Peters-Golden M, Shelly C
J Biol Chem. 1987 Aug 5;262(22):10594-600.
The sulfhydryl reactant N-ethylmaleimide (NEM) stimulates the release and cyclooxygenase metabolism of arachidonic acid in rat alveolar macrophages. Because both 5-lipoxygenation and leukotriene (LT) C4 synthesis represent sulfhydryl-dependent steps in the 5-lipoxygenase pathway, we examined the effect of NEM on 5-lipoxygenase, as well as cyclooxygenase, metabolism in resting and agonist-stimulated cells by reverse-phase high performance liquid chromatography and radioimmunoassay. NEM at 5-10 microM stimulated the synthesis of thromboxane, but not prostaglandin E2 or the 5-lipoxygenase products LTC4, LTB4, or 5-hydroxyeicosatetraenoic acid from endogenously released arachidonate. In the presence of exogenous fatty acid, however, NEM stimulated the synthesis of large quantities of LTB4. The effect of NEM on arachidonate metabolism stimulated by the calcium ionophore A23187 and the particulate zymosan was also investigated. NEM augmented arachidonate release and thromboxane synthesis stimulated by A23187 but inhibited A23187-induced LTC4 synthesis with an IC50 of approximately 4.3 microM. This inhibitory effect closely paralleled the ability of NEM to deplete intracellular glutathione (IC50 approximately 4.3 microM). Preincubation with the intracellular cysteine delivery agent L-2-oxothiazolidine-4-carboxylate augmented intracellular glutathione concentration and A23187-stimulated LTC4 synthesis and attenuated the capacity of NEM to deplete glutathione and inhibit LTC4 synthesis. While LTB4 and 5-hydroxyeicosatetraenoic synthesis were unaffected at these low NEM concentrations, LTB4 synthesis was inhibited at high concentrations (IC50 approximately 210 microM). Zymosan-induced eicosanoid synthesis was modulated by NEM in a similar fashion. Thus, NEM is an agonist of arachidonate metabolism with the capacity to modulate the spectrum of macrophage-derived eicosanoids by virtue of specific biochemical interactions with substrates and enzymes of the 5-lipoxygenase pathway.
巯基反应物N-乙基马来酰亚胺（NEM）可刺激大鼠肺泡巨噬细胞中花生四烯酸的释放及环氧化酶代谢。由于5-脂氧合作用和白三烯（LT）C4合成均代表5-脂氧合酶途径中依赖巯基的步骤，我们通过反相高效液相色谱法和放射免疫分析法，研究了NEM对静息及激动剂刺激细胞中5-脂氧合酶以及环氧化酶代谢的影响。5-10微摩尔的NEM可刺激血栓素的合成，但不会刺激内源性释放的花生四烯酸生成前列腺素E2或5-脂氧合酶产物LTC4、LTB4或5-羟基二十碳四烯酸。然而，在存在外源性脂肪酸的情况下，NEM可刺激大量LTB4的合成。我们还研究了NEM对钙离子载体A23187和颗粒状酵母聚糖刺激的花生四烯酸代谢的影响。NEM可增强A23187刺激的花生四烯酸释放和血栓素合成，但抑制A23187诱导的LTC4合成，其半数抑制浓度（IC50）约为4.3微摩尔。这种抑制作用与NEM耗尽细胞内谷胱甘肽的能力密切相关（IC50约为4.3微摩尔）。用细胞内半胱氨酸递送剂L-2-氧代噻唑烷-4-羧酸盐进行预孵育可提高细胞内谷胱甘肽浓度，增强A23187刺激的LTC4合成，并减弱NEM耗尽谷胱甘肽和抑制LTC4合成的能力。在这些低NEM浓度下LTB4和5-羟基二十碳四烯酸的合成不受影响，但在高浓度下（IC50约为210微摩尔）LTB4合成受到抑制。酵母聚糖诱导的类花生酸合成也以类似方式受到NEM的调节。因此，NEM是花生四烯酸代谢的激动剂，凭借与5-脂氧合酶途径的底物和酶的特异性生化相互作用而具有调节巨噬细胞衍生类花生酸谱的能力。
