Qayyum I, Zubrow A B, Ashraf Q M, Kubin J, Delivoria-Papadopoulos M, Mishra O P
Department of Pediatrics, Thomas Jefferson University, USA.
Neurochem Res. 2001 Oct;26(10):1163-9. doi: 10.1023/a:1012331108641.
Previous studies have shown that hypoxia induces nitric oxide synthase-mediated generation of nitric oxide free radicals leading to peroxynitrite production. The present study tests the hypothesis that hypoxia results in NO-mediated modification of Na+, K+-ATPase in the fetal brain. Studies were conducted in guinea pig fetuses of 58-days gestation. The mothers were exposed to FiO2 of 0.07% for 1 hour. Brain tissue hypoxia in the fetus was confirmed biochemically by decreased ATP and phosphocreatine levels. P2 membrane fractions were prepared from normoxic and hypoxic fetuses and divided into untreated and treated groups. The membranes were treated with 0.5 mM peroxynitrite at pH 7.6. The Na+, K+-ATPase activity was determined at 37 degrees C for five minutes in a medium containing 100 mM NaCl, 20 mM KCl, 6.0 mM MgCl2, 50 mM Tris HCl buffer pH 7.4, 3.0 mM ATP with or without 10 mM ouabain. Ouabain sensitive activity was referred to as Na+, K+-ATPase activity. Following peroxynitrite exposure, the activity of Na+, K+-ATPase in guinea pig brain was reduced by 36% in normoxic membranes and further 29% in hypoxic membranes. Enzyme kinetics was determined at varying concentrations of ATP (0.5 mM-2.0 mM). The results indicate that peroxynitrite treatment alters the affinity of the active site of Na+, K+-ATPase for ATP and decreases the Vmax by 35% in hypoxic membranes. When compared to untreated normoxic membranes Vmax decreases by 35.6% in treated normoxic membranes and further to 52% in treated hypoxic membranes. The data show that peroxynitrite treatment induces modification of Na+, K+-ATPase. The results demonstrate that peroxynitrite decreased activity of Na+, K+-ATPase enzyme by altering the active sites as well as the microenvironment of the enzyme. We propose that nitric oxide synthase-mediated formation of peroxynitrite during hypoxia is a potential mechanism of hypoxia-induced decrease in Na+, K+-ATPase activity.
以往研究表明，缺氧可诱导一氧化氮合酶介导的一氧化氮自由基生成，进而导致过氧亚硝酸根的产生。本研究旨在验证以下假说：缺氧会导致胎儿脑中一氧化氮介导的钠钾ATP酶修饰。研究选用妊娠58天的豚鼠胎儿。将孕母暴露于7%的氧浓度环境中1小时。通过检测ATP和磷酸肌酸水平降低，生化确认胎儿脑组织缺氧。从正常氧合和缺氧的胎儿中制备P2膜组分，并分为未处理组和处理组。将膜在pH 7.6条件下用0.5 mM过氧亚硝酸根处理。在含有100 mM氯化钠、20 mM氯化钾、6.0 mM氯化镁、50 mM Tris HCl缓冲液（pH 7.4）、3.0 mM ATP且有或无10 mM哇巴因的培养基中，于37℃测定钠钾ATP酶活性5分钟。哇巴因敏感活性被视为钠钾ATP酶活性。过氧亚硝酸根暴露后，正常氧合膜中豚鼠脑钠钾ATP酶活性降低36%，缺氧膜中进一步降低29%。在不同ATP浓度（0.5 mM - 2.0 mM）下测定酶动力学。结果表明，过氧亚硝酸根处理改变了钠钾ATP酶活性位点对ATP的亲和力，缺氧膜中Vmax降低35%。与未处理的正常氧合膜相比，处理后的正常氧合膜中Vmax降低35.6%，处理后的缺氧膜中进一步降至52%。数据表明，过氧亚硝酸根处理可诱导钠钾ATP酶修饰。结果证明，过氧亚硝酸根通过改变酶的活性位点以及微环境降低了钠钾ATP酶的活性。我们提出，缺氧期间一氧化氮合酶介导的过氧亚硝酸根形成是缺氧诱导钠钾ATP酶活性降低的潜在机制。
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