Brustovetsky N, Dubinsky J M
Department of Neuroscience, University of Minnesota, Minneapolis, Minnesota 55455, USA.
J Neurosci. 2000 Nov 15;20(22):8229-37. doi: 10.1523/JNEUROSCI.20-22-08229.2000.
Activation of the mitochondrial permeability transition may contribute to excitotoxic neuronal death (Ankarcrona et al., 1996; Dubinsky and Levi, 1998). However, cyclosporin A (CsA), a potent inhibitor of the permeability transition in liver mitochondria, only protects against neuronal injury by limited doses of glutamate and selected ischemic paradigms. The lack of consistent CsA inhibition of the mitochondrial permeability transition was analyzed with the use of isolated brain mitochondria. Changes in the permeability of the inner mitochondrial membrane were evaluated by monitoring mitochondrial membrane potential (Deltapsi), using the distribution of tetraphenylphosphonium, and by monitoring mitochondrial swelling, using light absorbance measurements. Metabolic impairments, large Ca(2+) loads, omission of external Mg(2+), or low doses of palmitic acid or the protonophore FCCP exacerbated Ca(2+)-induced sustained depolarizations and swelling and eliminated CsA inhibition. BSA restored CsA inhibition in mitochondria challenged with 50 microm Ca(2+), but not with 100 microm Ca(2+). CsA failed to prevent Ca(2+)-induced depolarization or to repolarize mitochondria when mitochondria were depolarized excessively. Similarly, CsA failed to prevent mitochondrial swelling or PEG-induced shrinkage after swelling when the Ca(2+) challenge produced a strong, sustained depolarization. Thus in brain mitochondria CsA may be effective only as an inhibitor of the permeability transition and the Ca(2+)-activated low permeability state under conditions of partial depolarization. In contrast, ADP plus oligomycin inhibited both permeabilities under all of the conditions that were tested. In situ, the neuroprotective action of CsA may be limited to glutamate challenges sufficiently toxic to induce the permeability transition but not so severe that mitochondrial depolarization exceeds threshold.
线粒体通透性转换的激活可能导致兴奋性毒性神经元死亡（安卡尔克隆纳等人，1996年；杜宾斯基和利维，1998年）。然而，环孢素A（CsA）是肝线粒体通透性转换的有效抑制剂，仅在有限剂量的谷氨酸和特定缺血模型中能保护神经元免受损伤。利用分离的脑线粒体分析了CsA对线粒体通透性转换缺乏一致抑制作用的情况。通过监测四苯基鏻的分布来监测线粒体膜电位（Δψ），以及通过光吸收测量来监测线粒体肿胀，从而评估线粒体内膜通透性的变化。代谢损伤、大量Ca(2+)负荷、去除细胞外Mg(2+)、低剂量的棕榈酸或质子载体FCCP会加剧Ca(2+)诱导的持续去极化和肿胀，并消除CsA的抑制作用。牛血清白蛋白（BSA）恢复了用50微摩尔Ca(2+)刺激的线粒体中CsA的抑制作用，但对用100微摩尔Ca(2+)刺激的线粒体无效。当线粒体过度去极化时，CsA无法阻止Ca(2+)诱导的去极化或使线粒体复极化。同样，当Ca(2+)刺激产生强烈、持续的去极化时，CsA无法阻止线粒体肿胀或肿胀后聚乙二醇（PEG）诱导的收缩。因此，在脑线粒体中，CsA可能仅在部分去极化条件下作为通透性转换和Ca(2+)激活的低通透性状态的抑制剂才有效。相比之下，在所有测试条件下，二磷酸腺苷（ADP）加寡霉素均能抑制两种通透性。在原位，CsA的神经保护作用可能仅限于对谷氨酸的刺激，这种刺激毒性足以诱导通透性转换，但又不至于严重到线粒体去极化超过阈值。
