一氧化氮抑制AngⅡ介导的心肌肥大反应的信号机制

Molecular mechanism of nitric oxide in preventing cardiomyocytes from hypertrophic response induced by angiotensinⅡ

本文主要利用培养的新生大鼠心肌细胞 ,从细胞学及分子生物学角度研究一氧化氮 (NO)信号系统在AngⅡ介导的心肌肥大反应中的作用及机制。实验以心肌细胞蛋白合成速率、心房钠尿肽 (ANP)的表达作为心肌肥大反应的指标 ,以硝酸盐及亚硝酸盐含量反映心肌细胞NO水平 ,以免疫印迹法测定MKP 1蛋白表达 ,以RT PCR测定eNOSmRNA水平。结果发现 :(1)L 精氨酸 (L Arg) 10、10 0 μmol/L分别增加心肌细胞NO水平 16 %及 31%,L Arg (10 0 μmol/L)还可增加心肌细胞eNOSmRNA表达 ,其作用可被NOS抑制剂 L NAME所抑制 ;(2 )L Arg (10 0 μmol/L)可降低AngⅡ (0 1μmol/L)诱导的心肌细胞ANPmRNA表达水平和蛋白合成速率 ,而在L Arg处理之前用针对MKP 1的反义寡核苷酸转染心肌细胞 ,蛋白合成速率明显增加 ,可取消L Arg的抑制作用 ,甚至超过AngⅡ组 ;(3)L Arg (10 0 μmol/L)明显增加MKP 1蛋白表达 ,比对照组增加 2 2 5 %,NOS抑制剂L NAME及蛋白激酶G(PKG)抑制剂KT 5 82 3皆可抑制L Arg诱导的MKP 1蛋白表达 ,分别抑制 88%、83%,而AngⅡ能增加L Arg诱导的MKP 1的表达 ,较对照组增加 36 5 %,增强了L Arg的作用。以上结果表明 ,NO抑制AngⅡ介导心肌肥大反应的机制可能是通过激活PKG ,促进MKP 1的表达 。

The aim of this study was to determine the molecular mechanism of nitric oxide (NO) in preventing cardiomyocytes from hypertrophic response induced by angiotensinⅡ (AngⅡ). Hypertrophic response of neonatal rat cardiomyocytes was assayed by protein synthesis rate and expression of atrial natriuretic peptide (ANP) mRNA. The level of NO was shown by the content of nitrate and nitrite in cardiac myocytes. The protein expression of MKP-1 and the gene expression of eNOS were measured with Western blotting and RT-PCR, respectively. The results are as follows. (1) L-arginine (L-Arg) induced a dose-dependent increase in NO by 16% and 31% at the concentrations of 10 μmol/L and 100 μmol/L, respectively. L-Arg also increased the gene expression of eNOS. However, these effects were inhibited by L-NAME, the inhibitor of NOS. (2) The gene expression and the protein synthesis of ANP induced by AngⅡ (0.1 μmol/L) were inhibited by L-Arg (100 μmol/L). The inhibitory action of L-Arg was abolished after pretreatment with antisense oligoneucleotide against MKP-1. (3) L-Arg (100 μmol/L) increased the protein expression of MKP-1 by 225%, which was inhibited by L-NAME, an NOS inhibitor, and KT-5823, a cGMP-dependent protein kinase (PKG) inhibitor. However, AngⅡ enhanced the effect induced by L-Arg. The above results show that NO may activate PKG, and thereby promote the protein expression of MKP-1 and inactivate MAPK, resulting in an inhibition of cardiomyocyte hypertrophic response induced by AngⅡ.