Phosphorylation of endothelial nitric oxide synthase in response to fluid shear stress
Phosphorylation of endothelial nitric oxide synthase in response to fluid shear stress
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DOI：
10.1161/01.res.79.5.984
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发表时间：
1996-11-01
影响因子：
20.1
通讯作者：
Harrison, DG
中科院分区：
文献类型：
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作者：
Corson, MA;James, NL;Harrison, DG
内皮细胞释放一氧化氮（NO）更有力地响应增加的剪切应力比激动剂，提高细胞内游离钙浓度（[Ca 2 +]（i））。为了确定内皮组成型NO合酶（ecNOS）的调节机制的差异，我们测量了牛主动脉内皮细胞暴露于剪切应力在层流室或处理与Ca 2+离子载体在静态培养的NO生产。累积NO产生的动力学变化显著：剪切应力（25达因/cm（2））刺激两相增加，在60分钟时为对照的13倍，而升高[Ca 2 +]（i）导致单相增加6倍。我们假设，激活蛋白激酶级联介导的早期阶段的流量依赖性NO的生产。免疫沉淀的ecNOS磷酸化流动开始后1分钟，增加了210%，而没有显着增加后Ca 2+离子载体治疗。虽然ecNOS没有酪氨酸磷酸化，早期阶段的流量依赖性NO的生产被阻断染料木素，酪氨酸激酶的抑制剂。为了确定流动依赖性NO产生的Ca 2+需求，我们用一种新的流动步骤方案测量了[Ca 2 +]（i），[Ca 2 +]（i）随着剪切应力的开始而增加，但不是在一个步骤增加之后。然而，剪切应力的逐步增加与NO产生速率和ecNOS磷酸化的强有力的双相增加相关。这些研究表明，剪切应力可以增加NO生产的情况下增加[Ca 2 +]（i）。并且他们提示ecNOS的磷酸化可能在施加增加的剪切应力期间重要地调节其活性。
Endothelial cells release nitric oxide (NO) more potently in response to increased shear stress than to agonists which elevate intracellular free calcium concentration ([Ca2+](i)). To determine mechanistic differences in the regulation of endothelial constitutive NO synthase (ecNOS), we measured NO production by bovine aortic endothelial cells exposed to shear stress in a laminar how chamber or treated with Ca2+ ionophores in static culture. The kinetics of cumulative NO production varied strikingly: shear stress (25 dyne/cm(2)) stimulated a biphasic increase over control that was 13-fold at 60 minutes, whereas raising [Ca2+](i) caused a monophasic 6-fold increase. We hypothesized that activation of a protein kinase cascade mediates the early phase of flow-dependent NO production. Immunoprecipitation of ecNOS showed a 210% increase in phosphorylation 1 minute after flow initiation, whereas there was no significant increase after Ca2+ ionophore treatment. Although ecNOS was not tyrosine-phosphorylated, the early phase of flow-dependent NO production was blocked by genistein, an inhibitor of tyrosine kinases. To determine the Ca2+ requirement for flow-dependent NO production, we measured [Ca2+](i) with a novel flow-step protocol, [Ca2+](i) increased with the onset of shear stress, but not after a step increase. However, the step increase in shear stress was associated with a potent biphasic increase in NO production rate and ecNOS phosphorylation. These studies demonstrate that shear stress can increase NO production in the absence of increased [Ca2+](i). and they suggest that phosphorylation of ecNOS may importantly modulate its activity during imposition of increased shear stress.