Faculty of Biology, M.V. Lomonosov Moscow State University, Moscow, Russia.
Faculty of Biology, M.V. Lomonosov Moscow State University, Moscow, Russia; Tomsk National Research University, Tomsk, Russia; Siberian Medical State University, Tomsk, Russia.
Cell Calcium. 2017 Dec;68:5-13. doi: 10.1016/j.ceca.2017.10.002. Epub 2017 Oct 9.
In rat vascular smooth muscle cells (RVSMC), 3-h Na,K-ATPase inhibition by ouabain or in K-free medium resulted in the inversion of the [Na]/[K] ratio and elevation up to 7-fold the content of Egr1, Atf3, Nr4a1 and Ptgs2 mRNAs. Ouabain increased the rate of 45Ca influx by 2-fold that was abolished by L-type voltage-gated Ca channel blocker nicardipine, but it was resistant to Na/Ca exchanger inhibitor KB-R7943. To study the role of Ca-mediated signaling in the expression of Na/K-sensitive genes we used intracellular Ca chelator BAPTA and incubated RVSMC in Ca-free medium. The elevation of Nr4a1 and Ptgs2 expression triggered by ouabain was diminished in Ca-depeleted cells as well as in the presence of nicardipine and calmodulin antagonists A-7 and W-7. Ptgs2 expression was also suppressed by inhibitor of Ca/calmodulin-dependent protein kinase (CaMKII) KN-93 whereas increment of Nr4a1 content triggered by ouabain was attenuated by inhibitor of Ca/calmodulin-dependent protein phosphatase (calcineurin, CaN) cyclosporin A. Neither Ca depletion nor above listed compounds had any impact on the augmented expression of Egr1 and Atf3 in ouabain-treated RVSMC. Our results strongly suggest that dissipation of transmembrane gradient of monovalent cations increases Ptgs2 and Nr4a1 transcription via augment Ca influx through L-type Ca channels that, in turn, leads to CaMKII-mediated phosphorylation of CREB and calcineurin-mediated dephosphorylation of NFAT, respectively. Additional experiments should be performed to identify intermediates of Na,K-mediated Ca-independent excitation-transcription coupling involved the regulation of Egr1 and Atf3 expression.
在大鼠血管平滑肌细胞（RVSMC）中，哇巴因或无钾介质 3 小时抑制 Na，K-ATP 酶导致[Na]/[K]比值反转，Egr1、Atf3、Nr4a1 和 Ptgs2 mRNA 的含量增加 7 倍。哇巴因使 45Ca 内流增加 2 倍，该作用被 L 型电压门控钙通道阻滞剂尼卡地平所阻断，但对钠钙交换体抑制剂 KB-R7943 无作用。为研究钙介导的信号在 Na/K 敏感基因表达中的作用，我们使用细胞内 Ca 螯合剂 BAPTA，并将 RVSMC 孵育在无钙介质中。哇巴因诱导的 Nr4a1 和 Ptgs2 表达升高在钙耗竭细胞中以及在尼卡地平存在和钙调蛋白拮抗剂 A-7 和 W-7 存在时均减弱。钙/calmodulin 依赖性蛋白激酶（CaMKII）抑制剂 KN-93 也抑制 Ptgs2 表达，而哇巴因诱导的 Nr4a1 含量增加被钙/calmodulin 依赖性蛋白磷酸酶（钙调神经磷酸酶，CaN）抑制剂环孢素 A 减弱。钙耗竭或上述化合物均不影响哇巴因处理的 RVSMC 中 Egr1 和 Atf3 表达的增加。我们的结果强烈表明，单价阳离子跨膜梯度的耗散通过增加通过 L 型钙通道的 Ca 内流来增加 Ptgs2 和 Nr4a1 转录，这反过来导致 CaMKII 介导的 CREB 磷酸化和钙调神经磷酸酶介导的 NFAT 去磷酸化。应进行额外的实验以鉴定涉及 Egr1 和 Atf3 表达调节的 Na，K 介导的 Ca 独立兴奋-转录偶联的中间产物。
