Acta Physiologica Sinica, June 25, 2003, 55(3): 265－272
Received 2002-11-01 Accepted 2003-01-22
This work was supported by the National Natural Sciences Foundation of China (No.30070280).
Corresponding author. Tel: +86-24-23056238; ; Fax: +86-24-23922184; Email: hanyal@mail.sy.ln.cn
Research Paper
Protein kinase C
and protein tyrosine kinase mediate lipopolysaccharide- and cytokine- induced
nitric oxide formation in vascular smooth muscle cells of rats
HAN Ya-Ling1,*, KANG Jian1, LI Shao-Hua2
1Department of Cardiology, General Hospital of Shenyang, The Institute of Cardiovascular Research, PLA, 110016;
2Department of Pathology and Laboratory Medicine, Robert Wood Johnson Medical School, New Jersey 08854, USA
Abstract: Rat aorta media, adventitia and cultured vascular smooth muscle cells (VSMCs) were used in this study to identify the source of nitric oxide (NO) generation from various cell types of vascular tissues and to elucidate the mechanisms involved in NO formation. Treatment of vascular media and VSMCs with lipopolysaccharide (LPS) or cytokines [tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β)] resulted in a dose-dependent increase of NO release. Inducible nitric oxide synthase (iNOS) in the stimulated VSMCs was significantly upregulated as shown by Western blot analysis. Protein kinase C (PKC) inhibitor 1-(5-Isoquinolinesulfonyl)- 2-methylpiperazine (H7) prevented LPS-, TNF-α- and IL-1β- induced NO production, whereas N-(2-guanidinoethyl)-5- isoquinoline- sulfonamide (HA1004), an H7 analogue with little activity towards PKC, had no inhibition effect. The role of PKC in LPS- and cytokine-induced on NO formation was confirmed by using another structurally distinct PKC inhibitor chelerythrine. Treatment of VSMCs with protein tyrosine kinase (PTK) inhibitor genistein or tyrphostin AG18 also reduced the NO production evoked by LPS, TNF-α or IL-1β and was associated with inhibition of iNOS protein expression. In contrast, PKC inhibitor chelerythrine did not affect iNOS expression. These results suggest that PTK mediates LPS- and cytokine-induced NO formation by upregulation of iNOS expression. PKC may be involved in the post-translational modification of iNOS or the regulation of the availability of iNOS substrates and cofactors.
Key words: nitric oxide; protein kinase C; protein tyrosine kinase; smooth muscle
蛋白激酶C和蛋白酪氨酸激酶介导脂多糖及细胞因子诱导大鼠血管平滑肌细胞一氧化氮的生成
韩雅玲1,*, 康建1, 李少华2
1沈阳军区总医院全军心血管内外科研究所心内科, 沈阳 110016;
2美国Rober Wood Johnson医学院病理与医学实验科, 美国新泽西州 08854
摘要: 采用Sprague-Dawley大鼠胸主动脉中膜、 外膜和培养的血管平滑肌细胞(VSMCs)作材料, 鉴定不同类型的血管组织经炎性介质刺激后其一氧化氮(NO)的产生来源, 阐明蛋白激酶C (PKC)和蛋白酪氨酸激酶(PTK)介导大鼠VSMCs生成NO的调控机制。大鼠VSMCs经脂多糖(LPS)和细胞因子(TNF-α, IL-1β)处理后, 以剂量依赖方式促进NO释放。采用Western Blot证实经刺激的VSMCs伴有iNOS表达上调。进一步实验表明PKC和PTK参与LPS和细胞因子诱导NO生成的胞内信号转导。用PKC抑制剂H7与VSMCs共培育, H7能明显减少LPS、 TNF-α和IL-1β诱导细胞NO的形成。白屈菜赤碱亦可抑制NO的生成, 但HA1004对VSMCs 的NO生成无抑制作用, 提示PKC参与NO的生成与调控。PTK抑制剂genistein和tyrphostin AG18均能抑制由LPS、 TNF-α和IL-1β引发VSMCs 释放NO, 同时伴iNOS蛋白表达下调, 而PKC抑制剂不能阻断iNOS的表达。上述观察结果提示, PKC介导LPS和细胞因子诱导细胞合成NO可能是通过iNOS翻译后加工; 而PTK则以上调iNOS表达而促增NO生成。
关键词: 一氧化氮; 蛋白激酶C; 蛋白酪氨酸激酶; 平滑肌
中图分类号: Q555.7;R34