NO-Zinc Signaling in Intact Pulmonary Endothelium

描述（由申请人提供）：尽管据报道缺氧性肺血管收缩（HPV）影响肺循环的所有部分，但对腺泡内小动脉（<50 μ m）的血管收缩知之甚少。我们的初步数据表明，缺氧诱导的NO生物合成的增加导致增强的细胞内游离锌，这反过来可能有助于在完整的离体灌注肺（IPL）的微血管的血管收缩。由于该解剖部位主要由内皮细胞组成，其壁中有孤立或不连续的平滑肌样细胞（例如周细胞），因此微循环内收缩事件的性质可能与近端肺血管的血管调节不同。我们建议使用当代的光学显微镜和荧光报告分子，包括一个绿色荧光蛋白（GFP）嵌合体的锌结合蛋白金属硫蛋白（MT）适合的荧光共振能量转移（FRET）分析的代谢状态的MT，以促进我们的第一次深入了解细胞内锌在肺血管调节中的作用。本修正提案的具体目标是：确定NO-Zn信号传导在介导分离的肺微血管内皮细胞和平滑肌细胞在缺氧中收缩的内在能力中的作用，通过药理学或遗传学抑制途径的不同组分（NOS抑制、TPEN介导的Zn螯合、MT表达的siRNA抑制）。二.通过监测灌注压对抑制已知的NO介导的对HPV的作用的血管舒张分支的反应和操纵缺氧诱导的NO介导的不稳定Zn的增加，评估NO-Zn信号传导在介导完整大鼠肺中的HPV中的作用。我们将通过来自TIE- 2-GFP和MT和eNOS无效小鼠的IPL的胸膜下血管的活体显微镜检查来检查NO-MT-Zn对微循环收缩的作用。此外，我们将使用内皮细胞定向的非病毒介导的FRET报告构建体的体细胞基因转移，和锌敏感的荧光团，然后通过光谱激光扫描共聚焦显微镜来揭示缺氧-NO-MT-Zn途径的元素。三.研究NO诱导的不稳定锌变化通过激活PKC调节HPV的途径，使用活细胞和活体成像方法观察MT +/+和MT -/-小鼠中PKC激活、锌稳态改变和PKC抑制背景下的血管反应性。

DESCRIPTION (provided by applicant): Although hypoxic pulmonary vasoconstriction (HPV) has been reported to affect all segments of the pulmonary circulation relatively little is known regarding vasoconstriction of small (<50um) intra-acinar arterioles. Our preliminary data suggest that hypoxic induced increases in NO biosynthesis result in enhanced intracellular free zinc that in turn may contribute to vasoconstriction of microvessels in the intact isolated perfused lung (IPL). Since this anatomic site is composed primarily of endothelial cells with solitary or discontinuous smooth muscle like cells (e.g. pericytes) in their wall, the nature of contractile events within the microcirculation are likely to be distinct from vasoregulation of proximal pulmonary vessels. We propose to use contemporary optical microscopy and fluorescent reporter molecules, including a green fluorescent protein (GFP) chimera of the zinc-binding protein metallothionein (MT) suitable for fluorescence resonance energy transfer (FRET) analysis of the metallation status of MT, to facilitate our first insights into the role of intracellular zinc in pulmonary vasoregulation. The specific aims of this amended proposal are to: I. Determine the role of NO-Zn signaling in mediating the intrinsic capacity of isolated pulmonary microvascular endothelial and smooth muscle cells to constrict in hypoxia by pharmacological or genetic inhibition of the distinct components of the pathway (NOS inhibition, TPEN mediated Zn chelation, siRNA inhibition of MT expression). II. Assess the role of NO-Zn signaling in mediating HPV in the intact rat lung by monitoring perfusion pressure responses to inhibition of the known vasodilatory limbs of NO mediated effects on HPV and manipulation of hypoxia-induced, NO-mediated increases in labile Zn. We will examine the role of NO- MT-Zn on constriction of the microcirculation via intravital microscopy of subpleural vessels of IPLs from TIE- 2-GFP and MT and eNOS null mice. Furthermore, we will use endothelial cell directed non-viral mediated somatic gene transfer of FRET reporter constructs, and zinc-sensitive fluorophores followed by spectral laser scanning confocal microscopy to reveal elements of hypoxia-NO-MT-Zn pathway. III. Investigate a pathway by which NO-induced changes in labile zinc modulate HPV via activation of PKC using live cell and intravital imaging approaches to visualize PKC activation, alterations in Zn homeostasis, and vascular reactivity against a background of PKC inhibition in MT +/+ and MT -/- mice.