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: 7594091
• 负责人: sheldon s miller
• 金额: $ 43.27万
• 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/7594091
• 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; Data; Dorzolamide; Drops; Electric Capacitance; Goals; Human; Hydration status; Isoenzymes; Journals; Lateral; Liquid substance; Membrane; Peer Review; Physiological; Proteins; Protons; Publishing; Research; Reverse Transcriptase Polymerase Chain Reaction; Role; Structure of retinal pigment epithelium; Surface; Techniques; Water; Western Blotting; absorption; apical membrane; carbonate dehydratase; 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. Twelve of the fourteen 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, and XIV in hfRPE cells by immunocytochemistry and Western blot. Immunocytochemical studies indicate that CA IV and CA IX are expressed at the apical surface of the RPE. 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. 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.

碳酸酐酶 (CA) 催化 CO2 的可逆水合，并间接参与水、碳酸氢盐和质子的运输。 已通过 RT-PCR 和定量 RT-PCR 在人胎儿 RPE 细胞中鉴定出 14 种同工酶中的 12 种。 我们通过免疫细胞化学和蛋白质印迹测定了 hfRPE 细胞中 CA II、IV、IX 和 XIV 的表达和亚细胞分布。 免疫细胞化学研究表明 CA IV 和 CA IX 在 RPE 顶端表面表达。 CA II 是细胞内蛋白，CA XIV 主要在侧膜上观察到，也存在于顶膜和基底膜上。 电容探针技术用于研究跨培养的人胎儿和成年牛 RPE 单层的跨上皮液体转运。 Dorzolamide 是一种 CA II 和 IX 的 CA 抑制剂，当应用于细胞顶端表面时，会导致人类胎儿和成年牛 RPE 的液体吸收可逆性减少。该数据表明 CA 对于调节 RPE 上的液体运输非常重要。 pH 实验表明，CA II 抑制剂（多佐胺）在应用于 hfRPE 顶膜时会导致 pH 值下降。 同样，当暴露于 hfRPE 的顶膜时，CA IX 抑制剂（化合物 17）会导致 pH 值下降和 TEP 强烈下降。 这些实验结果提供了与碳酸氢根转运代谢理论一致的信息。 进一步的研究将确定哪些 CA 有助于调节 RPE 的 pH 值和液体吸收。