REGULATION OF PULMONARY CIRCULATION IN FETUS AND NEWBORN

胎儿肺血管阻力(PVR)经历 出生时急剧减少，主要是对氧气的反应。血管 内皮细胞通过释放一氧化氮(NO)来调节这种反应。 这种适应的失败会导致持续性的肺动脉高压 新生儿(PPHN)，这是一种与高发病率和 死亡率。然而，氧气诱导一氧化氮的机制 合成是未知的。拟议中的实验调查了 假设(1)胎儿红细胞检测到肺泡的增加 PO2，增加ATP的合成和释放，(2)ATP及其 代谢产物腺苷刺激内皮细胞释放NO和(3) P2y和A2嘌呤受体促进NO释放 药物激活K+通道。将对红细胞和红细胞进行研究 132日龄胎羊肺动脉内皮细胞的分离 怀孕了。为了研究第一个假设，胎儿红细胞将通过 在代表动脉血氧饱和度的20和60毫米汞柱的血氧饱和度下孵化 在胎儿和新生儿中，在150和600毫米汞柱的二氧化碳分压下， 表示暴露于室内空气或氧气中的出生后肺泡氧分压。 细胞内三磷酸腺苷水平和三磷酸腺苷外流到胞外部位 将在这些PO2上进行量化。植物体内ATP合成机制的研究进展 RBC将通过将细胞暴露在这些PO2中而被研究 氧化抑制剂2，4-二硝基苯酚和抗霉素A 磷酸化。红细胞释放三磷酸腺苷的机制为 通过使用Band 4.5抑制剂对细胞进行预处理进行研究 核苷转运通道和带3阴离子转运通道 在这些PO2重复孵化。调查第二起案件 假说，0.01-2.0微米浓度的三磷酸腺苷和 腺苷将研究a)内皮型一氧化氮合酶(ENOS) 定量逆转录聚合酶链式反应检测基因转录， B)免疫印迹法检测eNOS蛋白水平；c) 培养胎儿NO_2~-~+-NO_3~-释放法测定eNOS活性 肺动脉内皮细胞。两种eNOS的量化 转录和活性是确定是否涉及三磷酸腺苷所必需的 出生时PVR的急剧下降和更缓慢的下降均可见 在出生后的早期生活中。为了研究第三种假设， 腺苷和三磷酸腺苷对内皮细胞释放一氧化氮的影响 在1，3-二丙基-7-甲基黄嘌呤、A2存在下研究 腺苷受体拮抗剂b)雪巴克隆蓝，一种P2y受体 拮抗剂c)K+ATP通道拮抗剂格列本脲和d) 四乙基氯化铵，非特异性K+通道拮抗剂。 这些研究将探讨三磷酸腺苷诱导NO释放的机制 来自内皮细胞。拟议的实验将提供新的 氧诱导肺血管扩张机制的信息，a 新生儿存活的关键一步。

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.