Regulation of aquaporin-2 trafficking in collecting duct

集合管中水通道蛋白 2 运输的调控

基本信息

批准号：
7092015
负责人：
Kay-Pong Daniel Yip
金额：
$ 16.3万
依托单位：
UNIVERSITY OF SOUTH FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-09-20 至 2008-06-30
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): The long term goals of this proposed study are to delineate the mechanisms and regulation of aquaporin-2 (AQP2) trafficking in kidney collecting duct at normal and pathophysiological conditions. The antidiuretic hormone (arginine vasopressin, AVP) regulates osmotic water permeability (Pf) of kidney collecting duct, conferring the precise control of renal excretion of water. AVP stimulates Pf in collecting duct by triggering the translocation and fusion of cytoplasmic AQP2 containing vesicles to apical membrane of principal cells. By developing confocal imaging techniques using styryl dye FM1-43 as a plasma membrane marker to monitor exocytosis, fluorescien sulfonate as volume marker to monitor Pf and fluo-4 as intracellular Ca 2+ concentration ([Ca2+]i) marker in perfused rat inner medullary collecting duct (IMCD), it was found that AVP induces [Ca2+]i oscillations in individual IMCD cells, and that AVP induced apical exocytosis and increase of Pf are Ca 2+ dependent. AVP induced Ca 2+ mobilization is cAMP-dependent but is not sensitive to protein kinase A inhibitor at a dose known to inhibit AQP-2 phosphorylation. Physiological dose of atrial natriuretic factor (ANF) inhibits AVP stimulated Pf and [Ca2+]i oscillations. The aims of this proposal are (1) to test whether AQP2 exocytosis is coupled to intracellular Ca2+ mobilization, (2) to test whether cAMP directly involved in Ca2+ mobilization and AQP2 exocytosis via cAMP-guanine-nucleotide-exchange factors (3) to elucidate the subcellular mechanisms and the physiological significance of [Ca2+]i oscillations in AVP induced exocytosis, (4) to determine the interactions between vasopressin and atrial natriuretic peptide in regulating [Ca2+]i and AQP2 exocytosis. All proposed studies will be conducted in live cells of perfused IMCD. Apical exocytosis is monitored by FM1-43 fluorescence. Pf is monitored by the emission of fluorescien sulfonate included in the luminal perfused. An UV pulse laser is used to manipulate [Ca2+]i via flash photolysis of photoactivatable intracellular Ca2+ chelators or caged signaling molecules without interrupting image acquisition. A reversible permeabilization procedure is used to delivery antibodies, inhibitory peptides, and impermeant caged compounds into IMCD cells. Complementary immunolocalization of AQP2 with fluorescence microscopy will be employed. This proposed study will provide new information on the mechanisms and regulation of the insertion and trafficking of AQP2 in intact cell of perfused IMCD. These may lead a better understand of the role of kidney collecting duct in water balance disorders.

描述（由申请人提供）：本研究的长期目标是阐明正常和病理生理条件下肾集合管中水通道蛋白 2 (AQP2) 运输的机制和调节。抗利尿激素（精氨酸加压素，AVP）调节肾集合管的渗透水渗透率（Pf），从而精确控制肾脏的水排泄。 AVP 通过触发含有 AQP2 的细胞质囊泡与主细胞顶膜的易位和融合来刺激集合管中的 Pf。通过开发共聚焦成像技术，使用苯乙烯基染料 FM1-43 作为质膜标记物来监测胞吐作用，荧光磺酸盐作为体积标记物来监测 Pf 和 Fluo-4 作为灌注大鼠内髓中的细胞内 Ca 2+ 浓度 ([Ca2+]i) 标记物集合管（IMCD）中，发现AVP诱导单个IMCD细胞中的[Ca2+]i振荡，并且AVP诱导顶端胞吐作用和 Pf 的增加是 Ca 2+ 依赖性的。 AVP 诱导的 Ca 2+ 动员是 cAMP 依赖性的，但对已知抑制 AQP-2 磷酸化剂量的蛋白激酶 A 抑制剂不敏感。生理剂量的心房钠尿因子 (ANF) 抑制 AVP 刺激的 Pf 和 [Ca2+]i 振荡。该提案的目的是 (1) 测试 AQP2 胞吐作用是否与细胞内 Ca2+ 动员相关，(2) 测试 cAMP 是否直接参与 Ca2+ 动员和通过 cAMP-鸟嘌呤核苷酸交换因子参与 AQP2 胞吐作用 (3) 阐明AVP 诱导的胞吐作用中 [Ca2+]i 振荡的亚细胞机制和生理意义，(4) 确定之间的相互作用加压素和心房钠尿肽调节 [Ca2+]i 和 AQP2 胞吐作用。所有拟议的研究都将在灌注 IMCD 的活细胞中进行。通过 FM1-43 荧光监测顶端胞吐作用。 Pf 通过管腔灌注中包含的荧光磺酸盐的发射来监测。 UV 脉冲激光用于通过可光激活的细胞内 Ca2+ 螯合剂或笼状信号分子的闪光光解来操纵 [Ca2+]i，而无需中断图像采集。可逆透化程序用于将抗体、抑制肽和不透性的笼状化合物递送至 IMCD 细胞中。将采用荧光显微镜对 AQP2 进行补充免疫定位。这项拟议的研究将为灌注 IMCD 的完整细胞中 AQP2 的插入和运输的机制和调节提供新的信息。这些可能有助于更好地了解肾集合管在水平衡紊乱中的作用。