Lidocaine depolarizes the mitochondrial membrane potential by intracellular alkalization in rat dorsal root ganglion neurons
Lidocaine depolarizes the mitochondrial membrane potential by intracellular alkalization in rat dorsal root ganglion neurons
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DOI：
10.1007/s00540-010-1079-y
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发表时间：
2011-04-01
影响因子：
2.8
通讯作者：
Tsuneyoshi, Isao
中科院分区：
文献类型：
--
作者：
Onizuka, Shin;Yonaha, Tetsu;Tsuneyoshi, Isao
线粒体膜电位（DeltaIm）是细胞凋亡的重要因素，由质子电化学梯度（DeltaAmuH（+））产生。因此，细胞内质子浓度（pH（in））是改变Δ Im的重要因素。然而，利多卡因对pH（in）的影响尚不清楚。为探讨线粒体对利多卡因的反应，我们同时用Delta Im、黄素腺嘌呤二核苷酸（FAD）和还原型烟酰胺腺嘌呤二核苷酸（NADH）荧光检测线粒体pH（in），计算FAD/NADH比值（氧化还原比），用荧光显微镜下Annexin-V FITC染色观察线粒体形态学变化和早期凋亡。用JC-1和HPTS比率荧光探针同时测定大鼠背根神经节（DRG）神经元Δ Im和pH（in）。同时测量FAD和NADH自发荧光，并计算FAD/NADH荧光比（氧化还原比）。用线粒体过氧化物酶-红色荧光探针测定超氧阴离子。利多卡因在1，5和10 mM进行了评价。10 mM利多卡因给药后观察到形态学变化和早期凋亡。利多卡因以剂量依赖性方式使Delta Im去极化，pH（in）增加。在低pH盐水（pH 6）中，在存在两种弱酸（乙酸盐和丙酸盐）的情况下，利多卡因未能降低Delta Im并增加pH（in）。另一方面，利多卡因以剂量依赖性方式降低细胞中的氧化还原比并增加超氧化物的水平。这些结果表明，利多卡因通过细胞内碱化使Delta Im去极化。这些结果可能表明利多卡因诱导的神经毒性的机制之一。
The mitochondrial membrane potential (Delta Im) is an important factor for apoptosis, and it is produced by the proton electrochemical gradient (Delta A mu H(+)). Therefore, the intracellular proton concentration (pH(in)) is an important factor for modifying the Delta Im. However, the effects of lidocaine on pH(in) are unclear. To investigate mitochondrial responses to lidocaine, therefore, we simultaneously measured pH(in) with Delta Im, flavin adenine dinucleotide (FAD), and reduced form of nicotinamide adenine dinucleotide (NADH) fluorescence, and calculated the FAD/NADH ratio (redox ratio), the superoxide production in mitochondria.Morphological change and early apoptosis were observed by annexin-V FITC staining under fluorescent microscope. The ratiometric fluorescent probe JC-1 and HPTS were used for the simultaneous measurements of Delta Im with pH(in) in rat dorsal root ganglion (DRG) neurons. FAD and NADH autofluorescence were simultaneously measured, and the FAD/NADH fluorescence ratio (redox ratio) was calculated. The superoxide was measured by mitosox-red fluorescent probe for mitochondrial superoxide. Lidocaine was evaluated at 1, 5, and 10 mM.Morphological change and early apoptosis were observed after 10 mM lidocaine administration. Lidocaine depolarized Delta Im with increased pH(in) in a dose-dependent manner. In low-pH saline (pH 6), in the presence of both the weak acids (acetate and propionate), lidocaine failed to depolarize Delta Im and increase pH(in). On the other hand, lidocaine decreased the redox ratio in the cell and increased the levels of superoxide in a dose-dependent manner.These results demonstrated that lidocaine depolarizes Delta Im by intracellular alkalization. These results may indicate one of the mechanisms responsible for lidocaine-induced neurotoxicity.