Regulation of pulmonary circulation in fetus and newborn

描述（由申请人提供）：胎儿的肺血管阻力在出生时迅速下降，以促进出生后生活中的气体交换。这种适应的改变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尚不清楚。先前的研究表明，出生时肺血管舒张是由胎儿红细胞释放ATP和ATP刺激内皮细胞释放一氧化氮（NO）促进的。应激蛋白Hsp90与内皮细胞NOS （eNOS）的关联促进了NO在生理刺激下的释放。抑制Hsp90-NOS相互作用似乎使NOS活性从释放NO到超氧化物（O2"），一种血管收缩剂。这些研究将探讨以下假设：(i)当正常胎儿肺血管暴露于ATP时，Hsp90与eNOS的关联促进NO的释放和血管舒张；（ii） PPHN压力负荷增加时，Hsp90与eNOS的分离使NOS活性的平衡从NO转向O2-； （iii） O2通过抑制血管平滑肌上的K通道而损害NO独立的血管舒张。这些假设将在由动脉导管收缩引起的肺动脉高压胎儿羔羊（PPHN的一个已建立的模型）和假动脉导管结扎的对照羔羊中进行研究。研究将在肺动脉隔离控制和PPHN羊羔来确定Hsp90-NOS交互的角色和Kv渠道在正常的放松反应ATP和非耦合号活动的作用不良反应PPHN研究内皮细胞从控制和PPHN羊羔将决定的影响ATP (a)的不平衡和O2 -释放以挪士,一半寿命(b)——以挪士协会和(c)丝氨酸- 1177以挪士的磷酸化,激活的一个标志。这些研究将(1)描述Hsp90-eNOS关联在ATP刺激NO释放中的作用；(2)确定当NOS被ATP激活时，Hsp90-eNOS关联的降低是否会导致O2的产生。提出的研究还将探讨PPHN中Hsp90- enos关联降低的两个潜在机制：(a)由于硝基酪氨酸的形成，Hsp90的酪氨酸磷酸化降低；(b)在压力负荷增加时，Hsp90被募集到细胞骨架蛋白、肌动蛋白和a-微管蛋白上，以保持其完整性。这些研究将为PPHN的适应性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.