333 - Nitrite Inhibits Mitochondrial Phosphodiesterase and Activates CAMP-PKA-AKAP1 Signaling to Modulate Mitochondrial Function and Cytoprotection in Normoxia

Abstract

Nitrite is an established regulator of hypoxic mitochondrial function that inhibits oxidative phosphorylation and mitochondrial oxidant product, but its effects on mitochondrial function in normoxia remain unclear. We previously demonstrated that nitrite activates protein kinase A (PKA) in normoxic cardiomyocytes and heart tissue and this activity is linked to cytoprotection after a future ischemic episode. However, the mechanism by which nitrite activates PKA and the effect of this activation on physiological mitochondrial function remains unexplored. Here, we show for the first time that nitrite inhibits the activity of the mitochondrially localized phosphodiesterase 2A (PDE2A), which increases mitochondrial cAMP levels (21% compared to control), leading to PKA activation. Additionally, nitrite increases the expression of A-kinase anchoring protein (AKAP1), which tethers PKA to the mitochondrial membrane. Consistent with the mitochondrial targeting of PKA, we show that nitrite induces the phosphorylation of Ser58 on mitochondrial complex IV-1 (a known PKA target) and increases its activity by 86%, leading to augmented basal and maximal respiration (48% and 29%, respectively compared to control). These data demonstrate that nitrite is a versatile signaling molecule that modulates not only nitrosative protein modification but also through the stimulation of phosphorylation. Further, these data show a novel mechanism by which nitrite selectively modulates mitochondrial PKA-dependent signaling through the inhibition of PDE. The implications of this pathway on metabolism and cytoprotection after ischemia will be discussed.