Schatz-Munding M, Ullrich V
Faculty of Biology, University of Konstanz, Federal Republic of Germany.
Eur J Biochem. 1992 Mar 1;204(2):705-12. doi: 10.1111/j.1432-1033.1992.tb16685.x.
Pretreatment of human polymorphonuclear leukocytes with the recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) enhances leukotriene biosynthesis in response to a receptor agonist (e.g. N-formyl-methionyl-leucyl-phenylalanine, fMLP) or a Ca(2+)-ionophore (e.g. ionomycin). This priming effect could be traced back to an elevated release of arachidonic acid from the phospholipid pools and hence an increased leukotriene biosynthesis by 5-lipoxygenase. Preincubation of polymorphonuclear leukocytes with GM-CSF did not influence the basal intracellular Ca2+ level and does not enhance cytosolic free calcium after stimulation with fMLP or ionomycin. Only a small increase in the second Ca2+ phase after receptor agonist stimulation was found. However, the Ca(2+)-threshold level necessary for the liberation of arachidonic acid by phospholipase A2 was decreased from 350-400 nM calcium in untreated cells to about 250 nM calcium in primed cells. This allows phospholipase A2 to be activated by a release of calcium from intracellular stores and by ionomycin concentrations which are ineffective in untreated cells. Protein biosynthesis inhibitors like actinomycin D (10 micrograms/ml) and cycloheximide (50 micrograms/ml) had no effect on the enhanced leukotriene biosynthesis in primed cells after stimulation with ionomycin. However, staurosporine (200 nM), an inhibitor of protein kinase C totally abolished the priming effect of GM-CSF after stimulation with ionomycin. The priming effect of GM-CSF could be mimicked by phorbol myristate acetate (PMA; 1 nM) and no additive or synergistic effect was found on leukotriene biosynthesis by simultaneous pretreatment with PMA and GM-CSF and stimulation with either fMLP or ionomycin. These results provide evidence that the enhanced arachidonic acid release in GM-CSF-primed polymorphonuclear leukocytes after stimulation with either fMLP or ionomycin involves activation of protein kinase C which, by a still unknown mechanism, reduces the Ca2+ requirement of phospholipase A2.
用重组人粒细胞 - 巨噬细胞集落刺激因子（rhGM - CSF）预处理人多形核白细胞，可增强其对白三烯生物合成的反应，这种反应是针对受体激动剂（如N - 甲酰 - 甲硫氨酰 - 亮氨酰 - 苯丙氨酸，fMLP）或钙离子载体（如离子霉素）的。这种启动效应可追溯到从磷脂池中花生四烯酸释放的增加，从而导致5 - 脂氧合酶介导的白三烯生物合成增加。用GM - CSF预孵育多形核白细胞不会影响基础细胞内Ca2 +水平，并且在用fMLP或离子霉素刺激后也不会增强胞质游离钙水平。仅在受体激动剂刺激后的第二个Ca2 +阶段发现有小幅增加。然而，磷脂酶A2释放花生四烯酸所需的Ca(2 +)阈值水平，从未经处理的细胞中的350 - 400 nM钙降低到预处理细胞中的约250 nM钙。这使得磷脂酶A2能够被细胞内储存释放的钙以及在未经处理的细胞中无效的离子霉素浓度激活。蛋白质生物合成抑制剂如放线菌素D（10微克/毫升）和环己酰亚胺（50微克/毫升）在用离子霉素刺激后对预处理细胞中增强的白三烯生物合成没有影响。然而，蛋白激酶C抑制剂星形孢菌素（200 nM）在用离子霉素刺激后完全消除了GM - CSF的启动效应。GM - CSF的启动效应可被佛波酯（PMA；1 nM）模拟，并且在用PMA和GM - CSF同时预处理并用fMLP或离子霉素刺激后，对白三烯生物合成未发现相加或协同效应。这些结果表明，在用fMLP或离子霉素刺激后，GM - CSF预处理的多形核白细胞中花生四烯酸释放的增强涉及蛋白激酶C的激活，其通过一种仍未知的机制降低了磷脂酶A2对钙的需求。
