Transduction of the bradykinin response in human fibroblasts: prolonged elevation of diacylglycerol level and its correlation with protein kinase C activation.
Transduction of the bradykinin response in human fibroblasts: prolonged elevation of diacylglycerol level and its correlation with protein kinase C activation.
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DOI：
10.1091/mbc.2.3.229
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发表时间：
1991-03
期刊：
影响因子：
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通讯作者：
B. Etscheid;K. Albert;M. Villereal;H. Palfrey
中科院分区：
文献类型：
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作者：
B. Etscheid;K. Albert;M. Villereal;H. Palfrey
刺激静止的人成纤维细胞的肽促分裂素缓激肽（BK）导致细胞的1，2-二酰甘油（DAG）的双相升高，通过测量总DAG质量或[3 H]花生四烯酸掺入估计。一个快速的初始瞬态，达到峰值后15秒，BK除了下降到接近基础水平，然后第二次上升到一个平台期，在此期间，DAG水平保持升高小于或等于45分钟。最初的DAG瞬态的来源似乎主要是polyphosphoinositides，因为这些磷脂被迅速水解后BK添加。这一短暂的时间与先前观察到的三磷酸肌醇积累和细胞内游离[Ca 2 +]在相同的细胞中观察到的动力学。用[3 H]肉豆蔻酸预孵育的培养物主要将标记掺入磷脂酰胆碱（PC）池中。随后在这些条件下添加BK仅导致[3 H]DAG相对缓慢地积累至平台水平，而没有初始瞬变。连同发现在BK刺激后PC减少的观察结果，这些观察结果表明DAG积累的晚期可能涉及包括PC在内的其他磷脂的分解。为了研究DAG升高的后果，我们检测了酸性80 kDa蛋白的磷酸化，其磷酸化仅依赖于蛋白激酶C（PK-C）的活化。80 kDa的成纤维细胞蛋白可以免疫沉淀的牛脑“豆蔻酰化和丙氨酸丰富的C-激酶底物”（MARCKS）和磷酸肽图谱的大脑和成纤维细胞MARCKS是相似的抗体。刺激[32 P]-预标记的成纤维细胞与血清，BK，加压素，或12-O-十四烷酰佛波醇乙酸酯，但不是表皮生长因子或钙离子载体，导致快速磷酸化的MARCKS。与BK或血清，这种磷酸化表现出一个初始的短暂的峰值在不到1分钟，然后再次上升到一个平台的水平，持续小于或等于45分钟。去除BK导致MARCKS磷酸化的快速下降。这些研究表明，BK刺激的人成纤维细胞中的双相DAG信号与PK-C的活化状态密切相关。然而，PK-C的持续激活似乎并不需要持续高水平的Ca 2+。
Stimulation of quiescent human fibroblasts with the peptide mitogen bradykinin (BK) led to a biphasic elevation in cellular 1,2-diacylglycerol (DAG), as estimated by either measurement of total DAG mass or [3H]arachidonate incorporation. A rapid initial transient that peaked 15 s after BK addition was followed by a decline to near basal levels then a second rise to a plateau phase during which DAG levels remained elevated for less than or equal to 45 min. The source of the initial DAG transient appeared to be primarily polyphosphoinositides as these phospholipids were rapidly hydrolyzed after BK addition. This transient correlates well temporally with previous observations of the kinetics of inositol trisphosphate accumulation and intracellular free [Ca2+] observed in the same cells. Cultures preincubated with [3H]myristic acid incorporated label predominantly into the phosphatidylcholine (PC) pool. Subsequent addition of BK under these conditions caused only a relatively slow accumulation of [3H]DAG to a plateau level, without an initial transient. Together with the observation that PC was found to decrease upon BK stimulation, these observations suggest that the late phase of DAG accumulation may involve breakdown of other phospholipids including PC. To investigate the consequences of DAG elevation we examined the phosphorylation of an acidic 80 kDa protein, whose phosphorylation is solely dependent on the activation of protein kinase C (PK-C). The 80 kDa fibroblast protein could be immunoprecipitated by an antibody to bovine brain "myristoylated and alanine-rich C-kinase substrate" (MARCKS) and phosphopeptide maps of brain and fibroblast MARCKS were similar. Stimulation of [32P]-prelabeled fibroblasts with serum, BK, vasopressin, or 12-O-tetradecanoyl phorbol acetate, but not epidermal growth factor or calcium ionophores, resulted in the rapid phosphorylation of MARCKS. With BK or serum this phosphorylation showed an initial transient peak at less than 1 min then rose again to a plateau level that was sustained for less than or equal to 45 min. Removal of BK resulted in a rapid decline in MARCKS phosphorylation. These studies show that the biphasic DAG signal in BK-stimulated human fibroblasts correlates well with the state of activation of PK-C. However, the persistent activation of PK-C does not appear to require continued high levels of Ca2+.