NO-Zinc Signaling in Intact Pulmonary Endothelium

描述（由申请人提供）：尽管有报道称缺氧性肺血管收缩（HPV）会影响肺循环的所有部分，但对于小的（<50um）腺泡内小动脉的血管收缩知之甚少。我们的初步数据表明，缺氧诱导的NO生物合成增加导致细胞内游离锌增加，进而可能导致完整离体灌注肺（IPL）微血管收缩。由于该解剖部位主要由内皮细胞组成，其壁上有孤立或不连续的平滑肌样细胞（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。我们建议使用现代光学显微镜和荧光报告分子，包括适合于荧光共振能量转移（FRET）分析MT金属化状态的锌结合蛋白金属硫蛋白（MT）的绿色荧光蛋白（GFP）嵌合体，以促进我们首次了解细胞内锌在肺血管调节中的作用。该修订提案的具体目的是：1 .确定NO-Zn信号在介导离体肺微血管内皮细胞和平滑肌细胞在缺氧条件下收缩的内在能力中的作用，通过药理学或遗传抑制该途径的不同组分（NOS抑制、TPEN介导的Zn螯合、siRNA抑制MT表达）。2。评估NO-Zn信号在完整大鼠肺中介导HPV的作用，通过监测灌注压反应来抑制已知的NO介导的血管舒张肢体对HPV的影响，以及操纵缺氧诱导的NO介导的不稳定Zn的增加。我们将通过活体显微镜检查NO- MT- zn对TIE- 2-GFP和MT和eNOS缺失小鼠IPLs胸膜下血管收缩的作用。此外，我们将使用内皮细胞定向非病毒介导的体细胞基因转移FRET报告结构，锌敏感荧光团和光谱激光扫描共聚焦显微镜来揭示缺氧- no - mt - zn途径的要素。3。在MT +/+和MT -/-小鼠的PKC抑制背景下，使用活细胞和活体成像方法观察PKC激活、Zn稳态改变和血管反应性，研究no诱导的不稳定锌通过PKC激活调节HPV的途径。

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.