Cellular targets and mechanisms of nitros(yl)ation: an insight into their nature and kinetics in vivo

Abstract

There is mounting evidence that the established paradigm of nitric oxide (NO) biochemistry, from formation through NO synthases, over interaction with soluble guanylyl cyclase, to eventual disposal as nitrite/nitrate, represents only part of a richer chemistry through which NO elicits biological signaling. Additional pathways have been suggested that include interaction of NO-derived metabolites with thiols and metals to form S-nitrosothiols (RSNOs) and metal nitrosyls. Despite the overwhelming attention paid in this regard to RSNOs, little is known about the stability of these species, their significance outside the circulation, and whether other nitros(yl)ation products are of equal importance. We here show that N-nitrosation and heme-nitrosylation are indeed as ubiquitous as S-nitrosation in vivo and that the products of these reactions are constitutively present throughout the organ system. Our study further reveals that all NO-derived products are highly dynamic, have fairly short lifetimes, and are linked to tissue oxygenation and redox state. Experimental evidence further suggests that nitroso formation occurs substantially by means of oxidative nitrosylation rather than NO autoxidation, explaining why S-nitrosation can compete effectively with nitrosylation. Moreover, tissue nitrite can serve as a significant extravascular pool of NO during brief periods of hypoxia, and tissue nitrate/nitrite ratios can serve as indicators of the balance between local oxidative and nitrosative stress. These findings vastly expand our understanding of the fate of NO in vivo and provide a framework for further exploration of the significance of nitrosative events in redox sensing and signaling. The findings also raise the intriguing possibility that N-nitrosation is directly involved in the modulation of protein function.

Fig. 1.

Relative blood and tissue concentrations of nitrate and nitrite (Upper) and individual nitros(yl)ation products normalized to protein concentration (Lower) within each compartment. RSNOs and RNNOs are found in every compartment whereas NO-heme compounds are only found in a select few tissues. Data are means ± SEM of 10–14 animals.

Fig. 2.

Kinetic changes in the concentration of different nitros(yl)ation products, as well as nitrate/nitrite in blood and tissues after NOS inhibition by l-NIO. (A) Changes in RSNO concentration in plasma and RBCs. (B) Change in individulal nitros(yl)ation products in the brain. *, P < 0.05. (C) Fractional changes in nitrate/nitrite across all tissues and blood. (D) Fractional changes in nitros(yl)ation products across all tissues and blood. (E) Changes in nitrate/nitrite ratio in different tissues. Depicted data are the means from 2–3 animals per time point for NOS inhibition and 14 animals for the controls. *, P < 0.05.

Fig. 3.

Fractional decrease in RSNO (RSNO_final/RSNO_basal) vs. fractional increase in total ascorbate (Asc_basal/Asc_final) in different rat tissues after administration of ascorbate with the drinking water (10 g/liter for 7 days). The results show that the changes in RSNO content in the various organs are correlated to the changes in intracellular ascorbate concentrations.

Fig. 4.

Dynamics of NO-related products during global hypoxia. (A and B) Representative time courses obtained for different products and tissues (n = 3–5). (A) Changes in total nitrosation in select tissues. B focuses on changes in individual nitros(yl)ation products in the brain. Total nitrosation in the brain increases by 40–50% but reveals that RSNOs and NO-hemes account for the increase whereas RNNOs actually decrease, suggestive of a crosstalk between species. (C) Changes in concentration of NO-related products in the brain observed after 3 and 10 min of global hypoxia (means ± SEM; n = 3–5).

Scheme 1.

Biochemical pathways of NO–target interactions in vivo.