CYTOKINE-INDUCED EXPRESSION OF NITRIC-OXIDE SYNTHASE RESULTS IN NITROSYLATION OF HEME AND NONHEME IRON PROTEINS IN VASCULAR SMOOTH-MUSCLE CELLS
CYTOKINE-INDUCED EXPRESSION OF NITRIC-OXIDE SYNTHASE RESULTS IN NITROSYLATION OF HEME AND NONHEME IRON PROTEINS IN VASCULAR SMOOTH-MUSCLE CELLS
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DOI：
10.1006/excr.1994.1275
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发表时间：
1994-09-01
影响因子：
3.7
通讯作者：
HANSSON, GK
中科院分区：
文献类型：
--
作者：
GENG, YJ;PETERSSON, AS;HANSSON, GK
一氧化氮合酶（NOS）催化L-精氨酸合成生物介质一氧化氮（NO）。我们分析了NOS诱导和活性在培养的大鼠血管平滑肌细胞（SMC），响应于NO的舒张和抑制线粒体呼吸。干扰素-γ和肿瘤坏死因子-α均诱导NOS mRNA的表达，两种细胞因子的组合具有协同作用。一种与鼠巨噬细胞NOS mRNA互补的内部寡核苷酸与来自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和巨噬细胞的诱导型NOS之间具有高度的同源性。NOS依赖的亚硝酸盐生产的分析表明，该酶需要NADPH作为辅因子，但不需要钙的活性。细胞因子治疗导致亚硝酰复合物的电子顺磁共振（EPR）信号特征的发展，表明由酶促合成NO的SMC分子的亚硝基化。从头NOS基因转录和蛋白质合成所需的精氨酸诱导的蛋白质亚硝基化，因为除了放线菌素D和放线菌酮废除了细胞因子的作用。在细胞因子治疗的早期阶段，当使用低剂量的细胞因子时，EPR信号由血红素-亚硝酰基复合物典型的三重超精细结构主导。随着孵育时间和/或细胞因子剂量的增加，EPR谱逐渐转换成类似于nonheme铁（II）-亚硝酰巯基复合物的模式。此后，EPR信号的形状不再改变，而信号强度增加定量与NO的合成，这表明，相当数量的NO合成可以被困在细胞内形成亚硝酰复合物与细胞内分子。总之，这些结果提供了直接的生化证据细胞因子诱导的NO合成和蛋白质亚硝基化SMC。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。(C)1994年出版社出版。
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 heme-nitrosyl 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 metabolismin blood vessels. (C) 1994 Academic Press, Inc.