Mitani Y, Dubyak G R, Ismail-Beigi F
Department of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4951, USA.
Am J Physiol. 1996 Jan;270(1 Pt 1):C235-42. doi: 10.1152/ajpcell.1996.270.1.C235.
Exposure of Clone 9 cells (a rat liver cell line expressing only the GLUT-1 isoform) to 5 mM azide or to 3 microM ionomycin for 12 h results in 3.7 +/- 0.3- and 4.9 +/- 0.4-fold increases in GLUT-1 mRNA content, respectively, suggesting the hypothesis that a rise in cytosolic free calcium concentration ([Ca2+]i) mediates the induction of GLUT-1 mRNA by azide. Five lines of evidence were employed to test this hypothesis. 1) Exposure of cells to 0-3 microM of ionomycin increased [Ca2+]i from 83 +/- 9 to 504 +/- 20 nM (half-maximal effect at 0.1 microM ionomycin), whereas half-maximal increase in GLUT-1 mRNA occurred at 1 microM ionomycin, with the increase in the mRNA being negligible at [Ca2+]i below 400 nM. Exposure of cells to 5 mM azide, however, increased [Ca2+]i to maximal value of 174 +/- 22 nM at 15 s, suggesting that the magnitude of the increase in [Ca2+]i by azide may not be adequate for the response. 2) The increase in GLUT-1 mRNA content by azide was fully preserved in cells preloaded with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). 3) GLUT-1 mRNA content increased within 30 min of exposure to ionomycin, whereas the mRNA increased after a "delay" period of 2 h in cells exposed to 5 mM azide. 4) A brief (2-min) rise in [Ca2+]i by ionomycin was sufficient to increase GLUT-1 mRNA content, whereas continuous exposure to azide for > 1 h was necessary for a subsequent induction of the mRNA. 5) Treatment with ionomycin, A-23187, and thapsigargin caused larger increases in glucose-regulated protein 78 and 94 and in 70-kDa heat shock protein mRNAs than in GLUT-1 mRNA, whereas treatment with azide resulted in greater induction of GLUT-1 mRNA. These results strongly suggest that, whereas increased [Ca2+]i enhances GLUT-1 mRNA expression and azide increases [Ca2+]i, the rise in [Ca2+]i does not mediate the induction of GLUT-1 mRNA in response to inhibition of oxidative phosphorylation.
将克隆9细胞（一种仅表达GLUT - 1亚型的大鼠肝细胞系）暴露于5 mM叠氮化物或3 μM离子霉素中12小时，结果GLUT - 1 mRNA含量分别增加了3.7±0.3倍和4.9±0.4倍，这提示了一种假说，即胞质游离钙浓度（[Ca2 + ]i）的升高介导了叠氮化物对GLUT - 1 mRNA的诱导。采用了五条证据来检验这一假说。1）将细胞暴露于0 - 3 μM离子霉素中，[Ca2 + ]i从83±9 nM增加到504±20 nM（在0.1 μM离子霉素时达到半数最大效应），而GLUT - 1 mRNA的半数最大增加发生在1 μM离子霉素时，当[Ca2 + ]i低于400 nM时，mRNA的增加可忽略不计。然而，将细胞暴露于5 mM叠氮化物中，15秒时[Ca2 + ]i增加到最大值174±22 nM，这表明叠氮化物引起的[Ca2 + ]i增加幅度可能不足以引发该反应。2）在预先加载1,2 - 双（2 - 氨基苯氧基）乙烷 - N,N,N',N' - 四乙酸（BAPTA）的细胞中，叠氮化物引起的GLUT - 1 mRNA含量增加完全得以保留。3）暴露于离子霉素后30分钟内GLUT - 1 mRNA含量增加，而暴露于5 mM叠氮化物的细胞中，mRNA在2小时的“延迟”期后增加。4）离子霉素引起的[Ca2 + ]i短暂（2分钟）升高足以增加GLUT - 1 mRNA含量，而持续暴露于叠氮化物超过1小时对于随后诱导mRNA是必要的。5）用离子霉素、A - 23187和毒胡萝卜素处理导致葡萄糖调节蛋白78和94以及70 kDa热休克蛋白mRNA的增加幅度大于GLUT - 1 mRNA，而用叠氮化物处理导致GLUT - 1 mRNA的诱导作用更强。这些结果有力地表明，虽然[Ca2 + ]i升高增强了GLUT - 1 mRNA表达且叠氮化物增加了[Ca2 + ]i，但[Ca2 + ]i的升高并不介导氧化磷酸化受抑制时GLUT - 1 mRNA的诱导。
