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, lucigenin-derived chemiluminescence (LDCL) has been widely used to assess the generation of superoxide by the NADPH oxidase of leukocytes. Previously, we suggested that lucigenin could interact with mitochondrial-derived superoxide in alveolar macrophages. The purpose of this study was to further demonstrate that LDCL is in fact a probe of mitochondrial superoxide 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 fluorescent indicator of mitochondrial membrane potential. We have also compared the effects of a spectrum of agents known to modulate mitochondrial respiration on both LDCL and cellular oxygen consumption. The agents examined included a Complex I inhibitor, rotenone; a Complex III inhibitor, antimycin a; and a Complex IV inhibitor, KCN. While these compound all inhibited oxygen consumption, only those that exert an effect prior to (rotenone) or at (antimycin a) the point of mitochondrial superoxide formation inhibited LDCL. KCN exhibits effects that are concetration dependent. At low concentrations (1–100 μM), a slight enhancement of LDCL is observed, while at higher concentrations (1–10 mM) both LDCL and oxygen consumption are inhibited. The ATP synthase inhibitor, oligomycin, was also observed to correspondingly inhibit oxygen consumption and LDCL. These inhibitor studies, taken together with the observation that lucigenin localizes to the mitochondria of alveolar macrophages, provides strong evidence that LDCL can be used to qualitatively asses the modulation of mitochondrial superoxide generation in mononuclear cells.