MECHANISMS OF HEPATIC ELECTROLYTE TRANSPORT

本提案中描述的研究将调查两个相关的 关于激素调节膜K+渗透性的假说;以及 最近描述的内向Na+耦合机制的表征 肝细胞中的HCO 3-转运。 这些代表了基本的，但很差的 理解直接和广泛影响专业化的机制， 肝脏的功能，包括肝细胞生成、小管胆汁 形成和膜溶质运输。 拟议的研究将 利用多种方法，但强调直接测量整个细胞 和单通道电流与膜片钳记录技术， 以前很少应用于这些或其他方面， 肝细胞功能 第一个假设的长期目标 是为了确定某种特定的受体 细胞表面的相互作用与膜K+（和其他）的变化 渠道活动，具体目标是：i）进一步评估 cAMP类似物激活特异性膜K+电流的机制 特别强调调节K+磷酸化的作用 通过cAMP依赖性蛋白激酶（PKA）的通道; ii）以确定是否 胰高血糖素和其它与腺苷酸环化酶连接的激素激活膜 K+电流只通过cAMP或是否cAMP独立机制 （三）调查造成这一现象的机制; 胰岛素的抑制作用和钙离子的调节作用 和/或Ca 2+依赖性激酶对K+电流的影响。 的长期目标 第二个假设是确定 膜H+ HCO 3-转运a它们与维持细胞内 pH和胆汁HCO 3分泌。 具体目标是：i）确定 内向Na+-耦合HCO 3-转运的化学计量学和调节 膜电位差对HCO 3-流入影响; ii） 研究这种机制可能的变构调节， 细胞内H+、激酶和某些胆汁酸;和iii）确定 功能意义和机制负责抑制 膜K+通道的H+。 这些肝细胞凋亡机制的研究 电解质转运直接导致胆汁淤积，细胞 肿胀，以及伴随大多数疾病的运输和代谢紊乱 肝脏疾病

The studies described in this proposal will investigate two related hypotheses regarding hormonal regulation of membrane K+ permeability; and characterization of a recently described mechanism for inward Na+-coupled HCO3-transport in hepatocytes. These represent fundamental yet poorly understood mechanisms that directly and broadly influence specialized functions of the liver including gluconeogenesis, canalicular 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 had 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 certain hormone-receptor interactions at the cell surface to changes in membrane K+ (and other) channel activity, and the specific aims are: i) to further evaluate the mechanisms for activation of specific membrane K+ currents by cAMP analogs with particular emphasis on the role of regulatory phosphorylation of K+ channels by cAMP-dependent protein kinases (PKA); ii) to determine whether glucagon and other hormones linked to adenylate cyclase activate membrane K+ currents exclusively through cAMP or whether cAMP-independent mechanisms are also involved; and iii) to investigate the mechanisms responsible for the inhibitory effects of insulin; and the modulatory effects of Ca2+ and/or Ca2+ -dependent kinases on K+ currents. The long term objective of the second hypothesis is to determine the mechanisms responsible for membrane H+HCO3- transport a they pertain to maintenance of intracellular pH and biliary HCO3- secretion. The specific aims are i) to determine the stoichiometry of inward Na+-coupled HCO3- transport and the regulatory influence of membrane potential difference on HCO3- influx; ii) to investigate possible allosteric regulation of this mechanism by intracellular H+, kinases, and certain bile acids; and iii) to determine the functional significance and mechanisms responsible for inhibition of membrane K+ channels by H+. Abnormalities of these mechanisms of hepatic electrolyte transport directly contribute to the cholestasis, cell swelling, and disordered transport and metabolism that accompanies most liver disease.