生理学报Acta Physiologica Sinica, April
研究论文
内皮素-1预处理对培养乳鼠心肌细胞低氧损伤的保护作用
潘燕霞1,2， 林丽1， 袁文俊1，*,唐朝枢3
1第二军医大学生理学教研室, 上海 200433; 2福建医科大学生理学教研室, 福州 350004; 3北京大学医学部生理和病理生理学系， 北京 100083
摘要: 实验观察了0.01-1 nmol/L内皮素-1 (ET-1)预处理对低氧孵育(3% O2-5% CO2 , 12 h) 的培养乳鼠心肌细胞乳酸脱氢酶(LDH)释放量、 培养液上清超氧化物歧化酶(SOD)活性以及丙二醛(MDA)含量的影响。用Fluo-3/AM负载培养的心肌细胞, 在激光扫描共聚焦显微镜下监测急性低氧的心肌细胞[Ca2+]i的变化和ET-1预处理对低氧所致[Ca2+]i变化的影响。结果如下: (1) 心肌细胞低氧孵育12 h后, 培养液上清LDH活力和MDA含量较常氧对照组明显升高, 分别为43.33±1.21 U/L vs 19.33±1.03 U/L和1.71±0.02 nmol/L vs 0.91±0.03 nmol/L (P<0.01), SOD活性为16.93±1.11 U/ml明显低于常氧对照组的33.48±1.15 U/ml (P<0.01); 0.01-1 nmol/L ET-1预处理呈浓度依赖性抑制低氧培养心肌细胞LDH释放, 减少培养液上清MDA含量、 提高SOD活性(P<0.01)。(2) 低氧灌流后29±1.5 s (n=23)心肌细胞自发性钙瞬变完全终止, [Ca2+]i升高了107±13.2% (P<0.001); 0.01-1 nmol/L ET-1能明显加快心肌细胞钙瞬变的频率(P<0.01); ET-1预处理后低氧所致钙瞬变终止的时间较单纯低氧组明显推迟, [Ca2+]i 过度升高被明显减轻 (P<0.01)。上述结果表明, 0.01-1 nmol/L ET-1预处理可减轻培养乳鼠心肌细胞的低氧损伤和抑制低氧所致[Ca2+]i的变化, 具有一定的细胞保护作用。
关键词: 内皮素; 低氧; 心肌细胞; 细胞内钙
中图分类号: Q463; R331.31
Preventive effect
of endothelin-1 pretreatment on hypoxia-induced injury in cultured neonatal rat
cardiomyocytes
PAN Yan-Xia1,2, LIN Li1, YUAN Wen-Jun1，*, TANG Chao-Shu3
1Department of Physiology, Second Military Medical University, Shanghai 200433; 2Department of Physiology, Fujian Medical University, Fuzhou 350004; 3Department of Physiology and Pathophysiology, Health Sciences Center, Peking University, Beijing 100083
Abstract: This study was designed to observe the effects of endothelin-1 (ET-1) pretreatment on hypoxia-induced injury and changes in [Ca2+]i in cultured neonatal rat cardiomyocytes. The activity of lactate dehydrogenase (LDH) and superoxide dismutase (SOD), and the content of malondialdehyde (MDA) in the supernatant were determined in the cultured cardiomyocytes subjected to a 12-h hypoxia induced by a 3% O2-5% CO2 atmosphere at 37℃ with or without ET-1 pretreatment. [Ca2+]i was measured with Ca2+-sensitive fluorescent probe fluo-3/AM under a laser scanning confocal microscope. Fluorescence intensity emitted from fluo-3/AM-loaded cells reflected the concentration of [Ca2+]i. The hypoxia model used in [Ca2+]i measurement was established by continously perfusing cardiomyocytes for 30 min with 95% N2-5% CO2 saturated DMEM solution containing 1 mmol/L Na2S2O4. Pretreatment with ET-1 consisted of three cycles of ET-1 perfusion (5 min for each) followed by ET-1-free DMEM solution (10 min for each) prior to hypoxia. The results showed that (1) after incubation in a 3% O2-5% CO2 hypoxic atmosphere for 12 h, the activity of LDH and the content of MDA in the supernatant significantly increased from 19.33±1.03 U/L to 43.33±1.21 U/L and from 0.91±0.03 nmol/L to 1.71±0.02 nmol/L, respectively, whereas the activity of SOD decreased from 33.48±1.15 U/ml to 16.93±1.11 U/ml (P<0.01). In hypoxic cardiomyocytes pretreated with 0.01-1 nmol/L ET-1, LDH release and supernatant MDA content were decreased, while SOD activity was enhanced dose-dependently (P<0.01). (2) The spontaneous calcium transient in cultured cardiomyocytes terminated at 29±1.5 s and [Ca2+]i increased by 107±13.2% during perfusion of hypoxic solution (P<0.001) at the end of 30 min. ET-1 (0.01-1 nmol/L) increased the frequency of [Ca2+]i transient in cultured cardiomyocytes in a dose-dependent manner (P<0.01). The termination of [Ca2+]i transient and the elevation of [Ca2+]i caused by hypoxia were postponed by pretreatment with 0.01-1 nmol/L ET-1 (P<0.01). These results show that pretreatment with 0.01-1 nmol/L ET-1 attenuates hypoxia-induced injury and [Ca2+]i changes in cultured neonatal cardiomyocytes, indicating a cyto-protective role of ET-1 pretreatment.
Key words: ET-1; hypoxia; cardiomyocytes; intracellular calcium