Na^+-K^+-ATP酶活性及其α_1催化亚基在鸡晶状体中的分布

Activity of Na^+-K^+-ATPase and distribution of catalytic subunit α_1 in chicken lenses

目的对Na+-K+-ATP酶催化亚基蛋白在鸡晶状体的上皮层、周边部和中央部纤维的分布和酶活性进行检测和比较。方法将部分胚胎第10天(E10)的鸡晶状体显微解剖为晶状体上皮部、周边部纤维和中央部纤维3个部分,剩余部分制备冰冻切片。利用免疫荧光染色和Western blot方法检测Na+-K+-ATP酶催化亚基(α)在晶状体各部分的表达,通过检测哇巴因敏感的ATP水解率测定Na+-K+-ATP酶的活性。结果在晶状体切片上,α1催化亚基蛋白在晶状体上皮细胞(LECs)膜上有很强的表达,而在晶状体纤维细胞膜的表达明显减弱,在周边部上皮细胞的表达有明显的极性,而在中央部上皮细胞中未见表达。Western blotting检测也再次证实α1催化亚基蛋白在LECs中的表达明显高于在晶状体纤维中的表达。上皮层Na+-K+-ATP酶的活性是周边部纤维酶活性的2倍,是中央部纤维酶活性的11倍。结论Na+-K+-ATP酶α1催化亚基蛋白在鸡晶状体的上皮细胞和纤维上表达;在鸡晶状体上Na+-K+-ATP酶及其活性的分布是不均一的。

Objective The catalytic subunits of Na^＋-K^＋-ATPase have been identified in the lenses of rat, bovine, porcine, newt and human. However,there have been no reports about it in chicken lenses. Present study was conducted to detect and compare the specific activity of Na^＋-K^＋-ATPase and examine the expression of Na^＋-K^＋-ATPase protein in epithelium, peripheral fibers and central fibers of chicken lens. Methods Embryonic day 10 （EIO） chicken lenses were microdissected into lens epithelium,peripheral fibers and central fibers. Cryosections of E10 lenses were prepared. Immunofluorescence staining and Western blotting assay were used to detect Na^＋-K^＋-ATPase catalytic subunit（α）.Na^＋-K^＋ -ATPase activity was determined by detecting the ouabain-sensitive rate of adenosine triphosphate （ATP） hydrolysis. Results In immunostaining sections, the catalytic subunit α1 presented the stronger green fluorescence in the plasma membrane of lens epithelium and weaker fluorescence in lens fibers. A polarized distribution of α1 catalytic subunit isoform was shown in peripheral fibers but not in central fibers. Western blotting assay indicated that the expression intensity of α1 protein was very high in lens epithelium but was iow in both peripheral and centrai fibers.Na^＋-K^＋-ATPase activity（ OD value） in the epithelium （0. 226 ± 0. 062） was two times higher than that in the peripheral fibers （0. 111 ±0.023,q =2.08,P 〉0.05） and about 11 times higher than that in the central fibers （0. 020 ± 0. 044,q = 5.34,P 〈 0.05 ） , and the activity of Na^＋-K^＋-ATPase in peripheral fibers was about 5.55 times higher than that of central fibers （q = 4.78,P 〈 O. 05 ）. Conclusion The catalytic subunit α1 of Na^＋-K^＋-ATPase exist in epithelial and fiber cells of E10 chicken lenses. The distribution and specific activity of Na^＋-K^＋-ATPase are not uniform in the lenses, showing a consistent outcome with the report in other animals.