Nitrite Differently Activates cGMP versus cAMP

Nitrite is an established signaling molecule that regulates mitochondrial function in hypoxia by inhibiting respiration and mitochondrial oxidant production, but its effects on mitochondrial function in normoxia remain unclear. We hypothesize that nitrite mediates differential signaling in hypoxia and normoxia leading to modulation of mitochondrial function. Here, we demonstrate that nitrite switches from stimulation of soluble guanylate cyclase-dependent cGMP generation in hypoxia to cAMP production in normoxic conditions. Nitrite (unlike nitric oxide) increases cAMP levels in H9C2 cardiomyocytes and in isolated mitochondria in normoxia leading to protein kinase A (PKA) activation outside and inside the mitochondria. This increase in cAMP levels is due to nitrite-dependent inhibition of phosphodiesterases (PDE2A and PDE4A), which degrades cAMP. In addition to increasing PKA activation, we observed that 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). Pharmacological inhibition of PKA attenuates nitrite-dependent increase in respiration, complex IV activity and phosphorylation. These data demonstrate that nitrite is a unique signaling molecule able to increase cAMP levels in normoxia and show a novel mechanism by which nitrite selectively modulates mitochondrial PKA-dependent signaling. Further, these data show that nitrite is a versatile signaling molecule that not only modulates nitrogen dependent post-translational modification, but also modulates phosphorylation.