VOLUME REGULATION IN NORMAL AND CATARACTOUS LENSES

描述(申请人提供)：晶状体产生溶质的内部循环，随后是液体，为这个无血管器官创造一个微循环系统。循环依赖于纤维细胞缝隙连接来传导细胞内的离子流量，也许还有液体，这些离子和液体从晶状体的中心流向表面。细胞外流量沿纤维细胞之间的间隙从晶状体表面向中心移动。细胞内和细胞外的通量由纤维细胞膜连接，纤维细胞膜表达非选择性阳离子通道、氯通道和水通道AQP0。溶质的跨膜通量产生小的跨膜渗透梯度，从而产生流体的循环。循环对中央纤维细胞的动态平衡至关重要，中央纤维细胞依靠向内定向的细胞外液体流动将必要的营养物质、氨基酸和抗氧化剂输送到晶状体中。我们已经花了很多年来描述晶状体循环的分子基础，并取得了巨大的进展(在Mathias等人，2008年进行了审查)，但仍然存在问题，如本提案的目标1和2所概述的。在最后的资助期间，我们开始整合我们所学到的知识，以了解年龄起病的中心性白内障的发生。根据我们的发现，这一提议的总体假设是：老年性核性白内障是晶状体循环受损的结果，这是由于运输蛋白的累积氧化损伤。该提案的目标3将直接解决这一假设。此外，我们需要了解正常功能的循环是如何依赖于晶状体运输参数的，以及这些参数如何被累积的氧化损伤所改变。目标1是进一步描述正常晶状体中的缝隙连接耦合。它涉及到检验两个假设。1)晶状体中的细胞内水流是由缝隙连接调节的。1b)缝隙连接耦合电导在赤道到两极之间的差异是通过MAP激酶途径对CX50通道进行调节的结果。目的2是进一步研究晶状体水通道的特征。它涉及到检验两个假设。除了提供水通道外，AQP0还具有特殊的作用。2B)AQP0受pH和[Ca~(2+)]i的生理调节。目的3研究累积氧化损伤对晶状体转运和细胞内稳态的影响。它涉及到检验两个假设。3a)晶状体中的氧化应激导致缝隙连接耦合电导降低，导致循环障碍和最终的白内障。3b)氧化介导的晶状体缝隙连接耦合的减少是通过激活PKC3来启动的。为了实现这些目标，我们将使用标准的生化技术和我们开发的几种专门技术。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。基于微电极的技术将被用来测量完整晶状体细胞内静水压力以及细胞内钠和钙的空间分布。大多数研究涉及转基因小鼠的晶状体，这些晶状体表达修饰的运输蛋白或其他酶。 公共卫生相关性：这项建议中描述的工作旨在加深我们对晶状体循环的理解，以及运输蛋白的累积氧化损伤如何损害循环并导致老年性中枢性白内障。我们的假设是，晶状体中缝隙连接的氧化损伤是引发一系列导致中枢性白内障的事件的第一步。如果是这样的话，有药物干预可以上调缝隙连接偶联，例如PKC3的抑制剂，这些可能会阻止白内障的发展。

DESCRIPTION (provided by applicant): The lens generates an internal circulation of solute, which is followed by fluid to create a micro circulatory system for this avascular organ. The circulation depends on fiber cell gap junctions to conduct intracellular fluxes of ions and perhaps fluid, which flow from the center to surface of the lens. Extracellular fluxes move from the surface to center of the lens along the spaces between fiber cells. The intracellular and extracellular fluxes are linked by fiber cell membranes, which express nonselective cation channels, chloride channels and the water channel AQP0. The transmembrane flux of solute creates small transmembrane osmotic gradients that generate the circulation of fluid. The circulation is critical for homeostasis in central fiber cells, which rely on the inwardly directed extracellular fluid flow to carry essential nutrients, amino acids and antioxidants into the lens. We have spent many years characterizing the molecular basis of the lens circulation and have made enormous progress (reviewed in Mathias et al., 2008), but there are still questions, as outlined in aims 1 and 2 of this proposal. In the last grant period, we began the process of putting together what we have learned to understand generation of the age-onset central cataract. Based on our findings, the overall hypothesis for this proposal is: The age-onset nuclear cataract is a consequence of compromise of the lens circulation due to cumulative oxidative damage to transport proteins. Aim 3 of this proposal will directly address this hypothesis. Moreover, we need to understand how a normally functioning circulation depends on lens transport parameters, and how these parameters can be altered by cumulative oxidative damage. Aim 1 is to further characterize gap junction coupling in the normal lens. It involves testing 2 hypotheses. 1a) Intracellular water flow in the lens is mediated by gap junctions. 1b) The equator to poles variation in gap junction coupling conductance is due to regulation of Cx50 channels through the MAP kinase pathway. Aim 2 is to further characterize lens water channels. It involves testing 2 hypotheses. 2a) AQP0 has a specialized role other than providing a water channel. 2b) AQP0 is physiologically regulated by pH and [Ca2+]i. Aim 3 is to characterize the effects of cumulative oxidative damage on lens transport and intracellular homeostasis. It involves testing 2 hypotheses. 3a) Oxidative stress in the lens causes reductions in gap junction coupling conductance, leading to compromise of the circulation and eventual cataracts. 3b) Oxidation-mediated reductions in lens gap junction coupling are initiated through activation of PKC3. To achieve these aims we will use standard biochemical techniques and several specialized techniques that were developed by us. 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. Microelectrode based techniques will be used to measure the spatial distribution of intracellular hydrostatic pressure, and intracellular sodium and calcium in intact lenses. Most studies involve lenses from genetically altered mice that express modified transport proteins or other enzymes. PUBLIC HEALTH RELEVANCE: The work described in this proposal is designed to further our understanding of the lens circulation, and how cumulative oxidative damage to transport proteins can compromise the circulation and lead to age-onset central cataracts. Our hypothesis is oxidative damage to gap junction coupling in the lens is the first step that initiates a cascade of events leading to central cataracts. If so, there are pharmacological interventions that can up regulate gap junction coupling, such as inhibitors of PKC3, and these could potentially prevent development of the cataract.