摘要
[目的]肌浆网钙泵抑制剂thapsigargin(TG)常被用于耗减肌浆网(sarcoplasmic reticulum,SR)的钙容量。本课题旨在探讨TG(100 nmol·L-1)对源自SR的胞内钙释放和SR钙容量耗减的时间效应以及SR内钙对SR钙释放的调节机理。[方法]局部场刺激作用于成年大鼠心肌细胞,使其产生动作电位,进而诱发胞内钙瞬变(action-potential-induced Ca2+ transients,ACT)。由ACT可估测胞内钙释放。SR钙容量可由咖啡因(20 mmol·L-1)诱发的钙瞬变(caffeine-induced Ca2+ transients,CCT)估测。实验结果用激光共聚焦显微镜系统记录。[结果]TG对ACT峰值(可衡量钙瞬变的大小)的时间效应如下:0 min,5.55±0.28(单位为标准钙荧光强度即F/F0,F0为静息钙荧光强度);10 min,4.89±0.36;17 min,2.58±0.38;25 min,2.11±0.19;35 min,2.22±0.17;50min,2.27±0.27。TG对CCT峰值的时间效应如下:0 min,6.73±0.17;10 min,6.60±0.59;17 min,6.24±0.44;25 min,3.99±0.28;35 min,2.24±0.37;50 min,1.14±0.01。以CCT为自变量,ACT为因变量的曲线呈“J”形。[结论]TG对心肌胞内钙释放和SR钙容量呈非线性的时间依赖效应。SR内钙部分耗减即可明显减少钙释放,说明SR内钙对胞内钙释放具有重要的调节作用。
[Objective] Thapsigargin (TG), a sarcoplasmic reticulum (SR) Ca2+-ATPase inhibitor, is commonly employed to deplete SR Ca2+ content. In this study, we investigated the time effects of TG (100 nmol·L-1) on SR Ca2+ content and SR Ca2+ release, and the relationship between them. [Methods] Local field stimulation was delivered to freshly isolated rat single cardiac myocytes to generate action potential-induced Ca2+ transients (ACT) for assessing intracellular Ca2+ release. Caffeine (20 mmol · L-1)-induced Ca2+ transients (CCT) was used to estimate the SR Ca2+ content. Results were recorded by confocal Ca2+ imaging with a Zeiss LSM-410 confocal microscopic system. [Results]Time-dependent effects of TG on ACT peak were: 5. 55±0. 28 (in F/ F0, the normalized fluorescence, where Fo denotes the resting fluorescence), 0 min; 4. 89 ± 0. 36, 10 min; 2. 58±0.38, 17 min; 2. 11 ±0. 19, 25 min; 2.22±0.17, 35 min; and 2.27±0.27,50 min. Time-dependent effects of TG on CCT peak were: 6.73±0. 17, 0 min; 6. 60 ±0.59, 10 min; 6. 24 ±0.44, 17 min; 3.99±0.28, 25 min; 2. 24 ±0.37, 35 min; and 1. 14 ±0. 01, 50 min. Curve of ACT as a function of CCT displayed a steep “J”-like shape. [Conclusion]TG demonstrates a nonlinear time-dependence on cardiac intracellular Ca2+ release and SR Ca2+ content. Partial depletion of SR Ca2+ content severely diminishes Ca2+ release, suggesting that SR Ca2+ release is tightly regulated by Ca2+ from the lumenal side of the SR.
出处
《中山大学学报（医学科学版）》
CAS
CSCD
北大核心
2004年第1期19-23,共5页
Journal of Sun Yat-Sen University:Medical Sciences
基金
国家重大基础研究发展基金资助项目(G2000057002)
西安交通大学重点基金资助项目(X160
082003)
广东省卫生厅"五个一"工程重点基金资助项目(413626)
