Shaikh Soni, Sarkar Jaganmay, Pramanik Asmita, Karmakar Kanchan, Chakraborti Sajal
Indian J Biochem Biophys. 2013 Oct;50(5):419-27.
There is growing evidence that ouabain, a cardiotonic steroid may promote growth of cardiac and vascular myocytes, indicating its novel role in cell growth and proliferation, without appreciable inhibition of the sodium pump. The mechanism(s) by which low dose of ouabain produces pulmonary artery smooth muscle cell proliferation, a prerequisite for right ventricular hypertrophy, is currently unknown. Here, we analyzed the effects of low dose of ouabain (10 nM) on increase in [Ca2+]i, m-calpain and protein kinase C (PKC) activities on pulmonary artery smooth muscle cell proliferation and determined their sequential involvement in this scenario. We treated bovine pulmonary artery smooth muscle cells with a low dose of ouabain (10 nM) and determined [Ca2+]i in the cells by fluorometric assay using fura2-AM, m-calpain activity by fluorometric assay using SLLVY-AMC as the substrate. PKC activity using an assay kit and assay of Na+/K+ ATPase activity spectrophotometrically. We purified m-calpain and PKCalpha by standard chromatographic procedure by HPLC and then studied cleavage of the purified PKCalpha by m-calpain using Western immunoblot method. Subsequently, we performed cell proliferation assay utilizing the redox dye resazunin. We used selective inhibitors of [Ca2+]i (BAPTA-AM), m-calpain (MDL28170), PKCalpha (Go6976) and determined their involvement in ouabain (10 nM)-mediated smooth muscle cell proliferation. Our results suggested that treatment of bovine pulmonary artery smooth muscle cells with a low dose of ouabain (10 nM) increased [Ca2+]i and subsequently stimulated m-calpain activity and proteolytically activated PKCalpha in caveolae (signaling microdomain also known as signalosomes) of the cells. Upon activation, PKCalpha increased the smooth muscle cell proliferation via Go/G1 to S/G2-M phase transition. Thus, [Ca2]i-mCalpain-PKCalpha signaling axis plays a crucial role during low dose of ouabain-mediated pulmonary artery smooth muscle cell proliferation.
越来越多的证据表明，强心甾类药物哇巴因可能促进心肌细胞和血管平滑肌细胞的生长，这表明其在细胞生长和增殖中具有新的作用，且对钠泵没有明显抑制作用。低剂量哇巴因导致肺动脉平滑肌细胞增殖（这是右心室肥大的一个先决条件）的机制目前尚不清楚。在此，我们分析了低剂量哇巴因（10 nM）对肺动脉平滑肌细胞增殖过程中细胞内钙离子浓度（[Ca2+]i）升高、m-钙蛋白酶和蛋白激酶C（PKC）活性的影响，并确定了它们在此过程中的先后作用。我们用低剂量哇巴因（10 nM）处理牛肺动脉平滑肌细胞，通过使用fura2-AM的荧光测定法测定细胞内[Ca2+]i，使用SLLVY-AMC作为底物的荧光测定法测定m-钙蛋白酶活性，使用检测试剂盒测定PKC活性，并通过分光光度法测定Na+/K+ ATP酶活性。我们通过高效液相色谱法的标准色谱程序纯化m-钙蛋白酶和PKCα，然后使用蛋白质免疫印迹法研究m-钙蛋白酶对纯化后的PKCα的切割作用。随后，我们利用氧化还原染料刃天青进行细胞增殖测定。我们使用细胞内钙离子浓度（[Ca2+]i）的选择性抑制剂（BAPTA-AM）、m-钙蛋白酶的选择性抑制剂（MDL28170）、PKCα的选择性抑制剂（Go6976），并确定它们在哇巴因（10 nM）介导的平滑肌细胞增殖中的作用。我们的结果表明，用低剂量哇巴因（10 nM）处理牛肺动脉平滑肌细胞会增加细胞内钙离子浓度（[Ca2+]i），随后刺激m-钙蛋白酶活性，并在细胞的小窝（也称为信号小体的信号微区）中蛋白水解激活PKCα。激活后，PKCα通过从G0/G1期到S/G2-M期的转变增加平滑肌细胞增殖。因此，[Ca2+]i-m-钙蛋白酶-PKCα信号轴在低剂量哇巴因介导的肺动脉平滑肌细胞增殖过程中起关键作用。
