Moseley A E, Dean W L, Delamere N A
Department of Biochemistry, University of Louisville, Kentucky, USA.
Invest Ophthalmol Vis Sci. 1996 Jul;37(8):1502-8.
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.
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.
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.
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.
晶状体上皮被认为能为整个晶状体细胞团进行钠钾转运。晶状体纤维的离子转运能力较差。作者测试了两种细胞类型中是否表达不同的钠钾 - ATP酶多肽，以及根据钠钾 - ATP酶α亚基的mRNA的存在情况判断这两种细胞是否都具备持续表达钠钾 - ATP酶所需的机制。
从成年大鼠晶状体上皮或纤维中分离出膜，并对钠钾 - ATP酶α1、α2和α3多肽进行蛋白质印迹实验。从成年大鼠晶状体上皮或纤维细胞中分离出总RNA，并对钠钾 - ATP酶α1、α2和α3 mRNA进行Northern印迹分析。一些实验使用新生（3日龄）大鼠晶状体的纤维细胞进行。
在成年大鼠晶状体上皮中检测到多种钠钾 - ATP酶同工型。根据Northern印迹条带强度判断，钠钾 - ATP酶α1和α2的mRNA比α3 mRNA更丰富。通过蛋白质印迹法，在100至108 kDa处观察到钠钾 - ATP酶α1、α2和α3多肽呈清晰条带。在纤维细胞中，仅检测到钠钾 - ATP酶α1免疫反应性多肽。根据免疫印迹密度判断，α1多肽在上皮和纤维细胞材料中的量相似。然而，在成年晶状体纤维中未发现钠钾 - ATP酶α亚基mRNA。为了测试在纤维细胞生长过程中是否发生钠钾 - ATP酶合成，用新生（3日龄）晶状体纤维的RNA进行Northern印迹分析；钠钾 - ATP酶α1 mRNA清晰可见。
成年大鼠晶状体上皮表达不止一种钠钾 - ATP酶催化亚基同工型，而在纤维细胞中仅能检测到α1同工型。在成年大鼠晶状体纤维细胞中，观察到α1多肽但未观察到α1 mRNA，这表明α1的持续合成较低。基于在新生晶状体纤维中检测到α1 mRNA，晶状体纤维在细胞伸长和生长过程中钠钾 - ATP酶的合成可能更高。
