Winkler J D, Sung C M, Hubbard W C, Chilton F H
Division of Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406.
Biochem J. 1993 May 1;291 ( Pt 3)(Pt 3):825-31. doi: 10.1042/bj2910825.
The present studies were conducted to understand better the regulation of phospholipase A2 (PLA2)-dependent mobilization of lipid mediators by arachidonic acid (C20:4). After stimulation of human neutrophils, g.l.c./m.s. analysis of non-esterified fatty acids indicated that the quantity of C20:4 increased as a function of time after stimulation, from undetectable quantities to > 800 pmol/10(7) cells. In contrast with C20:4, the quantities of other free fatty acids such as oleic and linoleic were high in resting cells and did not change after stimulation. Some 15% of the C20:4 released from cellular lipids remained cell-associated. To examine the effect of C20:4 on its own release, neutrophils were exposed to [2H8]C20:4, to differentiate it by g.l.c./m.s. from naturally occurring C20:4. In A23187-stimulated neutrophils, low concentrations (5-10 microM) of [2H8]C20:4 added just before A23187 increased the quantity of C20:4 produced by the cell, whereas higher concentrations (30-50 microM) decreased the quantity of C20:4 released from phospholipids. As other measures of PLA2 activity, the effects of C20:4 on production of platelet-activity factor (PAF) and leukotriene B4 (LTB4) were assessed. C20:4 treatment just before stimulation of neutrophils blocked PAF and LTB4 production in a concentration-dependent manner (IC50 10-20 microM). The effect of C20:4 was not blocked by the cyclo-oxygenase inhibitor naproxine (10 microM), nor could it be mimicked by 1 microM LTB4, 5-hydroxyeicosa-6,8,11,14-tetraenoic acid (5HETE), 5-hydroperoxyeicosa-6,8,11,14-tetraenoic acid (5HPETE) or 15-hydroxyeicosa-5,8,11,13-tetraenoic acid (15HETE). The 5-lipoxygenase (5LO) inhibitor zileuton induced a concentration-dependent decrease in PAF, with a maximal effect of a 50% decrease at 10-50 microM. The decrease in PAF by the 5LO inhibitor could not be circumvented by addition of 1 microM 5HETE, 5HPETE and LTB4, and may be attributed to the capacity of zileuton to increase the quantity of C20:4 in A23187-treated neutrophils. The inhibitory effect of C20:4 (20-40 microM) on PAF production could be antagonized by the protein kinase C inhibitor staurosporine (30 nM), but not by inhibitors of protein kinase A, tyrosine kinase or calmodulin kinase II. Taken together, these data demonstrate that C20:4 is selectively released from membrane phospholipids of A23187-stimulated neutrophils, and this C20:4 may play an important role in regulating the mobilization of C20:4 by altering PLA2 activity.
开展本研究是为了更好地理解花生四烯酸（C20:4）对磷脂酶A2（PLA2）依赖性脂质介质动员的调节作用。刺激人中性粒细胞后，对非酯化脂肪酸进行气相色谱/质谱分析表明，刺激后C20:4的量随时间增加，从检测不到的量增加到>800 pmol/10(7)个细胞。与C20:4不同，其他游离脂肪酸如油酸和亚油酸在静息细胞中的量很高，刺激后没有变化。从细胞脂质中释放的C20:4约有15%仍与细胞相关。为了研究C20:4对其自身释放的影响，将中性粒细胞暴露于[2H8]C20:4，以便通过气相色谱/质谱将其与天然存在的C20:4区分开来。在A23187刺激的中性粒细胞中，在A23187之前加入低浓度（5 - 10 microM）的[2H8]C20:4会增加细胞产生的C20:4的量，而较高浓度（30 - 50 microM）则会减少从磷脂中释放的C20:4的量。作为PLA2活性的其他测量指标，评估了C20:4对血小板活性因子（PAF）和白三烯B4（LTB4）产生的影响。在刺激中性粒细胞之前进行C20:4处理以浓度依赖性方式阻断PAF和LTB4的产生（IC50为10 - 20 microM）。C20:4的作用未被环氧化酶抑制剂萘普生（10 microM）阻断，1 microM的LTB4、5 - 羟基二十碳 - 6,8,11,14 - 四烯酸（5HETE）、5 - 氢过氧二十碳 - 6,8,11,14 - 四烯酸（5HPETE）或15 - 羟基二十碳 - 5,8,11,13 - 四烯酸（15HETE）也不能模拟其作用。5 - 脂氧合酶（5LO）抑制剂齐留通诱导PAF浓度依赖性降低，在10 - 50 microM时最大降低效果为50%。5LO抑制剂引起的PAF降低不能通过加入1 microM的5HETE、5HPETE和LTB4来规避，这可能归因于齐留通增加A23187处理的中性粒细胞中C20:4量的能力。C20:4（20 - 40 microM）对PAF产生的抑制作用可被蛋白激酶C抑制剂星形孢菌素（30 nM）拮抗，但不能被蛋白激酶A、酪氨酸激酶或钙调蛋白激酶II的抑制剂拮抗。综上所述，这些数据表明C20:4是从A23187刺激的中性粒细胞的膜磷脂中选择性释放的，并且这种C20:4可能通过改变PLA2活性在调节C20:4的动员中起重要作用。
