[Preventive effect of endothelin-1 pretreatment on hypoxia-induced injury in cultured neonatal rat cardiomyocytes].
This study was designed to observe the effects of endothelin-1 (ET-1) pretreatment on hypoxia-induced injury and changes in [Ca(2+)](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% O(2)-5% CO(2) atmosphere at 37 with or without ET-1 pretreatment. [Ca(2+)](i) was measured with Ca(2+)-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 [Ca(2+)](i). The hypoxia model used in [Ca(2+)](i) measurement was established by continously perfusing cardiomyocytes for 30 min with 95% N(2)-5% CO(2) saturated DMEM solution containing 1 mmol/L Na(2)S(2)O(4). 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% O(2)-5% CO(2) 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 [Ca(2+)](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 [Ca(2+)](i) transient in cultured cardiomyocytes in a dose-dependent manner (P<0.01). The termination of [Ca(2+)](i) transient and the elevation of [Ca(2+)](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 [Ca(2+)](i) changes in cultured neonatal cardiomyocytes, indicating a cyto-protective role of ET-1 pretreatment.
Pan
Yan-Xia
YX
Department of Physiology, Second Military Medical University, Shanghai, PR China.
Lin
Li
L
Yuan
Wen-Jun
WJ
Tang
Chao-Shu
CS
D000818:Animals; D000831:Animals, Newborn; D002118:Calcium / Q000378:metabolism; D015687:Cell Hypoxia; D002478:Cells, Cultured; D019332:Endothelin-1 / Q000494:pharmacology*; D019157:Ischemic Preconditioning, Myocardial / Q000379:methods*; D015428:Myocardial Reperfusion Injury / Q000503:physiopathology / Q000517:prevention & control*; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
D019332:Endothelin-1; D002118:Calcium