FURTHER EVIDENCE THAT LUCIGENIN-DERIVED CHEMILUMINESCENCE MONITORS MITOCHONDRIAL SUPEROXIDE GENERATION IN RAT ALVEOLAR MACROPHAGES

Lucigenin is well recognized for its ability to react with superoxide, yielding a product that emits chemiluminescence. Accordingly, lucigen in-derived chemiluminescence (LDCL) has been widely used to assess the generation of superoxide by the NADPH oxidase of leukocytes. Previous ly, we suggested that lucigenin could interact with mitochondrial-deri ved superoxide in alveolar macrophages. The purpose of this study was to further demonstrate that LDCL is in fact a probe of mitochondrial s uperoxide generation. Using fluorescence microscopy, we have observed that lucigenin accumulates at the mitochondria of alveolar macrophages and exhibits a localization similar to that of rhodamine 123, a fluor escent indicator of mitochondrial membrane potential. We have also com pared the effects of a spectrum of agents known to modulate mitochondr ial respiration on both LDCL and cellular oxygen consumption. The agen ts examined included a Complex I inhibitor, rotenone; a Complex III in hibitor, antimycin a; and a Complex IV inhibitor, KCN. While these com pounds all inhibited oxygen consumption, only those that exert an effe ct prior to (rotenone) or at (antimycin a) the point of mitochondrial superoxide formation inhibited LDCL. KCN exhibits effects that are con centration dependent. At low concentrations (1-100 mu M), a slight enh ancement of LDCL is observed, while at higher concentrations (1-10 mM) both LDCL and oxygen consumption are inhibited. The ATP synthase inhi bitor, oligomycin, was also observed to correspondingly inhibit oxygen consumption and LDCL. These inhibitor studies, taken together with th e observation that lucigenin localizes to the mitochondria of alveolar macrophages, provides strong evidence that LDCL can be used to qualit atively assess the modulation of mitochondrial superoxide generation i n mononuclear cells.