Touyz R M, El Mabrouk M, He G, Wu X H, Schiffrin E L
Experimental Hypertension Laboratory, Medical Research Council Multidisciplinary Group on Hypertension, Clinical Research Institute of Montreal, University of Montreal, Quebec, Canada.
Circ Res. 1999 Mar 19;84(5):505-15. doi: 10.1161/01.res.84.5.505.
This study investigates the role of extracellular signal-regulated kinases (ERKs) in angiotensin II (Ang II)-generated intracellular second messengers (cytosolic free Ca2+ concentration, ie, [Ca2+]i, and pHi) and in contraction in isolated vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR) and control Wistar Kyoto rats (WKY) using the selective mitogen-activated protein (MAP)/ERK inhibitor, PD98059. VSMCs from mesenteric arteries were cultured on Matrigel basement membrane matrix. These cells, which exhibit a contractile phenotype, were used to measure [Ca2+]i, pHi, and contractile responses to Ang II (10(-12) to 10(-6) mol/L) in the absence and presence of PD98059 (10(-5) mol/L). [Ca2+]i and pHi were measured by fura-2 and BCECF methodology, respectively, and contraction was determined by photomicroscopy. Ang II-stimulated ERK activity was measured by Western blot analysis using a phospho-specific ERK-1/ERK-2 antibody and by an MAPK enzyme assay. Ang II increased [Ca2+]i and pHi and contracted cells in a dose-dependent manner. Maximum Ang II-elicited contraction was greater (P<0.05) in SHR (41.9+/-5.1% reduction in cell length relative to basal length) than in WKY (28.1+/-3.0% reduction in cell length relative to basal length). Basal [Ca2+]i, but not basal pHi, was higher in SHR compared with WKY. [Ca2+]i and pHi effects of Ang II were enhanced (P<0.05) in SHR compared with WKY (maximum Ang II-induced response [Emax] of [Ca2+]i, 576+/-24 versus 413+/-43 nmol/L; Emax of pHi, 7.33+/-0.01 versus 7.27+/-0.03, SHR versus WKY). PD98059 decreased the magnitude of contraction and attenuated the augmented Ang II-elicited contractile responses in SHR (Emax,19. 3+/-3% reduction in cell length relative to basal length). Ang II-stimulated [Ca2+]i (Emax, 294+/-55 nmol/L) and pHi (Emax, 7. 27+/-0.04) effects were significantly reduced by PD98059 in SHR. Ang II-induced ERK activity was significantly greater (P<0.05) in SHR than in WKY. In conclusion, Ang II-stimulated signal transduction and associated VSMC contraction are enhanced in SHR. MAP/ERK inhibition abrogated sustained contraction and normalized Ang II effects in SHR. These data suggest that ERK-dependent signaling pathways influence contraction and that they play a role in vascular hyperresponsiveness in SHR.
本研究使用选择性丝裂原活化蛋白（MAP）/细胞外信号调节激酶（ERK）抑制剂PD98059，研究ERK在血管紧张素II（Ang II）产生细胞内第二信使（胞质游离Ca2+浓度，即[Ca2+]i，和细胞内pH值，即pHi）以及自发性高血压大鼠（SHR）和对照Wistar Kyoto大鼠（WKY）离体血管平滑肌细胞（VSMC）收缩中的作用。肠系膜动脉的VSMC在基质胶基底膜基质上培养。这些呈现收缩表型的细胞用于测量在不存在和存在PD98059（10(-5)mol/L）时，[Ca2+]i、pHi以及对Ang II（10(-12)至10(-6)mol/L）的收缩反应。[Ca2+]i和pHi分别通过fura-2和BCECF方法测量，收缩通过显微镜检查确定。使用磷酸化特异性ERK-1/ERK-2抗体通过蛋白质免疫印迹分析和MAPK酶测定法测量Ang II刺激的ERK活性。Ang II以剂量依赖性方式增加[Ca2+]i和pHi并使细胞收缩。最大Ang II引起的收缩在SHR中（相对于基础长度细胞长度减少41.9±5.1%）比在WKY中（相对于基础长度细胞长度减少28.1±3.0%）更大（P<0.05）。与WKY相比，SHR中的基础[Ca2+]i更高，但基础pHi无差异。与WKY相比，SHR中Ang II对[Ca2+]i和pHi的作用增强（P<0.05）（[Ca2+]i的最大Ang II诱导反应[Emax]，576±24对413±43nmol/L；pHi的Emax，7.33±0.01对7.27±0.03，SHR对WKY）。PD98059降低了收缩幅度并减弱了SHR中增强的Ang II引起的收缩反应（Emax，相对于基础长度细胞长度减少19.3±3%）。在SHR中，PD98059显著降低了Ang II刺激的[Ca2+]i（Emax，294±55nmol/L）和pHi（Emax，7.27±0.04）作用。Ang II诱导的ERK活性在SHR中比在WKY中显著更高（P<0.05）。总之，SHR中Ang II刺激的信号转导及相关的VSMC收缩增强。MAP/ERK抑制消除了SHR中的持续收缩并使Ang II作用正常化。这些数据表明ERK依赖性信号通路影响收缩，并且它们在SHR的血管高反应性中起作用。
