Zhu Jian-Hua, Liu Zhong, Huang Zhao-Yang, Li Shan
Department of Cardiology, the First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China.
Sheng Li Xue Bao. 2005 Oct 25;57(5):587-92.
The aim of this study was to investigate the effects of angiotensin II (Ang II) on extracellular signal-regulated protein kinase (ERK) signaling pathway in cultured vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats. VSMCs from SHR and WKY rats were treated with 1x10(-7) mmol/L Ang II for 24 h in the absence or presence of 30 min of pre-treatment of valsartan (1x10(-5) mmol/L) or PD98059 (1x10(-5)mmol/L), selective inhibitor of ERKs- dependent pathways, when they were cultured in 20% calf serum medium. VSMCs of SHR and WKY cultured in serum-free medium were used as control groups. Among the different treatments, VSMCs from the SHR and WKY were devided into four groups: (1) control, (2) Ang II, (3) Ang II + valsartan, (4) Ang II + PD98059. ERK activity in VSMCs was measured by immuno-precipitation. Proteins of total ERK (t-ERK), phosphorylated-ERK (p-ERK) and mitogen-activated protein kinases phosphatase-1 (MKP-1) in VSMCs were detected by Western blot. MKP-1 mRNA in VSMCs was measured by RT-PCR. In VSMCs from WKY or SHR rats, ERK activity, p-ERK, MKP-1 and MKP-1 mRNA in Ang II group were higher than those in control group (P<0.05). In both SHRs and WKYs, there were no significant differences in ERK activity, p-ERK, MKP-1 and MKP-1 mRNA among the control group, Ang II + valsartan group and Ang II + PD98059 group. ERK activity, p-ERK, MKP-1 and MKP-1 mRNA in SHRs were significantly higher than those in WKYs with same treatments (P<0.01). There was no significant difference in t-ERK among different groups and no difference in t-ERK between SHRs and WKYs (P>0.05). Our results show that Ang II activates VSMCs ERK signaling pathways via Ang II type 1 (AT(1)) receptors. Ang II increased ERK activity and p-ERK, but not t-ERK, accompanied by an increase in MKP-1 mRNA expression and protein. Among the different treatments, ERK activity and p-ERK were higher in SHR than in WKY. Valsartan and PD98059 blocked Ang II-stimulated ERK activation. These results suggest that ERK signaling pathway plays an important role in the pathogenesis of hypertension. The effect of Ang II on SHR and WKY VSMCs' ERK signaling pathway may be mediated by AT(1) receptors, enhancing ERK activity and the amount of p-ERK, and then increasing MKP-1 mRNA and its expression.
本研究旨在探讨血管紧张素II（Ang II）对自发性高血压大鼠（SHR）和Wistar-Kyoto（WKY）大鼠培养的血管平滑肌细胞（VSMC）中细胞外信号调节蛋白激酶（ERK）信号通路的影响。将SHR和WKY大鼠的VSMC在含20%小牛血清的培养基中培养，在不存在或存在缬沙坦（1x10(-5) mmol/L）或PD98059（1x10(-5)mmol/L，ERK依赖途径的选择性抑制剂）预处理30分钟的情况下，用1x10(-7) mmol/L Ang II处理24小时。将在无血清培养基中培养的SHR和WKY的VSMC用作对照组。在不同处理中，将SHR和WKY的VSMC分为四组：（1）对照组，（2）Ang II组，（3）Ang II + 缬沙坦组，（4）Ang II + PD98059组。通过免疫沉淀法测量VSMC中的ERK活性。通过蛋白质印迹法检测VSMC中总ERK（t-ERK）、磷酸化ERK（p-ERK）和丝裂原活化蛋白激酶磷酸酶-1（MKP-1）的蛋白质。通过RT-PCR测量VSMC中MKP-1 mRNA。在WKY或SHR大鼠的VSMC中，Ang II组的ERK活性、p-ERK、MKP-1和MKP-1 mRNA高于对照组（P<0.05）。在SHR和WKY中，对照组、Ang II + 缬沙坦组和Ang II + PD98059组之间的ERK活性、p-ERK、MKP-1和MKP-1 mRNA无显著差异。相同处理下，SHR的ERK活性、p-ERK、MKP-1和MKP-1 mRNA显著高于WKY（P<0.01）。不同组之间的t-ERK无显著差异，SHR和WKY之间的t-ERK也无差异（P>0.05）。我们的结果表明，Ang II通过1型血管紧张素II（AT(1)）受体激活VSMC的ERK信号通路。Ang II增加ERK活性和p-ERK，但不增加t-ERK，同时伴随着MKP-1 mRNA表达和蛋白质的增加。在不同处理中，SHR的ERK活性和p-ERK高于WKY。缬沙坦和PD98059阻断Ang II刺激的ERK激活。这些结果表明，ERK信号通路在高血压发病机制中起重要作用。Ang II对SHR和WKY VSMC的ERK信号通路的作用可能由AT(1)受体介导，增强ERK活性和p-ERK的量，进而增加MKP-1 mRNA及其表达。
