Phosphorylation of cAMP response element binding (CREB) protein during hypoxia in cerebral cortex of newborn piglets and the effect of nitric oxide synthase inhibition
Phosphorylation of cAMP response element binding (CREB) protein during hypoxia in cerebral cortex of newborn piglets and the effect of nitric oxide synthase inhibition
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DOI：
10.1016/s0306-4522(02)00275-0
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发表时间：
2002-01-01
期刊：
影响因子：
3.3
通讯作者：
Delivoria-Papadopoulos, M
中科院分区：
文献类型：
--
作者：
Mishra, OP;Ashraf, QM;Delivoria-Papadopoulos, M
以往的研究表明，缺氧导致CREB蛋白的磷酸化增加，该蛋白介导了基因的表达，包括促凋亡基因Bax的表达。我们还表明，低氧诱导的Bax蛋白的表达可以通过阻断一氧化氮合酶(NOS)来阻止。本研究验证了N-硝基-L-精氨酸抑制一氧化氮合酶可阻止缺氧诱导的新生仔猪神经元核内CREB蛋白磷酸化增加的假说。为了验证这一假设，用免疫印迹法检测了5头常氧(NX)、10头缺氧(HX)和5头HX-NNLA处理过的3-5日龄仔猪的CREB蛋白的磷酸化。缺氧前60min以上给予NNLA(40 mg/kg)或生理盐水。通过降低FiO(2)(0.15至0.05)60min来实现低氧，并通过三磷酸腺苷(ATP)和磷酸肌酸(PCr)水平进行生化记录。用不连续蔗糖梯度离心法分离纯化神经元核团。核蛋白经12%十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法分离，转移到硝酸纤维素膜上，与抗磷酸化CREB蛋白抗体反应，再与辣根过氧化物酶抗体偶联。用增强化学发光法检测蛋白质条带，并用图像密度法进行定量。蛋白质密度用吸光度(OD)×mm(2)表示。三磷酸腺苷水平(摩尔/克脑)：NX组为4.3+/-0.6，HX组为1.3+/-0.5(P<；0.001)，HX-NNLA组为1.1+/-0.2(P<；0.001 vs.NX和HX)。同样，聚合酶链式反应水平(pmoL/g脑)在NX组为3.8+/-0.6，HX组为0.7+/-0.2(P<；0.001)，HX-NNLA组为0.6+/-0.1(P<；0.001 vs.NX和HX)。磷酸化CREB蛋白的密度(OD x mm(2))在NX组为134.2+/-52.4，而HX-NNLA组为746.0+/-76.8(P<0.05)，HX-NNLA组为491.1+/-40.9(P<；0.05 HX)。(C)2002年IBRO。爱思唯尔科学有限公司出版。版权所有。
Previous studies have shown that hypoxia results in increased phosphorylation of CREB protein that mediates gene expression including that of the pro-apoptotic gene bax. We also have shown that hypoxia-induced expression of Bax protein is prevented by blocking nitric oxide synthase (NOS). The present study tests the hypothesis that inhibition of NOS by N-nitro-L-arginine (NNLA) will prevent the hypoxia-induced increased phosphorylation of CREB protein in neuronal nuclei of newborn piglets. To test this hypothesis, phosphorylation of CREB protein was assessed by immunoblotting neuronal nuclear proteins from five normoxic (Nx), 10 hypoxic (Hx) and five Hx-NNLA-treated 3-5-day-old piglets. NNLA (40 mg/kg) or saline was infused over 60 min prior to induction of hypoxia. Hypoxia was achieved by reducing the FiO(2) (0.15 to 0.05) for 60 min and documented biochemically by ATP and phosphocreatine (PCr) levels. Neuronal nuclei were isolated using discontinuous sucrose gradient centrifugation and purified. Nuclear proteins were separated on 12% sodium dodecylsulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, reacted with anti-phosphorylated CREB protein antibody and conjugated with horseradish peroxidase antibody. Protein bands were detected using the enhanced chemiluminescence method and quantitated by imaging densitometry. Protein density was expressed as absorbance (OD) X mm(2). ATP levels (mumol/g brain) were 4.3 +/- 0.6 in the Nx group, 1.3 +/- 0.5 in the Hx group (P < 0.001) and 1.1 +/- 0.2 in the Hx-NNLA group (P < 0.001 vs. Nx and Hx). Similarly, PCr levels (pmol/g brain) were 3.8 +/- 0.6 in the Nx group, 0.7 +/- 0.2 in the Hx group (P < 0.001) and 0.6 +/- 0.1 in the Hx-NNLA group (P < 0.001 vs. Nx and Hx). Density of phosphorylated CREB protein (OD x mm(2)) was 134.2 +/- 52.4 in the Nx group compared to 746.0 +/- 76.8 in the Hx group (P < 0.05) and 491.1 +/- 40.9 in the Hx-NNLA group (P < 0.05 Hx).The data show that NOS inhibition attenuates the hypoxia-induced increase in CREB protein phosphorylation in the cerebral cortex of newborn piglets. (C) 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved.