Na,K-ATPase isoforms in rat lens epithelium and fiber cells

Purpose. To determine whether different Na,K-ATPase isoform polypeptides are expressed in rat lens epithelium and fiber cells and whether both cell types contain the machinery needed for ongoing Na,K-ATPase isoform expression as evidenced by the presence of mRNA for Na,K-ATPase alpha subunit isoforms. Methods. Membrane material was isolated from adult rat lens epithelium or fiber cells and neonatal (3-day old) fibers and Western blots were conducted for Na,K-ATPase α1, α2 and α3 polypeptides. Total RNA was isolated from adult rat lens epithelium or fiber cells and neonatal fibers and Northern blots were conducted for Na,K-ATPase α1, α2 and α3 mRNA. Results. Judged by Northern blot band intensity, mRNA for Na,K-ATPase α1 and α2 was more abundant than α3 mRNA in epithelium. Also in epithelium, Western blot analysis revealed the presence of Na,K-ATPase α1 and α2 polypeptides as distinct bands at ∼105kDa; Na,K-ATPase α3 polypeptide was observed as a diffuse band at 60kDa suggesting possible proteolysis. In fiber cells, only Na,K-ATPase α1 immunoreactive polypeptide was detected. However, Na,K-ATPase α subunit mRNA was not found in adult lens fibers. To test whether Na,K-ATPase synthesis takes place during fiber cell growth, Northern blots were conducted using total RNA from neonatal lens fibers; Na,K-ATPase α1 mRNA was clearly visible. Conclusions. Adult rat lens epithelium expresses more than one isoform of Na,K-ATPase catalytic subunit while only the α1 isoform can be detected in fibers. In adult rat lens fiber cells the detection of α1 polypeptide but no α1 mRNA suggestes that ongoing α1 synthesis is low. Based on the detection of α1 mRNA in neonatal lens fibers, we propose that Na,K-ATPase synthesis by lens fibers may occur mainly during cell elongation and growth.