Matsuda N, Mori T, Nakamura H, Shigekawa M
Department of Surgery, Tottori University School of Medicine, Yonago, Japan.
J Surg Res. 1995 Dec;59(6):712-8. doi: 10.1006/jsre.1995.1228.
This study investigated the selective effects of intracellular (pHi) or extracellular change in pH on reoxygenation-induced Ca2+ overload in simulated myocardial hypoxia. Experiments were performed in cultured cardiomyocytes isolated from the ventricle of neonatal ICR mouse. A model of chemical hypoxia with 2 mM sodium cyanide was developed to mimic the ATP depletion of hypoxia. This chemical hypoxia was "reoxygenated" and the dynamics in intracellular Ca2+ concentration ([Ca2+]i) and pHi were monitored using the fluorescent dyes fura-2 and 2', 7'-bis (2-carboxyethyl)-5(6)-carboxyfluorescein, respectively. During a 40-min chemical hypoxia, pHi progressively fell from 7.2 to 6.6. Reoxygenation with control solution caused rapid recovery of pHi and a marked increase in [Ca2+]i (1884 +/- 136 nM). Intracellular acidotic reoxygenation produced by lactate apparently prolonged the time course of pHi recovery and significantly suppressed reoxygenation-induced Ca2+ overload (1170 +/- 118 nM, P = 0.008). Extracellular acidotic reoxygenation with 2-(N-morpholino) ethanesulfonic acid (pK = 5.96) buffer somewhat suppressed the Ca2+ overload; however, the maximal value of [Ca2+]i was not reduced significantly compared with the control (1790 +/- 122 nM, P = 0.130). In addition, inhibition of Na(+)-H+ exchange by amiloride potentiated prolongation of intracellular acidosis during reoxygenation and resulted in a minimal increase in [Ca2+]i (985 +/- 102 nM, P = 0.004). These results suggest that reoxygenation-induced Ca2+ overload is closely correlated with intracellular pH in the initial phase of reoxygenation, and the protective effects of extracellular acidosis is principally mediated by intracellular acidification of reoxygenated cardiomyocytes.
本研究调查了细胞内（pHi）或细胞外pH变化对模拟心肌缺氧复氧诱导的Ca2+超载的选择性影响。实验在从新生ICR小鼠心室分离的培养心肌细胞中进行。建立了用2 mM氰化钠的化学缺氧模型以模拟缺氧时的ATP耗竭。这种化学缺氧进行“复氧”，并分别使用荧光染料fura-2和2'，7'-双（2-羧乙基）-5（6）-羧基荧光素监测细胞内Ca2+浓度（[Ca2+]i）和pHi的动态变化。在40分钟的化学缺氧期间，pHi从7.2逐渐降至6.6。用对照溶液复氧导致pHi快速恢复和[Ca2+]i显著增加（1884±136 nM）。乳酸产生的细胞内酸中毒复氧明显延长了pHi恢复的时间进程，并显著抑制复氧诱导的Ca2+超载（1170±118 nM，P = 0.008）。用2-（N-吗啉代）乙磺酸（pK = 5.96）缓冲液进行细胞外酸中毒复氧在一定程度上抑制了Ca2+超载；然而，与对照相比，[Ca2+]i的最大值没有显著降低（1790±122 nM，P = 0.130）。此外，氨氯地平抑制Na(+)-H+交换增强了复氧期间细胞内酸中毒的延长，并导致[Ca2+]i的最小增加（985±102 nM，P = 0.004）。这些结果表明，复氧诱导的Ca2+超载在复氧初始阶段与细胞内pH密切相关，细胞外酸中毒的保护作用主要由复氧心肌细胞的细胞内酸化介导。
