MITOGEN-STIMULATED DIACYLGLYCEROL METABOLISM

这些研究的主要目标是分析动力学和生化 体外培养成纤维细胞中甘油二酯代谢的研究 刺激这种脂质生成的不同条件。 最近，有证据表明，凝血酶可以激活 至少两种刺激脂代谢的偶联机制 在培养的成纤维细胞中：(1)对α-凝血酶有反应的R1 而不是伽马-凝血酶，(B)被胰凝乳杆菌酶灭活，以及(C) 刺激多聚磷脂酰肌醇的水解和释放 花生四烯酸；和(2)R2，它(A)对两种α-凝血酶都有反应 和γ-凝血酶，(B)对胰凝乳酶不敏感，(C)不 刺激多聚磷脂酰肌醇的水解和释放 花生四烯酸。激活R1和R2中的任何一个会导致 细胞二酰甘油水平升高。PDGF激活R1样蛋白 而EGF激活R2样活性。受刺激的人 甘油二酯的代谢将在以下条件下进行分析 区分R1和R2。最近，使用了一种高度敏感的 一种能够准确测量二酰甘油质量分数的方法 已经研究了二酰甘油生产的动力学 α-凝血酶和表皮生长因子。我们已经开始研究这种剂量依赖性。 α-凝血酶刺激的二甘油对凝乳酶和胰凝乳酶的敏感性 制作。我们现在计划扩展这些研究，并包括一项 伽玛血栓和血小板衍生生长因子对甘油二酯的影响 甘油二酯的生产及分子种类鉴定 受到这些分子的刺激。同时检测EGF的刺激作用 或PDGF，将进行实验以检验两者之间的关系 受体占有率和二酰甘油刺激之间的关系。这个 凝血酶的高亲和力受体的作用也将被研究 确定其在激活R1和R2中的潜在作用。总而言之， 实验被设计成定性和定量地 分析在以下条件下刺激的甘油二酯 区分R1和R2，并检查cell 磷脂酰肌醇作为这些二酰甘油的潜在来源。这些 分析将提供有关以下方面的宝贵信息： 刺激性二甘油对短期和长期的调节作用 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.