Intracellular Na+ directly modulates Na+,K+-ATPase gene expression in normal rat kidney epithelial cells.
BACKGROUND
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
In NRK, ouabain causes an increase in [Na+]i, which directly modulates Na+,K+-ATPase alpha1- and beta1-mRNA accumulation.
Muto
S
S
Department of Nephrology, Endocrinology and Metabolism, and Biology, Jichi Medical School, Minamikawachi, Tochigi, Japan. smuto@jichi.ac.jp
Nemoto
J
J
Okada
K
K
Miyata
Y
Y
Kawakami
K
K
Saito
T
T
Asano
Y
Y
D000818:Animals; D002118:Calcium / Q000502:physiology; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000502:physiology; D015870:Gene Expression / Q000502:physiology*; D007425:Intracellular Membranes / Q000378:metabolism*; D007527:Isoenzymes / Q000235:genetics; D007668:Kidney / Q000166:cytology / Q000502:physiology*; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D010042:Ouabain / Q000494:pharmacology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D012016:Reference Values; D012964:Sodium / Q000502:physiology*; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics*
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D019384:Nucleic Acid Synthesis Inhibitors; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D003609:Dactinomycin; D010042:Ouabain; D003513:Cycloheximide; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D002118:Calcium