The effect of erythropoietin on interleukin-1β mediated increase in nitric oxide synthesis in vascular smooth muscle cells
The effect of erythropoietin on interleukin-1β mediated increase in nitric oxide synthesis in vascular smooth muscle cells
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DOI：
10.1097/00004872-199917090-00003
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发表时间：
1999-09-01
影响因子：
4.9
通讯作者：
Asano, Y
中科院分区：
文献类型：
--
作者：
Akimoto, T;Kusano, E;Asano, Y
目的 近期，我们观察到重组人促红细胞生成素（rHuEPO）可抑制培养的大鼠血管平滑肌细胞（VSMC）中白细胞介素（IL）-1β诱导的一氧化氮（NO）产生和诱导型一氧化氮合酶（iNOS）表达，并探讨rHuEPO的抑制作用机制。结合免疫沉淀和蛋白质印迹分析磷脂酶 C (PLC) 的酪氨酸磷酸化。通过髓磷脂碱性蛋白（MBP4-14）的磷酸化测定来分析蛋白激酶C（PKC）活性，将VSMC与测试剂一起孵育24小时，并测量作为稳定NO代谢物的亚硝酸盐。分别采用Northern印迹法和Western印迹法分析iNOS mRNA和蛋白表达。结果RT-PCR分析显示EpoR mRNA表达；此外，它可能在VSMC中选择性剪接，rHuEPO诱导PLC-gamma 1的酪氨酸磷酸化和PKC的激活，rHuEPO不仅抑制IL-1β诱导的亚硝酸盐产生，而且抑制iNOS mRNA和蛋白的表达。 rHuEPO 的这些抑制作用在 PKC 抑制剂、钙磷蛋白 C (1 mu mol/l) 或十字孢菌素 (10 nmol/l) 存在下被逆转，佛波醇肉豆蔻酸酯乙酸酯激活 PKC 抑制亚硝酸盐的产生。 rHuEPO对IL-1β诱导的亚硝酸盐产生的抑制作用在PKC耗尽的细胞中或在抗EpoR抗体存在的情况下也被消除。结论rHuEPO通过EpoR抑制iNOS mRNA和蛋白表达来抑制IL-1β诱导的NO产生，并且PLC-gamma 1和PKC途径可能参与其中。 I Hypertens 1999, 17:1249-1256 (C) Lippincott Williams & Wilkins。
Objective Recently, we observed that recombinant human erythropoietin (rHuEPO) inhibits the interleukin (IL)-1 beta induced nitric oxide (NO) production and inducible NO synthase (iNOS) expression in cultured rat vascular smooth muscle cells (VSMC), The mechanisms of these inhibitory effects of rHuEPO were evaluated,Methods Reverse transcription-polymerase chain reaction (RT-PCR) was performed to identify a specific erythropoietin receptor (EpoR), Tyrosine phosphorylation of phospholipase C (PLC) was analyzed by combination of immunoprecipitation and Western blotting. Protein kinase C (PKC) activities were analyzed by phosphorylation assay of myelin basic protein (MBP4-14), VSMC were incubated with test agents for 24 h and nitrite as a stable NO metabolite was measured. iNOS mRNA and protein expression was analyzed by Northern and Western blotting, respectively.Results RT-PCR analysis revealed that EpoR m-RNA was expressed; furthermore, it might be alternatively spliced in VSMC, rHuEPO induced tyrosine phosphorylation of PLC-gamma 1 and activation of PKC, rHuEPO inhibited not only IL-1 beta induced nitrite production, but also the expression of iNOS mRNA and protein. These inhibitory effects of rHuEPO were reversed in the presence of PKC inhibitors, calphostin C (1 mu mol/l) or staurosporine (10 nmol/l), PKC activation by phorbol myristate acetate inhibited nitrite production. The inhibitory effect of rHuEPO on IL-1 beta induced nitrite production was also eliminated in PKC depleted cells or in the existence of anti-EpoR antibody.Conclusion rHuEPO inhibits IL-1 beta induced NO production by suppressing iNOS mRNA and protein expressions through EpoR, and the PLC-gamma 1 and PKC pathway may be involved. I Hypertens 1999, 17:1249-1256 (C) Lippincott Williams & Wilkins.