Proinsulin C-peptide increases nitric oxide production by enhancing mitogen-activated protein-kinase-dependent transcription of endothelial nitric oxide synthase in aortic endothelial cells of Wistar rats
Proinsulin C-peptide increases nitric oxide production by enhancing mitogen-activated protein-kinase-dependent transcription of endothelial nitric oxide synthase in aortic endothelial cells of Wistar rats
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DOI：
10.1007/s00125-003-1232-3
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发表时间：
2003-12-01
期刊：
影响因子：
8.2
通讯作者：
Saito, M
中科院分区：
文献类型：
--
作者：
Kitamura, T;Kimura, K;Saito, M
目标/假设。最近的研究表明，胰岛素原C肽可能通过产生一氧化氮(NO)来改善血管功能。为了阐明C-肽诱导血管产生NO的分子机制，我们研究了C-肽对大鼠主动脉内皮细胞NO产生和NO合成酶表达的影响，并与丝裂原活化蛋白激酶(MAPK)的激活有关。从雌性Wistar大鼠分离主动脉内皮细胞，进行融合培养，血清饥饿24 h后给予C肽处理。用DAF-2荧光染料法检测一氧化氮的生成，用Western印迹和RT-PCR方法分别半定量检测内皮型一氧化氮合酶(ENOS)蛋白及其mRNA的相对含量。用蛋白印迹法检测MAPK活性相关的磷酸化水平，并用体外蛋白激酶分析法检测MAPK的活性。C肽刺激细胞3h可使细胞产生倍增的NO，NO合成酶抑制剂N-G-硝基-L-精氨酸甲酯(L-NAME)可抑制这种作用。刺激还可增加eNOS的mRNA和蛋白含量，对转录抑制剂放线菌素D敏感，但不影响诱导型一氧化氮合酶基因的表达。C-肽还可诱导细胞外信号调节激酶(ERK，也称为p44/42MAPK)的快速磷酸化和激活，但不能诱导p38MAPK的快速磷酸化和激活。在用ERK抑制剂PD98059预处理的细胞中，C肽诱导的NO产生增加，eNOS呈剂量依赖性地被抑制，C肽诱导的ERK磷酸化也受到抑制。我们的结果表明，C-肽通过ERK依赖的eNOS基因转录上调而增加eNOS蛋白含量，从而增加NO的产生。这可以解释C-肽在血管系统中的一些作用，表明C-肽在血管动态平衡中起着关键作用。
Aims/hypothesis. Recent studies have suggested that proinsulin C-peptide improves vascular functions, possibly through nitric oxide (NO) production. To clarify the molecular mechanisms of vascular NO production induced by C-peptide, we examined the effects of C-peptide on NO production and NO synthase expression in rat aortic endothelial cells in connection with mitogen-activated protein kinase (MAPK) activation.Methods. Aortic endothelial cells were isolated from female Wistar rats, cultured to confluence, and serum-starved for 24 h before treatment with C-peptide. Nitric oxide production was measured by the DAF-2 fluorescence dye method and relative amounts of endothelial nitric oxide synthase (eNOS) protein and its mRNA were semi-quantified by western blot and RT-PCR analyses respectively. Activation of MAPK was estimated by western blot detection of activity-related phosphorylation and in vitro kinase assay.Results. Stimulation of cells with C-peptide for 3 h doubled NO production, which was suppressed by the NO synthase inhibitor, N-G-nitro-L-arginine methyl ester (L-NAME). Stimulation also increased mRNA and protein contents of eNOS in a manner sensitive to the transcription inhibitor actinomycin D. It did not affect inducible NO synthase mRNA. C-peptide also induced rapid phosphorylation and activation of extracellular signal-regulated kinase (ERK, also known as p44/42MAPK), but not of p38MAPK. In cells pretreated with the ERK inhibitor PD98059 the C-peptide-elicited increase of NO production and eNOS was abrogated in a dose-dependent manner; suppression of ERK phosphorylation induced by C-peptide also occurred.Conclusions/interpretation. Our results show that C-peptide increases NO production by increasing eNOS protein contents through ERK-dependent up-regulation of eNOS gene transcription. This could explain some actions of C-peptide on the vasculature, indicating a pivotal role for C-peptide in vascular homeostasis.