P32: Potential role for 8-Nitro-cGMP in nitrite-mediated hypoxic signaling

Abstract

Background Nitrite has been shown to elicit both acute and chronic nitric oxide (NO)-dependent effects, especially at lower oxygen tensions.In this paradigm, nitrite reduction by deoxyhemoglobin and deoxymyoglobin activates NO-signaling to mediate several functions including hypoxic vasodilation.The mechanism underlying this however remains unclear, with a major challenge being how to reconcile nitrite-derived NO-signaling in an NO-scavenging environment, considering the high heme concentrations (∼10 mM) and rapid reaction rates between heme and NO ( k ∼ 10 7 M −1 s −1 ).Recent studies also implicate the derivative of cGMP, 8-nitro-cGMP, as a novel second messenger in NO-dependent signaling.Both cGMP and 8-nitro-cGMP activates PKG, however, unlike cGMP, 8-nitro-cGMP is resistant to PDE5 degradation and has the potential to elicit NO-induced signal transduction via a new chemical modification of proteins referred to as S -guanylation.To date, nitrite-dependent NO formation and 8-nitro-cGMP-dependent activation of NO-signaling have been considered independent of each other.In this study, we proposed a link between these two signaling paradigms.We hypothesized that 8-nitro-cGMP is a critical mediator of nitrite-dependent activation of hypoxic NO-signaling and tested if nitrite promotes hypoxic NO-signaling via 8-nitro-cGMP. Methods First, 8-nitro-cGMP formation in rat aorta exposed to nitrite at high O 2 (21%) and low O 2 (1%) tensions was assessed by immunostaining.The role of 8-nitro-cGMP in nitrite-mediated vasodilation was assessed using ex vivo aortic vessel dilation as an index of nitrite-dependent NO-signaling at high O 2 (21%) and low O 2 (1%) tensions, with and without the PDE5 inhibitors, sildenafil or zaprinast. Results The highest intensity of staining for 8-nitro-cGMP was observed in the vessels treated with nitrite at low O 2 (1%) tension and in the presence of RBC, indicating a nitrite-induced formation of 8-nitro-cGMP under hypoxic conditions.Nitrite stimulated vasodilation at 1% O 2 tension in a dose-dependent manner and this response was significantly improved in the presence of RBCs.Treatment of the aortic vessel segments with the PDE5 inhibitors, sildenafil or zaprinast improved RBC-dependent nitrite-mediated vasodilation by ∼2-fold at 21% O 2 .However, PDE5 inhibition had no effect on nitrite-dependent vasodilation at 1% O 2. Moreover, NO-dependent vasodilation using MAHMA NONOate was sensitive to PDE5 inhibition at both 21% and 1% O 2 .These data suggest a hypoxia-dependent shift from a PDE5-sensitive to PDE5-insensitive mechanism for nitrite-dependent vasodilation. Conclusion Collectively, these data indicate the possible role for 8-nitro-cGMP in nitrite-mediated hypoxic signaling paradigms. Disclosure Nothing to disclose.