Characterization of Na,K-ATPase isoform expression and activity in MDCK and Caco-2 epithelial cells.
The asymmetric distribution of the Na,K-ATPase in the plasma membrane of epithelial cells is essential to the establishment of the transepithelial Na+ gradient that supports the vectorial transport of ions and solutes. To investigate the changes that occur during the development of polarity, we have characterized Na,K-ATPase expression and activity in two epithelial cell culture lines, Madin Darby canine kidney (MDCK) and Caco-2 cells. RNA and immunoblot analysis of both cell lines demonstrate that only the alpha 1 and beta 1 isoforms are expressed in nonpolarized and polarized cultures. Interestingly, alpha 1 and beta 1 message increases in MDCK cells with the development of polarity, yet there is little change in the amount of protein for either subunit. In contrast, alpha 1 and beta 1 polypeptide expression increases in Caco-2 cells with the development of polarity, even though the amount of both transcripts decreases. The lack of correlation between the changes that occur at the level of the message and protein suggest that appropriate expression is mediated in part by a combination of transcriptional and translational events. Furthermore, while there was a slight decrease in activity in polarized MDCK cells, there was a 1.9 fold increase in Na,K-ATPase activity in the polarized Caco-2 cells as compared to nonpolarized cells. These results demonstrate that the regulation of Na,K-ATPase alpha 1 and beta 1 isoform expression is mediated by a combination of transcriptional and translational events during the development of polarity in both cell lines.
Grindstaff
K K
KK
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110, USA.
Blanco
G
G
Mercer
R W
RW
D000818:Animals; D018938:Caco-2 Cells / Q000166:cytology / Q000201:enzymology*; D002460:Cell Line / Q000201:enzymology; D016764:Cell Polarity / Q000502:physiology; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D004847:Epithelial Cells; D004848:Epithelium / Q000201:enzymology; D015870:Gene Expression / Q000502:physiology; D006801:Humans; D015151:Immunoblotting; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D007686:Kidney Tubules, Distal / Q000166:cytology; D011506:Proteins / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D000254:Sodium-Potassium-Exchanging ATPase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
D007527:Isoenzymes; D011506:Proteins; D012333:RNA, Messenger; D000254:Sodium-Potassium-Exchanging ATPase