REGULATION OF SODIUM PUMPS IN THE KIDNEY

Na，K-ATP酶是一种重要的膜蛋白， 在所有动物细胞中发现， 细胞外K+偶联ATP水解。 Na，K-的调节 ATP酶是细胞容积、液体和电解质所必需的 稳态，电兴奋性，并驱动Na+耦合共- 营养物质的运输和阳离子的反向运输。 的 本提案的总体目标是了解钠泵 使用肾细胞系调节肾中的代谢 （MDCK）和近端小管的原代培养物。 我们已经表明 低K+处理和Na+/H+交换激活 增加Na，K-ATP酶的合成， 转录）;以及甲状腺激素（T3） 调节近曲小管细胞Na，K-ATP酶的合成。 的 第一个目的是确定T3和离子刺激是否直接作用或 间接：确定T3诱导Na，K-ATP酶合成是否 其次是离子通量的变化，通过比较 离子通量的变化与α和β mRNA的变化 T3之后;并确定离子和T3是否增加mRNA 直接或间接地通过翻译 抑制剂的 第二个目的是确定Na，K-ATPase中的步骤 由离子和T3调节的代谢：比较亚基的变化 mRNA水平对肽合成速率的变化， 丰度和Na，K-ATP酶活性，以确定是否额外 涉及翻译前、共或翻译后调节步骤。 的 第三个目的是鉴定离子/T3敏感的调节区域， Na，K-ATP酶亚基基因：确定上游区域 从阿尔法和贝塔结构基因能够 当放置在一个离子/T3调节器的上游时， 通常不受信号调节的异源基因;和 确定调控序列是否含有结合位点， 假定的反式作用调节因子。 第四个目标是 确定（Na+）或（H+）是否是调节Na，K-ATP酶的信号 通过检测细胞内Na+和H+的变化， 独立地增加Na，K-ATP酶亚基的合成。 实现这些目标将有助于解释 调节肾脏Na，K-ATP酶表达的机制， 可能影响对体液和电解质失衡的理解 在疾病状态下。

Na,K-ATPase, the sodium pump, is an integral membrane protein found in all animal cells which exchanges intracellular Na+ for extracellular K+ coupled to ATP hydrolysis. Regulation of Na,K- ATPase is necessary for cell volume, fluid and electrolyte homeostasis, electrical excitability, and to drive Na+ coupled co- transport of nutrients and countertransport of cations. The overall aim of this proposal is to understand how sodium pump metabolism is regulated in the kidney using a kidney cell line (MDCK) and primary cultures of proximal tubule. We have shown that low K+ treatment and activation of Na+/H+ exchange increases Na,K-ATPase synthesis pretranslationally (at transcription) in MDCK cells; and that thyroid hormone (T3) regulates Na,K-ATPase synthesis in proximal tubule cells. The first aim is to determine if T3 and ionic stimuli act directly or indirectly: determine if T3 induction of Na,K-ATPase synthesis is secondary to changes in ion fluxes by comparing the timecourse of change in ion fluxes to changes in alpha and beta mRNAs following T3; and determine if the ions and T3 increase mRNAs directly or secondary to an intermediary by employing translation inhibitors. The second aim is to identify steps in Na,K-ATPase metabolism regulated by ions and T3: compare changes in subunit mRNA levels to changes in peptide synthesis rates, peptide abundance and Na,K-ATPase activity to determine if additional pre-, co-, or post-translational regulatory steps are involved. The third aim is to identify the ion/T3 sensitive regulatory regions of the Na,K-ATPase subunit genes: determine if regions upstream from the alpha and beta structural genes are capable of conferring ionic/T3 regulation when placed upstream from a heterologous gene not normally regulated by the signals; and determine if the regulatory sequences contain binding sites for putative trans-acting regulatory factors. The fourth aim is to determine if (Na+) or (H+) are signals that regulate Na,K-ATPase expression by examining if changing intracellular Na+ and H+ independently increases synthesis of Na,K-ATPase subunits. Accomplishing these aims will contribute to an explanation of the mechanisms regulating Na,K-ATPase expression in the kidney and may impact the understanding of fluid and electrolyte imbalance in disease states.