通过阴离子通道阻滞剂DIDS在小鼠腹膜中性粒细胞中增加自由基的产生。

B S Wang, Y J Chen, S H Liu, S Y Lin-Shiau
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引用次数: 0

摘要

DIDS(4,4 '-二异硫氰基二苯乙烯- 2,2 '-二磺酸)是公认的阴离子通道阻滞剂。在这项研究中,我们证明了DIDS显著增强小鼠腹膜中性粒细胞自由基的产生。通过鲁米诺-化学发光(LCL)监测系统,DIDS显著提高了LCL, SOD、叠氮化钠(NaN3)、EGTA和BAPTA-AM均能抑制LCL,而staurosporine (STP)仅能轻微抑制LCL。通过TG消耗内质网(ER)-Ca2+储存对DIDS增强的LCL没有影响,但通过fura-2染色监测,DIDS显著增加了细胞内游离钙的量。这些结果表明,DIDS可能增加线粒体Ca2+释放介导的自由基产生。phorboll -12-肉豆酸酯-13-乙酸酯(PMA)和DIDS均可诱导中性粒细胞p47-phox向膜组分转移,而STP预处理可抑制这种转移。由于自由基的产生可以降低细胞质pH (pHi),我们进一步研究了DIDS是否能够诱导细胞内酸化。结果表明,DIDS确实降低了pHi,而na3和nan预处理也抑制了pHi,但二苯二胺(DPI)没有抑制pHi。这些发现使我们提出了一个有效的假设,即DIDS主要诱导超氧化物的产生,并伴随着通过Ca2+依赖线粒体而不是NADPH氧化酶介导的pHi降低。使用亲脂性荧光染料DiOC6(3),我们发现DIDS降低了过渡线粒体膜电位。NaN3在降低线粒体膜电位的过程中拮抗DIDS,而STP和吡啶二硫代氨基甲酸酯(PDTC)则没有拮抗作用。综上所述,所有这些发现表明,线粒体阴离子通道可能通过改变线粒体过渡膜电位和Ca2+释放来调节自由基的产生和中性粒细胞的细胞内酸化。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
An increase in free radical production by means of an anion channel blocker DIDS in mouse peritoneal neutrophils.

DIDS (4, 4'-diisothiocyanostilbene-2, 2'-disulfonic acid) has been recognized as an anion channel blocker. In this study, we demonstrated that DIDS significantly enhanced the production of free radicals in mouse peritoneal neutrophils. By means of a luminol-chemiluminescence (LCL) monitoring system, DIDS markedly increased LCL which could be suppressed by SOD, sodium azide (NaN3), EGTA and BAPTA-AM and only slightly inhibited by staurosporine (STP). Depletion of the endoplasmic reticulum (ER)-Ca2+ store by means of thapsigargin (TG) had no effects on DIDS-enhanced LCL, but DIDS significantly increased the amount of intracellular free calcium as monitored by means of fura-2 staining. These results indicate that DIDS may enhance free radical production mediated by Ca2+ release from the mitochondria. Both phorbol-12-myristate-13-acetate (PMA) and DIDS can induce increased translocation of p47-phox of the neutrophil to the membrane fraction, which is inhibited by STP pretreatment. Since free radical generation could reduce the cytoplasmic pH (pHi), we further examined whether DIDS was capable of inducing intracellular acidification. The result indicated that DIDS certainly lowered the pHi which was also suppressed by pretreatment with either NaN3 or NaCN, but not by diphenyleneiodonium (DPI). These findings lead us to propose a working hypothesis that DIDS mainly induces superoxide production accompanied by decreasing pHi mediated through a Ca2+ -dependent effect on the mitochondria rather than on NADPH oxidase. Using the lipophilic fluorescent dye DiOC6(3), we showed that DIDS decreased the transitional mitochondrial membrane potential. NaN3, but not STP or pyrrolidine dithiocarbamate (PDTC), antagonized DIDS in the course of decreasing the mitochondrial membrane potential. Taken together, all of these findings imply a possible role of anion channels of the mitochondria in modulating free radical production and intracellular acidification of neutrophils through alteration of the mitochondrial transition membrane potential and Ca2+ -release.

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