 具体目的是：i）评价膜电位的变化 差异和离子渗透性，其表征了 与Ca 2+动员和cAMP生成偶联的受体; ii） 确定负责cAMP和胰高血糖素的单个K+通道 K+外排的依赖性增加;评估cAM的相对作用- 依赖性蛋白激酶和GTP结合蛋白在通道激活中的作用， 并研究其抑制作用的机制， 胰岛素; iii）评估胰岛素与胰岛素之间的刺激和抑制相互作用， Ca ~（2+）和蛋白激酶C对Ca ~（2+）渗透性阳离子的调节 通道;和iv）评估肝受体的补体 由细胞外ATP激活 第二个长期目标 Na+/HCO 3-共转运在调节 膜酸/碱运输，具体目的是i）确定 Na+/HCO 3-共转运的化学计量，ii）评估 膜电位差和细胞内H、Ca 2+和激酶 作为潜在的调节因素;和iii）评估机制 其有助于膜电位的pH依赖性调节 通过测量[H+]对K+开放的影响， 等渠道 这些机制的建立有助于 直接和重要的是胆汁淤积，细胞肿胀， 伴随大多数肝脏疾病的运输和代谢紊乱。

The studies described in this proposal will investigate two related hypotheses regarding hormonal regulation of membrane ion permeability, and characterization of a recently described mechanism for Na+/HCO3- cotransport in hepatocytes. These represent fundamental yet poorly understood mechanisms that directly and broadly influence many specialized functions of the liver including gluconeogenesis, bile formation, and membrane solute transport. The proposed studies will utilize a variety of methods but emphasize direct measurement of whole cell and single channel currents with patch clamp recording techniques which have little prior application to these or other aspects of hepatocellular function. The long term objective of the first hypothesis is to define the mechanisms which link hormone-receptor interactions to regulated changes in membrane K+ and other ion channel activity, and the Specific Aims are: i) to evaluate the changes in membrane potential difference and ion permeability which characterize activation of receptors coupled to Ca2+ mobilization and cAMP generation; ii) to identify individual K+ channels responsible for cAMP- and glucagon- dependent increases in K+ efflux; assess the relative roles of cAM- dependent protein kinases and GTP-binding proteins in channel activation, and investigate the mechanisms responsible for the inhibitory effects of insulin; iii) to evaluate stimulatory and inhibitory interactions between Ca2+ and protein kinase C in regulation of Ca2+ permeable cation channels; and iv) to evaluate the complement of hepatic receptor(s) activated by extracellular ATP. The long term objective of the second hypothesis to assess the role of Na+/HCO3-cotransport in regulation of membrane acid/base transport, and the Specific Aims are i) to determine the stoichiometry of Na+/HCO3-cotransport, ii) assess the roles of membrane potential difference and of intracellular H, Ca2+, and kinases as potential regulatory factors; and iii) to evaluate the mechanisms which contribute to pHi-dependent modulation of membrane potential difference through measurement of the effects of [H+] on opening of K+ and other channels. Abnormalities of these mechanisms contribute directly and importantly to the cholestasis, cell swelling, and disordered transport and metabolism that accompany most liver diseases.