Li F, Sugishita K, Su Z, Ueda I, Barry W H
Cardiology Division, University of Utah School of Medicine, 50 N Medical Drive, Salt Lake City, Utah 84132, USA.
J Mol Cell Cardiol. 2001 Dec;33(12):2145-55. doi: 10.1006/jmcc.2001.1477.
ATP depletion due to ischemia or metabolic inhibition (MI) causes Na(+) and Ca(2+) accumulation in myocytes, which may be in part due to opening of connexin-43 hemichannels. Halothane (H) has been shown to reduce conductance of connexin-43 hemichannels and to protect the heart against ischemic injury. We therefore investigated the effect of halothane on [Ca(2+)]i and [Na(+)]i in myocytes during MI. Isolated rabbit left ventricular myocytes were loaded with 4 microM fluo-3 AM for 30 min, or with 5 microM sodium green AM for 60 min at 37 degrees C. After washing, the myocytes were exposed to: (1) Normal HEPES solution; (2) MI solution (2 mM NaCN, 20 mM 2-deoxy-D-glucose and 0-glucose); or (3) MI+H (0.95 mM, 4.7 mM) for 60 min. Propidium iodide (PI, 25 microM) was added to all samples before data acquisition. The fluorescence intensity was measured by flow cytometry with 488 nm excitation and 530 nm emission for fluo-3 or sodium green, and 670 nm for PI. The [Ca(2+)]i and [Na(+)]i were then calculated by calibration. In some experiments, the effect of 10 microM tetrodotoxin (TTX) and 20 microM nifedipine (NIF) were studied. Metabolic inhibition for 60 min caused a significant increase in [Ca(2+)]i and [Na(+)]i in myocytes when compared to controls, which was significantly reduced by halothane in a dose-dependent fashion. In the presence of TTX and NIF, halothane also significantly reduced the rise in the [Ca(2+)]i and [Na(+)]i in myocytes subjected to MI. 1-heptanol, another gap junction blocker, had similar effects. Thus, halothane reduced [Ca(2+)]i and [Na(+)]i overload produced by MI in myocytes. This effect is not solely due to block of voltage-gated Na(+) and Ca(2+) channels, and is likely mediated by inhibiting the opening of connexin-43 hemichannels.
缺血或代谢抑制（MI）导致的三磷酸腺苷（ATP）耗竭会使心肌细胞内钠离子（Na⁺）和钙离子（Ca²⁺）蓄积，这可能部分归因于连接蛋白43半通道的开放。已证实氟烷（H）可降低连接蛋白43半通道的电导，并保护心脏免受缺血性损伤。因此，我们研究了氟烷对MI期间心肌细胞内钙离子浓度（[Ca²⁺]i）和钠离子浓度（[Na⁺]i）的影响。将分离的兔左心室心肌细胞在37℃下用4微摩尔的氟-3乙酰甲酯（fluo-3 AM）加载30分钟，或用5微摩尔的钠绿乙酰甲酯（sodium green AM）加载60分钟。洗涤后，将心肌细胞暴露于：（1）正常的羟乙基哌嗪乙磺酸（HEPES）溶液；（2）MI溶液（2毫摩尔的氰化钠、20毫摩尔的2-脱氧-D-葡萄糖和0葡萄糖）；或（3）MI + H（0.95毫摩尔、4.7毫摩尔）60分钟。在采集数据前，向所有样本中加入碘化丙啶（PI，25微摩尔）。通过流式细胞术测量荧光强度，激发波长为488纳米，发射波长为530纳米用于检测氟-3或钠绿，670纳米用于检测PI。然后通过校准计算[Ca²⁺]i和[Na⁺]i。在一些实验中，研究了10微摩尔的河豚毒素（TTX）和20微摩尔的硝苯地平（NIF）的作用。与对照组相比，60分钟的代谢抑制导致心肌细胞内[Ca²⁺]i和[Na⁺]i显著增加，氟烷以剂量依赖的方式显著降低了这种增加。在存在TTX和NIF的情况下，氟烷也显著降低了MI心肌细胞内[Ca²⁺]i和[Na⁺]i的升高。另一种缝隙连接阻滞剂1-庚醇也有类似作用。因此，氟烷降低了MI在心肌细胞中产生的[Ca²⁺]i和[Na⁺]i过载。这种作用并非仅仅归因于阻断电压门控的Na⁺和Ca²⁺通道，可能是通过抑制连接蛋白43半通道的开放介导的。
