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.
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