A noninvasive fluorimetric procedure for measurement of membrane potential - Quantification of the NADPH oxidase-induced depolarization in activated neutrophils
A noninvasive fluorimetric procedure for measurement of membrane potential - Quantification of the NADPH oxidase-induced depolarization in activated neutrophils
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DOI：
10.1074/jbc.274.37.26098
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发表时间：
1999-09-10
影响因子：
4.8
通讯作者：
Grinstein, S
中科院分区：
文献类型：
--
作者：
Jankowski, A;Grinstein, S
NADPH 氧化酶的产电活性与活化的中性粒细胞质膜的去极化有关。然而，这种去极化的程度和后果仍然未知。中性粒细胞不适合通过电流钳对膜电位进行电生理测定。相反，去极化的发生是通过使用电位敏感荧光染料来推断的。然而，此类染料会分配到细胞内细胞器中，并可能产生错误的结果，特别是因为 NADPH 氧化酶主要存在于分泌颗粒中，据称它在分泌颗粒中被激活。我们使用二氢罗丹明证实了氧化酶产物的细胞内生成，二氢罗丹明在氧化时转化为荧光罗丹明 123。罗丹明 123 在中性粒细胞和分化的 HL60 细胞的内膜细胞器内积累，与初级颗粒标记 CD63 共定位；为了在不受细胞器干扰的情况下估计表面膜电位，我们设计了一种基于电压驱动的跨质膜摄取 Mn2+ 的方法。通过钙释放激活通道对 Mn2+ 的摄取被测量为毒胡萝卜素处理的细胞中 Indo-1 荧光猝灭的速率。当使用导电离子载体控制膜电位以及激活 NADPH 氧化酶时，发现 Mn2+ 流入速率会发生变化。使用 Na+ 离子载体 SQI-Pr 构建正电位范围内的校准曲线。使用此校准，发现佛波酯激活的中性粒细胞的膜电位达到+58 +/- 6 mV，与静息电位相比，持续去极化超过100 mV。当用二亚苯基碘鎓抑制 NADPH 氧化酶时，去极化大大减弱。总之，这些结果表明 NADPH 氧化酶可以产生质膜的大量去极化，这应该足以激活各种电压门控通道，包括向外整流的 H+ 电导。
The electrogenic activity of the NADPH oxidase is associated with depolarization of the plasma membrane in activated neutrophils, The magnitude and consequences of this depolarization, however, remain unknown. Neutrophils are not amenable to electrophysiological determinations of membrane potential by current clamp. Instead, the occurrence of depolarization has been inferred from the use of potential-sensitive fluorescent dyes. However, such dyes partition into intracellular organelles and may yield erroneous results, particularly because the NADPH oxidase resides largely in secretory granules, where it has been claimed to become activated. We confirmed the intracellular generation of oxidase products using dihydrorhodamine, which is converted to the fluorescent rhodamine 123 when oxidized. Rhodamine 123 accumulated inside endomembrane organelles in both neutrophils and in differentiated HL60 cells, where it co-localized with the primary granule marker CD63; To estimate the surface membrane potential without interference from organelles, we devised a method based on the voltage-driven uptake of Mn2+ across the plasmalemma. The uptake of Mn2+ through calcium release-activated channels was measured as the rate of Indo-1 fluorescence quenching in thapsigargin-treated cells. The rate of Mn2+ influx was found to vary when the membrane potential was manipulated using conductive ionophores and also when the NADPH oxidase was activated. A calibration curve in the positive potential range was constructed using the Na+ ionophore SQI-Pr. Using this calibration, the membrane potential of phorbol ester-activated neutrophils was found to reach +58 +/- 6 mV, a sustained depolarization of over 100 mV compared with the resting potential. The depolarization was greatly diminished when the NADPH oxidase was inhibited with diphenylene iodonium. Together, these results indicate that the NADPH oxidase can generate a large depolarization of the plasmalemma, which should suffice to activate a variety of voltage-gated channels, including the outwardly rectifying H+ conductance.