Muto S, Nemoto J, Okada K, Miyata Y, Kawakami K, Saito T, Asano Y
Department of Nephrology, Endocrinology and Metabolism, and Biology, Jichi Medical School, Minamikawachi, Tochigi, Japan.
Kidney Int. 2000 Apr;57(4):1617-35. doi: 10.1046/j.1523-1755.2000.00006.x.
In a wide variety of cell systems, increases in cell Na+ ([Na+]i) lead to an induction of N+,K+-ATPase mRNA expression. On the other hand, the increase in [Na+]i can also induce a rise in cell Ca2+ ([Ca2+]i) through a secondary inhibition of Na+/Ca2+ exchange and a decrease in cell pH (pHi) through a secondary inhibition of Na+/H+ exchange. It is not known whether [Na+]i, [Ca2+]i, and/or pHi directly modulate N+,K+-ATPase mRNA expression.
We used normal rat kidney epithelial cells (NRK) to examine the effects of ouabain on N+,K+-ATPase alpha1- and beta1-mRNA accumulation by Northern blot analysis and the relationship between the mRNA accumulation and [Na+]i, [Ca2+]i, or pHi. [Na+]i, [Ca2+]i, and pHi were measured using a Na+-sensitive fluorescent dye (SBFI), a Ca2+-sensitive fluorescent dye (Fura-2), and a pH-sensitive fluorescent dye (BCECF), respectively.
Ouabain (1 mmol/L) significantly increased [Na+]i. Upon addition of ouabain, alpha1-mRNA levels increased to 2. 3 times the control level at three hours, with maximum 3.3-fold elevations at 12 hours. beta1-mRNA levels also increased to 2.4 times the control level at 3 hours, with a maximum 3.3-fold increase at 12 hours. The ouabain-mediated alpha1- and beta1-mRNA induction was inhibited by both the RNA transcription inhibitor (actinomycin D) and the protein synthesis inhibitor (cycloheximide). Ouabain at three hours caused an increase in [Ca2+]i. Similar increases in [Ca2+]i, which were elicited by the Ca2+ ionophore (ionomycin) in the presence of extracellular Ca2+, had no effect on alpha1- or beta1-mRNA levels. In Ca2+-free medium treated with EGTA, ouabain at three hours caused a significant increase in [Na+]i without any changes in [Ca2+]i, and also increased alpha1- and beta1-mRNA levels. Ouabain at three hours caused a significant decrease in pHi. Similar decreases in pHi, which were elicited by the specific inhibitor of Na+/H+ exchange (ethylisopropylamiloride), caused no effect on alpha1- or beta1-mRNA levels. Exposure of NRK to the Na+ ionophore (monensin) in the absence of extracellular Ca2+ increased [Na+]i and alpha1- and beta1-mRNA levels. The increases in alpha1- and beta1-mRNA levels upon addition of ouabain were associated with significant increases in alpha1- and beta1-subunit proteins.
In NRK, ouabain causes an increase in [Na+]i, which directly modulates Na+,K+-ATPase alpha1- and beta1-mRNA accumulation.
在多种细胞系统中，细胞内钠离子浓度（[Na⁺]i）升高会导致N⁺,K⁺-ATP酶mRNA表达增加。另一方面，[Na⁺]i升高还可通过对钠钙交换的继发性抑制使细胞内钙离子浓度（[Ca²⁺]i）升高，并通过对钠氢交换的继发性抑制使细胞内pH值（pHi）降低。目前尚不清楚[Na⁺]i、[Ca²⁺]i和/或pHi是否直接调节N⁺,K⁺-ATP酶mRNA表达。
我们使用正常大鼠肾上皮细胞（NRK），通过Northern印迹分析来检测哇巴因对N⁺,K⁺-ATP酶α1和β1 mRNA积累的影响，以及mRNA积累与[Na⁺]i、[Ca²⁺]i或pHi之间的关系。分别使用钠敏感荧光染料（SBFI）、钙敏感荧光染料（Fura-2）和pH敏感荧光染料（BCECF）来测量[Na⁺]i、[Ca²⁺]i和pHi。
哇巴因（1 mmol/L）显著升高了[Na⁺]i。加入哇巴因后，α1 mRNA水平在3小时时增加到对照水平的2.3倍，在12小时时最高升高3.3倍。β1 mRNA水平在3小时时也增加到对照水平的2.4倍，在12小时时最高增加3.3倍。哇巴因介导的α1和β1 mRNA诱导被RNA转录抑制剂（放线菌素D）和蛋白质合成抑制剂（环己酰亚胺）所抑制。3小时时的哇巴因导致[Ca²⁺]i升高。在细胞外存在钙离子的情况下，由钙离子载体（离子霉素）引起的类似[Ca²⁺]i升高对α1或β1 mRNA水平没有影响。在用EGTA处理的无钙培养基中，3小时时的哇巴因使[Na⁺]i显著升高，而[Ca²⁺]i没有任何变化，同时也增加了α1和β1 mRNA水平。3小时时的哇巴因导致pHi显著降低。由钠氢交换特异性抑制剂（乙基异丙基氨氯吡咪）引起的类似pHi降低对α1或β1 mRNA水平没有影响。在无细胞外钙离子的情况下，将NRK暴露于钠载体（莫能菌素）中会增加[Na⁺]i以及α1和β1 mRNA水平。加入哇巴因后α1和β1 mRNA水平的升高与α1和β1亚基蛋白的显著增加相关。
在NRK中，哇巴因导致[Na⁺]i升高，这直接调节了N⁺,K⁺-ATP酶α1和β1 mRNA的积累。
