培养的牛视网膜色素上皮细胞中的钠钾ATP酶活性

Na+,K+-ATPase activity in cultured bovine retinal pigment epithelium.

作者信息

Shimura M, Kakazu Y, Oshima Y, Tamai M, Akaike N

机构信息

Department of Ophthalmology, School of Medicine, Tohoku University, Sendai, Japan.

出版信息

Invest Ophthalmol Vis Sci. 1999 Jan;40(1):96-104.

PMID:9888432

Abstract

PURPOSE

To characterize electrogenic Na+,K+-ATPase activity in cultured bovine retinal pigment epithelium (RPE).

METHODS

Cultured bovine RPE cells from passages 3 through 5 were dissociated enzymatically. Na+,K+-adenosine triphosphatase (ATPase)-activated currents (Ip) were measured by using a nystatin perforated-patch recording technique under voltage- clamp conditions. In the presence of suitable blockers for known voltage-dependent Na+, K+, and Ca2+ conductances, the Ip was activated in a concentration-dependent manner by adding K to the external solution.

RESULTS

The median effective concentration (EC50) and Hill coefficient for external K+ concentration ([K+]o) were 1.06 mM and 2.55, respectively. The Ip showed no significant voltage dependency. A large outward shift of holding current was observed when [Na+]o, was removed. In the presence of [Na+]o, the addition of K+ to the external solution induced Ip, even when the internal solution did not contain Na+, suggesting the existence of a continuous Na+ influx across the plasma membrane in the presence of [Na+]o,. When Na+ was removed from the external and internal solutions, a transient Ip was observed, indicating that the transient Ip was activated by the intracellular residual Na+. The Ip was concentration-dependently suppressed by ouabain. The 50% inhibitory concentration (IC50) value and Hill coefficient for ouabain were 5.98 microM and 1.12, respectively.

CONCLUSIONS

The present study is the first to reported the functional properties of electrogenic Na+,K+-ATPase activity in cultured bovine RPE.

摘要

目的

表征培养的牛视网膜色素上皮（RPE）中的电生钠钾ATP酶活性。

方法

对第3至5代培养的牛RPE细胞进行酶解。在电压钳制条件下，使用制霉菌素穿孔膜片钳记录技术测量钠钾三磷酸腺苷酶（ATP酶）激活电流（Ip）。在存在已知电压依赖性钠、钾和钙电导的合适阻滞剂的情况下，通过向外部溶液中添加钾，Ip以浓度依赖性方式被激活。

结果

外部钾浓度（[K+]o）的半数有效浓度（EC50）和希尔系数分别为1.06 mM和2.55。Ip没有明显的电压依赖性。当去除[Na+]o时，观察到保持电流有一个大的向外偏移。在存在[Na+]o的情况下，即使内部溶液不含钠，向外部溶液中添加钾也会诱导Ip，这表明在存在[Na+]o时，跨质膜存在持续的钠内流。当从外部和内部溶液中去除钠时，观察到一个短暂的Ip，表明短暂的Ip是由细胞内残留的钠激活的。Ip被哇巴因浓度依赖性抑制。哇巴因的50%抑制浓度（IC50）值和希尔系数分别为5.98 microM和1.12。