The Physiological Role of Carbonic Anhydrases in the Retinal Pigment Epithelium

碳酸酐酶在视网膜色素上皮中的生理作用

• 批准号: 7734633
• 负责人: sheldon s miller
• 金额: $ 15.94万
• 依托单位: NATIONAL EYE INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7734633
• 关键词: Adherent Culture; Adult; Apical; Bicarbonates; Bos taurus; Carbon Dioxide; Carbonic Anhydrase II; Carbonic Anhydrase IV; Carbonic Anhydrase Inhibitors; Cattle; Cell surface; Cells; Co-Immunoprecipitations; Cultured Cells; Data; Dorzolamide; Drops; Electric Capacitance; Future; Glaucoma; Goals; Human; Hydration status; Isoenzymes; Lateral; Liquid substance; Membrane; Modeling; Physiological; Proteins; Protons; Reverse Transcriptase Polymerase Chain Reaction; Role; Sodium-Bicarbonate Symporters; Structure of retinal pigment epithelium; Surface; Techniques; Von Hippel-Lindau Syndrome; Water; Western Blotting; absorption; apical membrane; carbonate dehydratase; carbonic anhydrase XII; carbonic anhydrase XIV; compound 17; fetal; immunocytochemistry; inhibitor/antagonist; research study; theories

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.

碳酸酐酶（CA）催化CO2的可逆水合作用，并间接参与水、碳酸氢盐和质子的运输。 用RT-PCR和定量RT-PCR方法在人胚胎RPE细胞中鉴定出16种同工酶中的14种。 我们用免疫细胞化学和Western blot方法检测了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 XII在RPE的顶端表面表达。 CA IX在顶膜和基底侧膜上表达。 CA II是一种细胞内蛋白，CA XIV主要在侧膜上观察到，也存在于顶膜和基底膜上。 免疫共沉淀实验表明CA IX和碳酸氢钠协同转运蛋白1和3（NBC 1，3）之间存在弱的正相互作用。 用电容探针技术研究了体外培养的人胎儿和成年牛视网膜色素上皮细胞单层的跨上皮液体转运。多佐胺是CA II和IX的CA抑制剂，当应用于细胞的顶端表面时，可引起人胎牛和成年牛RPE的液体吸收可逆性降低。该数据表明CA对于调节穿过RPE的流体运输是重要的。 pH实验表明，CA II抑制剂（多佐胺）在应用于hfRPE的顶端膜时引起pH下降。 同样，当暴露于hfRPE的顶膜时，CA IX抑制剂（化合物17）引起pH下降和强烈的TEP下降。 这些实验结果提出的信息是与碳酸氢盐运输代谢子理论相一致的。 进一步的研究将确定这些CA中的哪一个有助于调节pH值和通过RPE的液体吸收。 我们的研究将有助于我们对人类青光眼的理解和未来的治疗。 我们的hfRPE细胞培养将为研究von Hippel-Lindau（VHL）病提供一个模型系统。