Schatz-Munding M, Hatzelmann A, Ullrich V
Faculty of Biology, University of Konstanz, Federal Republic of Germany.
Eur J Biochem. 1991 Apr 23;197(2):487-93. doi: 10.1111/j.1432-1033.1991.tb15936.x.
We have addressed the question why in the presence of a Ca2+ ionophore human polymorphonuclear leukocytes generate leukotrienes in high yields, but in only low amounts after stimulation by receptor agonists like fMLF (fM, formylmethionine), leukotriene B4 or platelet-activating factor (PAF), although a significant release of intracellular calcium can be measured. Using ionomycin we can show that from the two enzymes involved, phospholipase A2 and 5-lipoxygenase, the first requires a threshold level of about 350-400 nM calcium whereas 5-lipoxygenase shows a linear dependence on calcium and saturates at this concentration. Our data indicate that the Ca2+ requirement of phospholipase A2 can only be met by an additional influx of extracellular calcium, whereas 5-lipoxygenase will operate already at levels provided by intracellular stores. Consequently, the complexing of extracellular calcium by EGTA stops phospholipase A2 activity immediately, whereas added arachidonate can be still adequately metabolized by intracellular Ca2+ release triggered by fMLF or PAF. Interestingly, PAF shows a stronger extracellular component in its Ca2+ transient than fMLF, and also generates more 5-lipoxygenase metabolites. However, a clear correlation between the amount of 5-lipoxygenase metabolites and the extracellular Ca2+ signal was lacking, since maximal activity was achieved before the bulk of the extracellular calcium was monitored. Ca2+ influx after PAF stimulation could be blocked after 2 min by EGTA, but a further increase in the formation of 5-lipoxygenase metabolites was observed. In contrast ionomycin-elicited 5-lipoxygenase activity could be stopped at any time shortly after EGTA addition.
为什么在存在钙离子载体的情况下，人多形核白细胞能高产率地生成白三烯，但在受到受体激动剂如fMLF（fM，甲酰甲硫氨酸）、白三烯B4或血小板活化因子（PAF）刺激后，生成量却很低，尽管可以检测到细胞内钙的显著释放。使用离子霉素我们可以表明，在参与的两种酶，即磷脂酶A2和5-脂氧合酶中，第一种酶需要约350 - 400 nM的钙阈值水平，而5-脂氧合酶对钙呈线性依赖，并在该浓度下达到饱和。我们的数据表明，磷脂酶A2对钙的需求只能通过细胞外钙的额外流入来满足，而5-脂氧合酶在细胞内钙库提供的水平下就可以发挥作用。因此，EGTA对细胞外钙的络合会立即停止磷脂酶A2的活性，而添加的花生四烯酸仍可被fMLF或PAF触发的细胞内钙释放充分代谢。有趣的是，PAF在其钙瞬变中显示出比fMLF更强的细胞外成分，并且还产生更多的5-脂氧合酶代谢产物。然而，5-脂氧合酶代谢产物的量与细胞外钙信号之间缺乏明显的相关性，因为在监测到大部分细胞外钙之前就已经达到了最大活性。PAF刺激后2分钟，EGTA可以阻断钙流入，但观察到5-脂氧合酶代谢产物的形成进一步增加。相比之下，离子霉素引发的5-脂氧合酶活性在添加EGTA后不久的任何时候都可以停止。
