Ishizawa Keisuke, Izawa Yuki, Ito Hiroyuki, Miki Chieko, Miyata Kayoko, Fujita Yoshiko, Kanematsu Yasuhisa, Tsuchiya Koichiro, Tamaki Toshiaki, Nishiyama Akira, Yoshizumi Masanori
Department of Pharmacology, University of Tokushima Graduate School of Medicine, Japan.
Hypertension. 2005 Oct;46(4):1046-52. doi: 10.1161/01.HYP.0000172622.51973.f5. Epub 2005 Aug 8.
The nongenomic effects of aldosterone have been implicated in the pathogenesis of various cardiovascular diseases. Aldosterone-induced nongenomic effects are attributable in part to the activation of extracellular signal-regulated kinase 1/2 (ERK1/2), a classical mitogen-activated protein (MAP) kinase. Big MAP kinase 1 (BMK1), a newly identified MAP kinase, has been shown to be involved in cell proliferation, differentiation, and survival. We examined whether aldosterone stimulates BMK1-mediated proliferation of cultured rat aortic smooth muscle cells (RASMCs). Mineralocorticoid receptor (MR) expression and localization were evaluated by Western blotting analysis and fluorolabeling methods. ERK1/2 and BMK1 activities were measured by Western blotting analysis with the respective phosphospecific antibodies. Cell proliferation was determined by Alamar Blue colorimetric assay. Aldosterone (0.1 to 100 nmol/L) dose-dependently activated BMK1 in RASMCs, with a peak at 30 minutes. To clarify whether aldosterone-induced BMK1 activation is an MR-mediated phenomenon, we examined the effect of eplerenone, a selective MR antagonist, on aldosterone-induced BMK1 activation. Eplerenone (0.1 to 10 micromol/L) dose-dependently inhibited aldosterone-induced BMK1 activation in RASMCs. Aldosterone also stimulated RASMC proliferation, which was inhibited by eplerenone. Aldosterone-mediated phenomena were concluded to be attributable to a nongenomic effect because cycloheximide failed to inhibit aldosterone-induced BMK1 activation. Transfection of dominant-negative MAP kinase/ERK kinase 5 (MEK5), which is an upstream regulator of BMK1, partially inhibited aldosterone-induced RASMC proliferation, which was almost completely inhibited by MEK inhibitor PD98059. In addition to the classical steroid activity, rapid nongenomic effects induced by aldosterone may represent an alternative etiology for vascular diseases such as hypertension.
醛固酮的非基因组效应与多种心血管疾病的发病机制有关。醛固酮诱导的非基因组效应部分归因于细胞外信号调节激酶1/2（ERK1/2）的激活，ERK1/2是一种经典的丝裂原活化蛋白（MAP）激酶。大MAP激酶1（BMK1）是一种新发现的MAP激酶，已被证明参与细胞增殖、分化和存活。我们研究了醛固酮是否刺激培养的大鼠主动脉平滑肌细胞（RASMCs）中BMK1介导的增殖。通过蛋白质印迹分析和荧光标记方法评估盐皮质激素受体（MR）的表达和定位。用各自的磷酸特异性抗体通过蛋白质印迹分析测量ERK1/2和BMK1的活性。通过阿拉玛蓝比色法测定细胞增殖。醛固酮（0.1至100 nmol/L）在RASMCs中剂量依赖性地激活BMK1，在30分钟时达到峰值。为了阐明醛固酮诱导的BMK1激活是否是一种MR介导的现象，我们研究了选择性MR拮抗剂依普利酮对醛固酮诱导的BMK1激活的影响。依普利酮（0.1至10 μmol/L）在RASMCs中剂量依赖性地抑制醛固酮诱导的BMK1激活。醛固酮还刺激RASMC增殖，这被依普利酮抑制。由于放线菌酮未能抑制醛固酮诱导的BMK1激活，因此醛固酮介导的现象被认为归因于非基因组效应。转染显性负性MAP激酶/ERK激酶5（MEK5），它是BMK1的上游调节因子，部分抑制醛固酮诱导的RASMC增殖，而MEK抑制剂PD98059几乎完全抑制该增殖。除了经典的类固醇活性外，醛固酮诱导的快速非基因组效应可能代表高血压等血管疾病的另一种病因。
