Isoform-specific up-regulation by ouabain of Na+,K+-ATPase in cultured rat astrocytes.
There are two alpha-subunit isoforms (alpha1 and alpha2) and two beta-subunit isoforms (beta1 and beta2) of Na+,K+-ATPase in astrocytes, but the functional heterodimer composition is not known. Ouabain (0.5-1.0 mM) increased the levels of alpha1 and beta1 mRNAs, whereas it decreased those of alpha2 and beta2 mRNAs in cultured rat astrocytes. The increases in alpha1 and beta1 mRNAs were observed at 6-48 h after addition of the inhibitor. Immunochemical analyses showed that ouabain increased alpha1 and beta1, but not alpha2 and beta2, proteins, and that the isoforms in control and ouabain-treated cultures were of glial origin. Low extracellular K+ and monensin (20 microM) mimicked the effect of ouabain on alpha1 mRNA. The ouabain-induced increase in alpha1 mRNA was blocked by the protein synthesis inhibitor cycloheximide (10 microM), the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (30 microM), and the calcineurin inhibitor FK506 (1 nM). These findings indicate that chronic inhibition of Na+,K+-ATPase up-regulates the alpha1 and beta1, but not alpha2 and beta2, isoforms in astrocytes, suggesting a functional coupling of alpha1beta1 complex. They also suggest that intracellular Na+, Ca2+, and calcineurin may be involved in ouabain-induced up-regulation of the enzyme in astrocytes.
Hosoi
R
R
Department of Pharmacology, Faculty of Pharmaceutical Sciences, Osaka University, Japan.
Matsuda
T
T
Asano
S
S
Nakamura
H
H
Hashimoto
H
H
Takuma
K
K
Baba
A
A
D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology / Q000201:enzymology*; D002614:Chelating Agents / Q000494:pharmacology; D003513:Cycloheximide / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D007527:Isoenzymes / Q000096:biosynthesis*; D007700:Kinetics; D010042:Ouabain / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D000254:Sodium-Potassium-Exchanging ATPase / Q000096:biosynthesis*; D016559:Tacrolimus / Q000494:pharmacology; D013997:Time Factors; D014158:Transcription, Genetic / Q000187:drug effects*
D002614:Chelating Agents; D007527:Isoenzymes; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D010042:Ouabain; D003513:Cycloheximide; D000254:Sodium-Potassium-Exchanging ATPase; D016559:Tacrolimus