DIABETES AND SIGNAL TRANSDUCTION ABNORMALITIES

糖尿病和信号传导异常

• 批准号: 2518390
• 负责人: JOHN R. WILLIAMSON
• 金额: $ 14.6万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-09-01 至 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2518390
• 关键词: DNA replication; G protein; autocrine; biological signal transduction; calcium flux; collagen; diabetic nephropathy; digital imaging; eicosanoid metabolism; enzyme activity; glycosylation; hormone regulation /control mechanism; hyperglycemia; immunofluorescence technique; kidney cell; laboratory rat; microinjections; mixed tissue /cell culture; paracrine; phospholipase A2; phospholipase C; protein tyrosine kinase; receptor coupling; renal glomerulus; single cell analysis

The overall hypothesis behind this proposal is that diabetic complications in renal cells are due to specific malfunctions in signal transduction mechanisms induced by hyperglycemia and abnormal glucose metabolism. Prolonged hyperglycemia has two major consequences in affected tissues. One is that there is an elevated level of diacylglycerol by de novo synthesis from glucose, while the second is an accumulation of advanced glycation end products (AGEs). These changes cause abnormal activation of protein kinase C and an expansion of the extracellular matrix. The effects of high glucose and AGE products on signaling pathways activated in response to mitogenic and non-mitogenic agonists will be correlated with functional changes in glomerular endothelial and mesangial cells in solo or co-culture conditions. Powerful new techniques involving microinjection of reagents and digital Ca2+ image analysis will be applied to study the changes in cell signaling at the single cell level. Effects of two classes of hormones, those that interact with receptors coupled to G-proteins and those that contain or recruit tyrosine kinases, will be investigated by using intracellular free Ca2+, PLA2 activity, DNA synthesis and secretion of type IV collagen as functional parameters. Steps in receptor-activated signal transduction pathways will be probed by microinjection or use of fluorescent indicators, caged compounds, antibodies and specific inhibitors. The following specific aims will be addressed, l) the specificity of receptor-G-protein coupling will be determined in glomerular endothelial and mesangial cells, 2) the mechanism and regulation of hormone-induced Ca2+ oscillations will be investigated in endothelial and mesangial cells, 3) the effects of high glucose and AGE products will be measured on agonist-induced Ca2+ mobilization, DNA synthesis, phospholipase A2 activity and secretion of type IV collagen, and 4) effects of high glucose and AGE products on cell-cell signaling mechanisms will be assessed in co-cultures of endothelial and mesangial cells An important goal of the present work will be to define the interplay of autocrine and paracrine factors between endothelial and mesangial cells under conditions simulating the diabetic state. This information will be critical for the development of future pharmacological approaches for the prevention and treatment of diabetic nephropathy.

这一建议背后的总体假设是，糖尿病并发症 在肾细胞中是由于信号转导中的特定故障 高血糖和糖代谢异常引起的机制。 长期高血糖在受影响的组织中有两个主要后果。 一个是，有一个高水平的甘油二酯的从头 第一种是葡萄糖的合成，而第二种是高级葡萄糖的积累。 糖基化终产物（AGEs）。 这些变化会导致 蛋白激酶C和细胞外基质的扩张。 的 高糖和AGE产物对激活的信号通路的影响 对促有丝分裂激动剂和非促有丝分裂激动剂的反应将是相关的 肾小球内皮细胞和系膜细胞的功能变化， 单独或共培养条件。 强大的新技术， 采用微量注射法和数字化钙离子图像分析技术 在单细胞水平上研究细胞信号的变化。 影响 两类激素，那些与受体相互作用， G蛋白和那些含有或招募酪氨酸激酶的蛋白，将被 用细胞内游离Ca ~（2+）、PLA_2活性、DNA IV型胶原的合成和分泌作为功能参数。 受体激活的信号转导途径的步骤将通过以下方法进行探测： 显微注射或使用荧光指示剂，笼状化合物， 抗体和特异性抑制剂。 以下具体目标将是 解决，l）受体-G-蛋白偶联的特异性将是 在肾小球内皮细胞和系膜细胞中确定，2）机制 并研究了钙离子诱导的钙离子振荡的调控 在内皮细胞和系膜细胞中，3）高糖和AGE的作用 将测量激动剂诱导的Ca 2+动员、DNA IV型胶原合成、磷脂酶A2活性和分泌， 高糖和AGE产物对细胞-细胞信号传导的影响 将在内皮细胞和系膜细胞的共培养物中评估其机制。 细胞目前工作的一个重要目标将是定义 内皮细胞和巨噬细胞自分泌和旁分泌因子相互作用 模拟糖尿病状态条件下的系膜细胞。 这 这些信息对于未来药理学的发展至关重要。 糖尿病肾病的预防和治疗方法。