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血管紧张素Ⅱ上调自发性高血压大鼠和Wistar--Kyoto大鼠血管平滑肌细胞外信号调节激酶的信号转导

朱建华, 刘忠, 黄朝阳, 李闪

浙江大学医学院附属第一医院.浙江,杭州 310003

摘要

该文研究血管紧张素Ⅱ (angiotensinⅡ，AngⅡ)对自发性高血压大鼠(spontaneously hypertensive rat，SHR)和Wistar--Kyoto (WKY)大鼠血管平滑肌细胞(vascular smooth muscle cells, VSMCs)细胞外信号调节激酶(extracellular signal--regulated protein kinases，ERKs)信号途径的影响。体外培养SHR和WKY大鼠的VSMCs，先在培养基中加入终浓度为1×10~(-5) mmol/L的缬沙坦或1×10~(-5) mmol/L的PD98059或不加药物，再给予1×10~(-7) mmol/L的AngⅡ刺激24 h后收集细胞，以无血清培养基培养的VSMCs作对照。用免疫沉淀法测定ERK活性；用Western--blot方法检测总ERK (total ERK，t--ERK)、磷酸化ERK (phosphorylated-ERK，p--ERK)及丝裂素活化蛋白激酶磷酸酶--1 (mitogen--activated protein kinases phosphatase--1, MKP--1)水平；用RT-PCR法半定量测定MKP--1 mRNA的含量。结果显示：(1) SHR和WKY大鼠AngⅡ刺激组VSMCs中ERK活性、p-ERK、MKP--1及MKP--1 mRNA水平均明显高于对照组({sl P}<0.05); SHR和WKY大鼠AngⅡ+缬沙坦组和AngⅡ+PD98059组的上述指标与对照组比较均无显著性差异。 (2) SHR大鼠VSMCs中ERK活性、p--ERK、MKP--1及MKP-1 mRNA均显著高于相同干预的WKY大鼠({sl P}<0.01)。 (3) SHR和WKY大鼠之间以及对照组、AngⅡ刺激组、AngⅡ+缬沙坦组和AngⅡ+PD98059组间VSMCs中t--ERK水平均无显著性差异。以上结果表明，AngⅡ可能主要通过其1型(AngⅡ type 1，AT_(1))受体激活SHR和WKY大鼠VSMCs中ERK途径，增加ERK活性和p-ERK蛋白水平，继而引起MKP--1及 MKP--1 mRNA水平升高。

关键词： 血管紧张素Ⅱ; 丝裂素活化蛋白激酶; 细胞外信号调节激酶; 血管平滑肌细胞; 大鼠; 自发性高血压

Effects of angiotensin Ⅱ on extracellular signal--regulated protein kinases signaling pathway in cultured vascular smooth muscle cells from Wistar--Kyoto rats and spontaneously hypertensive rats

Zhu Jianhua, Liu Zhong, Huang Zhaoyang, Li Shan

Department of Cardiology, the First Affiliated Hospital, College of Medicine, Zhejiang University.Hangzhou 310003,Zhejiang

Abstract

The aim of this study was to investigate the effects of angiotensinⅡ (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 1×10~(-7) mmol/L AngⅡfor 24h in the absence or presence of 30 min of pre-treatment of valsartan (1×10~(-5) mmol/L) or PD98059 (1×10~(-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) AngⅡ, (3) AngⅡ+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 AngⅡgroup 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, AngⅡ+valsartan group and AngⅡ+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 AngⅡactivates VSMCs ERK signaling pathways via AngⅡtype 1 (AT_(1)) receptors. Ang Ⅱincreased 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 AngⅡ-stimulated ERK activation. These results suggest that ERK signaling pathway plays an important role in the pathogenesis of hypertension. The effect of AngⅡon 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.

Key words: angiotensinⅡ;mitogen-activated protein kinases;extracellular signal-regulated protein kinase;Vascular smooth muscle cells;Rats;spontaneously hypertensive

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引用本文：

朱建华, 刘忠, 黄朝阳, 李闪. 血管紧张素Ⅱ上调自发性高血压大鼠和Wistar--Kyoto大鼠血管平滑肌细胞外信号调节激酶的信号转导 [J]. 生理学报 2005; 57 (5): .

Zhu Jianhua, Liu Zhong, Huang Zhaoyang, Li Shan. Effects of angiotensin Ⅱ on extracellular signal--regulated protein kinases signaling pathway in cultured vascular smooth muscle cells from Wistar--Kyoto rats and spontaneously hypertensive rats . Acta Physiol Sin 2005; 57 (5): (in Chinese with English abstract).