Human corneal endothelial cell expression of Na+,K+-adenosine triphosphatase isoforms
Human corneal endothelial cell expression of Na+,K+-adenosine triphosphatase isoforms
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DOI：
10.1001/archopht.121.6.840
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发表时间：
2003-06-01
影响因子：
--
通讯作者：
Pepose, JS
中科院分区：
文献类型：
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作者：
Huang, B;Blanco, G;Pepose, JS
目的：测定人角膜内皮细胞（HCEC）中Na+，K+-腺苷三磷酸酶（ATPase）α亚基和不同同工酶的表达。方法：对经猿猴病毒40转化永生化的HCEC制备物进行Na+，K+-ATPase ut亚基表达的免疫印迹和RNA分析。Na+，K+-ATP酶活性通过以下方法测定：构建哇巴因抑制人角膜内皮细胞中 Na+,K+-ATPase 活性的剂量反应曲线。 结果：信使 RNA 分析和免疫印迹研究均表明 HCEC 表达 ATPase 催化 α1 和 α3，但不表达 α2 和 α4 亚基。与 α1 亚基相比，HCEC 中表达的 α3 亚基数量有限。 Na+,K+-ATPase 活性的生化分析揭示了 2 个独立活性的 Na+,K+-ATPase 同工酶，一个低亲和力位点的哇巴因抑制常数 (K-i) 的动力学参数在微摩尔范围内，一个高亲和力位点的常数 K-i 在纳摩尔范围内。这两个位点可能分别与 HCEC 中表达的 α1 和 α3 异构体相关。结论：人角膜内皮细胞表达 Na+,K+-ATPase 的 α1 和 α3 异构体，并且两种多肽在这些细胞中均具有催化能力。确定 HCEC 中 Na+,K+-ATP 酶的成分是阐明调节角膜内皮离子泵功能的机制以及与角膜水肿相关的角膜疾病发病机制的重要一步。
Objective: To determine the expression of alpha subunits and different isozymes of Na+,K+-adenosine triphosphatase (ATPase) in human corneal endothelial cells (HCECs).Methods: Immunoblot and RNA analysis of Na+,K+-ATPase ut subunit expression were performed in preparations from HCECs that had been immortalized by transformation with simian virus 40. Na+,K+-ATPase activity was determined by constructing dose-response curves for the ouabain inhibition of Na+,K+-ATPase activity in human corneal endothelial cells.Results: Both messenger RNA analysis and immunoblot studies indicated that HCECs express ATPase catalytic alpha1 and alpha3, but not alpha2 and alpha4, subunits. A limited amount of alpha3 subunit was expressed in HCECs compared with the alpha1 subunit. Biochemical analyses of Na+,K+-ATPase activity revealed 2 independently active Na+,K+-ATPase isoenzymes, a low-affinity site with a kinetic parameter for ouabain inhibition constant (K-i) in the micromolar range and a high-affinity site with a constant K-i in the nanomolar range. These 2 sites may be associated with alpha1 and alpha3 isoforms, respectively, expressed in HCECs.Conclusions: Human corneal endothelial cells express alpha1 and alpha3 isoforms of Na+,K+-ATPase, and both polypeptides are catalytically competent in these cells. Defining the components of Na+,K+-ATPase in HCECs is an important step toward elucidating the mechanisms that regulate corneal endothelial ionic pump function as well as the pathogenesis of corneal diseases associated with corneal edema.