Mori M, Gotoh T, Nagasaki A, Takiguchi M, Sonoki T
Department of Molecular Genetics, Kumamoto University School of Medicine, Japan.
J Inherit Metab Dis. 1998;21 Suppl 1:59-71. doi: 10.1023/a:1005357608129.
Nitric oxide (NO) is synthesized from arginine by nitric-oxide synthase (NOS), and citrulline that is generated can be recycled to arginine by argininosuccinate synthase (AS) and argininosuccinate lyase (AL). Rats were injected with bacterial lipopolysaccharide (LPS) and expression of the inducible isoform of NOS (iNOS), AS and AL was analysed. In RNA blot analysis, iNOS mRNA was induced by LPS in the lung, heart, liver and spleen, and less strongly in the skeletal muscle and testis. AS and AL mRNAs were induced in the lung and spleen. Kinetic studies showed that iNOS mRNA increased rapidly in both spleen and lung, reached a maximum 2-5 h after the treatment, and decreased thereafter. On the other hand, AS mRNA increased more slowly and reached a maximum in 6-12 h (by about 10-fold in the spleen and 2-fold in the lung). AL mRNA in the spleen and lung increased slowly and remained high up to 24 h. In immunohistochemical analysis, macrophages in the spleen that were negative for iNOS and AS before LPS treatment were strongly positive for both iNOS and AS after this treatment. As iNOS, AS and AL were co-induced in rat tissues and cells, citrulline-arginine recycling seems to be important in NO synthesis under the conditions of stimulation. Arginine is a common substrate of NOS and arginase. Rat peritoneal macrophages were cultured in the presence of LPS and expression of iNOS and livertype arginase (arginase I) was analysed. mRNAs for iNOS and arginase I were induced by LPS in a dose-dependent manner. iNOS mRNA appeared 2 h after LPS treatment and increased up to a near-maximum at 8-12 h. On the other hand, arginase I mRNA began to increase after 4 h with a lag time and reached a maximum at 12 h. Immunoblot analysis showed that iNOS and arginase I proteins were also induced. Induction of iNOS and arginase I mRNAs were also observed in LPS-injected rats in vivo. Thus, arginase I appears to have an important role in downregulating NO synthesis in murine macrophages by decreasing the availability of arginine. A cDNA for human arginase II, an arginase isozyme, was isolated. A polypeptide of 354 amino acid residues including the putative NH2-terminal presequence for mitochondrial import was predicted. It was 59% identical with arginase I. mRNA for human arginase II was present in the kidney and other tissues but was not detected in the liver. Arginase II mRNA was co-induced with iNOS mRNA in murine macrophage-like RAW 264.7 cells by LPS. This induction was enhanced by dexamethasone and dibutyrul cAMP, and was prevented by interferon-gamma. These results indicate that NO synthesis is regulated by arginine-synthesizing and -degrading enzymes in a complicated manner.
一氧化氮（NO）由一氧化氮合酶（NOS）催化精氨酸合成，生成的瓜氨酸可通过精氨琥珀酸合成酶（AS）和精氨琥珀酸裂解酶（AL）再循环为精氨酸。给大鼠注射细菌脂多糖（LPS），分析诱导型一氧化氮合酶（iNOS）、AS和AL的表达。在RNA印迹分析中，LPS可诱导肺、心脏、肝脏和脾脏中iNOS mRNA表达，在骨骼肌和睾丸中诱导作用较弱。AS和AL mRNA在肺和脾脏中被诱导。动力学研究表明，脾脏和肺中iNOS mRNA迅速增加，处理后2 - 5小时达到峰值，随后下降。另一方面，AS mRNA增加较慢，6 - 12小时达到峰值（脾脏中约增加10倍，肺中增加2倍）。脾脏和肺中的AL mRNA增加缓慢，直至24小时仍保持高水平。免疫组织化学分析显示，LPS处理前脾脏中iNOS和AS阴性的巨噬细胞，处理后iNOS和AS均呈强阳性。由于iNOS、AS和AL在大鼠组织和细胞中共同被诱导，在刺激条件下瓜氨酸 - 精氨酸循环在NO合成中似乎很重要。精氨酸是NOS和精氨酸酶的共同底物。在LPS存在的条件下培养大鼠腹腔巨噬细胞，分析iNOS和肝型精氨酸酶（精氨酸酶I）的表达。LPS以剂量依赖的方式诱导iNOS和精氨酸酶I的mRNA表达。LPS处理后2小时iNOS mRNA出现，8 - 12小时增加至接近最大值。另一方面，精氨酸酶I mRNA在4小时后开始增加，有滞后时间，12小时达到最大值。免疫印迹分析表明iNOS和精氨酸酶I蛋白也被诱导。在体内注射LPS的大鼠中也观察到iNOS和精氨酸酶I mRNA的诱导。因此，精氨酸酶I似乎通过降低精氨酸的可用性在下调小鼠巨噬细胞中NO合成方面发挥重要作用。分离出人类精氨酸酶II（一种精氨酸酶同工酶）的cDNA。预测其为一个354个氨基酸残基的多肽，包括推测的用于线粒体导入的NH2末端前序列。它与精氨酸酶I有59%的同源性。人类精氨酸酶II的mRNA存在于肾脏和其他组织中，但在肝脏中未检测到。在小鼠巨噬细胞样RAW 264.7细胞中，LPS可共同诱导精氨酸酶II mRNA和iNOS mRNA表达。地塞米松和二丁酰环磷腺苷可增强这种诱导作用，而干扰素 - γ可抑制这种诱导。这些结果表明，NO合成受到精氨酸合成和降解酶的复杂调控。
