Ghosh S, Freitag A C, Martin-Vasallo P, Coca-Prados M
Department of Ophthalmology, Yale University School of Medicine, New Haven, Connecticut 06510.
J Biol Chem. 1990 Feb 15;265(5):2935-40.
We have investigated the localization and pattern of expression of the three alpha subunit isoforms of Na,K-ATPase in the transporting ciliary epithelium of the bovine eye. Using specific cDNA probes and antisera to the alpha 1, alpha 2, and alpha 3 isoforms of Na,K-ATPase, we demonstrated that mRNAs and polypeptides for the three distinct forms of the Na,K-ATPase alpha subunit (alpha 1, alpha 2, and alpha 3) were expressed in the ciliary epithelium in vivo. Immunochemical localization of the three alpha isoforms of Na,K-ATPase in two ultrastructurally different regions of the ciliary epithelium (namely, the pars plicata and pars plana) revealed that the three alpha isoforms of Na,K-ATPase were distributed in a distinct fashion in the basolateral plasma membrane domains of nonpigmented (NPE) and pigmented (PE) cells. The NPE cells in the pars plicata showed an immunoreactive signal to all the three alpha isoforms; in the pars plana, they showed immunoreactive signals only for the alpha 1 and alpha 2 isoforms but not for alpha 3. The PE cells, in both the pars plana and pars plicata regions, showed an immunoreactive signal only for the alpha 1 isoform; immunoreactive signals were not detected for alpha 2 and alpha 3. To verify the differential immunostaining patterns of NPE and PE cells, specific antibodies for each of the three alpha subunit isoforms of Na,K-ATPase were applied to immunoblots containing microsomal fractions from flow cytometric-sorted cells (NPE and PE). Our results indicate that alpha 1, alpha 2, and alpha 3 polypeptides were present in microsomal fractions of NPE cells of the pars plicata and pars plana and that the alpha 1 polypeptide was the only polypeptide present in the PE cells from both regions of the ciliary epithelium. These results also revealed that the alpha 3 isoform epitope recognized by the monoclonal antibody McB-X3.1 in the pars plicata is not readily accessible in the pars plana. A cell line was established from the ciliary epithelium of a bovine eye by viral transformation with simian virus 40. In culture, this cell line expressed all three alpha isoforms at the mRNA and polypeptide levels, suggesting that the line may have derived from the NPE layer.
我们研究了牛眼运输性睫状体上皮中钠钾ATP酶三种α亚基同工型的定位和表达模式。使用针对钠钾ATP酶α1、α2和α3同工型的特异性cDNA探针和抗血清，我们证明了钠钾ATP酶α亚基三种不同形式（α1、α2和α3）的mRNA和多肽在体内睫状体上皮中表达。对睫状体上皮两个超微结构不同区域（即睫状突和平坦部）中钠钾ATP酶三种α同工型的免疫化学定位显示，钠钾ATP酶三种α同工型以不同方式分布于非色素（NPE）和色素（PE）细胞的基底外侧质膜结构域。睫状突中的NPE细胞对所有三种α同工型均显示免疫反应信号；在平坦部，它们仅对α1和α2同工型显示免疫反应信号，而对α3无反应。在平坦部和睫状突区域的PE细胞均仅对α1同工型显示免疫反应信号；未检测到α2和α3的免疫反应信号。为验证NPE和PE细胞的差异免疫染色模式，将针对钠钾ATP酶三种α亚基同工型各自的特异性抗体应用于包含流式细胞仪分选细胞（NPE和PE）微粒体部分的免疫印迹。我们的结果表明，α1、α2和α3多肽存在于睫状突和平坦部NPE细胞的微粒体部分，且α1多肽是睫状体上皮两个区域PE细胞中唯一存在的多肽。这些结果还显示，单克隆抗体McB-X3.1在睫状突中识别的α3同工型表位在平坦部不易接近。通过用猴病毒40进行病毒转化，从牛眼睫状体上皮建立了一个细胞系。在培养中，该细胞系在mRNA和多肽水平均表达所有三种α同工型，表明该细胞系可能源自NPE层。
