Ghosh S, Hernando N, Martín-Alonso J M, Martin-Vasallo P, Coca-Prados M
Department of Ophthalmology and Visual Science, Yale University School of Medicine, New Haven, Connecticut 06510.
J Cell Physiol. 1991 Nov;149(2):184-94. doi: 10.1002/jcp.1041490203.
The Na+,K(+)-ATPase alpha 1, alpha 2, and alpha 3 subunit isoforms have been shown to be differentially expressed in the nonpigmented (NPE) and pigmented (PE) cells of the ocular ciliary epithelium (CE) (Martin-Vasallo et al., J. Cell. Physiol., 141:243-252, 1989; Ghosh et al., J. Biol. Chem., 265:2935-2940, 1990). In this study we analyzed and compared the pattern of expression of the multiple Na+,K(+)-ATPase alpha (alpha 1, alpha 2, alpha 3) subunit genes with the pattern of expression of the Na+,K(+)-ATPase beta (beta 1, beta 2) subunit genes along the bovine CE. We have selected three regions in the CE, referred to as 1) the anterior region of the pars plicata, near the iris; 2) the middle region of the pars plicata; and 3) the posterior region of the pars plana, near the ora serrata. Using isoform-specific cDNA probes and antibodies for the Na+,K(+)-ATPase alpha 1, alpha 2, alpha 3, beta 1, and beta 2 subunits on Northern and Western blot analysis, we found that mRNA and polypeptides are expressed in all three CE regions with different abundance. The pattern of expression of alpha and beta isoforms detected along the NPE cell layers suggests a gradient of alpha 1, alpha 2, alpha 3, beta 1, and beta 2 mRNAs and polypeptides that correlates with decreasing Na+,K(+)-ATPase activity from the most anterior region at the pars plicata towards the posterior region at the ora serrata. We also found marked differences in the pattern of immunolocalization of Na+,K(+)-ATPase alpha 1, alpha 2, alpha 3, beta 1, and beta 2 subunit isoforms in different regions of the CE. In the anterior region, NPE cells stained intensely at the basal lateral membrane with specific monoclonal and polyclonal antibodies for each of the alpha (alpha 1, alpha 2, alpha 3) and beta (beta 1, beta 2) Na,K-ATPase isoforms. In the middle and posterior regions of the CE, NPE cells showed lower or absent levels of staining with alpha 1, alpha 2, alpha 3, and beta 1 antibodies, although staining with beta 2 was abundant. In contrast, PE cells throughout the CE were stained at the basal lateral membrane by antibodies to alpha 1 and beta 1, while no staining signals were detected with the rest of the antibodies (i.e. alpha 2, alpha 3, and beta 2). Our results support the conclusion that the three alpha and two beta isoforms of the Na+,K(+)-ATPase are differentially expressed in the two cell layers that make up the CE.(ABSTRACT TRUNCATED AT 250 WORDS)
已证实，Na⁺,K⁺-ATP酶α1、α2和α3亚基同工型在眼睫状体上皮（CE）的无色素（NPE）细胞和色素（PE）细胞中存在差异表达（Martin-Vasallo等人，《细胞生理学杂志》，141:243 - 252，1989；Ghosh等人，《生物化学杂志》，265:2935 - 2940，1990）。在本研究中，我们分析并比较了多个Na⁺,K⁺-ATP酶α（α1、α2、α3）亚基基因的表达模式与Na⁺,K⁺-ATP酶β（β1、β2）亚基基因沿牛CE的表达模式。我们在CE中选取了三个区域，分别为：1）靠近虹膜的皱襞部前部区域；2）皱襞部中部区域；3）靠近锯齿缘的扁平部后部区域。通过在Northern和Western印迹分析中使用针对Na⁺,K⁺-ATP酶α1、α2、α3、β1和β2亚基的同工型特异性cDNA探针和抗体，我们发现mRNA和多肽在所有三个CE区域均有表达，但丰度不同。沿NPE细胞层检测到的α和β同工型的表达模式表明，α1、α2、α3、β1和β2 mRNA及多肽存在梯度变化，这与从皱襞部最前部区域到锯齿缘后部区域Na⁺,K⁺-ATP酶活性降低相关。我们还发现，Na⁺,K⁺-ATP酶α1、α2、α3、β1和β2亚基同工型在CE不同区域的免疫定位模式存在显著差异。在前部区域，NPE细胞在基底外侧膜处被针对每个α（α1、α2、α3）和β（β1、β2）Na,K-ATP酶同工型特异性的单克隆和多克隆抗体强烈染色。在CE的中部和后部区域，NPE细胞用α1、α2、α3和β1抗体染色水平较低或无染色，尽管用β2抗体染色丰富。相比之下，整个CE中的PE细胞在基底外侧膜处被抗α1和β1抗体染色，而用其余抗体（即α2、α3和β2）未检测到染色信号。我们的结果支持以下结论：构成CE的两个细胞层中，Na⁺,K⁺-ATP酶的三种α同工型和两种β同工型存在差异表达。（摘要截短于250字）
