MOLECULAR BIOLOGY OF SUGAR CATARACT IN LENS CELLS

晶状体细胞中糖性白内障的分子生物学

• 批准号: 2654643
• 负责人: Patrick Ross Cammarata
• 金额: $ 30.61万
• 依托单位: UNIVERSITY OF NORTH TEXAS HLTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-01 至 2000-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2654643
• 关键词: aldehyde reductase; animal tissue; diabetic cataract; diacylglycerols; enzyme activity; enzyme deficiency; gene expression; hyperglycemia; immunoelectron microscopy; immunofluorescence technique; lens; membrane transport proteins; molecular cloning; molecular pathology; organ culture; oxidoreductase inhibitor; scintillation counter; sorbitol; tissue /cell culture; western blottings

As a common complication of diabetes, sugar cataract represents a significant public health problem with no known effective treatment other than surgery. Hyperglycemia has been implicated in the etiology of sugar cataract formation, but the metabolic pathways and their modulation by genetic and biochemical factors have yet to be determined. The conversion of tissue glucose to sorbitol by the enzyme aldose reductase (AR), has been implicated as a potential mechanism leading to sugar cataract in the lens. Over the last six years, studies from this laboratory have defined metabolic links between hyperglycemia, polyol pathway activation and myo- inositol (MI) depletion. Recent studies from this laboratory have shown that hypertonic stress stimulates gene expression of the Na+ /M1 cotransporter and AR, increases protein synthesis and elevates both M1 uptake activity and polyol formation. The intracellular accumulation of these compatible, reciprocal, organic osmolytes operate to maintain osmotic balance and protect the lens against the perturbing effects of high intracellular concentrations of electrolytes. However, if polyol accumulation continues over a protracted period of time, it divests the lens of essential M1 by restricting M1 uptake activity via multiple inhibitory mechanisms; (1) polyol-mediated suppression of m1 carrier protein uptake activity, (2) polyol-mediated downregulation of the Na+ /M1 cotransporter mRNA and (3) polyol-activated efflux of Mi from cell to medium. In this regard hypertonicity is analogous to hyperglycemia, in that the former (by virtue of induction of AR mRNA), like the latter (by virtue of increased substrate availability) elicits an increase in intracellular polyol. The direct impairment of the M1 transport system by either means represents a plausible common mechanism that could account for the depletion of lenticular MI. This competing renewal application proposed detailed study of the mechanisms, implications and consequences of polyol- and non-polyol-induced M1 depletion in lens cell culture and lens organ culture. The depletion of intracellular M1 without alteration of intracellular polyol content will be achieved by employing a feeding regimen with L-glucose, a potent competitive inhibitor of M1 uptake, a novel technique shown by us to promote the depletion of intracellular M1 in lens cell culture without raising polyol content. In the first specific aim the molecular mechanisms which regulate the expression of the Na+/M1 cotransporter in hypertonicity and hyperosmotic diabetic conditions will be examined. In the second specific aim the interrelationship of polyol and M1 osmoregulatory mechanisms will be carefully defined. The third specific aim will identify and characterize the regulatory mechanism which modulates M1 efflux, currently the least understood mechanism of polyol-induced M1 depletion. The fourth specific aim will define the kinetics of M1 uptake in intact bovine lenses and correlate these results with known kinetic parameters from lens cell cultures. The final specific aim will utilize immunoelectron microscopy to localize the Na+/M1 cotransporter tot eh basolateral or apical aspect of lens epithelial cells in the intact lens. The studies proposed in this competitive renewal grant application will yield new information on the molecular and cellular regulation of the Na+/myo-inositol cotransporter and M1 uptake activity and provide new insight into a mechanism we believe contributes to the formation of sugar cataract.

作为糖尿病的常见并发症，糖白内障代表 重大的公共卫生问题没有已知的有效治疗其他 比手术。 高血糖与糖的病因有关 白内障形成，但代谢途径及其调节 遗传和生化因素尚未确定。 转换 通过酶还原酶（AR）的组织葡萄糖到山梨糖醇的 被视为导致晶状体糖白内障的潜在机制。 在过去的六年中，该实验室的研究定义了 高血糖，多元醇途径激活与肌醇之间的代谢联系 肌醇（MI）耗竭。 该实验室的最新研究表明 高渗应力刺激Na+ /M1的基因表达 共转运蛋白和AR增加蛋白质的合成并升高两个M1 摄取活性和多元醇形成。 细胞内积累 这些兼容的，相互的，有机渗透器可保持渗透性 平衡并保护镜头免受高高的扰动影响 电解质的细胞内浓度。 但是，如果多元 积累持续了一段时间的时间，它剥离了 通过多个限制M1吸收活性的必需M1镜头 抑制机制； （1）多元介导的M1载体抑制 蛋白质摄取活性，（2）多元介导的Na+ /M1的下调 Mi从细胞到MI的多元激活的流出物的共转运蛋白mRNA和（3） 中等的。 在这方面，高渗性类似于高血糖，在 前者（凭借AR mRNA的诱导），就像后者一样（由 底物可用性增加的优点）提高了 细胞内多元醇。 M1运输系统的直接损害 两种方式代表一个合理的共同机制，可以解释 凸耳Mi的耗竭。 此竞争续约申请 提议的详细研究对机制，含义和后果的详细研究 透镜细胞培养和晶状体中多元醇和非聚醇诱导的M1耗竭 器官文化。 细胞内M1的耗竭，没有改变 细胞内多元含量将通过采用喂养来实现 L-葡萄糖的方案是M1摄取的有效竞争抑制剂，A 我们显示的新技术促进细胞内M1的耗竭 透镜细胞培养，而无需提高多元元素含量。 在第一个特定 瞄准调节Na+/M1表达的分子机制 在高渗性和高渗性糖尿病状况方面的共转运蛋白将是 检查。 在第二个特定目标中，多元元的相互关系和 将仔细定义M1渗透调节机制。 第三个特定 AIM将识别和表征调节的调节机制 M1外排，目前最不理理解的多元醇诱导的M1机制 消耗。 第四个特定目标将定义M1摄取的动力学 在完整的牛透镜中，并将这些结果与已知动力学相关联 来自镜头细胞培养物的参数。 最终的特定目标将利用 免疫电子显微镜将Na+/M1共转运蛋白定位 完整晶状体中晶状体上皮细胞的基底外侧或顶端。 在此竞争更新授予申请中提出的研究将 产生有关分子和细胞调节的新信息 Na+/肌醇互醇共转运蛋白和M1摄取活动，并提供新的 洞悉我们认为有助于形成糖的机制 白内障。