Nitric Oxide Interaction with Red Blood Cells

说明(申请人提供)：一氧化氮(NO)通过其合成和失活之间的平衡来实现其生物学功能。NO的生物合成受到高度调控，并且有很好的文献记载，而对其失活的了解要少得多。NO失活的主要途径包括与含氧血红蛋白[HbFe(11)O2]的反应和各种自由基。传统观点认为，HbFe(11)O2对NO的失活不受调控，因为人们认为NO可以自由快速地扩散到红细胞膜上。最近的研究表明，NO进入红细胞的转运是由膜骨架蛋白控制的。红细胞消耗NO的速率可以通过细胞骨架结合蛋白(如Band 3)干扰细胞骨架网络来调节。亚硝酸铁-血红蛋白[HbFe(11)NO](-0.1%)的形成增加了NO的消耗率。这一调节因子具有重要的生理和病理意义，因为HbFe(11)NO是在缺氧过程中形成的，并已在各种条件下在人类体内检测到。因此，这项应用的目的是研究硝基Hb介导的调节的生化机制，并确定它们的生理/病理作用。假设HbFe(11)NO处于“超级T”状态，与带3结合，并将其群体转移到二聚体形式，从而疏松细胞骨架网络。由于HbFe(11)NO可在低氧状态下在肺内产生，HbFe(11)NO调节的NO消耗可能参与了缺氧性肺血管收缩。此外，根据初步数据，进一步假设HbFe(11)NO可减弱NO介导的冠状动脉扩张。具体目标1将使用生化和生物物理技术来研究这种调控的机制，这些技术探测细胞骨架和带3蛋白的状态。具体目标2将利用分离的猪肺微血管和冠状动脉微血管集中于这一调节的功能作用。总而言之，这些结果将建议临床相关性和潜在的干预措施。

DESCRIPTION (provided by applicant): Nitric oxide (NO) achieves its biological functions through a balance between its synthesis and inactivation. The biosynthesis of NO is highly regulated and well documented, whereas its inactivation is much less understood. The major pathways for NO inactivation include reactions with oxygenated hemoglobin [HbFe (ll) O2] and various free radicals. Conventional wisdom suggests that NO inactivation by HbFe (ll) O2 is not regulated, since NO is thought to diffuse freely and rapidly across the red blood cell (RBC) membrane. Recent findings have shown that NO transport into RBCs is controlled by the membrane skeleton proteins. The rate of NO consumption by RBCs can be modulated by perturbing the cytoskeleton network through cytoskeleton binding proteins such as Band 3. In particular. formation of iron-nitrosyl-hemoglobin [HbFe (ll) NO] (-0.1%) increased the NO consumption rate. This regulator is of physiological and pathological importance as HbFe (ll) NO is formed during hypoxia and has been detected in humans under various conditions. The purpose of this application is thus to investigate the biochemical mechanisms underlying the nitrosylHb-mediated regulations and to determine their physiological/pathological roles. It is hypothesized that HbFe (ll) NO in the "super T" state binds to Band 3 and shifts its population to the dimer form, which loosens the cytoskeleton network. Since HbFe (ll) NO may be produced in the lungs under hypoxia, the HbFe (ll) NO regulated NO consumption may participate in hypoxic pulmonary vasoconstriction. Moreover, based on preliminary data, it is further hypothesized that HbFe (ll) NO attenuates the NO-mediated coronary vasodilation. Specific aim 1 will investigate the mechanisms involved in this regulation using biochemical and biophysical techniques which probe the state of cytoskeleton and Band 3 protein. Specific aim 2 will focus on the functional roles of this regulation using isolated porcine pulmonary and coronary microvessels. Together, these results will suggest clinical relevance and potential interventions.