DIABETES AND SIGNAL TRANSDUCTION ABNORMALITIES

糖尿病和信号传导异常

• 批准号: 2148825
• 负责人: JOHN R. WILLIAMSON
• 金额: $ 13.88万
• 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/2148825
• 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.

该提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的特定故障引起的 高血糖和异常葡萄糖代谢引起的机制。 长时间高血糖在受影响的组织中有两个主要后果。 一个是从头开始有二酰基甘油的水平 葡萄糖的合成，而第二个是高级的积累 糖基终产物（年龄）。 这些变化导致异常激活 蛋白激酶C和细胞外基质的膨胀。 这 高葡萄糖和年龄产品对激活信号通路的影响 响应有丝分裂和非有丝分裂的激动剂将相关 随着肾小球内皮和肾小球细胞的功能变化 独奏或共培养条件。 强大的新技术涉及 将应用试剂的显微注射和数字CA2+图像分析 研究单细胞水平上细胞信号的变化。 效果 在两类激素中，与受体相互作用的激素耦合到 G蛋白和包含或招募酪氨酸激酶的蛋白将是 通过使用细胞内游离Ca2+，PLA2活性，DNA进行了研究 IV型胶原蛋白作为功能参数的合成和分泌。 受体激活的信号转导途径的步骤将通过 显微注射或使用荧光指示剂，笼子化合物， 抗体和特定抑制剂。 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肾小球细胞中确定，2）机制 将研究激素诱导的Ca2+振荡的调节 在内皮细胞和膜细胞中，3）高葡萄糖和年龄的影响 产品将以激动剂诱导的Ca2+动员，DNA进行测量 合成，磷脂酶A2活性和IV型胶原蛋白的分泌， 4）高葡萄糖和年龄产品对细胞细胞信号的影响 机制将在内皮和肾小管培养中进行评估 细胞本工作的重要目标是定义 内分泌和旁分泌因子之间的相互作用 在模拟糖尿病状态的条件下，肾小球细胞。 这 信息对于未来药理的发展至关重要 预防和治疗糖尿病性肾病的方法。