Regulation of a Novel Intestinal NHE Isoform (NHE8)

描述（由申请人提供）：胃肠道在成熟过程中经历显著的形态和功能变化，以满足日益增长的营养转运需求。胃肠道的主要功能是运输营养物质和液体，以维持足够的电解质稳态。1岁婴儿的胃肠道每天处理约3升液体，导致每天仅排出50克粪便，表明早期生命期间胃肠道的复杂运输能力。我们以前的研究表明，肠道钠/氢交换器（NHE 2和NHE 3）的表达在生命早期是低的，并随着成熟显着增加。此外，NHE 2和NHE 3的敲除小鼠模型的表型显示液体和电解质平衡的最小扰动，表明在此成熟期存在另一种NHE。我们的研究表明，NHE 8在胃肠道上皮细胞的顶膜中表达，在生命早期处于高水平，并在成年后逐渐降低。这一定位与NHE 8在肾上皮顶端表达的发现相似。因此，拟定的研究旨在检验以下假设：NHE 8代表一种独特的新型NHE，其在哺乳和断奶发育期间的电解质稳态中非常重要。我们计划探索五个具体目标，以确定NHE 8的生理作用及其在生理和病理条件下的调节。具体目标1旨在表征NHE 8的功能和药理学特性。我们的第二个具体目标是确定NHE 8的表达沿着大鼠肠道和NHE 2/3基因敲除小鼠的头尾轴和隐窝绒毛轴。我们还将寻求进一步加强我们的假设，即NHE 8表达在肠上皮细胞的刷状缘膜上。在具体目标3中，我们计划研究在基础条件下以及受EGF和糖皮质激素调节的NHE 8调控的转录机制。我们的初步数据表明，NHE 8基因的表达是由这些生理效应。EGF和糖皮质激素对于胃肠道的功能成熟是重要的，并且已经显示出调节肠NHE。此外，已知的肠道NHE在炎症期间受到调节，并且我们计划对我们的初步观察进行后续研究，即NHE 8在内毒素血症（例如LPS施用）期间和通过TNF α暴露受到调节。因此，我们的具体目标4旨在确定TNF α对NHES蛋白和基因表达的急性和慢性影响的机制。具体目标5旨在定义第二信使（环核苷酸和细胞内钙）对NHES进行急性调节的机制。总体而言，拟议的研究可能会对我们理解控制生命早期电中性NaCl吸收的分子机制及其与肠道内稳态各种扰动的关系产生重大影响。

DESCRIPTION (provided by applicant): The gastrointestinal tract undergoes significant morphological and functional changes during maturation to meet the increasing demands for nutrient transport. The major function of the gastrointestinal tract is to transport nutrients and fluids to maintain adequate electrolyte homeostasis. The gastrointestinal tract of a 1- year-old infant handles approximately 3 liters of fluid per day, resulting in only 50 grams of daily stool output, indicating the complex transport capacity of the gastrointestinal tract during early life. Our previous studies have shown that expression of the intestinal sodium/hydrogen exchangers (NHE2 and NHE3) is low during early life, and increases significantly with maturation. Moreover, the phenotypes of knockout mouse models of NHE2 and NHE3 show minimal perturbations in fluid and electrolyte balance, indicating the presence of another NHE(s) during this period of maturation. Our study demonstrates that NHE8 is expressed in the apical membrane of epithelial cells in the gastrointestinal tract, at high levels during early life, and decreasing into adulthood. This localization is similar to the finding that NHE8 is expressed apically in the renal epithelium. Therefore, the proposed studies are designed to test the hypothesis that NHE8 represents a unique, novel NHE, which is important in electrolyte homeostasis during the suckling and weaning periods of development. We plan to explore five specific aims to determine the physiological role of NHE8 and its regulation in physiological and pathological conditions. Specific Aim 1 is designed to characterize functional and pharmacological properties of NHE8. Our second Specific Aim is designed to determine NHE8 expression along the cephalo-caudal and crypt-villus axes in the rat intestine and in NHE2/3 knock-out mice. We will also seek to further strengthen our supposition that NHE8 is expressed on the brush-border membrane of intestinal epithelial cells. In Specific Aim 3, we plan to investigate the transcriptional mechanisms of NHE8 regulation under basal conditions and as regulated by EGF and glucocorticoids. Our preliminary data show that NHE8 gene expression is regulated by these physiological effectors. EGF and glucocorticoids are important for functional maturation of the gastrointestinal tract, and have been shown to regulate intestinal NHEs. Furthermore, known intestinal NHEs are regulated during inflammation and we plan to follow up on our preliminary observation that NHE8 is regulated during endotoxemia (e.g. LPS administration) and by TNFa exposure. Therefore our Specific Aim 4 is designed to determine the mechanism responsible for acute and chronic effects of TNFa on NHES protein and gene expression. Specific Aim 5 is designed to define the mechanisms involved in the acute regulation of NHES by second messengers (cyclic nucleotides and intracellular calcium). Overall, the proposed studies are likely to have a significant impact on our understanding of the molecular mechanisms controlling electroneutral NaCI absorption in early life and its relationship to various perturbations of intestinal homeostasis.