Wagner J G, Ganey P E, Roth R A
Department of Pharmacology, Michigan State University, East Lansing, USA.
Hepatology. 1996 Apr;23(4):803-10. doi: 10.1002/hep.510230422.
Polymorphonuclear leukocytes (PMNS) have been implicated as cellular mediators of hepatic injury in models of inflammation in vivo. In vitro, hepatocyte killing by activated PMNs is mediated in part by proteases, but the role of nitric oxide is unknown. NO is produced by PMNs and hepatocytes and can act either to damage or protect in various models of toxicity. Therefore, we tested the hypothesis that NO is important in PMN-mediated hepatocyte killing in vitro. Freshly isolated hepatocytes from rat liver and PMNs elicited from rat peritoneum were cultured together or alone for 16 hours. Both cell types spontaneously released NO, estimated as its stable breakdown product, nitrite. Accumulation of nitrite in medium from hepatocyte cultures was augmented threefold by incubation with L-arginine and was completely inhibited by treatment with the nitric oxide synthase (NOS) inhibitor NG-methyl-L-arginine (NMA). Nitrite release in PMN cultures was unaffected by L-arginine addition and only partially inhibited by NMA. In PMN:hepatocyte cocultures (10:1), accumulation of nitrite was additive relative to cells cultured separately. Incubation with NMA blocked nitrite production completely in cocultures, whereas L-arginine caused a two-fold increase in nitrite. Addition of PMN stimulants, N-formyl-methionyl-leucyl-phenylalanine (FMLP), or phorbol myristate acetate (PMA), caused increased release of alanine aminotransferase (ALT) activity into medium from hepatocytes cultured with PMNs but not from hepatocytes cultured alone; this indicated that injury to hepatocytes was due to activated PMNS. However, neither FMLP nor PMA significantly altered nitrite release from cocultures. Despite the alterations in NO production induced by addition of NMA or L-arginine, neither agent altered the release of ALT from hepatocytes in coculture with activated PMNs. Thus, PMNs and hepatocytes provided NO in vitro, but neither suppression nor elevation of NO production affected PMN-mediated hepatocyte killing. Accordingly, NO is not involved in the mechanisms by which FMLP-or PMA-stimulated PMNs mediate hepatocyte injury in vitro.
在体内炎症模型中，多形核白细胞（PMN）被认为是肝损伤的细胞介质。在体外，活化的PMN对肝细胞的杀伤作用部分由蛋白酶介导，但一氧化氮的作用尚不清楚。PMN和肝细胞均可产生NO，在各种毒性模型中，NO既可起到损伤作用，也可起到保护作用。因此，我们验证了以下假设：在体外，NO在PMN介导的肝细胞杀伤过程中起重要作用。将从大鼠肝脏新鲜分离的肝细胞和从大鼠腹膜中提取的PMN一起或单独培养16小时。两种细胞类型均自发释放NO，以其稳定的分解产物亚硝酸盐进行估算。与L-精氨酸孵育后，肝细胞培养物培养基中亚硝酸盐的积累增加了三倍，而用一氧化氮合酶（NOS）抑制剂NG-甲基-L-精氨酸（NMA）处理可完全抑制亚硝酸盐的积累。添加L-精氨酸对PMN培养物中亚硝酸盐的释放没有影响，NMA仅能部分抑制其释放。在PMN与肝细胞的共培养物（10:1）中，亚硝酸盐的积累相对于单独培养的细胞是相加的。用NMA孵育可完全阻断共培养物中亚硝酸盐的产生，而L-精氨酸可使亚硝酸盐增加两倍。添加PMN刺激剂N-甲酰甲硫氨酰亮氨酰苯丙氨酸（FMLP）或佛波酯肉豆蔻酸酯（PMA），可使与PMN共培养的肝细胞培养基中丙氨酸转氨酶（ALT）活性的释放增加，但单独培养的肝细胞则不会；这表明肝细胞损伤是由活化的PMN引起的。然而，FMLP和PMA均未显著改变共培养物中亚硝酸盐的释放。尽管添加NMA或L-精氨酸会改变NO的产生，但两种试剂均未改变与活化的PMN共培养的肝细胞中ALT的释放。因此，PMN和肝细胞在体外均可产生NO，但抑制或提高NO的产生均不影响PMN介导的肝细胞杀伤作用。因此，NO不参与FMLP或PMA刺激的PMN在体外介导肝细胞损伤的机制。
