Regulation of aquaporin-2 trafficking in collecting duct

集合管中水通道蛋白 2 运输的调节

• 批准号: 6804110
• 负责人: Kay-Pong Daniel Yip
• 金额: $ 16.69万
• 依托单位: UNIVERSITY OF SOUTH FLORIDA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-20 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6804110
• 关键词: adrenal medulla; antibody; arginine vasopressin; atrial natriuretic peptide; calcium channel; calcium ion; confocal scanning microscopy; cyclic AMP; enzyme activity; exocytosis; flash photolysis; fluorescence microscopy; fluorescence resonance energy transfer; guanine nucleotide exchange factors; intracellular transport; ion transport; laboratory rat; lasers; myosin light chain kinase; photoactivation; protein kinase A; protein protein interaction; protein transport; ryanodine; water channel; water solubility

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）：这项拟议的研究的长期目标是描述在正常和病理生理条件下肾脏收集导管中Aquaporin-2（AQP2）运输的机制和调节。抗利尿激素（精氨酸加压素，AVP）调节肾脏收集管道的渗透性水渗透性（PF），从而赋予了水的肾脏排泄的精确控制。 AVP通过触发含有囊泡的细胞质AQP2的易位和融合来刺激PF收集导管。通过使用Styryl Dye FM1-43作为质膜标记来开发共斑影像技术，以监测胞吐作用，荧光磺酸盐作为体积标记，以监测PF和Fluo-4作为细胞内Ca 2+浓度（[CA2+] I）在灌注灌注内髓内收集的ca2+Inducs（Induce）（I can）在单个IMCD细胞中，AVP诱导的顶胞外增生和PF的增加依赖于Ca 2+。 AVP诱导的Ca 2+动员是cAMP依赖性的，但对蛋白激酶A的抑制剂不敏感，该抑制剂已知可抑制AQP-2磷酸化。心房纳地纳特因子（ANF）的生理剂量抑制了AVP刺激的PF和[Ca2+] I振荡。 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诱导的胞吐作用，（4）确定加压素和心房脂肪肽之间在调节[Ca2+] I和AQP2胞胞菌病之间的相互作用。所有提出的研究将在灌注IMCD的活细胞中进行。通过FM1-43荧光监测顶端胞吐作用。通过腔内灌注中包含的荧光磺酸盐的发射来监测PF。 UV脉冲激光器用于通过光活化细胞内Ca2+螯合剂或笼子信号分子的光闪光分析来操纵[Ca2+] i，而无需中断图像采集。可逆的透化过程用于递送抗体，抑制性肽和不渗透的笼子化合物进入IMCD细胞。将采用AQP2与荧光显微镜的互补免疫定位。这项拟议的研究将提供有关AQP2在灌注IMCD完整细胞中插入和运输的机理和调节的新信息。这些可能会更好地了解肾脏收集管道在水平衡障碍中的作用。