P-glycoprotein expression in human retinal pigment epithelium.
P-glycoprotein expression in human retinal pigment epithelium.
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发表时间：
2002-11
期刊：
影响因子：
2.2
通讯作者：
B. Kennedy;N. Mangini
中科院分区：
文献类型：
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作者：
B. Kennedy;N. Mangini
目的视网膜色素上皮（RPE）是一种控制视网膜下腔水化和组成的转运上皮细胞单层。P-糖蛋白是一种ATP结合盒转运蛋白，已知可转运多种疏水化合物。P-糖蛋白在屏障上皮细胞中的表达表明，它可能具有正常的保护功能，可能清除敏感区室（如视网膜下腔）中的潜在有害物质。本研究旨在检测P-糖蛋白在正常人视网膜色素上皮中的表达和活性。方法采用RT-PCR和直接测序法检测培养的人RPE细胞中mdr 1 mRNA的表达。在Western印迹中采用P-糖蛋白特异性抗体来鉴定培养的人RPE和建立的RPE细胞系（D407）中的P-糖蛋白。还使用抗P-糖蛋白抗体通过免疫组织化学将蛋白质定位在天然人RPE/脉络膜的冷冻、甲醛固定切片中。最后，在培养的人RPE单层中进行罗丹明摄取以评估P-糖蛋白活性。使用抑制性抗体4 E3和逆转蛋白121和205来阻断转运活性。结果P-糖蛋白在未暴露于任何已知的P-糖蛋白诱导剂的人RPE组织中表达并具有活性。RT-PCR得到一个546 bp的产物，其序列与已发表的人P-糖蛋白mdr 1亚型的数据100%相同。蛋白质印迹证实了在蛋白质水平上的表达，在约220和165 kD处观察到特异性条带。在天然组织中，P-糖蛋白免疫反应主要是膜相关的，与本地化的顶端和基底侧细胞膜。最后，在人RPE中表达的P-糖蛋白是有活性的。稳态罗丹明积累增加的化合物的存在下，据报道，阻止P-糖蛋白介导的罗丹明外排。结论：人RPE，不暴露于诱导剂处理，表达P-糖蛋白与本地化的顶端和基底细胞表面。基底外侧P-糖蛋白对神经视网膜有保护作用，有助于清除视网膜下腔中的有害物质。P-糖蛋白也在顶端表面上的发现表明P-糖蛋白在RPE中可能具有另外的作用。
PURPOSE The retinal pigment epithelium (RPE) is a transporting epithelial monolayer that controls hydration and composition of the subretinal space. P-glycoprotein is an ATP-binding cassette transport protein known to transport a wide range of hydrophobic compounds. The expression of P-glycoprotein in barrier epithelial cells suggests that it could serve a normal protective function, possibly clearing potentially harmful substances from sensitive compartments, like the subretinal space. The present study is designed to determine the expression and activity of P-glycoprotein in normal human RPE. METHODS RT-PCR and direct sequencing were employed to examine the presence of mdr1 mRNA in cultured human RPE. P-glycoprotein-specific antibodies were employed in Western blotting to identify P-glycoprotein in cultured human RPE and in an established RPE cell line (D407). Anti-P-glycoprotein antibodies were also used to localize the protein in frozen, formaldehyde-fixed sections of native human RPE/choroid by immunohistochemistry. Finally, rhodamine uptake was performed in cultured human RPE monolayers to assess P-glycoprotein activity. The inhibitory antibody 4E3 and reversins 121 and 205 were used to block transport activity. RESULTS P-glycoprotein is expressed, and is active, in human RPE tissue not exposed to any known inducers of P-glycoprotein. RT-PCR yielded a 546 bp product that was 100% identical in sequence to published data for the mdr1 isoform of human P-glycoprotein. Western blotting demonstrated expression at the protein level, with specific bands observed at about 220 and 165 kD. In native tissue, P-glycoprotein immunoreactivity was predominantly membrane associated, with localization to both apical and basolateral cell membranes. Finally, P-glycoprotein expressed in human RPE is active. Steady-state rhodamine accumulation was increased in the presence of compounds reported to block P-glycoprotein mediated rhodamine efflux. CONCLUSIONS Human RPE, not exposed to inducer treatment, expresses P-glycoprotein with localization to both apical and basal cell surfaces. Basolateral P-glycoprotein could serve a protective function for the neural retina helping to clear unwanted substances from subretinal space. The finding that P-glycoprotein is also on the apical surface suggests possible additional roles for P-glycoprotein in the RPE.