Regulation mechanism of eNOS Ser633 phosphorylation induced by hypoxia/reoxygenation in H9C2 cell
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摘要: 目的:初步探讨缺氧/复氧 (H/R) 损伤过程心肌细胞株H9C2中内皮型一氧化氮合酶 (eNOS) Ser633磷酸化水平的变化及其可能的调控机制。方法:采用缺氧4h培养后恢复常氧条件培养12、16或24h造成H9C2细胞H/R损伤。选取缺氧4h/复氧12h (H4/R12) 进行后续实验。① H/R后用钙离子泵抑制剂毒胡萝卜素 (thapsigargin, TG), 1.0×10-6 mol/L处理细胞1h;用PI3K/Akt抑制剂LY294002 (5.0×10-5 mol/L) 预处理1h, 再进行H/R及TG处理1h。② 用PP2A/PP1抑制剂冈田酸Okadaic acid (OA) 低剂量 (5×10-8 mol/L)、高剂量 (1×10-6 mol/L) 预处理细胞30 min, 再进行H/R。用Western blot检测eNOS总蛋白及eNOS Ser633磷酸化水平, 化学比色法检测培养基中一氧化氮 (NO) 含量。结果:① H/R损伤后心肌细胞eNOS Ser633磷酸化水平降低 (P＜0.05);TG明显上调eNOS Ser633磷酸化水平 (P＜0.05), LY294002预处理可抑制TG的上调作用 (P＜0.05);低剂量OA、高剂量OA预处理均可上调H/R损伤后eNOS Ser633磷酸化水平 (P＜0.05), 且高、低剂量组间无明显差异。② H/R损伤后培养基中NO含量减少 (P＜0.05);TG、低剂量OA、高剂量OA处理均可增加H/R损伤后培养基中NO含量 (P＜0.05)。结论:H/R损伤可明显降低H9C2细胞中eNOS Ser633水平, 这可能是由于H/R过程中既有PI3K/Akt通路被抑制, 又有PP2A活性增强所致。Abstract: Objective: To investigate changes of endothelial nitric oxide synthase (eNOS) Ser633 phosphorylation during hypoxia/reoxygenation (H/R) injury in H9 C2 cells and its potential regulation mechanism.Method: After 4 hours cultivating under hypoxia condition, H/R injury was caused in H9 C2 cells by recovering the normoxia conditions for 12, 16 and 24 hours, respectively.Follow-up experiments were proceeded under the condition of hypoxia for 4 hours and reoxygenation for 12 hours (H4/R12). (1) Cells were treated by thapsigargin (TG, 1.0×10-6 mol/L), a calcium ion pump inhibitor, for 1 hour after H/R.Cells were pretreated by LY294002 (5.0×10-5 mol/L), a PI3 K/Akt inhibitor, for 1 hour, then cells were treated H/R or TG for 1 hour. (2) H9 C2 cells were pretreated by Okadaic acid (OA), a PP2 A/PP1 inhibitor, respectively with low dose (5×10-8 mol/L) and high dose (1×10-6 mol/L) for 30 minutes, then were treated H/R.Expression of eNOS and phosphorylation of eNOS Ser633 were detected by Western blot.The content of nitric oxide (NO) in the medium was detected by chemical colorimetry.Result: (1) Phosphorylation of eNOS Ser633 decreased after H/R (P＜0.05), which were significantly up-regulated by TG treatment (P＜0.05).LY294002 pretreatment inhibited the up regulation of eNOS Ser633 phosphorylation by TG (P＜0.05).Phosphorylation of eNOS Ser633 increased after treated with low dose OA or high dose OA (P＜0.05), there was no significant difference in phosphorylation level of eNOS Ser633 between high dose OA and low dose OA. (2) The content of NO in the medium reduced after H/R treatment (P＜0.05), which could be increased after TG, low dose OA and high dose OA treatment (P＜0.05).Conclusion: H/R injury can significantly reduced phosphorylation of eNOS Ser633 in H9 C2 cells, which may be due to the inhibition of PI3 K/Akt signaling pathway and enhanced activity of PP2 A.
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Key words:
- cardiomyocyte /
- hypoxia/reoxygenation /
- endothelial nitric oxide synthase /
- PP2A /
- PI3K/Akt
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