Bacteria-enhanced inducible nitric oxide synthase (iNOS) overproduces nitric oxide (NO) leading to mitochondrial and cellular damage. In mammals, arginase (ARG), the enzyme consuming the same substrate l-arginine with iNOS, was believed to inhibit iNOS activity by competing the substrate. But in fish, this conception has been widely challenged. In this study, the gene expression using real-time quantitative PCR (RT-qPCR) technology showed that when stimulated by Aeromonas hydrophila (A. hydrophila), grass carp (gc) iNOS was up-regulated in head kidney monocytes/macrophages (M0/MФ), and its changes were not detected in the whole tissue of liver or spleen, showing a high degree of cell-specific expression pattern. At the same time, gcARG2 had a high basal expression in tissues and was up-regulated by A. hydrophila stimulation. Next, phthalaldehyde-primaquine reaction was first used in the determination of intracellular urea in fish cells. It was found that the induced gcARG2 led to an increase in the intracellular urea content. Moreover, urea and NO production in M0/MФ were increased in a substrate dose-dependent manner from 30 to 100 μM of l-arginine and reached the highest yield at 300 and 3000 μM of l-arginine, respectively. Furthermore, head kidney M0/MФ was cultured in RPMI1640 medium containing physiological concentration (500 μM) of l-arginine to evaluate the effect of ARG. Under A. hydrophila stimulation, treatment with the arginase inhibitor S-(2-boronoethyl)-l-cysteine (BEC) showed that inhibition of arginase could further enhance the NO production stimulated by A. hydrophila. This in turn led to a cumulation in peroxynitrite (ONOO-) content and an injury of the mitochondrial membrane potential. Our study showed for the first time that fish ARG in head kidney M0/MФ can limit excessive production of NO and harmful products by iNOS to maintain mitochondrial and cellular homeostasis.

细菌增强的诱导型一氧化氮合酶 (iNOS) 过量产生一氧化氮 (NO)，导致线粒体和细胞损伤。在哺乳动物中，精氨酸酶 (ARG)（与 iNOS 消耗相同底物l-精氨酸的酶）被认为通过竞争底物来抑制 iNOS 活性。但在鱼类中，这一概念受到了广泛的挑战。本研究采用实时定量PCR（RT-qPCR）技术的基因表达表明，当受到嗜水气单胞菌（A. Hydrophila）刺激时，草鱼（gc）头肾单核细胞/巨噬细胞（M0）iNOS上调。 /MФ)，在肝或脾的整个组织中未检测到其变化，表现出高度的细胞特异性表达模式。同时，gcARG2在组织中具有高基础表达，并在嗜水气单胞菌刺激下上调。接下来，邻苯二甲醛-伯氨喹反应首次用于鱼细胞内尿素的测定。结果发现，诱导的gcARG2导致细胞内尿素含量增加。此外，M0/MФ中的尿素和NO产量以底物剂量依赖性方式从30μM L-精氨酸增加到100μM L-精氨酸，并分别在300μM和3000μM L-精氨酸时达到最高产量。此外，在含有生理浓度（500μM）l-精氨酸的RPMI1640培养基中培养头肾M0/MФ以评价ARG的效果。在嗜水气单胞菌刺激下，用精氨酸酶抑制剂S-(2-硼乙基)-l-半胱氨酸(BEC)处理表明，精氨酸酶的抑制可以进一步增强嗜水气单胞菌刺激的NO产生。这反过来导致过氧亚硝酸盐（ONOO-）含量累积和线粒体膜电位损伤。我们的研究首次表明，鱼头肾M0/MФ中的ARG可以限制iNOS过度产生NO和有害产物，以维持线粒体和细胞稳态。