title: An increase in free radical production by means of an anion channel blocker DIDS in mouse peritoneal neutrophils.
abstract: 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.
authors.affiliation: Institutes of Toxicology, College of Medicine, National Taiwan University, Taipei, ROC.
authors.forename: B S
authors.initials: BS
authors.lastname: Wang
authors.forename: Y J
authors.initials: YJ
authors.lastname: Chen
authors.forename: S H
authors.initials: SH
authors.lastname: Liu
authors.forename: S Y
authors.initials: SY
authors.lastname: Lin-Shiau
mesh_terms: D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology*; D000818:Animals; D000838:Anions; D001202:Ascitic Fluid / Q000166:cytology*; D002118:Calcium / Q000032:analysis; D004789:Enzyme Activation / Q000187:drug effects; D005609:Free Radicals; D006863:Hydrogen-Ion Concentration; D007473:Ion Channels / Q000037:antagonists & inhibitors*; D008163:Luminescent Measurements; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D008813:Mice, Inbred ICR; D008928:Mitochondria / Q000502:physiology / Q000648:ultrastructure; D019255:NADPH Oxidases / Q000378:metabolism; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*
chemical_list: D000838:Anions; D005609:Free Radicals; D007473:Ion Channels; D019255:NADPH Oxidases; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D002118:Calcium