Baas A S, Berk B C
Department of Internal Medicine, University of Washington, Seattle 98195, USA.
Circ Res. 1995 Jul;77(1):29-36. doi: 10.1161/01.res.77.1.29.
Increased generation of active oxygen species such as H2O2 and O2- may be important in vascular smooth muscle cell growth associated with atherosclerosis and restenosis. In previous work, we showed that H2O2 stimulated vascular smooth muscle cell growth and proto-oncogene expression. In the present study, we compared the effects of H2O2 and O2- on cultured rat aortic vascular smooth muscle cell growth and signal transduction. O2- was generated in a concentration-dependent manner by the naphthoquinolinedione LY83583. Vascular smooth muscle cell growth, as measured by [3H]thymidine incorporation, was stimulated by 200 mumol/L H2O2 (110% increase versus 0.1% serum) and 1 mumol/L LY83583 (175% increase) to levels comparable to 10 ng/mL platelet-derived growth factor (210% increase). Since activation of mitogen-activated protein kinase (MAP kinase) is one of the earliest growth factor signal events, the activity of MAP kinase was measured by changes in mobility on Western blot and by phosphorylation of myelin basic protein. There was a concentration-dependent increase in MAP kinase activity by LY83583 (maximum, 10 mumol/L) but not by H2O2. The time course for activation of MAP kinase by LY83583 showed a maximum at 5 to 10 minutes with return to baseline by 20 minutes. Activation of MAP kinase by LY83583 was protein kinase C dependent. Expression of MAP kinase phosphatase-1 (MKP-1), a transcriptionally regulated redox-sensitive protein tyrosine/threonine phosphatase, was also measured. Although H2O2 induced MKP-1 mRNA to a greater extent than did LY83583, the increased MKP-1 expression could not explain the inability of H2O2 to stimulate MAP kinase, because mRNA levels were not detected until 60 minutes.(ABSTRACT TRUNCATED AT 250 WORDS)
诸如过氧化氢（H2O2）和超氧阴离子（O2-）等活性氧物质生成增加，在与动脉粥样硬化和再狭窄相关的血管平滑肌细胞生长中可能起重要作用。在先前的研究中，我们发现H2O2可刺激血管平滑肌细胞生长及原癌基因表达。在本研究中，我们比较了H2O2和O2-对培养的大鼠主动脉血管平滑肌细胞生长及信号转导的影响。萘醌二酮LY83583以浓度依赖的方式生成O2-。通过[3H]胸苷掺入法测定，200μmol/L H2O2（与0.1%血清相比增加110%）和1μmol/L LY83583（增加175%）刺激血管平滑肌细胞生长至与10 ng/mL血小板衍生生长因子相当的水平（增加210%）。由于丝裂原活化蛋白激酶（MAP激酶）的激活是最早的生长因子信号事件之一，MAP激酶的活性通过蛋白质印迹法中迁移率的变化及髓鞘碱性蛋白的磷酸化来测定。LY83583（最大浓度为10μmol/L）可使MAP激酶活性呈浓度依赖性增加，而H2O2则无此作用。LY83583激活MAP激酶的时间进程在5至10分钟时达到最大值，20分钟时恢复至基线水平。LY83583对MAP激酶的激活依赖于蛋白激酶C。还测定了MAP激酶磷酸酶-1（MKP-1）的表达，MKP-1是一种受转录调控的氧化还原敏感蛋白酪氨酸/苏氨酸磷酸酶。尽管H2O2比LY83583更能诱导MKP-1 mRNA表达，但MKP-1表达增加并不能解释H2O2无法刺激MAP激酶的原因，因为直到60分钟才检测到mRNA水平。（摘要截短于250字）
