Rizzolo L J
Department of Anatomy and Cell Biology, Emory University School of Medicine, Atlanta, GA.
Exp Eye Res. 1990 Oct;51(4):435-46. doi: 10.1016/0014-4835(90)90156-o.
The polarity of retinal pigmented epithelia (RPE) from chicken embryos was studied in primary cell culture. Since cultured RPE approximates the morphological polarity of RPE in vivo, we investigated whether this polarity extends to the distribution of plasma membrane proteins that are peculiar to RPE. In contrast to other epithelia, the Na+,K(+)-ATPase of RPE is located in the apical rather than basolateral plasma membrane. To examine this property, we cultured RPE on extracellular matrix-coated filters. Primary cultures were compared to embryonic RPE in situ using electron microscopy and indirect immunofluorescence of frozen sections. The viability and morphology of RPE was improved by using a serum-free medium containing a bovine pituitary extract in conjunction with an extracellular matrix coating derived from Engelbreth-Holm-Swarm tumors. Cultured RPE mimicked the morphology of RPE in vivo with microvilli and junctional complexes on the apical pole and infoldings along the basolateral plasma membrane. Functional tight junctions formed as demonstrated by an EDTA-sensitive, transepithelial electrical resistance, and by the retention of [3H]inulin added to the apical chamber. In 2 hr, only 4-6% of the [3H]inulin crossed the monolayer, compared to 24% in control filters. Despite these features of polarity, the Na+,K(+)-ATPase was detected in both apical and basolateral membranes by immunofluorescence. In embryonic eyes in which the neural retina was removed, the Na+,K(+)-ATPase was confined to the apical membrane. In addition, the polarity of cultured RPE was probed with vesicular stomatitis virus. In contrast to other epithelia, budding virus particles were observed emerging from the apical, as well as basolateral, domain further suggesting the cultured cells were only partially polarized. These data indicate that structural criteria are inadequate to determine if cultured RPE have become polarized in the same manner as the epithelium in vivo.
在原代细胞培养中研究了鸡胚视网膜色素上皮（RPE）的极性。由于培养的RPE接近体内RPE的形态极性，我们研究了这种极性是否延伸到RPE特有的质膜蛋白分布。与其他上皮细胞不同，RPE的Na +，K（+）-ATP酶位于顶端质膜而非基底外侧质膜。为了研究这一特性，我们将RPE培养在细胞外基质包被的滤膜上。使用电子显微镜和冰冻切片的间接免疫荧光将原代培养物与原位胚胎RPE进行比较。通过使用含有牛垂体提取物的无血清培养基并结合源自Engelbreth-Holm-Swarm肿瘤的细胞外基质包被，RPE的活力和形态得到改善。培养的RPE模仿体内RPE的形态，顶端极有微绒毛和连接复合体，基底外侧质膜有褶皱。如通过EDTA敏感的跨上皮电阻以及添加到顶端腔室的[3H]菊粉的保留所证明的那样，形成了功能性紧密连接。在2小时内，只有4-6％的[3H]菊粉穿过单层，而对照滤膜中为24％。尽管有这些极性特征，但通过免疫荧光在顶端和基底外侧膜中均检测到Na +，K（+）-ATP酶。在去除神经视网膜的胚胎眼中，Na +，K（+）-ATP酶局限于顶端膜。此外，用水泡性口炎病毒探测培养的RPE的极性。与其他上皮细胞不同，观察到出芽的病毒颗粒从顶端以及基底外侧区域出现，这进一步表明培养的细胞仅部分极化。这些数据表明，结构标准不足以确定培养的RPE是否已以与体内上皮相同的方式极化。
