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Roles of intracellular calcium and nitric oxide in vascular smooth muscle cell - searching for new molecular target(s) of anti-atherosclerotic drugs -

血管平滑肌细胞内钙和一氧化氮的作用-寻找抗动脉粥样硬化药物的新分子靶点-

基本信息

批准号：
18590247
负责人：
NISHIO Matomo
金额：
$ 2.41万
依托单位：
Kanazawa Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

项目摘要

Preventing and treating atherosclerosis is an important clinical issue since its development is the primary cause of cardiovascular diseases. The aberrant proliferation of vascular smooth muscle cells (SMCs) has been accepted as a key event in the pathophysiology of atherosclerosis. To search for new molecular target(s) of anti-atherosclerotic drugs, we intended to regulate SMC growth by controlling intracellular calcium signaling and nitric oxide (NO)-mediated signaling. Calcium channel blockers (CCBs) are useful for this purpose, because some of these drugs exhibit anti-atherosclerotic action via mechanism(s) other than L type Ca^<2+> channel blockade. Human epidermoid carcinoma A431 cell line which lacks the L type Ca^<2+> channel was used as a target cell line. Analysis of cell growth demonstrated that dihydropyridine CCBs such as amlodipine inhibit the growth of A431 cells. Antiproliferative CCBs specifically attenuated the capacitative Ca^<2+> entry evoked by Ca^<2+> store deplet … More ion and phospholipase C-coupled receptor stimulation. Reverse transcription-polymerase chain reaction analysis showed that A431 cells express mRNAs of canonical transient receptor potential 1 and 5 (TRPC1, TRPC5), molecular candidates for store-operated and receptor-activated cation channels. Cell cycle analysis demonstrated that amlodipine induced Gi cell cycle arrest in A431 cells. Under this condition, amlodipine decreased the phosphorylation of retinoblastoma protein, a regulator of G1 to S phase transition, and kinase activities associated with cell cycle regulators such as cyclin Dl and cyclin-dependent kinase 4 (CDK4), whereas increased CDK inhibitor p21^<waf1/Cip1> protein expression. These results demonstrated that amlodipine caused GI cell cycle arrest and growth inhibition in A431 cells through induction of p21^<waf1/Cip1> (Yoshida J., et al., Biochem. Pharmacol., 73: 943-953, 2007). Therefore, p21^<waf1/Cip1> might be a key molecule for the inhibition of SMC growth, because its negative role in the progression of atherosclerosis is suggested. In this study, we established a method for quantifying the nanomolar levels of nitrite (NO_2), a metabolite of nitric oxide (NO), in biological samples (Ishibashi T., et al., Tohoku J. Exp. Med., 215:2008, in press). This sensitive method is applicable for exploring mechanism(s) underlying the NO-releasing effect of amlodipine and SMC growth inhibition by NO. Less

动脉粥样硬化是心血管疾病的主要原因，因此预防和治疗动脉粥样硬化是一个重要的临床问题。血管平滑肌细胞（SMC）的异常增殖已被认为是动脉粥样硬化病理生理学中的关键事件。为了寻找抗动脉粥样硬化药物的新的分子靶点，我们试图通过调控细胞内钙信号和一氧化氮（NO）介导的信号来调节SMC的生长。钙通道阻滞剂（CCB）可用于此目的，因为这些药物中的一些通过L型Ca^2+通道阻滞以外的机制表现出抗动脉粥样硬化作用。使用缺乏L型Ca^<2+>通道的人表皮样癌A431细胞系作为靶细胞系。细胞生长分析表明，二氢吡啶类CCBs，如NH 4，可抑制A431细胞的生长。抗增殖CCBs特异性减弱由Ca^<2+>库耗竭引起的容量性Ca^<2 +>内流 ...更多信息离子和磷脂酶C偶联受体刺激。逆转录-聚合酶链反应分析表明，A431细胞表达典型的瞬时受体电位1和5（TRPC 1，TRPC 5）的mRNA，储存操作和受体激活的阳离子通道的分子候选人。细胞周期分析表明，在A431细胞中，顺铂诱导Gi细胞周期阻滞。在此条件下，RNP降低视网膜母细胞瘤蛋白（G1至S期转变的调节剂）的磷酸化，以及与细胞周期调节剂（如细胞周期蛋白D1和细胞周期蛋白依赖性激酶4（CDK 4））相关的激酶活性，而增加CDK抑制剂p21^<waf 1/Cip 1>蛋白表达。这些结果表明，在A431细胞中，通过诱导p21 - 1 <waf 1/Cip 1>，Gl引起GI细胞周期停滞和生长抑制（Yoshida J.，例如，生物化学和药理学，73：943-953，2007）。因此，p21^<waf 1/Cip 1>可能是抑制SMC生长的关键分子，因为它在动脉粥样硬化的进展中起负性作用。在这项研究中，我们建立了一种定量生物样品中一氧化氮（NO）代谢产物亚硝酸盐（NO_2）纳摩尔水平的方法（Ishibashi T.，例如，东北J. Exp.医学、215：2008，印刷中）。这种灵敏的方法适用于探索NO释放作用的机制和NO抑制SMC生长的机制。