Signaling of S-nitrosoalbumin in Pulmonary Endothelium

描述(由申请人提供)： 白蛋白在血管内皮细胞中通透性的分子决定因素对毛细血管溶质和水交换以及血液物质的输送有着深远的影响。基于固定组织和培养细胞的常规成像，很明显，受体(Gp60)介导的天然白蛋白在肺循环中发生跨细胞作用，这一过程是整个肺血管通透性的重要组成部分。S-亚硝酸白蛋白(SNO-Alb)被认为是人体血浆中含量最丰富、最稳定的循环NO载体，具有与NO相似的血管扩张和抗聚集作用。鉴于受体介导的天然白蛋白跨细胞作用的重要性，值得注意的是，目前还没有关于SNO-Alb在肺内皮细胞内的分布和作用的信息。我们推测，SNO-Alb将通过与天然白蛋白类似的受体介导的小窝相关的细胞转运，然后作为NO的胞内来源，可能通过NO释放(和激活可溶性鸟苷酸环酶)和/或S(反式)亚硝化来调节内皮细胞的代谢和通透性。我们建议使用全光谱共聚焦和多光子激光扫描和全内反射荧光显微镜的综合多模成像方法来确定活体培养的大鼠肺微血管内皮细胞(RPVEC)和分离的灌流大鼠肺(IPL)中SNO-白蛋白转运的性质。因此，这项建议的具体目的是：1)确定SNO-白蛋白通过RPVEC转运的细胞机制以及SNO-白蛋白在IPL完整肺内皮细胞中的转运机制；2)确定SNO-Alb在肺内皮细胞内转运所激活的信号通路；3)确定SNO-Alb对RPVEC和大鼠肺上皮细胞通透性的影响；4)通过2D凝胶差示荧光凝胶电泳、生物素开关和MATI TOF质谱分析，确定SNO-白蛋白经SNO-Alb亚硝化后的蛋白质谱。

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.