P25 Exogenous and endogenous H2S stimulates mitochondrial electron transport and cellular bioenergetics in hepatocytes

Background In the field of environmental toxicology, H 2 S is viewed as a mitochondrial toxin: an inhibitor of mitochondrial electron transport by inhibiting cytochrome C oxidase (Complex IV).In bacteria and multicellular organisms living in oxygen-deprived environments, however, H 2 S can function as an electron donor and a source of energy. Recently, several studies suggested that this role of H 2 S as an electron donor and an inorganic source of energy may also exist in mammalian cells. The goal of our study was to define the relative role of these two processes in hepatic mitochondria and in hepatocytes. Methods Using Extracellular Flux Analysis technique (Seahorse XF24, a technique which measures oxygen consumption in a micro-chamber), in isolated rat liver mitochondria and cultured murine hepatocytes, we investigated the relative role of H 2 S and the Krebs-cycle-derived metabolite succinate in the regulation of mitochondrial electron transport and oxidative phosphorylation. In addition, we investigated the effect of the H 2 S precursor l -cysteine on mitochondrial H 2 S production, and on mitochondrial and cellular bioenergetics. H 2 S production from isolated mitochondria was measured by the methylene blue method. Results Succinate, at concentrations between 5 and 20 mM, induced a dose-dependent increase in mitochondrial electron transport. H 2 S, on its own (in the presence of ADP, but in the absence of succinate) was unable to stimulate mitochondrial electron transport. On the other hand, in the presence of 5 or 10 mM succinate (which partially stimulates mitochondrial electron transport), low concentrations of H 2 S (0.1–1 μM) elicited a significant increase in mitochondrial activity. Higher concentrations of H 2 S (3–30 μM) suppressed mitochondrial activity. In rat liver mitochondria, as well as in cultured hepatocytes, the H 2 S precursor l -cysteine also induced a similar biphasic effect on energetics: at lower concentrations (10–30 μM) it increased mitochondrial reserve capacity and oxygen consumption rate, while at higher concentrations (100–1000 μM) it became inhibitory. The l -cysteine-induced production of H 2 S in isolated mitochondria was confirmed by the methylene blue method. Conclusion H 2 S and l -cysteine appear to have a physiological role as a positive modulator of mitochondrial electron transport and its actions are dependent on a basal activity of the Krebs cycle. We hypothesize that both Krebs-cycle-derived electron donors and exogenously administered or endogenously produced H 2 S, as an electron donor, converge at Coenzyme Q. At higher concentrations of H 2 S or l -cysteine the inhibitory effect on Complex IV becomes predominant and mitochondrial function becomes suppressed; this finding is consistent with the inhibitory effect of H 2 S on Complex IV. The fact that the bioenergetic effects of H 2 S are most pronounced at intermediate concentrations of succinate may suggest that the bioenergetic effects of H 2 S may be most important a secondary/back-up/emergency ’fuel’ in cells where bioenergetic function is slightly impaired. The current result may help explain some of the biphasic or opposing biological effects of hydrogen sulfide on cell viability in physiology and pathophysiology.