MITOGEN-STIMULATED DIACYLGLYCEROL METABOLISM

这些研究的主要目标是分析动力学和生化 培养的成纤维细胞中二酰基甘油代谢的方面 条件差异刺激这种脂质的产生。 最近，有证据表明凝血酶激活 至少两种用于刺激脂质代谢的偶联机制 在培养的成纤维细胞中：（1）R1，其（a）响应于α-凝血酶 而不是γ-凝血酶，（B）被胰凝乳蛋白酶灭活，和（c） 刺激多磷酸肌醇的水解和 花生四烯酸;和（2）R2，其（a）响应于α-凝血酶 和γ-凝血酶，（B）是胰凝乳蛋白酶不敏感的，和（c）不 刺激聚磷酸肌醇的水解和 花生四烯酸 R1和R2的激活导致 细胞甘油二酯水平升高。 PDGF激活R1样 EGF激活R2活性。 受激 将在以下条件下分析二酰基甘油的代谢， R1和R2的区别 最近，使用高度敏感的 本发明提供了一种能够准确测量甘油二酯质量的测定方法， 已经研究了由以下刺激的二酰基甘油生产的动力学： α-凝血酶和EGF。 我们已经开始研究剂量依赖性 α-凝血酶刺激的甘油二酯的胰凝乳蛋白酶敏感性 生产 我们现正计划扩展这些研究， γ-血栓和PDGF刺激的二酰甘油的评价 生产和鉴定二酰基甘油的分子种类 受到这些分子的刺激。 在研究EGF的作用时， 或PDGF，将进行实验以检查 受体占有率和甘油二酯的刺激之间的关系。 的 凝血酶的高亲和力受体的作用也将被检查， 确定其在R1和R2激活中的潜在作用。总的来说， 已经设计了实验来定性和定量地 分析在以下条件下刺激的二酰基甘油， 区分R1和R2，并检查细胞 磷酸肌醇作为这些二酰基甘油的潜在来源。 这些 分析将提供有关以下方面作用的宝贵信息： 刺激甘油二酯调节短期和长期 激素和生长因子的反应。

The primary goal of these studies is to analyze kinetic and biochemical aspects of diacylglycerol metabolism in cultured fibroblasts under conditions which differentially stimulate the generation of this lipid. Recently, evidence has been presented indicating that thrombin activates at least two coupling mechanisms for the stimulation of lipid metabolism in cultured fibroblasts: (1) R1 which (a) responds to alpha-thrombin but not gamma-thrombin, (b) is inactivated by chymotrypsin, and (c) stimulates both the hydrolysis of polphosphoinositides and release of arachidonic acid; and (2) R2 which (a) responds to both alpha-thrombin and gamma-thrombin, (b) is chymotrypsin-insensitive and (c) does not stimulate the hydrolysis of polyphosphoinositides and release of arachidonic acid. Activation of either R1 and R2 results in the elevation of cellular diacylglycerol levels. PDGF activates R1-like activities while EGF activates R2-like activities. The stimulated metabolism of diacylglycerol will be analyzed under conditions which differentiate between R1 and R2. Recently, using a highly sensitive assay capable of accurately measuring mass amounts of diacylglycerol, we have examined the kinetics of diacylglycerol production stimulated by alpha-thrombin and EGF. We have begun to examine the dose-dependence and chymotrypsin sensitivity of alpha-thrombin-stimulated diacylglycerol production. We now plan to extend these studies and to include an evaluation of gamma-thrombi and PDGF stimulated diacylglycerol production and to identify the molecular species of the diacylglycerols stimulated by these molecules. While examining the stimulation by EGF or PDGF, experiments will be conducted to examine the relationship between receptor occupancy and the stimulation of diacylglycerol. The role of the high affinity receptor for thrombin will also be examined to determine its potential role in the activation of R1 and R2. In summary, experiments have been designed to qualitatively and quantitatively analyze the diacylglycerols stimulated under conditions which differentiate between R1 and R2, and to examine cellular phosphoinositides as a potential source of these diacylglycerols. These analyses will provide valuable information concerning the role of stimulated diacylglycerol in modulating short term and long term responses by hormones and growth factors.