The Physiological Role of Carbonic Anhydrases in the Retinal Pigment Epithelium

碳酸氢酶(CA)催化二氧化碳的可逆水合反应，并间接参与水、碳酸氢盐和质子的运输。用RT-PCR和定量RT-PCR方法在人胎视网膜色素上皮细胞中鉴定了16种同工酶中的14种。我们用免疫细胞化学和免疫印迹法检测了CA II、IV、IX、XII和XIV在hfRPE细胞中的表达和亚细胞分布。免疫细胞化学研究表明，CA IV和CA X II在RPE的顶面表达。CA IX在顶膜和基侧膜上均有表达。CAⅡ是一种细胞内蛋白，CA XIV主要分布于侧膜，也存在于顶膜和基底膜。免疫共沉淀实验表明，CA IX与碳酸氢钠共转运蛋白1和3(Nbc1，3)之间存在弱的正相互作用。用电容探针技术研究了培养的人胎儿和成年牛RPE的跨上皮液跨膜转运。多唑胺是CA II和IX的CA抑制剂，当应用于细胞的顶端表面时，可导致人胎儿和成年牛RPE对液体的可逆性吸收减少。这一数据表明，CA对于调节RPE中的流体传输是重要的。PH实验表明，CA II抑制剂(多唑胺)应用于hfRPE的顶膜时，会引起pH下降。同样，CA IX抑制剂(化合物17)暴露在hfRPE的顶膜上时，会引起pH下降和强烈的TEP下降。这些实验结果提供了与重碳酸盐运输代谢理论相一致的信息。进一步的研究将确定这些CA中的哪一个有助于调节RPE中流体的pH值和吸收。我们的研究将有助于我们对人类青光眼的认识和未来的治疗。我们的hfRPE细胞培养为研究von Hippel-Lindau(VHL)病提供了一个模型系统。

Carbonic anhydrases (CAs) catalyze the reversible hydration of CO2 and are indirectly involved in the transport of water, bicarbonate, and protons. Fourteen of the sixteen isozymes have been identified by RT-PCR and quantitative RT-PCR in human fetal RPE cells. We have determined the expression and subcellular distribution of CA II, IV, IX, XII and XIV in hfRPE cells by immunocytochemistry and Western blot. Immunocytochemical studies indicate that CA IV and CA XII are expressed at the apical surface of the RPE. CA IX is expressed on both apical and basal-lateral membranes. CA II is an intracellular protein and CA XIV is mainly observed on the lateral membrane and is also present at the apical and basal membranes. Co-immunoprecipitation experiments have shown the weak positive interaction between CA IX and sodium bicarbonate cotransporter 1 and 3 (NBC1, 3). The capacitance probe technique was used to study transepithelial fluid transport across monolayers of cultured human fetal and adult bovine RPE. Dorzolamide, a CA inhibitor of CA II and IX, caused a reversible decrease in fluid absorption across human fetal and adult bovine RPE when applied to the apical surface of the cells. This data indicates that CAs are important for regulating fluid transport across the RPE. pH-experiments shows that CA II inhibitor (dorzolamide) causes a pH-drop when applied to the apical membrane of the hfRPE. Likewise, CA IX inhibitor (compound 17) causes a pH-drop and a strong TEP-drop when exposed to the apical membrane of the hfRPE. These experimental results present information that are consistent with the bicarbonate-transport metabolon theory. Further studies will determine which of these CAs help regulate pH and absorption of fluid across the RPE. Our study will benefit our understanding and future treatment of human glaucoma. Our hfRPE cell culture would provide a model sysytem for studying von Hippel-Lindau (VHL) disease.