Cellular/molecular Na,K-ATPase regulation in choroid plexus

描述（由申请人提供）：一个长期目标是了解钠泵（Na，K-ATP酶）调节的生化和细胞机制，钠泵是脑脊液（CSF）分泌的驱动力。另一个是了解这种调节是如何与分泌上皮独特的形态学变化相协调的。我们有数据表明，在顶端膜的磷酸化事件协调与分泌细胞形态的调节Na，K-ATP酶的调节。脑脊液是由脑室中的脉络丛产生的，并通过其他结构流出。在临床上，当流出途径被阻断时，会导致脑积水（婴儿）或颅内高压（成人）。我们将研究降低脉络丛Na，K-ATP酶活性的内源性机制，包括口渴、加压素、5-羟色胺和卡巴胆碱。拟议的研究有三个互补的重点。第一个目标使用生物化学，原代培养和细胞遗传操作的工具来测试脉络丛Na，K-ATP酶受激酶介导的辅助蛋白磷酸化磷脂酶（FXYD 1）的调节，并改变酶的动力学的假设。比较野生型和磷莱曼基因敲除小鼠的培养物，可以将直接和间接效应分开。第二个目的是使用显微镜和生物化学的工具，以调查是否需要重新分配从微绒毛到根尖下室的调节。第三个目的是使用野生型，磷脂酶原基因敲除，和脑积水的小鼠来测试的假设，磷脂酶原是一个强制性的联系，以调节Na，K-ATP酶和脉络膜细胞活化状态。本研究旨在验证以下假说：1）Na，K-ATP酶和脉络膜细胞形态学改变通过大分子复合物协同调节：2）Phospholemman基因敲除小鼠对抑制Na，K-ATP酶和改变脉络膜细胞形态学的生理信号的反应缺陷;和3）发展脑积水的小鼠品系显示增强的磷膜蛋白磷酸化、增加的细胞形态变化和Na，K-ATP酶的减少。相关性：需要更好地了解CSF分泌器官中的这些基本机制，以促进减少CSF分泌的新方法的发现，以取代手术分流或至少在手术前稳定脆弱的婴儿。

DESCRIPTION (provided by applicant): One long term objective is to understand the biochemical and cellular mechanisms of the regulation of the sodium pump (Na,K-ATPase), which is the driving force for cerebrospinal fluid (CSF) secretion. The other is to understand how that regulation is coordinated with the unique morphological changes in the secretory epithelium. We have data that indicate that phosphorylation events at the apical membrane coordinate the regulation of Na,K-ATPase with the regulation of secretory cell morphology. CSF is produced by the choroid plexuses in the brain's ventricles, and it flows out through other structures. Clinically when the outflow pathways are blocked, hydrocephalus (infants) or intracranial hypertension (adults) results. There are endogenous mechanisms that reduce Na,K-ATPase activity in the choroid plexus that we will investigate, including thirst, vasopressin, serotonin, and carbachol. The proposed research has three complementary thrusts. The first aim uses the tools of biochemistry, primary cultures, and genetic manipulation of cells to test the hypothesis that choroid plexus Na,K-ATPase is regulated by kinase-mediated phosphorylation of an accessory protein, phospholemman (FXYD1), with a change in the kinetics of the enzyme. Comparison of cultures from wild type and phospholemman knockout mice will allow direct and indirect effects to be separated. The second aim uses the tools of microscopy and biochemistry to investigate whether the regulation entails redistribution from microvilli to a subapical compartment. The third aim uses wild type, phospholemman knockout, and hydrocephalic mice to test the hypothesis that phospholemman is an obligatory link to regulation of both Na,K-ATPase and choroid cell activation state. The aims test several hypotheses: 1) that there is concerted regulation of Na,K-ATPase and morphological change at the apical brush border through macromolecular complexes; 2) that the phospholemman knockout mouse has defective responses to physiological signals to inhibit Na,K-ATPase and change choroid cell morphology; and 3) that a mouse strain that develops hydrocephalus shows enhanced phospholemman phosphorylation, increased cell morphology changes, and reductions in Na,K-ATPase. Relevance: A better understanding of these basic mechanisms in the CSF-secreting organ is needed to facilitate new discoveries of ways to reduce CSF secretion, either to replace surgical shunts or to at least stabilize fragile infants prior to surgery.