Vajda Szilvia, Mándi Miklós, Konràd Csaba, Kiss Gergely, Ambrus Attila, Adam-Vizi Vera, Chinopoulos Christos
Neurobiochemical Group, Department of Medical Biochemistry, Hungarian Academy of Sciences, Szentagothai Knowledge Center, Semmelweis University, Budapest, Hungary.
FEBS J. 2009 May;276(10):2713-24. doi: 10.1111/j.1742-4658.2009.06995.x.
Massive amounts of Ca(2+) can accumulate in mitochondria, owing to its complexation with matrix phosphate. Under conditions in which the mitochondrial uniporter is the foremost pathway for Ca(2+) efflux, the release of sequestered Ca(2+) by protonophoric uncouplers is invariably demonstrated. This has been recently ascribed to matrix acidification, which results in the dissociation of the Ca(2+)-phosphate complex. In the present study, we compared the effect of stepwise depolarization on Ca(2+) release induced by either the complex III inhibitor stigmatellin or an uncoupler in energized Ca(2+)-loaded rat liver mitochondria in the presence of phosphate, at extramitochondrial pH (pH(o)) 6.8 and pH(o) 7.8. Both poisons were examined in the presence and absence of oligomycin. Prior to Ca(2+) loading, mitochondria were allowed to phosphorylate 0.5 mm ADP. Opening of the permeability transition pore was additionally hampered by cyclosporin A, and was monitored by changes in light scattering. Na(+) was excluded from the medium, preventing Na(+)/Ca(2+) exchange. At both pH(o) values, Delta pH was in the range 0.11-0.15. Complete depolarization by uncoupling with or without oligomycin resulted in an approximately pH 0.05 acidic shift, but there was none in the case of stigmatellin plus oligomycin. At pH(o) 6.8 and in the presence of oligomycin, the uncoupler-induced Ca(2+) release started in the -80 to -50 mV range, whereas in the absence of oligomycin, the release occurred at approximately -15 mV. Stigmatellin induced minor Ca(2+) release only in the presence of oligomycin, starting at approximately -4 mV. At pH(o) 7.8, the uncoupler-induced Ca(2+) release started at approximately -11 mV, irrespective of the presence or absence of oligomycin. Unexpectedly, at this alkaline pH and in the presence of oligomycin, stigmatellin induced substantial Ca(2+) release, starting at approximately -10 mV. From the above findings, we conclude that matrix acidification cannot be the sole explanation for uncoupler-induced Ca(2+) release from mitochondria.
由于钙离子与线粒体基质中的磷酸盐络合，大量的Ca(2+)可在线粒体中积累。在线粒体单向转运体是Ca(2+)外流的主要途径的条件下，质子载体解偶联剂释放被隔离的Ca(2+)的现象总是会出现。最近这被归因于基质酸化，它导致Ca(2+)-磷酸盐复合物的解离。在本研究中，我们比较了在磷酸盐存在下，在细胞外pH（pH(o)）为6.8和pH(o)为7.8时，逐步去极化对由复合物III抑制剂柱晶白霉素或解偶联剂诱导的Ca(2+)释放的影响，该实验使用的是充满能量且加载了Ca(2+)的大鼠肝线粒体。两种毒物都在有和没有寡霉素的情况下进行了检测。在加载Ca(2+)之前，让线粒体磷酸化0.5 mM的ADP。环孢菌素A额外阻碍了通透性转换孔的开放，并通过光散射的变化进行监测。培养基中排除了Na(+)，以防止Na(+)/Ca(2+)交换。在两个pH(o)值下，ΔpH在0.11 - 0.15范围内。有或没有寡霉素时通过解偶联实现的完全去极化导致pH值大约有0.05的酸性偏移，但柱晶白霉素加寡霉素的情况下没有这种情况。在pH(o) 6.8且存在寡霉素时，解偶联剂诱导的Ca(2+)释放在-80至-50 mV范围内开始，而在没有寡霉素时，释放在大约-15 mV时发生。柱晶白霉素仅在存在寡霉素时诱导少量Ca(2+)释放，大约在-4 mV时开始。在pH(o) 7.8时，解偶联剂诱导的Ca(2+)释放在大约-11 mV时开始，无论是否存在寡霉素。出乎意料的是，在这个碱性pH且存在寡霉素时，柱晶白霉素在大约-10 mV时开始诱导大量Ca(2+)释放。从上述发现中，我们得出结论，基质酸化不能是解偶联剂诱导线粒体释放Ca(2+)的唯一解释。
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