Regulation of superoxide flashes by transient and steady mitochondrial calcium elevations

The mitochondria play essential roles in both intracellular calcium and reactive oxygen species signaling. As a newly discovered universal and fundamental mitochondrial phenomenon, superoxide flashes reflect transient bursts of superoxide production in the matrix of single mitochondria. Whether and how the superoxide flash activity is regulated by mitochondrial calcium remain largely unknown. Here we demonstrate that elevating mitochondrial calcium either by the calcium ionophore ionomycin or by increasing the bathing calcium in permeabilized HeLa cells increases superoxide flash incidence, and inhibition of the mitochondrial calcium uniporter activity abolishes the flash response. Quantitatively, the superoxide flash incidence is correlated to the steady-state mitochondrial calcium elevation with 1.7-fold increase per 1.0 ΔF/F 0 of Rhod-2 signal. In contrast, large mitochondrial calcium transients (e.g., peak ΔF/F 0 ∼ 2.8, duration ∼ 2 min) in the absence of steady-state elevations failed to alter the flash activity. These results indicate that physiological levels of sustained, but not transient, mitochondrial calcium elevation acts as a potent regulator of superoxide flashes, but its mechanism of action likely involves a multi-step, slow-onset process.