Isoforms of Na,K-ATPase in rat lens epithelium and fiber cells.
OBJECTIVE
The lens epithelium is thought to conduct Na-K transport for the entire lens cell mass. Lens fibers have a poor ion transport capacity. The authors tested whether different Na,K-ATPase polypeptides are expressed in the two cell types and whether both cells have the machinery needed for ongoing Na,K-ATPase expression as judged by the presence of mRNA for the Na,K-ATPase alpha subunit.


METHODS
Membranes were isolated from adult rat lens epithelium or fibers, and Western blot experiments were conducted for Na,K-ATPase alpha 1, alpha 2, and alpha 3 polypeptides. Total RNA was isolated from adult rat lens epithelium or fiber cells, and Northern analysis was conducted for Na,K-ATPase alpha 1, alpha 2, and alpha 3 mRNA. Some experiments were conducted using fiber cells from neonatal (3-day-old) rat lenses.


RESULTS
Multiple isoforms of Na,K-ATPase were detected in adult rat lens epithelium. Judged by Northern blot band intensity, mRNA for Na,K-ATPase alpha 1 and alpha 2 was more abundant than for alpha 3 mRNA. By Western blot, Na,K-ATPase alpha 1, alpha 2, and alpha 3 polypeptides were observed as sharp bands at 100 to 108 kDa. In fiber cells, only Na,K-ATPase alpha 1 immunoreactive polypeptide was detected. Judged by immunoblot density, the amount of alpha 1 polypeptide was similar in both epithelium and fiber cell material. However, Na,K-ATPase alpha subunit mRNA was not found in adult lens fibers. To test whether Na,K-ATPase synthesis takes place during fiber cell growth, Northern blot analysis was conducted with RNA from neonatal (3-day-old) lens fibers; Na,K-ATPase alpha 1 mRNA was clearly visible.


CONCLUSIONS
Adult rat lens epithelium expresses more than one isoform of Na,K-ATPase catalytic subunit, whereas only the alpha 1 isoform can be detected in fiber cells. In adult rat lens fiber cells, the observation of alpha 1 polypeptide, but no alpha 1 mRNA, suggests that ongoing alpha 1 synthesis is low. Based on the detection of alpha 1 mRNA in neonatal lens fibers, Na,K-ATPase synthesis by lens fibers may be higher during cell elongation and growth.
Moseley
A E
AE
Department of Biochemistry, University of Louisville, Kentucky, USA.
Dean
W L
WL
Delamere
N A
NA
D000818:Animals; D000831:Animals, Newborn; D015152:Blotting, Northern; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology; D002462:Cell Membrane / Q000201:enzymology; D004848:Epithelium / Q000201:enzymology; D007527:Isoenzymes / Q000378:metabolism*; D007908:Lens, Crystalline / Q000166:cytology / Q000201:enzymology*; D012313:RNA / Q000302:isolation & purification; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
D007527:Isoenzymes; D012333:RNA, Messenger; D012313:RNA; D000254:Sodium-Potassium-Exchanging ATPase