Signaling of S-nitrosoalbumin in Pulmonary Endothelium

S-亚硝基白蛋白在肺内皮细胞中的信号传导

• 批准号: 7094203
• 负责人: LI-MING ZHANG
• 金额: $ 12.66万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-12 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7094203
• 关键词: albumins; biological signal transduction; caveolas; confocal scanning microscopy; intravital microscopy; isolation perfusion; laboratory rat; lung; mass spectrometry; matrix assisted laser desorption ionization; nitroso compounds; protein transport; pulmonary circulation; tissue /cell culture; transcytosis; transfection; two dimensional gel electrophoresis; vascular endothelium; vascular endothelium permeability

DESCRIPTION (provided by applicant): The molecular determinants of albumin permeability in the vascular endothelium have profound effects on capillary solute and water exchange and delivery of bloodborne substances. Based on conventional imaging in fixed tissue and culture cells, it is apparent that a receptor (gp60) mediated transcytosis of native albumin occurs within the pulmonary circulation and this process is an important component of overall pulmonary vascular permeability. S-nitrosoalbumin (SNO-Alb) is considered as the most abundant physiological stable circulating NO carrier in human plasma with vasodilatory and antiaggregation properties similar to NO. In light of the importance of receptor mediated transcytosis of native albumin, it is noteworthy that there is no information on the disposition and effect of SNO-Alb within the pulmonary endothelium. We hypothesize that SNO-Alb will be transported by similar receptor-mediated caveolae-associated transcytosis as native albumin, and then, by serving as an intracellular source of NO, will potentially modulate endothelial cell metabolism and permeability via NO release (and activation of soluble guanylyl cyclase) and/or S(trans)-nitrosation. We propose to use a comprehensive multimode imaging approach involving full spectral confocal and multiiphoton laser scanning and total internal reflection fluorescence microscopy to determine the nature of SNO-albumin transport in live cultured rat pulmonary microvascular endothelium (RPVEC) and isolated perfused rat lungs (IPL). Accordingly, the specific aims of this proposal are to: 1} identify the cellular mechanisms by which SNO-albumin is transported across RPVEC and mechanism of transport of SNO-albumin in the intact pulmonary endothelium of IPL; 2) identify signaling pathways activated by SNO-alb transport within pulmonary endothelium; 3) Determine the effect of SNO-Alb on permeability in RPVEC and in rat IPL; 4) Determine the spectrum of proteins that are S-nitrosated by SNO-albumin via 2D gel differential fluorescence gel electrophoresis, biotin switch and MALTI TOF mass spectrometry.

描述（由申请人提供）： 血管内皮白蛋白渗透性的分子决定因素对毛细血管溶质和水交换以及血液传播物质的递送具有深远的影响。基于固定组织和培养细胞的常规成像，很明显，受体（gp 60）介导的天然白蛋白转胞吞作用发生在肺循环内，该过程是整体肺血管通透性的重要组成部分。S-亚硝基白蛋白（SNO-Alb）被认为是人体血浆中最丰富的生理稳定的循环NO载体，具有与NO相似的血管扩张和抗聚集特性。鉴于受体介导的天然白蛋白转胞吞作用的重要性，值得注意的是，没有关于SNO-Alb在肺内皮内的分布和作用的信息。我们假设SNO-Alb将通过与天然白蛋白类似的受体介导的小窝相关的转胞吞作用转运，然后通过充当NO的细胞内来源，将通过NO释放（和可溶性鸟苷酸环化酶的激活）和/或S（反式）-亚硝化潜在地调节内皮细胞代谢和渗透性。我们建议使用一个全面的多模式成像方法，包括全光谱共聚焦和多光子激光扫描和全内反射荧光显微镜，以确定在活培养的大鼠肺微血管内皮细胞（RPVEC）和离体灌注大鼠肺（IPL）的SNO-白蛋白运输的性质。因此，该提议的具体目的是：1）鉴定SNO-白蛋白转运穿过RPVEC的细胞机制和SNO-白蛋白在IPL的完整肺内皮中的转运机制; 2）鉴定肺内皮内由SNO-Alb转运激活的信号传导途径; 3）确定SNO-Alb对RPVEC和大鼠IPL中的渗透性的影响; 4）通过2D凝胶示差荧光凝胶电泳、生物素开关和MALTI TOF质谱确定SNO-白蛋白S-亚硝化的蛋白质谱。