OP21 Role of phosphodiesterase inhibition and modulation of mitochondrial cAMP levels in the bioenergetic effect of hydrogen sulfide in isolated mitochondria

Abstract

Introduction Several lines of studies demonstrate the biphasic bioenergetic effect of hydrogen sulfide (H 2 S): at lower concentrations, endogenously produced and exogenously administered H 2 S stimulates mitochondrial electron transport and supports oxidative phosphorylation, whereas at higher concentration, H 2 S inhibits mitochondrial respiration by inhibiting mitochondrial Complex IV (cytochrome C oxidase).In addition, it has recently been demonstrated that in the mitochondrial matrix there is a distinct cAMP pool, which is not related to the cytosolic cAMP pool.Furthermore, a soluble adenylyl cyclase enzyme (sAC) has been localized to the mitochondrial matrix, which is involved in the maintenance of the intra-mitochondrial cAMP levels.Given the recently emerging evidence that (a) H 2 S is an endogenous inhibitor of cAMP and cGMP phosphodiesterases and that (b) intramitochondrial cAMP and PKA act an endogenous stimulators of mitochondrial electron transport and oxidative phosphorylation, the aim of the current study was to explore the potential role of mitochondrial cAMP and its downstream effector, protein kinase A (PKA) in the stimulatory bioenergetic effect of H 2 S in vitro . Methods The presence of PDE2A (a combined cAMP- and cGMP-degrading phosphodiesterase) and of PKA was investigated by western blotting of native isolated rat liver mitochondria, as well as its partially digested forms to yield mitoplasts and post-mitoplasts.In addition, freshly isolated rat liver mitochondria were subjected to H 2 S donor NaHS and analyzed by Extracellular Flux Analysis (Seahorse) in the absence or presence of various modulators of mitochondrial cAMP/PKA, followed by the measurement of oxygen consumption rate in the absence or presence of ADP, as well as a sequential administration of specific modulators (the ATP synthase inhibitor oligomycin, the mitochondrial uncoupler FCCP and the Complex III inhibitor antimycin) of mitochondrial electron transport chain complexes.The effect of the H 2 S donor NaHS on the activity of recombinant PDEA2 (an enzyme involved in the degradation of cAMP) was measured in a cell-free assay. Results Western blot analysis confirmed the presence of PDE2A (a combined cAMP- and cGMP-degrading phosphodiesterase) and of PKA in the mitochondrial preparations.Addition of the membrane-permeable cAMP analog 8-bromo-cAMP or the H 2 S donor NaHS to isolated rat liver mitochondria both increased oxygen consumption rate (OCR) and ATP turnover, as well as the maximal mitochondrial respiration elicited by the uncoupling agent FCCP. The positive bioenergetic effects of 8-bromo-cAMP and of H 2 S were both attenuated by the selective PKA inhibitor Rp-cAMP. H 2 S exerted a concentration-dependent inhibitory effect on the activity of recombinant PDE2A by H 2 S in vitro . Conclusions The current data indicate that H 2 S, in addition to acting as a direct electron donor to the mitochondria, stimulates mitochondrial electron transport and cellular bioenergetic effects by activating a secondary pathway that involves inhibition of phosphodiesterase activity, enhancement of cAMP levels and stimulation of mitochondrial PKA. Based on recent publications, the likely mode of the enhancement of mitochondrial electron transport by PKA may involve the direct phosphorylation of specific electron transport chain components.