DIABETES AND SIGNAL TRANSDUCTION ABNORMALITIES

这一提议背后的总体假设是糖尿病并发症 是由于信号转导的特殊故障造成的 高血糖和糖代谢异常诱导的机制。 长期的高血糖在受影响的组织中有两个主要后果。 其一，从头算起，二酰甘油的水平升高。 从葡萄糖合成，而第二个是积累高级 糖基化终产物(AGEs)。这些变化会导致异常激活 蛋白激酶C和细胞外基质的扩张。这个 高糖和AGE产物对激活的信号通路的影响 对有丝分裂激动剂和非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功能改变 单独或联合培养条件。强大的新技术，包括 将应用微量注射试剂和数字钙图像分析 在单细胞水平上研究细胞信号的变化。效应 两类激素，那些与偶联的受体相互作用的激素 G蛋白和那些含有或招募酪氨酸激酶的蛋白，将是 用细胞内游离钙、磷脂酶A2活性、DNA 以IV型胶原的合成和分泌为功能参数。 受体激活的信号转导通路中的步骤将通过 微量注射或使用荧光指示剂、笼状化合物、 抗体和特定的抑制物。以下是具体目标 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的表达，2)机制 并将研究激素诱导的钙振荡的调节 在内皮细胞和系膜细胞中，3)高糖和AGE的影响 产品将通过激动剂诱导的钙动员，DNA进行测量 IV型胶原的合成、磷脂酶A2活性及分泌 4)高糖和AGE产物对细胞-细胞信号转导的影响 内皮细胞和系膜细胞共培养的机制将被评估 细胞当前工作的一个重要目标将是定义 内皮细胞和血管内皮细胞间自分泌和旁分泌因子的相互作用 系膜细胞在模拟糖尿病状态的条件下。这 信息将对未来药理学的发展至关重要 糖尿病肾病的防治方法。

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.