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

描述(申请人提供)：一个长期目标是了解钠泵(Na，K-ATPase)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中独特的形态变化相协调的。我们有数据表明，顶膜上的磷酸化事件协调了Na，K-ATPase的调节和分泌细胞形态的调节。脑脊液是由脑室中的脉络丛产生的，它通过其他结构流出。临床上，当流出通路被阻塞时，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中Na，K-ATPase活性的内源性机制，包括口渴、加压素、5-羟色胺和卡巴胆碱。这项拟议的研究有三个互补的推动力。第一个目的是利用生物化学、原代培养和细胞的遗传操作等工具来验证脉络丛Na，K-ATPase受一种辅助蛋白磷脂酰肌醇(FXYD1)的激酶调节的假说，该酶的动力学发生变化。对野生型和磷脂酶基因敲除小鼠的培养进行比较，可以将直接和间接影响分开。第二个目的是使用显微镜和生化工具来研究这种调节是否需要从微绒毛重新分布到顶下室。第三个目的是使用野生型、磷脂酶基因敲除和脑积水小鼠来验证磷脂酶是调节Na，K-ATPase和脉络膜细胞激活状态的必备环节的假设。AIMS验证了几个假说：1)通过大分子复合体，Na，K-ATPase和顶刷边缘的形态变化存在协同调节；2)Phopolemman基因敲除小鼠对抑制Na，K-ATPase和改变脉络膜细胞形态的生理信号反应缺陷；3)发生脑积水的小鼠品系表现出增强的磷脂酰化，细胞形态变化和Na，K-ATPase减少。相关性：需要更好地了解脑脊液分泌器官中的这些基本机制，以促进新的发现减少脑脊液分泌的方法，无论是替换手术分流，还是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.