Touyz R M, Schiffrin E L
Medical Research Council Multidisciplinary Research Group on Hypertension, Clinical Research Institute of Montreal, Quebec, Canada.
Hypertension. 1997 Aug;30(2 Pt 1):222-9. doi: 10.1161/01.hyp.30.2.222.
Angiotensin II (Ang II), a potent vasoactive peptide with mitogenic potential, influences vascular smooth muscle cell contraction and growth through receptor-linked pathways that increase intracellular free Ca2+ concentration ([Ca2+]i) and pH (pHi). Activation of these second messengers by Ang II may involve tyrosine kinase-dependent signaling pathways. This study determined the role of tyrosine kinases in Ang II-stimulated pHi, and in simultaneously measured contractile and [Ca2+]i responses, as well as growth in cultured vascular smooth muscle cells from mesenteric arteries of Wistar-Kyoto rats. pHi was determined by fluorescent digital imaging using 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM). Vascular smooth muscle cell [Ca2+]i and contractile responses were assessed simultaneously by fura 2 methodology and by photomicroscopy in cells grown on rat tail collagen gels. Cell growth was determined by DNA and protein synthesis as measured by [3H]thymidine and [3H]leucine incorporation, respectively. The Ang II receptor subtypes (AT1 or AT2) through which Ang II mediates effects were assessed with [Sar1,Ile8]Ang II (a nonselective subtype antagonist), losartan (a selective AT1 antagonist), and PD 123319 (a selective AT2 antagonist). To determine whether tyrosine kinases influence Ang II-stimulated responses, cells were pretreated with 10(-5) mol/L tyrphostin A-23 (a specific tyrosine kinase inhibitor). Ang II increased pHi in a dose-dependent manner (pD2, 9.2+/-0.2) and significantly increased vascular smooth muscle cell contraction (30%) and [Ca2+]i (pD2, 7.4+/-0.1). Ang II (10(-7) mol/L) increased DNA ([3H]thymidine incorporation) and protein synthesis ([3H]leucine incorporation). [Sar1,Ile8]Ang II and losartan but not PD 123319 abolished Ang II-elicited responses. Tyrphostin A-23 significantly attenuated Ang II-stimulated pHi responses; it also inhibited [Ca2+]i and contractile responses and cell growth. The inactive analogue tyrphostin A-1 did not alter Ang II-stimulated actions. These results provide novel evidence for a role of tyrosine kinases in Ang II-mediated pHi responses in vascular smooth muscle cells and indicate that tyrosine kinases participate in the regulation of signal transduction associated with AT1 receptor subtype-mediated contraction and growth.
血管紧张素II（Ang II）是一种具有促有丝分裂潜能的强效血管活性肽，它通过增加细胞内游离钙离子浓度（[Ca2+]i）和pH值（pHi）的受体连接途径影响血管平滑肌细胞的收缩和生长。Ang II对这些第二信使的激活可能涉及酪氨酸激酶依赖性信号通路。本研究确定了酪氨酸激酶在Ang II刺激的pHi、同时测量的收缩反应和[Ca2+]i反应以及来自Wistar-Kyoto大鼠肠系膜动脉的培养血管平滑肌细胞生长中的作用。使用2',7'-双（2-羧乙基）-5（6）-羧基荧光素乙酰氧基甲酯（BCECF-AM）通过荧光数字成像测定pHi。通过fura 2方法和在大鼠尾胶原凝胶上生长的细胞中的显微镜检查同时评估血管平滑肌细胞的[Ca2+]i和收缩反应。分别通过[3H]胸苷和[3H]亮氨酸掺入测定DNA和蛋白质合成来确定细胞生长。用[Sar1，Ile8]Ang II（一种非选择性亚型拮抗剂）、氯沙坦（一种选择性AT1拮抗剂）和PD 123319（一种选择性AT2拮抗剂）评估Ang II介导作用所通过的Ang II受体亚型（AT1或AT2）。为了确定酪氨酸激酶是否影响Ang II刺激的反应，细胞用10（-5）mol/L酪氨酸磷酸化抑制剂A-23（一种特异性酪氨酸激酶抑制剂）进行预处理。Ang II以剂量依赖性方式增加pHi（pD2，9.2±0.2），并显著增加血管平滑肌细胞收缩（30%）和[Ca2+]i（pD2，7.4±0.1）。Ang II（10（-7）mol/L）增加DNA（[3H]胸苷掺入）和蛋白质合成（[3H]亮氨酸掺入）。[Sar1，Ile8]Ang II和氯沙坦而非PD 123319消除了Ang II引发的反应。酪氨酸磷酸化抑制剂A-23显著减弱了Ang II刺激的pHi反应；它还抑制了[Ca2+]i和收缩反应以及细胞生长。无活性类似物酪氨酸磷酸化抑制剂A-1没有改变Ang II刺激的作用。这些结果为酪氨酸激酶在血管平滑肌细胞中Ang II介导的pHi反应中的作用提供了新证据，并表明酪氨酸激酶参与与AT1受体亚型介导的收缩和生长相关的信号转导调节。
