REGULATION OF PULMONARY CIRCULATION IN FETUS AND NEWBORN

胎儿的肺血管阻力（PVR）经历了一个 出生时急剧下降，主要是对氧气的反应。血管 内皮通过释放一氧化氮（NO）调节这种反应。 这种适应的失败导致持续性肺动脉高压， 新生儿（PPHN），一种与高发病率相关的疾病， mortality. 然而，氧气诱导一氧化氮的机制 合成是未知的。 拟议的实验研究 假设：（1）胎儿红细胞检测肺泡上皮细胞的增加， 增加ATP的合成和释放;（2）ATP及其代谢产物 代谢产物腺苷刺激内皮细胞释放NO;（3） P2 γ和A2嘌呤受体促进NO释放 药物激活K+通道。 将对RBC进行研究， 132 d胎羊肺动脉内皮细胞 怀孕 为了研究第一种假设，胎儿红细胞将通过 在代表动脉PO 2的20和60 mmHg的PO 2下孵育 在胎儿和新生儿中，在150和600 mmHg的PO 2下， 代表出生后暴露于室内空气或O2的肺泡PO 2。 细胞内ATP水平和ATP外排到细胞外位点 将在这些PO 2下定量。 ATP合成的机制 通过将细胞暴露于这些PO 2，在存在 2，4-二硝基苯酚和抗霉素A的氧化抑制剂 磷酸化 从RBC释放ATP的机制将是 通过用带4.5的抑制剂预处理细胞来研究 核苷转运通道和带3阴离子转运通道， 在这些氧分压下重复孵育。 去调查第二个 假设，0.01-2.0 μ M浓度的ATP和 将研究腺苷对a）内皮型一氧化氮合酶（eNOS） 通过定量逆转录PCR评估基因转录， B）通过Western免疫印迹分析测量的eNOS蛋白水平，和c） NO ~（2-）+NO ~（3-）释放法测定培养胎儿内皮型一氧化氮合酶活性 肺动脉内皮细胞 两种eNOS的定量 转录和活性是确定ATP是否参与 在出生时PVR急性下降和观察到的更缓慢的下降中， 出生后早期的生活。 为了研究第三个假设， 腺苷和ATP对内皮细胞释放NO影响将被 在a）1，3-二丙基-7-甲基黄嘌呤，A2 腺苷受体拮抗剂B）汽巴蓝，P2 γ受体 拮抗剂c）格列本脲，一种K+ATP通道拮抗剂，和d） 四乙基氯化铵，一种非特异性K+通道拮抗剂。 这些研究将探讨ATP诱导NO释放的机制 从内皮细胞。 这些实验将提供新的 氧诱导肺血管舒张机制的信息，a 这是新生儿生存的关键一步。

The pulmonary vascular resistance (PVR) in the fetus undergoes a dramatic decrease at birth, primarily in response to oxygen. Vascular endothelium modulates this response by release of nitric oxide (NO). Failure of this adaption leads to persistent pulmonary hypertension of the newborn (PPHN), a condition associated with high morbidity and mortality. However, the mechanism by which oxygen induces nitric oxide synthesis is not know. The proposed experiments investigate the hypotheses that (1) fetal erythrocytes detect the increase in alveolar PO2 and increase the synthesis and release of ATP, (2) ATP and its metabolite adenosine stimulate endothelial cells to release NO and (3) stimulation of NO release is facilitated by P2y and A2 purinoceptor medicated activation of K+ channels. Studies will be done on RBCs and pulmonary artery endothelial cells isolated from fetal lambs at 132 d gestation. To investigate the first hypothesis, fetal RBCs will by incubated at Po2s of 20 and 60 mmHg which represent the arterial PO2s in the fetus and in the newborn and at PO2s of 150 and 600 mmHg which represent the postnatal alveolar PO2s with exposure to room air or O2. The intracellular levels of ATP and ATP efflux into extracellular site will be quantitated at these PO2s. The mechanism of ATP synthesis in RBC will be investigated by exposing cells to these PO2s in the presence of 2,4-dinitrophenol and antimycin A, inhibitors of oxidative phosphorylation. The mechanism of ATP release from RBC will be investigated by pretreatment of cells with inhibitors of band 4.5 nucleoside transport channel and band 3 anionic transport channel and repeating the incubation at these PO2s. To investigate the second hypothesis, the effects of 0.01-2.0 muM concentrations of ATP and adenosine will be studied on a) endothelial nitric oxide synthase (eNOS) gene transcription assessed by quantitative reverse transcription PCR, b) eNOS protein level measured by Western immunoblot analysis and c) eNOS activity as measured by NO2-+NO3- release in cultured fetal pulmonary artery endothelial cells. Quantitation of both eNOS transcription and activity are necessary to determine if ATP is involved in both acute decrease in PVR at birth and more gradual decrease seen over early postnatal life. To investigate the third hypothesis, the effect of adenosine and ATP on NO release by endothelial cells will be studied in the presence of a) 1,3 dipropyl-7 mehtylxanthine, an A2 adenosine receptor antagonist b) cibacron blue, a P2y receptor antagonist c) glybenclamide, a K+ATP channel antagonist and d) tetraethyl ammonium chloride, a non-specific K+ channel antagonist. These studies will investigate the mechanism of ATP induced NO release from endothelial cells. The proposed experiments will provide new information on mechanisms of O2 induced pulmonary vasodilation, a critical step for survival of newborn.