VOLUME REGULATION IN NORMAL AND CATARACTOUS LENSES

描述：晶状体有一个由离子传输产生的内部循环，它以相同的模式产生液体运动。离子的循环依赖于纤维细胞缝隙连接来传导细胞内向外的Na~(2+)和Ca~(2+)通量，而流体的循环依赖于AQPO在渗透梯度存在的情况下携带水穿过纤维细胞膜。这两个运输系统对中央纤维细胞的动态平衡至关重要，中央纤维细胞依靠内部定向的细胞外循环将必要的营养物质和抗氧化剂(如葡萄糖和抗坏血酸)输送到晶状体中。我们计划研究晶状体纤维细胞缝隙连接和水通道，它们与钙稳态的关系，以及运输的损害如何导致钙稳态的丧失和白内障。缝隙连接通道的基本亚基是连接蛋白。晶状体纤维细胞表达Cx46和CX50两种连接蛋白亚型。我们的第一个目标是回答这个问题：1)CX46和CX50在晶状体中的生理作用是什么？膜上的水通道形成了一个叫做水通道蛋白的蛋白质家族。晶状体上皮细胞表达AQP1，纤维细胞表达AQPO。我们的第二个目标是回答这个问题：2)为什么晶状体表达从AQP1切换到AQPO(即AQPO在纤维细胞中发挥什么独特的生理作用)？我们发现，膜转运的损害会导致钙稳态的丧失和核性白内障。我们的最后一个目标是回答这个问题：3)氧化对晶状体透明度的影响是由膜运输蛋白的损伤、随之而来的钙稳态丧失和蛋白分解增加引起的吗？功能缝隙结通道的分布将使用全透镜阻抗技术进行评估。对分离的纤维细胞膜小泡的渗透研究将确定水的渗透性及其受pH和钙的调节。一种新开发的测量完整晶状体细胞内钙空间分布的技术将被用来确定受损的转运对钙稳态的影响。大多数研究涉及表达修饰运输蛋白的转基因小鼠的晶状体。

DESCRIPTION: The lens has an internal circulation that is generated by ion transport, which creates fluid movement in the same pattern. The circulation of ions depends on fiber cell gap junctions to conduct intracellular, outwardly directed fluxes of Na2+ and Ca2+, whereas the circulation of fluid depends on AQPO to carry water across fiber cell membranes in the presence of an osmotic gradient. Both transport systems are critical for homeostasis in central fiber cells, which rely on the inwardly directed extracellular circulation to convect essential nutrients and antioxidants, like glucose and ascorbate, into the lens. We plan to study lens fiber cell gap junctions and water channels, their relationship to calcium homeostasis and how compromise of transport can lead to loss of calcium homeostasis and cataract. The basic subunit of a gap junction channel is the connexin. Lens fiber cells express 2 connexin isoforms, Cx46 and Cx50. Our first aim is to answer the question: 1) What are the physiological roles of Cx46 and Cx50 in the lens? Membrane water channels form a family of proteins called aquaporins. The lens expresses 2 isoforms of aquaporins, AQP1 in the epithelium and AQPO in fiber cells. Our second aim is to answer the question: 2) Why does the lens switch expression from AQP1 to AQPO (ie what unique physiological role does AQPO fulfill in fiber cells)? We have found that compromise of membrane transport leads to loss of calcium homeostasis and a nuclear cataract. Our last aim is to answer the question: 3) Are oxidative effects on lens transparency initiated by damage to membrane transport proteins, consequent loss of calcium homeostasis and increased proteolysis? The distribution of functional gap junction channels will be evaluated using whole lens impedance techniques. Osmotic studies of isolated fiber cell membrane vesicles will determine water permeability and its regulation by pH and calcium. A newly developed technique for measuring the spatial distribution of intracellular calcium in intact lenses will be used to determine the effects of compromised transport on calcium homeostasis. Most studies involve lenses from genetically altered mice that express modified transport proteins.