Regulation of pulmonary circulation in fetus and newborn

描述(申请人提供)：胎儿的肺血管阻力在出生时迅速下降，以促进出生后的气体交换。这种适应的改变会导致新生儿持续性肺动脉高压(PPHN)，这是一种与发病率和死亡率增加相关的疾病。PPHN中这种改变的适应机制仍不清楚。先前的研究表明，出生时的肺血管扩张是通过胎儿红细胞释放三磷酸腺苷和三磷酸腺苷刺激内皮细胞释放一氧化氮(NO)来促进的。应激蛋白Hsp90与内皮型一氧化氮合酶(ENOS)结合可促进NO在生理刺激下的释放。Hsp90-NOS相互作用的抑制似乎将NOS活性从NO释放到血管收缩物质超氧化物(O2“)。这些研究将探讨如下假设：(I)当正常胎肺血管暴露于ATP时，Hsp90与eNOS的结合促进了NO的释放和血管扩张，(Ii)在PPHN的压力负荷增加时，Hsp90与eNOS的解离将NOS活性的平衡从NO转移到O2-，以及(Iii)O2通过抑制血管平滑肌上的K通道而损害NO的独立血管扩张。这些假说将在已建立的PPHN模型--动脉导管狭窄所致的胎羊中进行研究，并在假结扎动脉管的对照羊中进行研究。我们将在对照和PPHN羔羊分离的肺动脉上进行研究，以确定Hsp90-NOS相互作用和Kv通道在ATP正常松弛反应中的作用，以及解偶联NOS活性在PPHN反应受损中的作用。在对照和PPHN羔羊的内皮细胞上的研究将确定ATP对(A)eNOS释放NO和O2-的平衡，(B)Hsp90-eNOS结合和(C)eNOS的丝氨酸-1177磷酸化的影响。这些研究将(1)阐明Hsp90-eNOS结合在ATP刺激NO释放中的作用；(2)确定当NOS被ATP激活时，Hsp90-eNOS结合减弱是否导致O2的产生。拟议的研究还将探讨PPHN中Hsp90-eNOS结合减少的两种可能机制：(A)由于硝基酪氨酸的形成而减少Hsp90的酪氨酸磷酸化；(B)Hsp90重新聚集到细胞骨架蛋白、肌动蛋白和a-微管蛋白中，以在增加的压力负荷下保持它们的完整性。这些研究将为PPHN的适应受损机制提供新的信息。

DESCRIPTION (provided by applicant): The pulmonary vascular resistance in the fetus undergoes a rapid decrease at birth to facilitate gas exchange during postnatal life. Alteration in this adaptation leads to Persistent Pulmonary Hypertension of Newborn (PPHN), a condition associated with increased morbidity and mortality. The mechanism of this altered adaptation in PPHN remains unknown. Previous studies have shown that pulmonary vasodilation at birth is facilitated by release of ATP from fetal RBC and stimulation of nitric oxide (NO) release from endothelial cells by ATP. Association of Hsp90, a stress protein, with endothelial NOS (eNOS) facilitates release of NO in response to physiological stimuli. Inhibition of Hsp90-NOS interaction appears to uncouple NOS activity from release of NO to superoxide (O2"), a vasoconstrictor. The proposed studies will investigate the hypotheses that (i) association of Hsp90 with eNOS facilitates NO release and vasodilation when normal fetal pulmonary vessels are exposed to ATP, (ii) dissociation of Hsp90 from eNOS during increased pressure load in PPHN shifts the balance of NOS activity from NO to O2- and (iii) O2 impairs No independent vasodilation by inhibition of K channels on vascular smooth muscle. The hypotheses will be investigated in fetal lambs with pulmonary hypertension induced by constriction of ductus arteriosus, an established model of PPHN, and in control lambs with sham ligation of ductus arteriosus. Studies will be done in pulmonary arteries isolated from control and PPHN lambs to determine the role of Hsp90-NOS interaction and Kv channels in normal relaxation response to ATP and the role of uncoupled NOS activity in the impaired response in PPHN Studies in endothelial cells from control and PPHN lambs will determine the effect of ATP on (a) balance of NO and O2- release from eNOS, (b) Hsp90 - eNOS association and (c) serine-1177-phosphorylation of eNOS, a marker of its activation. These studies will (1) delineate the role of Hsp90-eNOS association in stimulation of NO release by ATP and (2) determine if decreased Hsp90-eNOS association results in generation of O2 when NOS is activated by ATP. The proposed studies will also address two potential mechanisms for decreased Hsp90-eNOS association in PPHN: (a) decreased tyrosine phosphorylation of Hsp90 due to nitrotyrosine formation and (b) recruitment of Hsp90 to cytoskeleton proteins, actin and a-tubulin to preserve their integrity during increased pressure load. These studies will provide new information on mechanisms of impaired adaptation in PPHN.