Ryoyama K, Nomura T, Nakamura S
Department of Experimental Therapeutics, Kanazawa University, Japan.
Cancer Immunol Immunother. 1993 Nov;37(6):385-91. doi: 10.1007/BF01526795.
We examined whether inhibitors of the arachidonic acid cascade inhibited nitric oxide (NO) production, as measured by nitrite concentration, either in macrophages or by their cytosolic fractions. Nitrite production by peritoneal macrophages from mice receiving OK-432 treatment was significantly inhibited by phospholipase A2 inhibitors [dexamethasone and 4-bromophenacyl bromide (4-BPB)], lipoxygenase inhibitors [nordihydroguaiaretic acid (NDGA) and ketoconazole] and a glutathione S-transferase (leukotrienes LTA4-LTC4) inhibitor (ethacrynic acid). However, caffeic acid and esculetin, inhibitors of 5- and 12-lipoxygenase respectively, were not inhibitory. On the other hand, indomethacin, a cyclooxygenase inhibitor, slightly inhibited whereas another inhibitor, ibuprofen, did not. Inhibition of the nitrite production by dexamethasone, 4-BPB, NDGA and ethacrynic acid was also demonstrated when the macrophages were restimulated ex vivo with OK-432 or with lipopolysaccharide. The inhibitory activity of dexamethasone, NDGA and ethacrynic acid was significantly reduced by ex vivo restimulation with OK-432, whereas that of 4-BPB was hardly affected. Furthermore, the inhibitory activity of dexamethasone, NDGA and ethacrynic acid was much higher when the macrophages were continuously exposed to the agents than when they were pulsed. Meanwhile, inhibition by 4-BPB was almost the same with either treatment. In addition, the inhibitory activity of these agents was not blocked with L-arginine, a substrate of NO synthases, or with arachidonate metabolites (LTB4, LTC4 and LTE4). Ethacrynic acid and 4-BPB, but not dexamethasone and NDGA, also inhibited nitrite production by the cytosolic fractions from OK-432-restimulated peritoneal macrophages, and the inhibitory activity of 4-BPB was superior to that of ethacrynic acid. These agents, however, did not inhibit nitrite production from sodium nitroprusside, a spontaneous NO-releasing compound. These results indicate that dexamethasone, 4-BPB, NDGA and ethacrynic acid inhibited the production of NO by macrophages through at least two different mechanisms: one was inhibited by dexamethasone, NDGA and ethacrynic acid and the other by 4-BPB. Furthermore, 4-BPB and ethacrynic acid directly inhibited the activity of the NO synthase in macrophages, suggesting that the agents work by binding to the active site(s) of the enzyme.
我们研究了花生四烯酸级联反应的抑制剂是否会抑制一氧化氮（NO）的生成（通过亚硝酸盐浓度来测定），无论是在巨噬细胞中还是在其胞质组分中。用磷脂酶A2抑制剂[地塞米松和4-溴苯甲酰溴（4-BPB）]、脂氧合酶抑制剂[去甲二氢愈创木酸（NDGA）和酮康唑]以及谷胱甘肽S-转移酶（白三烯LTA4-LTC4）抑制剂（依他尼酸）可显著抑制接受OK-432处理的小鼠腹腔巨噬细胞产生亚硝酸盐。然而，分别作为5-脂氧合酶和12-脂氧合酶抑制剂的咖啡酸和七叶亭并无抑制作用。另一方面，环氧化酶抑制剂吲哚美辛有轻微抑制作用，而另一种抑制剂布洛芬则无此作用。当巨噬细胞在体外再次用OK-432或脂多糖刺激时，地塞米松、4-BPB、NDGA和依他尼酸对亚硝酸盐生成的抑制作用也得到了证实。用OK-432进行体外再刺激可显著降低地塞米松、NDGA和依他尼酸的抑制活性，而4-BPB的抑制活性几乎不受影响。此外，当巨噬细胞持续暴露于这些药物时，地塞米松、NDGA和依他尼酸的抑制活性比脉冲处理时高得多。同时，4-BPB在两种处理方式下的抑制作用几乎相同。此外，这些药物的抑制活性不会被一氧化氮合酶的底物L-精氨酸或花生四烯酸代谢产物（LTB4、LTC4和LTE4）所阻断。依他尼酸和4-BPB，但不是地塞米松和NDGA，也抑制了OK-432再刺激的腹腔巨噬细胞胞质组分产生亚硝酸盐，且4-BPB的抑制活性优于依他尼酸。然而，这些药物并不抑制自发释放NO的化合物硝普钠产生亚硝酸盐。这些结果表明，地塞米松、4-BPB、NDGA和依他尼酸通过至少两种不同机制抑制巨噬细胞产生NO：一种机制受地塞米松、NDGA和依他尼酸抑制，另一种受4-BPB抑制。此外，4-BPB和依他尼酸直接抑制巨噬细胞中一氧化氮合酶的活性，这表明这些药物通过与该酶的活性位点结合起作用。
