Cougnon Marc, Benammou Samia, Brouillard Franck, Hulin Philippe, Planelles Gabrielle
Institut National de la Santé et de la Recherche Médicale Unité 467, Université Paris V, Faculté de Médecine Necker-Enfants Malades, 75730 Paris Cedex 15, France.
Am J Physiol Cell Physiol. 2002 Jun;282(6):C1445-53. doi: 10.1152/ajpcell.00410.2001.
To investigate the effects of reactive oxygen species (ROS) on NH4+ permeation in Xenopus laevis oocytes, we used intracellular double-barreled microelectrodes to monitor the changes in membrane potential (V(m)) and intracellular pH (pH(i)) induced by a 20 mM NH4Cl-containing solution. Under control conditions, NH4Cl exposure induced a large membrane depolarization (to V(m) = 4.0 +/- 1.5 mV; n = 21) and intracellular acidification [reaching a change in pH(i) (DeltapH(i)) of 0.59 +/- 0.06 pH units in 12 min]; the initial rate of cell acidification (dpH(i)/dt) was 0.06 +/- 0.01 pH units/min. Incubation of the oocytes in the presence of H2O2 or beta-amyloid protein had no marked effect on the NH4Cl-induced DeltapH(i). By contrast, in the presence of photoactivated rose bengal (RB), tert-butyl-hydroxyperoxide (t-BHP), or xanthine/xanthine oxidase (X/XO), the same experimental maneuver induced significantly greater DeltapH(i) and dpH(i)/dt. These increases in DeltapH(i) and dpH(i)/dt were prevented by the ROS scavengers histidine and desferrioxamine, suggesting involvement of the reactive species (1)DeltagO2 and.OH. Using the voltage-clamp technique to identify the mechanism underlying the ROS-measured effects, we found that RB induced a large increase in the oocyte membrane conductance (G(m)). This RB-induced G(m) increase was prevented by 1 mM diphenylamine-2-carboxylate (DPC) and by a low Na+ concentration in the bath. We conclude that RB, t-BHP, and X/XO enhance NH4+ influx into the oocyte via activation of a DPC-sensitive nonselective cation conductance pathway.
为了研究活性氧（ROS）对非洲爪蟾卵母细胞中NH4+通透的影响，我们使用细胞内双管微电极监测含20 mM NH4Cl溶液诱导的膜电位（V(m)）和细胞内pH（pH(i)）的变化。在对照条件下，暴露于NH4Cl会引起较大的膜去极化（至V(m)=4.0±1.5 mV；n = 21）和细胞内酸化[在12分钟内pH(i)变化（ΔpH(i)）达到0.59±0.06个pH单位]；细胞酸化的初始速率（dpH(i)/dt）为0.06±0.01个pH单位/分钟。在H2O2或β-淀粉样蛋白存在的情况下孵育卵母细胞，对NH4Cl诱导的ΔpH(i)没有明显影响。相比之下，在光活化的孟加拉玫瑰红（RB）、叔丁基过氧化氢（t-BHP）或黄嘌呤/黄嘌呤氧化酶（X/XO）存在的情况下，相同的实验操作会诱导明显更大的ΔpH(i)和dpH(i)/dt。ROS清除剂组氨酸和去铁胺可阻止ΔpH(i)和dpH(i)/dt的这些增加，表明活性物种单线态氧（1）ΔgO2和羟基自由基（·OH）参与其中。使用电压钳技术来确定ROS测量效应背后的机制，我们发现RB会导致卵母细胞膜电导（G(m)）大幅增加。这种RB诱导的G(m)增加可被1 mM二苯胺-2-羧酸盐（DPC）和浴液中低Na+浓度阻止。我们得出结论，RB、t-BHP和X/XO通过激活DPC敏感的非选择性阳离子电导途径增强NH4+流入卵母细胞。
