Regulation of Na+,K(+)-ATPase and the Na+/K+/Cl- co-transporter in the renal epithelial cell line NBL-1 under osmotic stress.
The long-term adaptation of the Na+,K(+)-ATPase to hypertonicity was studied using the bovine renal epithelial cell line NBL-1. Na+,K(+)-ATPase activity measured in intact cells as the ouabain-sensitive fraction of Rb+ uptake was stimulated (40% above controls) after incubating the cells in hypertonic medium. This stimulation was not correlated with significant changes in the amount of Na+,K(+)-ATPase alpha 1 subunit protein. Nevertheless, the amount of alpha 1 but not beta 1 subunit mRNA progressively increased after hypertonic shock (3-4-fold above basal values). These results suggest that the alpha 1 subunit gene is modulated by medium osmolarity, although this does not necessarily involve enhanced translation of the mRNA into active alpha 1 protein. Indeed, the increase in the biological activity of the Na+,K(+)-ATPase is abolished when the electrochemical Na+ transmembrane gradient is depleted by monensin, which is consistent with a post-translational effect on the activity of the sodium pump. A furosemide-sensitive component of Rb+ uptake, attributable to Na+/K+/Cl- co-transporter activity, was very low when cells were cultured in a regular medium, but was greatly induced after hypertonic shock. This induction could not be blocked by cycloheximide. Colcemide addition slightly reduced the absolute increase in Na+/K+/Cl- co-transporter activity, while cytochalasin B significantly potentiated the effect triggered by hypertonic shock. It is concluded: (i) that in NBL-1 cells the alpha 1 but not the beta 1 subunit of the Na+,K(+)-ATPase is encoded by an osmotically sensitive gene, and (ii) that the Na+/K+/Cl- co-transporter, although an osmotically sensitive carrier, is induced by a mechanism that is independent of protein synthesis but may rely, in an undetermined manner, on the structure of the cytoskeletal network.
Department de Bioquímica i Biologia Molecular, Universitat de Barcelona, Spain.
A
A
Ferrer-Martinez
F J
FJ
Casado
A
A
Felipe
M
M
Pastor-Anglada
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D002460:Cell Line; D004848:Epithelium / Q000378:metabolism; D005786:Gene Expression Regulation; D007668:Kidney / Q000378:metabolism*; D009997:Osmotic Pressure; D012333:RNA, Messenger / Q000032:analysis; D028021:Sodium-Potassium-Chloride Symporters; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
D002352:Carrier Proteins; D012333:RNA, Messenger; D028021:Sodium-Potassium-Chloride Symporters; D000254:Sodium-Potassium-Exchanging ATPase