Oxygen dependence of nitric oxide-mediated signaling

Abstract

Nitric oxide (•NO) is a biologically important short-lived free radical signaling molecule. Both the enzymatic synthesis and the predominant forms of cellular metabolism of •NO are oxygen-dependent. For these reasons, changes in local oxygen concentrations can have a profound influence on steady-state •NO concentrations. Many proteins are regulated by •NO in a concentration-dependent manner, but their responses are elicited at different thresholds. Using soluble guanylyl cyclase (sGC) and p53 as model •NO-sensitive proteins, we demonstrate that their concentration-dependent responses to •NO are a function of the O2 concentration. p53 requires relatively high steady-state •NO concentrations (>600 nM) to induce its phosphorylation (P-ser-15), whereas sGC responds to low •NO concentrations (<100 nM). At a constant rate of •NO production (liberation from •NO-donors), decreasing the O2 concentration (1%) lowers the rate of •NO metabolism. This raises steady-state •NO concentrations and allows p53 activation at lower doses of the •NO donor. Enzymatic •NO production, however, requires O2 as a substrate such that decreasing the O2 concentration below the K m for O2 for nitric oxide synthase (NOS) will decrease the production of •NO. We demonstrate that the amount of •NO produced by RAW 264.7 macrophages is a function of the O2 concentration. Differences in rates of •NO production and •NO metabolism result in differential sGC activation that is not linear with respect to O2. There is an optimal O2 concentration (≈5-8%) where a balance between the synthesis and metabolism of •NO is established such that both the •NO concentration and sGC activation are maximal.

Keywords: Autooxidation; BH4, tetrahydrobiopterin; DETA/NO, (Z)-1-[N-(2-aminoethyl)–N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate; FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide; Km, Michaelis constant; LPS, lipopolysaccharide; NADPH, nicotinamide adenine dinucleotide phosphate, reduced; NO2−, nitrite; NO3−, nitrate; Nitric oxide; Nitric oxide synthase; O2, oxygen; ODQ, 1H-[1,2,4]Oxadiazolo[4,3–a]quinoxalin-1-one; Oxygen; P-Ser-15, phospho-serine 15; Sper/NO, (Z)-1-[N-[3–aminopropyl]–N-[4-(3-aminopropylammonio)butyl]-amino]diazen-1-ium-1,2-diolate; cGMP, cyclic guanosine monophosphate; eNOS, endothelial nitric oxide synthase; iNOS, inducible nitric oxide synthase; nNOS, neuronal nitric oxide synthase; p53; sGC; sGC, soluble guanylyl cyclase; •NO, nitric oxide.

Graphical abstract

Fig. 1

Signaling effects of •NO are a function of its steady-state concentration. MCF-7 cells were treated with increasing concentrations of Sper/NO (0–100 μM) at 21% O₂. (A) Measurement of [•NO]_ss in media (•NO selective electrode, ∼1 mm above monolayer). (B) Immunoblot demonstrating p53 P-Ser-15 accumulation (t=2 h). (C) Measurement of cGMP production as an indicator of sGC activation (t=30 min). n=3, ^⁎⁎ indicates p<0.01 with respect to untreated controls which are arbitrarily set to 1.0.

Fig. 2

Oxygen concentration influences •NO consumptive process. (A) MCF-7 cells were placed in suspension and stirred in a reaction vessel at 37 °C with an •NO-selective electrode. •NO metabolism (disappearance) was measured after the additions of bolus amounts of an •NO-saturated stock solution at 21% O₂ or 1% O₂. (B) Measurement of [•NO]_ss in the media of cells treated with 500 μM of the •NO-donor DETA/NO (•NO-selective electrode, ∼1 mm above monolayer) at 21% O₂ or 1% O₂. (C,D) Calculated half-life of •NO via autooxidation from 50–1000 nM starting concentrations at 1% and 21% O₂. (E) Disappearance of 200 nM •NO at 21% O₂ in the presence or absence of cells.

Fig. 3

Low O₂ decreases the dose of •NO-donor compound required for p53 activation. MCF-7 cells were treated with increasing concentrations of Sper/NO (0–100 μM) at 21% O₂ or 1% O₂ for 2 h. Immunoblot demonstrating accumulation of p53-Ser-15 phosphorylation. n=3, ^⁎⁎indicates p<0.01 with respect to untreated controls which are arbitrarily set to 1.0.

Fig. 4

The rate of •NO synthesis is a function of both the NOS and O₂ concentrations. RAW 264.7 cells were cultured at different O₂ concentrations and stimulated with LPS (t=0 h). The rate of •NO synthesis was measured by quantifying NO₂⁻/NO₃⁻ accumulation in the media by chemiluminescence. (A) Contour plot demonstrating the temporal relationship between changing O₂ concentrations and rates of •NO synthesis. (B) Immunoblot demonstrating increased iNOS expression as a function of decreasing O₂ concentrations (t=10 h). (C) (columns 1, 2, 4, & 5) Cells were grown at 21% O₂ or 1% O₂ and NO₂⁻/NO₃⁻ was measured at 16 h and 24 h. (columns 3 & 6) Cells were grown at 21% O₂ or 1% O₂. After 16 h the cells at 21% O₂ were moved to 1% O₂ and the cells at 1% O₂ were moved to 21% O₂. Both sets of cells were incubated for an additional eight hours (24 h total) and NO₂⁻/NO₃⁻ was measured. Values given at 24 h are the addition of the 16 h and additional 8 h reads. n=3, ^⁎ indicates 0.01<p<0.05; ^⁎⁎indicates p<0.01 with respect to untreated controls which are arbitrarily set to 1.0.

Fig. 5

Signaling effects of endogenously produced •NO are affected by O₂ concentration. (A) Activated RAW 264.7 cells were cocultured in serum free media with MCF-7 cells at 21% O₂. Coculture with unactivated RAW cells; activated RAW cells±the iNOS inhibitor aminoguanidine (AG); or the sGC inhibitor ODQ. After 30 min the RAW 264.7 cells were removed and cGMP production was measured in the MCF-7 cells by ELISA. (B) Increasing amounts of activated RAW 264.7 cells were cocultured in serum free media with MCF-7 cells over a range of O₂ concentrations (0.5–21%). After 30 min the RAW 264.7 cells were removed and cGMP production was measured in the MCF-7 cells by ELISA. Contour plot demonstrating the relationship between cGMP production, O₂ concentration, and different densities of •NO-producing RAW 264.7 cells.