Geng Y J, Petersson A S, Wennmalm A, Hansson G K
Department of Clinical Chemistry, Gothenburg University, Sahlgren's Hospital, Sweden.
Exp Cell Res. 1994 Sep;214(1):418-28. doi: 10.1006/excr.1994.1275.
Nitric oxide synthase (NOS) catalyzes the synthesis of the biomediator, nitric oxide (NO), from L-arginine. We have analyzed NOS induction and activity in cultured rat vascular smooth muscle cells (SMC), which respond to NO by relaxation and inhibition of mitochondrial respiration. Both interferon-gamma and tumor necrosis factor-alpha induced the expression of NOS mRNA and a combination of the two cytokines had a synergistic effect. An internal oligonucleotide complementary to murine macrophage NOS mRNA hybridized to polymerase chain reaction (PCR) products derived from SMC NOS but not brain NOS. Direct sequencing of the PCR products showed a high degree of homology between inducible NOS from SMC and macrophages. Analysis of NOS-dependent nitrite production demonstrated that the enzyme requires NADPH as a cofactor but not calcium for its activity. Cytokine treatment resulted in the development of electron paramagnetic resonance (EPR) signals characteristic for nitrosyl complexes, indicating nitrosylation of SMC molecules by enzymatically synthesized NO. De novo NOS gene transcription and protein synthesis are required for the cytokine-induced protein nitrosylation since addition of actinomycin D and cycloheximide abolished the cytokine effect. At an early stage of cytokine treatment and when low doses of cytokines were used, the EPR signal was dominated by a triplet hyperfine structure typical for hemenitrosyl complexes. With increasing incubation time and/or cytokine dose, the EPR spectra were gradually converted into a pattern resembling that of nonheme iron(II)-nitrosyl thiol complexes. Thereafter, the EPR signal shape no longer changed while the signal intensity increased quantitatively with NO synthesis, suggesting that considerable amounts of NO synthesized could be trapped in the cells by formation of nitrosyl complexes with intracellular molecules. Together, these results provide direct biochemical evidence for cytokine induction of NO synthesis and protein nitrosylation in SMC. This may represent an important second messenger system for cytokine effects on cellular metabolism in blood vessels.
一氧化氮合酶（NOS）催化由L-精氨酸合成生物介质一氧化氮（NO）。我们分析了培养的大鼠血管平滑肌细胞（SMC）中NOS的诱导和活性，这些细胞通过舒张和抑制线粒体呼吸对NO作出反应。干扰素-γ和肿瘤坏死因子-α均诱导NOS mRNA的表达，两种细胞因子联合使用具有协同作用。与鼠巨噬细胞NOS mRNA互补的内部寡核苷酸与源自SMC NOS而非脑NOS的聚合酶链反应（PCR）产物杂交。PCR产物的直接测序显示，SMC和巨噬细胞中的诱导型NOS之间具有高度同源性。对NOS依赖性亚硝酸盐生成的分析表明，该酶的活性需要NADPH作为辅助因子，但不需要钙。细胞因子处理导致电子顺磁共振（EPR）信号出现亚硝酰基复合物特有的特征，表明酶促合成的NO使SMC分子发生亚硝基化。细胞因子诱导的蛋白质亚硝基化需要从头进行NOS基因转录和蛋白质合成，因为添加放线菌素D和环己酰亚胺可消除细胞因子的作用。在细胞因子处理的早期阶段以及使用低剂量细胞因子时，EPR信号以血红素亚硝酰基复合物典型的三重态超精细结构为主。随着孵育时间的增加和/或细胞因子剂量的增加，EPR光谱逐渐转变为类似于非血红素铁（II）-亚硝酰基硫醇复合物的模式。此后，EPR信号形状不再改变，而信号强度随NO合成定量增加，这表明通过与细胞内分子形成亚硝酰基复合物，大量合成的NO可被困在细胞中。总之，这些结果为细胞因子诱导SMC中NO合成和蛋白质亚硝基化提供了直接的生化证据。这可能代表了细胞因子对血管细胞代谢作用的重要第二信使系统。
