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: 2331623
• 负责人: Patrick Ross Cammarata
• 金额: $ 27.82万
• 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 1999-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2331623
• 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，糖性白内障代表了一种 重大公共卫生问题，且没有已知有效治疗方法 而不是手术 高血糖与糖的病因学有关 白内障的形成，但代谢途径及其调制 遗传和生化因素尚待确定。 转换 组织葡萄糖通过醛糖还原酶（AR）转化为山梨糖醇， 暗示其为导致透镜中的糖性白内障的潜在机制。 在过去的六年里，这个实验室的研究已经确定了 高血糖症、多元醇途径激活和肌营养不良之间的代谢联系。 肌醇（MI）耗竭。 该实验室最近的研究表明， 高渗应激刺激Na+ /M1基因表达， 协同转运蛋白和AR，增加蛋白质合成，并提高M1 吸收活性和多元醇形成。 的细胞内积累 这些相容的、相互作用的有机渗压剂起作用以维持渗透压 平衡并保护透镜免受高温的扰动影响 细胞内电解质浓度。 然而，如果多元醇 积累持续了很长一段时间，它剥夺了 通过限制M1摄取活性，通过多种途径抑制必需M1的透镜 抑制机制：（1）多元醇介导的抑制m1载体 蛋白质摄取活性，（2）多元醇介导的Na+ /M1的下调 协同转运蛋白mRNA和（3）多元醇激活的Mi从细胞流出， 介质 在这方面，高渗类似于高血糖， 前者（通过AR mRNA的诱导），后者（通过 由于增加的底物可用性）， 胞内多元醇 M1运输系统的直接损害 任何一种方法都代表了一种合理的共同机制，可以解释 晶状体MI的消失。 这种竞争性的续期申请 拟议详细研究的机制、影响和后果 多元醇和非多元醇诱导的透镜细胞培养物和透镜中的M1消耗 器官培养 细胞内M1的消耗而不改变细胞内的 细胞内多元醇含量将通过采用补料来实现 方案与L-葡萄糖，一个强大的竞争性抑制剂M1摄取， 我们所展示的促进细胞内M1耗竭的新技术， 不提高多元醇含量的透镜细胞培养。 在第一个具体的 目的探讨Na+/M1表达调控的分子机制 在高渗性和高渗性糖尿病条件下的协同转运蛋白将是 考察 在第二个具体目标中，多元醇和 将仔细界定M1的监管机制。 第三特定 目的是确定和描述调节机制， M1外排，目前对多元醇诱导M1的机制了解最少 耗尽 第四个具体目标将定义M1摄取的动力学 并将这些结果与已知的动力学 来自透镜细胞培养物的参数。 最终的具体目标将利用 免疫电镜定位Na+/M1协同转运蛋白 完整透镜中透镜上皮细胞的基底面或顶面。 在此竞争性更新补助金申请中提出的研究将 产生新的信息的分子和细胞调控的 Na+/肌醇协同转运蛋白和M1摄取活性，并提供新的 深入了解我们认为有助于糖形成的机制 白内障