Souza M M, Gross S, Boyle R T, Lieberman M
Department of Cell Biology, Duke University Medical Center, Durham, NC, USA.
Mol Cell Biochem. 2000 Jul;210(1-2):173-83. doi: 10.1023/a:1007154412805.
Previous studies in chick embryo cardiac myocytes have shown that the inhibition of Na+/K+-ATPase with ouabain induces cell shrinkage in an isosmotic environment (290 mOsm). The same inhibition produces an enhanced RVD (regulatory volume decrease) in hyposmotic conditions (100 mOsm). It is also known that submitting chick embryo cardiomyocytes to a hyperosmotic solution induces shrinkage and a concurrent intracellular alkalization. The objective of this study was to evaluate the involvement of intracellular pH (pHi), intracellular Ca2+ ([Ca2+]i) and Na+/K+-ATPase inhibition during hyposmotic swelling. Changes in intracellular pH and Ca2+ were monitored using BCECF and fura-2, respectively. The addition of ouabain (100 microM) under both isosmotic and hyposmotic stimuli resulted in a large increase in [Ca2+]i (200%). A decrease in pHi (from 7.3 +/- 0.09 to 6.4 +/- 0.08, n = 6; p < 0.05) was only observed when ouabain was applied during hyposmotic swelling. This acidification was prevented by the removal of extracellular Ca2+. Inhibition of Na+/H+ exchange with amiloride (1 mM) had no effect on the ouabain-induced acidification. Preventing the mitochondrial accumulation of Ca2+ using CCCP (10 microM) resulted in a blockade of the progressive acidification normally induced by ouabain. The inhibition of mitochondrial membrane K+/H+ exchange with DCCD (1 mM) also completely prevented the acidification. Our results suggest that intracellular acidification upon cell swelling is mediated by an initial Ca2+ influx via Na+/Ca2+ exchange, which under hyposmotic conditions activates the K+ and Ca2+ mitochondrial exchange systems (K+/H+ and Ca2+/H+).
先前在鸡胚心肌细胞中的研究表明，在等渗环境（290 mOsm）中用哇巴因抑制钠钾ATP酶会诱导细胞皱缩。在低渗条件（100 mOsm）下，同样的抑制作用会使调节性容积减小（RVD）增强。还已知将鸡胚心肌细胞置于高渗溶液中会诱导细胞皱缩并同时发生细胞内碱化。本研究的目的是评估低渗肿胀过程中细胞内pH（pHi）、细胞内Ca2+（[Ca2+]i）和钠钾ATP酶抑制作用的参与情况。分别使用BCECF和fura-2监测细胞内pH和Ca2+的变化。在等渗和低渗刺激下添加哇巴因（100 microM）都会导致[Ca2+]i大幅增加（200%）。仅在低渗肿胀期间应用哇巴因时才观察到pHi降低（从7.3±0.09降至6.4±0.08，n = 6；p < 0.05）。去除细胞外Ca2+可防止这种酸化。用氨氯吡咪（1 mM）抑制钠氢交换对哇巴因诱导的酸化没有影响。使用CCCP（10 microM）阻止Ca2+在线粒体中的积累会导致通常由哇巴因诱导的渐进性酸化受阻。用DCCD（1 mM）抑制线粒体膜钾氢交换也完全防止了酸化。我们的结果表明，细胞肿胀时的细胞内酸化是由通过钠钙交换的初始Ca2+内流介导的，在低渗条件下，该内流会激活钾和钙的线粒体交换系统（钾氢和钙氢）。
