Cell-specific expression of the human Na+,K(+)-ATPase beta 2 subunit isoform in the nonpigmented ciliary epithelium.
Cell-specific expression of the human Na+,K(+)-ATPase beta 2 subunit isoform in the nonpigmented ciliary epithelium.
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DOI：
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发表时间：
1995-12
影响因子：
4.4
通讯作者：
M. Coca‐Prados;M. Fernandez-Cabezudo;J. Sánchez-Torres;J. Crabb;S. Ghosh
中科院分区：
文献类型：
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作者：
M. Coca‐Prados;M. Fernandez-Cabezudo;J. Sánchez-Torres;J. Crabb;S. Ghosh
目的研究人眼睫状体上皮细胞Na+，K（+）-ATP酶和H+，K（+）-ATP酶β 2亚基亚型的表达模式及β 2亚基的细胞特异性分布。方法从人眼组织中提取总RNA，以人Na+，K（+）-ATP酶亚基亚型（β 1和β 2）或H+，K（+）-ATP酶亚基（α和β）的cDNA探针进行北方印迹分析。针对人β 2同种型的氨基和羧基末端区域产生抗体。用聚合酶链反应检测睫状体非色素上皮细胞（NPE）β 2亚单位的表达。结果除透镜仅表达β 1外，所有眼组织中均存在不同水平的Na+，K（+）-ATP酶β 1和β 2亚基亚型转录本。在眼睛中未检测到H+，K（+）-ATP酶α或β亚基的转录本。同种型β 2特异性抗肽抗体V15 E（N-末端）和A18 R（C-末端）识别睫状上皮中的55- 60-kDa蛋白质和N-聚糖酶处理后的32 kDa核心蛋白。睫状上皮内的免疫细胞化学定位表明Na+，K（+）-ATP酶β 2亚型优先在NPE细胞中表达。聚合酶链反应扩增和Southern印迹分析证实Na+，K（+）-ATP酶β 2亚型在人NPE细胞系ODM-2中表达。结论Na+，K（+）-ATP酶β 2亚基在人眼组织中广泛表达，而H+，K（+）-ATP酶β 2亚基在人眼组织中不表达。β 2亚型在NPE细胞内的有限细胞分布代表了与Na+，K（+）-ATP酶的多种α亚基亚型相关的重要差异基因标志物。
PURPOSE To evaluate the patterns of expression of beta subunit isoforms of the Na+,K(+)-ATPase and H+,K(+)-ATPase in the human eye and to determine the cell-specific distribution of the beta 2 subunit in the human ciliary epithelium. METHODS Total RNA extracted from human ocular tissues was screened by Northern blot analysis with cDNA probes for the human Na+,K(+)-ATPase subunit isoforms (beta 1 and beta 2) or the H+,K(+)-ATPase (alpha and beta) subunits. Antibodies were raised to the amino and carboxyl terminal regions of the human beta 2 isoform. Polymerase chain reaction was used to verify the expression of beta 2 subunit in nonpigmented ciliary epithelial cells (NPE). RESULTS Transcripts for the Na+,K(+)-ATPase beta 1 and beta 2 subunit isoforms were present at different levels in all the ocular tissues except the lens, which expressed only beta 1. No transcripts for the alpha or beta subunits of the H+,K(+)-ATPase were detected in the eye. Isoform beta 2 specific anti-peptide antibodies V15E (N-terminus) and A18R (C-terminus) recognized a 55- to 60-kDa protein in the ciliary epithelium and the core protein of 32 kDa after N-glycanase treatment. Immunocytochemical localization within the ciliary epithelium indicates that the Na+,K(+)-ATPase beta 2 isoform is expressed preferentially in the NPE cells. The expression of Na+,K(+)-ATPase beta 2 isoform in the human NPE cell line, ODM-2, was confirmed by polymerase chain reaction amplification and Southern blot analysis. CONCLUSIONS The Na+,K(+)-ATPase beta 2 subunit isoform, but not H+,K(+)-ATPase, was expressed widely in ocular tissues of the human eye. The restricted cellular distribution of beta 2 isoform within the NPE cells represents an important differential gene marker associated with the multiple alpha subunit isoforms of Na+,K(+)-ATPase.