Ramana Kota V, Chandra Deepak, Wills Nancy K, Bhatnagar Aruni, Srivastava Satish K
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, 6-644 Basic Science Building, Galveston, TX 77555-0647, USA.
J Diabetes Complications. 2004 May-Jun;18(3):177-82. doi: 10.1016/S1056-8727(03)00003-5.
We have determined the abundance of the chloride channel, ClC-3, and Na(+)/Ca(2+) exchanger proteins in isolated rat lens cortex fiber cells by immunofluorescence method using polyclonal anti-ClC-3 antibodies and monoclonal antibodies against the canine cardiac Na(+)/Ca(2+) exchanger protein. These proteins were also quantified in the lens cortex of streptozotocin-injected rats by Western blots. Also, mRNA for ClC-3 was determined by Northern blot analysis. The isolated rat lens cortical fibers expressed basal levels of ClC-3 and Na(+)/Ca(2+) exchanger proteins. As compared to controls, the ClC-3 protein in the lens cortex of diabetic rats (blood glucose>400 mg%) increased by 2.5-fold in 7 days and 4.5-fold in 14 days. However, the ClC-3 protein decreased to near-normal values in 40 days. The changes in ClC-3 mRNA closely followed the protein levels. Similarly, as compared to controls, on Day 7, the Na(+)/Ca(2+) exchanger protein in the diabetic rat lens cortex increased by 3.5-fold and on Day14 by 5.5-fold. Subsequently, it decreased to control levels on Day 40. Treatment with the antioxidant, Trolox (2 mg/kg body weight), prevented the initial increase in ClC-3 and Na(+)/Ca(2+) exchanger proteins. The up-regulation of ClC-3 and Na(+)/Ca(2+) exchanger proteins during the early stages of diabetes and its prevention by antioxidants suggests that the proteins regulating ion transport may have a pathophysiological role in the development of diabetic cataracts.
我们使用多克隆抗ClC-3抗体和针对犬心脏钠钙交换蛋白的单克隆抗体，通过免疫荧光法测定了分离的大鼠晶状体皮质纤维细胞中氯离子通道ClC-3和钠钙交换蛋白的丰度。这些蛋白质也通过蛋白质印迹法在链脲佐菌素注射大鼠的晶状体皮质中进行了定量。此外，通过Northern印迹分析测定了ClC-3的mRNA。分离的大鼠晶状体皮质纤维表达基础水平的ClC-3和钠钙交换蛋白。与对照组相比，糖尿病大鼠（血糖>400mg%）晶状体皮质中的ClC-3蛋白在7天内增加了2.5倍，在14天内增加了4.5倍。然而，40天时ClC-3蛋白降至接近正常的值。ClC-3 mRNA的变化与蛋白质水平密切相关。同样，与对照组相比，在第7天，糖尿病大鼠晶状体皮质中的钠钙交换蛋白增加了3.5倍，在第14天增加了5.5倍。随后，在第40天降至对照水平。用抗氧化剂Trolox（2mg/kg体重）治疗可防止ClC-3和钠钙交换蛋白的最初增加。糖尿病早期ClC-3和钠钙交换蛋白的上调及其被抗氧化剂预防表明，调节离子转运的蛋白质可能在糖尿病性白内障的发生发展中具有病理生理作用。
