Increased iNOS activity in vascular smooth muscle cells from diabetic rats: Potential role of Ca2+/calmodulin-dependent protein kinase II delta 2 (CaMKIIδ2)
Increased iNOS activity in vascular smooth muscle cells from diabetic rats: Potential role of Ca2+/calmodulin-dependent protein kinase II delta 2 (CaMKIIδ2)
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DOI：
10.1016/j.atherosclerosis.2012.10.062
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发表时间：
2013-01-01
期刊：
影响因子：
5.3
通讯作者：
Pandolfi, Assunta
中科院分区：
文献类型：
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作者：
Di Pietro, Natalia;Di Tomo, Pamela;Pandolfi, Assunta
目的：诱导型一氧化氮合酶 (iNOS) 表达可能因细胞因子血浆水平增加而导致糖尿病血管损伤。除了转录调控外，Ca2+/CaMKII 可能在翻译后控制的 iNOS 活性中发挥作用。因此，我们研究了Ca2+/CaMKII delta(2)信号通路在调节糖尿病大鼠主动脉血管平滑肌细胞(VSMC)中脂多糖(LPS)诱导的iNOS活性中的作用。方法和结果：用20μg/ml LPS刺激从10只糖尿病大鼠(DR)和10只对照大鼠(CR)获得的VSMC。 24小时后，DR-VSMC中的iNOS蛋白水平比CR-VSMC增加了1.37倍（p < 0.05；蛋白质印迹），而DR-VSMC中的iNOS活性（将L-(H-3)-精氨酸转化为L-(H-3)-瓜氨酸）和细胞内硝基酪氨酸水平（免疫荧光）大约是CR-VSMC中的2.7倍。有趣的是，与 CR-VSMC 相比，LPS 在 DR 中更显着地增加细胞内 Ca2+ 水平（荧光视频成像）。这与磷酸化引起的 CaMKII 激活、免疫共沉淀 iNOS/CaMKII delta(2)（Western Blot）量减少以及 iNOS 活性增加有关。 CaMKII 抑制剂 KN-93 消除了所有 LPS 效应。结论：这些结果表明 Ca2+/CaMKII delta(2) 信号通路可能是糖尿病 iNOS 活性的重要调节因子，因此有助于开发糖尿病血管并发症的创新治疗策略。 (C) 2012 Elsevier Ireland Ltd. 保留所有权利。
Objective: Inducible nitric oxide synthase (iNOS) expression may be increased by cytokine plasma levels contributing to vascular damage in diabetes. Besides transcriptional regulation, Ca2+/CaMKII may play a role in post-translationally controlled iNOS activity. We accordingly investigated the involvement of the Ca2+/CaMKII delta(2) signaling pathway in regulating lipopolysaccharide (LPS)-induced iNOS activity in cultured aortic vascular smooth muscle cells (VSMCs) from diabetic rats.Methods and results: VSMCs obtained from 10 diabetic rats (DR) and 10 control rats (CR) were stimulated with 20 mu g/ml LPS. After 24 h, iNOS protein levels were 1.37 fold increased in DR- vs CR-VSMCs (p < 0.05; Western Blot), while iNOS activity (conversion L-(H-3)-arginine into L-(H-3)-citrulline) and intracellular nitrotyrosine levels (immunofluorescence) were about 2.7 fold greater in DR-than in CR-VSMCs. Interestingly, LPS increased intracellular Ca2+ levels (Fluorescence video imaging) more markedly in DR-than in CR-VSMCs. This was associated with CaMKII activation by phosphorylation, a decreased amount of co-immunoprecipitating iNOS/CaMKII delta(2) (Western Blot) and increased iNOS activity. The CaMKII inhibitor KN-93 abolished all the LPS-effects.Conclusion: These results indicate that the Ca2+/CaMKII delta(2) signaling pathway may be an important regulator of iNOS activity in diabetes, and hence contribute to the potential development of innovative therapeutic strategies for vascular complications in diabetes. (C) 2012 Elsevier Ireland Ltd. All rights reserved.