Yamamoto K, Ikeda U, Okada K, Saito T, Kawakami K, Shimada K
Jichi Medical School, Tochigi, Japan.
Cardiovasc Res. 1994 Jul;28(7):957-62. doi: 10.1093/cvr/28.7.957.
Na/K-ATPase (Na,K pump) plays an important role in the regulation of intracellular ion composition. The purpose of this study is to determine whether Na+ regulates the levels of mRNA coding for Na/K-ATPase alpha and beta isoforms in cultured rat vascular smooth muscles cells.
Na/K-ATPase alpha and beta isoform mRNA expression was evaluated by northern blot analysis. The alpha 1 subunit content was determined by ELISA. A luciferase reporter gene assay was performed to study whether the alpha 1 promoter might contain Na+ responsive elements.
Na/K-ATPase alpha 1 and beta 1 isoform mRNAs, but not alpha 2 and alpha 3 isoform mRNAs, were expressed in cultured rat vascular smooth muscle cells. Exposure of the cells to 10 microM veratridine, an Na+ channel activator, resulted in two- and threefold increases in alpha 1 and beta 1 mRNA accumulation, respectively, with a maximum at 60 min. The veratridine induced alpha 1 and beta 1 mRNA accumulation was still observed even in the absence of extracellular Ca2+. The increase in alpha 1 mRNA accumulation caused by 10 microM veratridine was associated with a significant increase in alpha 1 subunit protein accumulation. Transfection experiments with chimeric plasmids containing 5'-flanking sequences of alpha 1 isoform gene and luciferase reporter gene showed that 10 microM veratridine caused a threefold increase in luciferase activity.
These results suggest that Na+ stimulates the transcription of Na/K-ATPase alpha 1 and beta 1 isoform genes in vascular smooth muscle cells. The transfection study further supports the premise that Na+ responsive elements are located within the 5'-flanking sequences of alpha 1 isoform gene.
钠钾ATP酶（钠钾泵）在调节细胞内离子组成中起重要作用。本研究的目的是确定钠离子是否调节培养的大鼠血管平滑肌细胞中编码钠钾ATP酶α和β亚型的mRNA水平。
通过Northern印迹分析评估钠钾ATP酶α和β亚型mRNA的表达。采用酶联免疫吸附测定法测定α1亚基含量。进行荧光素酶报告基因测定以研究α1启动子是否可能含有钠离子反应元件。
培养的大鼠血管平滑肌细胞中表达钠钾ATP酶α1和β1亚型mRNA，但不表达α2和α3亚型mRNA。将细胞暴露于10微摩尔藜芦碱（一种钠离子通道激活剂），导致α1和β1 mRNA积累分别增加两倍和三倍，在60分钟时达到最大值。即使在没有细胞外钙离子的情况下，仍可观察到藜芦碱诱导的α1和β1 mRNA积累。10微摩尔藜芦碱引起的α1 mRNA积累增加与α1亚基蛋白积累的显著增加相关。用含有α1亚型基因5'侧翼序列和荧光素酶报告基因的嵌合质粒进行转染实验表明，10微摩尔藜芦碱使荧光素酶活性增加了三倍。
这些结果表明，钠离子刺激血管平滑肌细胞中钠钾ATP酶α1和β1亚型基因的转录。转染研究进一步支持了钠离子反应元件位于α1亚型基因5'侧翼序列内的前提。
