Effect of anoxia on intracellular ATP, Na+i, Ca2+i, Mg2+i, and cytotoxicity in rat hepatocytes.
Effect of anoxia on intracellular ATP, Na+i, Ca2+i, Mg2+i, and cytotoxicity in rat hepatocytes.
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DOI：
10.1016/s0021-9258(19)50477-x
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发表时间：
1992-04
期刊：
影响因子：
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通讯作者：
Antonio GasbarriniSQII;A. Borle;Hassan FarghaliQ;C. Bender;Antonio Francavillap;David Van ThielQ
中科院分区：
文献类型：
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作者：
Antonio GasbarriniSQII;A. Borle;Hassan FarghaliQ;C. Bender;Antonio Francavillap;David Van ThielQ
在琼脂糖凝胶线中保存并用 Krebs-Henseleit 碳酸氢盐缓冲液 (KHB) 灌注的新鲜分离的大鼠肝细胞中研究了缺氧的影响。使用水母发光蛋白测量胞质游离钙 (Ca2+i)，使用 SBFI 测量细胞内钠 (Na+i)，使用 BCECF 测量细胞内 pH (pHi)，通过乳酸氧化为丙酮酸期间 NADH 吸光度的增加测量乳酸脱氢酶 (LDH)，通过 31P NMR 光谱实时测量 ATP，并根据 β-ATP 相对于化学位移的化学位移测量细胞内游离 Mg2+ (Mg2+i)。 NMR 光谱中的 α-ATP。通过用 95% N2、5% CO2 饱和的 KHB 灌注细胞来诱导缺氧。缺氧 1 小时后，β-ATP 下降 66%，2 小时后下降 85%，而 Pi/ATP 比率从 2.75 增加到 28.3，增加了 10 倍。在对照条件下，静息胞质游离钙为 127 +/- 6 nM。缺氧会在两个不同的阶段增加 Ca2+i：第一次上升发生在 15 分钟内，并达到平均值 389 +/- 35 nM（p 小于 0.001）。 1 小时后，第二个峰值达到最大值 1.45 +/- 0.12 microM（p 小于 0.001）。在缺氧的第一个小时内，Na+i 从 15.9 +/- 2.4 mM 增加到 32.2 +/- 1.2 mM（p 小于 0.001），Mg2+i 从 0.51 +/- 0.05 翻倍到 1.12 +/- 0.01 mM（p 小于 0.001），pHi 从 7.41 +/- 0.03 下降至 7.06 +/- 0.1（p 小于 0.001）。 LDH 释放在缺氧的第一个小时内增加了一倍，在缺氧的第二个小时内增加了 6 倍。复氧后，ATP、Ca2+i、Mg2+i、Na+i 和 LDH 在 45 分钟内恢复到接近对照水平。为了确定LDH释放的增加是否与Ca2+i的增加有关，以及Ca2+i的增加是否是由Ca2+流入引起的，在缺氧期间用不含Ca(2+)的KHB（+ 0.1 mM EGTA）灌注细胞。在无Ca(2+)培养基中缺氧2小时后，β-ATP再次下降90%，但Ca2+i在第一个初始峰值后下降到低于对照水平，LDH释放仅增加2.7倍。在复氧过程中，Ca2+i、ATP、Na+i 和 LDH 在 45 分钟内恢复到接近对照水平。这些结果表明，缺氧引起的Ca2+i升高是由于Ca2+从细胞外液流入引起的，LDH释放和细胞损伤可能与由此引起的Ca2+i升高有关。
The effects of anoxia were studied in freshly isolated rat hepatocytes maintained in agarose gel threads and perfused with Krebs-Henseleit bicarbonate buffer (KHB). Cytosolic free calcium (Ca2+i) was measured with aequorin, intracellular sodium (Na+i) with SBFI, intracellular pH (pHi) with BCECF, lactic dehydrogenase (LDH) by the increase in NADH absorbance during lactate oxidation to pyruvate, ATP by 31P NMR spectroscopy in real time, and intracellular free Mg2+ (Mg2+i) from the chemical shift of beta-ATP relative to alpha-ATP in the NMR spectra. Anoxia was induced by perfusing the cells with KHB saturated with 95% N2, 5% CO2. After 1 h of anoxia, beta-ATP fell 66%, and 85% after 2 h, while the Pi/ATP ratio increased 10-fold from 2.75 to 28.3. Under control conditions, the resting cytosolic free calcium was 127 +/- 6 nM. Anoxia increased Ca2+i in two distinct phases: a first rise occurred within 15 min and reached a mean value of 389 +/- 35 nM (p less than 0.001). A second peak reached a maximum value of 1.45 +/- 0.12 microM (p less than 0.001) after 1 h. During the first hour of anoxia, Na+i increased from 15.9 +/- 2.4 mM to 32.2 +/- 1.2 mM (p less than 0.001), Mg2+i doubled from 0.51 +/- 0.05 to 1.12 +/- 0.01 mM (p less than 0.001), and pHi decreased from 7.41 +/- 0.03 to 7.06 +/- 0.1 (p less than 0.001). LDH release doubled during the first hour and increased 6-fold during the second hour of anoxia. Upon reoxygenation, ATP, Ca2+i, Mg2+i, Na+i, and LDH returned near the control levels within 45 min. To determine whether the increased LDH release was related to the rise in Ca2+i, and whether the increased Ca2+i was caused by Ca2+ influx, the cells were perfused with Ca(2+)-free KHB (+ 0.1 mM EGTA) during the anoxic period. After 2 h of anoxia in Ca(2+)-free medium, beta-ATP again fell 90%, but Ca2+i, after the first initial peak, fell below control levels, and LDH release increased only 2.7-fold. During reoxygenation, Ca2+i, ATP, Na+i, and LDH returned near the control levels within 45 min. These results suggest that the rise in Ca2+i induced by anoxia is caused by an influx of Ca2+ from the extracellular fluid, and that LDH release and cell injury may be related to the resulting rise in Ca2+i.