Cui Guangming, Dean William L, Delamere Nicholas A
Department of Ophthalmology and Visual Sciences, University of Louisville School of Medicine, Louisville, Kentucky 40202, USA.
Invest Ophthalmol Vis Sci. 2002 Aug;43(8):2714-20.
Earlier studies from this laboratory demonstrated the ability of lens epithelium to synthesize new Na,K-adenosine triphosphatase (Na,K-ATPase) catalytic subunit (alpha) polypeptide under conditions of increased ion permeability. In the present study, the authors considered whether continuous synthesis of Na,K-ATPase protein is necessary for maintenance of Na,K-ATPase activity in lens cells.
Na,K-ATPase activity was measured by quantifying the ouabain-sensitive rate of ATP hydrolysis in cultured human lens epithelial cells (HLE-B3) permeabilized with digitonin. The abundance of Na,K-ATPase alpha subunit was determined by Western blot analysis. Synthesis of Na,K-ATPase alpha1 polypeptide was investigated by measuring 35S-methionine incorporation.
Na,K-ATPase activity was reduced to less than 20% of the control level in HLE-B3 cells exposed to 100 microM cycloheximide for 24 hours. However, as judged by Western blot density, the abundance of Na,K-ATPase alpha1 and alpha3 subunit in cycloheximide-treated cells was 90% and 84% of the control level, respectively. 35S-methionine incorporation experiments revealed detectable labeling of Na,K-ATPase alpha1 subunit polypeptide within 30 minutes, consistent with alpha1 polypeptide synthesis. Na,K-ATPase alpha1 polypeptide labeling was also detected in the epithelium of intact rat lenses that had been allowed to incorporate 35S-methionine. Cycloheximide abolished 35S-methionine incorporation into Na,K-ATPase alpha1 subunit polypeptide of HLE-B3 cells. When added during the chase phase of the experiment, cycloheximide was found to slow the disappearance of labeled alpha1 polypeptide, consistent with a reduced rate of polypeptide degradation.
The results suggest that a continuous cycle of Na,K-ATPase alpha1 synthesis and degradation may occur in lens epithelial cells. Cycloheximide appeared to inhibit Na,K-ATPase protein synthesis and degradation. The observed reduction of Na,K-ATPase activity after treatment with cycloheximide indicates that even though Na,K-ATPase remains abundant, Na,K-ATPase becomes inactivated when protein synthesis is inhibited.
本实验室早期的研究表明，晶状体上皮细胞在离子通透性增加的条件下能够合成新的钠钾 - 三磷酸腺苷酶（Na，K - ATPase）催化亚基（α）多肽。在本研究中，作者探讨了持续合成Na，K - ATPase蛋白对于维持晶状体细胞中Na，K - ATPase活性是否必要。
通过定量用洋地黄皂苷通透处理的培养人晶状体上皮细胞（HLE - B3）中哇巴因敏感的ATP水解速率来测量Na，K - ATPase活性。通过蛋白质印迹分析确定Na，K - ATPaseα亚基的丰度。通过测量³⁵S - 甲硫氨酸掺入来研究Na，K - ATPaseα1多肽的合成。
在暴露于100μM环己酰亚胺24小时的HLE - B3细胞中，Na，K - ATPase活性降低至对照水平的20%以下。然而，通过蛋白质印迹密度判断，环己酰亚胺处理细胞中Na，K - ATPaseα1和α3亚基的丰度分别为对照水平的90%和84%。³⁵S - 甲硫氨酸掺入实验显示在30分钟内可检测到Na，K - ATPaseα1亚基多肽的标记，这与α1多肽合成一致。在已掺入³⁵S - 甲硫氨酸的完整大鼠晶状体上皮中也检测到Na，K - ATPaseα1多肽标记。环己酰亚胺消除了³⁵S - 甲硫氨酸掺入HLE - B3细胞的Na，K - ATPaseα1亚基多肽中。当在实验的追踪阶段添加时，发现环己酰亚胺减缓了标记的α1多肽的消失，这与多肽降解速率降低一致。
结果表明，晶状体上皮细胞中可能发生Na，K - ATPaseα¹合成和降解的持续循环。环己酰亚胺似乎抑制Na，K - ATPase蛋白的合成和降解。用环己酰亚胺处理后观察到的Na，K - ATPase活性降低表明，即使Na，K - ATPase仍然丰富，但当蛋白质合成受到抑制时，Na，K - ATPase会失活。
