Key bioactive reaction products of the NO/H2S interaction are S/N-hybrid species, polysulfides, and nitroxyl

Abstract

Experimental evidence suggests that nitric oxide (NO) and hydrogen sulfide (H2S) signaling pathways are intimately intertwined, with mutual attenuation or potentiation of biological responses in the cardiovascular system and elsewhere. The chemical basis of this interaction is elusive. Moreover, polysulfides recently emerged as potential mediators of H2S/sulfide signaling, but their biosynthesis and relationship to NO remain enigmatic. We sought to characterize the nature, chemical biology, and bioactivity of key reaction products formed in the NO/sulfide system. At physiological pH, we find that NO and sulfide form a network of cascading chemical reactions that generate radical intermediates as well as anionic and uncharged solutes, with accumulation of three major products: nitrosopersulfide (SSNO(-)), polysulfides, and dinitrososulfite [N-nitrosohydroxylamine-N-sulfonate (SULFI/NO)], each with a distinct chemical biology and in vitro and in vivo bioactivity. SSNO(-) is resistant to thiols and cyanolysis, efficiently donates both sulfane sulfur and NO, and potently lowers blood pressure. Polysulfides are both intermediates and products of SSNO(-) synthesis/decomposition, and they also decrease blood pressure and enhance arterial compliance. SULFI/NO is a weak combined NO/nitroxyl donor that releases mainly N2O on decomposition; although it affects blood pressure only mildly, it markedly increases cardiac contractility, and formation of its precursor sulfite likely contributes to NO scavenging. Our results unveil an unexpectedly rich network of coupled chemical reactions between NO and H2S/sulfide, suggesting that the bioactivity of either transmitter is governed by concomitant formation of polysulfides and anionic S/N-hybrid species. This conceptual framework would seem to offer ample opportunities for the modulation of fundamental biological processes governed by redox switching and sulfur trafficking.

Keywords: gasotransmitter; nitric oxide; nitroxyl; redox; sulfide.

Fig. 1.

Sulfide affects NO bioavailability in vivo and in vitro. (A and B) Continuous i.v. infusion of sodium hydrosulfide (2.8 µmol/kg per min NaHS in PBS, pH 7.4) progressively decreases blood pressure (BP) in rats. (A) Original recording depicting progressive decrease in BP during ongoing sulfide infusion; incisions (arrows) are caused by interruption of pressure recording during blood collection. (B) Changes in mean arterial blood pressure (MAP; n = 5; ANOVA P = 0.0256) and (Inset) heart rate (HR). *Dunnett's P < 0.05 vs. baseline. (C) Gradual increases in circulating nitroso species (RXNO) levels in RBCs (n = 3; ANOVA P = 0.0032). *Dunnett's P < 0.05 vs. baseline. (D) Concomitant transient decrease followed by an increase in NO-heme levels during continuous sulfide infusion (2.8 µmol/kg per min NaHS in PBS, pH 7.4; n = 3; ANOVA P = 0.0126). *Tuckey P < 0.05 vs. baseline. (E) Sulfide (10 µM Na₂S) decreases Sper/NO (100 µM)-mediated sGC activation in RFL-6 cells pretreated with the phosophdiesterase inhibitor 3-isobutyl-1-metylxanthine (IBMX). The scheme represents the experimental setup (n = 6; ANOVA P < 0.001). CTRL, control. *Tuckey P < 0.01 vs. untreated. # t test P < 0.05 (F) Equimolar concentrations of sulfide (33.4 µM) scavenge NO released form NO donors (33.4 µM DETA/NO) as assessed by time-resolved chemiluminescence detection under both aerated and (Inset) deaerated conditions, whereas excess sulfide (334 µM) transiently elevates NO release (representative of n = 3 independent experiments); DETA/NO, diethylenetriamine NONOate.

Fig. 2.

The reaction of NO with sulfide leads to formation of three major products, which we assign to be SSNO⁻ (λ_max = 412 nm), HS_n⁻ (λ_max = 290–300 nm), and SULFI/NO (λ_max = 259 nm). (A) Reaction of aqueous solutions of NO (200 µM) with sulfide (2 mM) under deaerated conditions in buffer at pH 7.4 leads to formation of a peak with λ_max = 412 nm (SSNO⁻) and increases in absorbance at λ_max < 300 nm (HS_n⁻). Products with λ_max < 250 nm are not discernable from sulfide because of the high concentration of HS⁻ (λ_max = 230 nm) in these experiments; spectra were taken at the reaction start (blue) and every 5 s after the addition of NO. (Inset) kinetics of SSNO⁻ formation. (B) Reaction of the NO donor DEA/NO (1 mM) with sulfide (10 mM) under aerated conditions forms SSNO⁻ and HS_n⁻. The blue line indicates the spectrum before the addition of sulfide. (Inset) Kinetics of formation of SSNO⁻. (C and D) The yield of SSNO⁻ formation depends on both (C) sulfide concentration and (D) the rate of NO release; all spectra were taken 10 min after the start of the reaction. (E) SSNO⁻ (λ_max = 412 nm), HS_n⁻ (λ_max = 290–300 nm), and SULFI/NO (λ_max = 259 nm) are formed in the reaction of sulfide with the S-nitrosothiol SNAP (1 mM SNAP + 10 mM Na₂S; 10 min); SULFI/NO is detectable after removal of sulfide by gassing with N₂ for 10 min. (F) Addition of HS_n⁻ (12.5–200 µM) increases the rate of formation of SSNO⁻ from the reaction of SNAP (200 µM) and sulfide (2 mM); the induction period observed at no/low added HS_n⁻ points to an autocatalytic effect of HS_n⁻ (n = 3). All spectra in A–E are representative of 3–10 independent experiments. Au, arbitrary unit; DEA/NO, dietylamine/NONOate; SNAP, S-nitroso-N-acetyl-penicillamine.

Fig. 3.

Identification by ESI-HRMS of SULFI/NO and SSNO⁻ as S/N-hybrid species formed by the reaction of sulfide with DEA/NO or SNAP. (A) Formation of SSNO⁻ from DEA/NO (1 mM)/sulfide (2 mM) and SNAP (1 mM)/sulfide (2 mM) incubates. (Left) Mass and (Center) fragmentation spectra of SSNO⁻ (compound 1) from DEA-NO-sulfide incubates; (Right) shift in m/z of SSNO⁻ using an equimolar mixture of ¹⁴N/¹⁵N-labeled SNAP with sulfide. (B) Formation of SULFI/NO (compound 2) from DEA/NO (1 mM)/sulfide (2 mM) and SNAP (1 mM)/sulfide (2 mM) incubates. (Left) Mass and (Center) fragmentation spectra of SULFI/NO; (Right) m/z shifts of one and two by reacting an equimolar mixture of ¹⁵N/¹⁴N-SNAP (1 mM) with sulfide (2 mM). (C) Extracted ion chromatograms showing SNAP consumption accompanied by formation of SULFI/NO and SSNO⁻ together with polysulfides (compound 3; n = 2–7), including monoprotonated tri-, tetra-, and pentasulfide (HS₃⁻, HS₄⁻, and HS₅⁻, respectively), sulfite (HSO₃⁻), sulfate (HSO₄⁻), and thiosulfate (HS₂O₃⁻). SI Appendix, Table S7 has details on predicted molecular masses. A.U., arbitrary unit; DEA/NO, dietylamine NONOate; SNAP, S-nitroso-N-acetyl-penicillamine; m/z, mass-to-charge ratio.

Fig. 4.

NO and HNO bioactivity of SSNO⁻ and SULFI/NO in vitro. The scheme shows that the release of NO and HNO from SSNO⁻ and SULFI/NO leads to activation of sGC in cells. (A) Kinetics of NO release from SSNO⁻ after incubation of SNAP (0.1 or 1 mM) and Na₂S (1 or 10 mM) for 1 min as determined by chemiluminescence (final dilution of 1:100). (Inset) NO release from authentic SULFI/NO (100 µM) in the absence of sulfide. (B) Release of nitroxyl (HNO) as assessed by P-Rhod fluorescence from increasing concentrations of SULFI/NO (blue) and SSNO⁻ (orange; 1 mM SNAP, 10 mM Na₂S; gassed). ΔFI, fluorescence intensity-background. (C) The reaction of DEA/NO (10–200 µM) with sulfide (100 µM) generates N₂O over prolonged periods of time. (D) HNO scavenging by triphenylphosphine reveals that part of the N₂O formed during the DEA/NO/sulfide reaction originates from HNO dimerization/dehydration. ***Dunnett’s P < 0.001. (E) SSNO⁻ (20 µM) activates sGC in RFL-6 cells in both the presence and the absence of SOD, whereas equivalent concentrations of SULFI/NO (10 µM) activate sGC only in the presence of SOD after conversion of HNO into NO (n = 6–12; one-way ANOVA, P < 0.001). **Dunnett’s P < 0.01; ***Dunnett’s P < 0.001; ^§t test vs. untreated P < 0.001 (paired t test P = 0.0056); ^#P < 0.01 vs. 10 µM SULFI/NO without SOD. n.s., nonsignificant. (F) Higher concentrations of SULFI/NO (100 µM) activate sGC even in the absence of added SOD, an effect that is abolished by the NO scavenger cPTIO (500 µM) and the HNO scavenger Cys (1 mM; n = 5–10; one-way ANOVA, P < 0.001; F = 27.14). *Sidak’s P < 0.05 vs. CTRL; ***Sidak’s P < 0.001 vs. CTRL; ^#P < 0.01 vs. 100 µM SULFI/NO; ^##P < 0.001 vs. 100 µM SULFI/NO. cPTIO, 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide; CTRL, control; Cys, cysteine; DEA/NO, diethylamine NONOate; IBMX, 3-isobutyl-1-metylxanthine; SOD, superoxide dismutase; TXPTS, triarylphosphine.

Fig. 5.

SSNO⁻ and SULFI/NO show distinct bioactivity in rats in vivo. (A) i.v. Injection of the SSNO⁻ mix (dose range of 0.03–3 µmol/kg) leads to significant dose-dependent decreases of mean arterial pressure (MAP), whereas only the highest dose of SULFI/NO (3 µmol/kg) decreases MAP (n = 3 per group; RM two-way ANOVA, P < 0.01 for treatment and doses). *Sidak’s P < 0.01; **Sidak’s P < 0.01. At comparable doses, NaHS (1.8 and 3.5 µmol/kg) does not significantly affect blood pressure. (B) The SSNO⁻ mix (dose range of 0.03–3 µmol/kg) does not affect cardiac function, whereas higher doses of SULFI/NO (3 µmol/kg) increase cardiac contractility, which was assessed by changes in velocity time index (VTI), cardiac output (CO), and peak flow velocity (PFV; n = 3 per group; RM two-way ANOVA, P < 0.01) (SI Appendix, Tables S3–S5). (Inset) NaHS (dose range of 1.8–18 µmol/kg) does not affect VTI. ^#t Test P < 0.05. (C) Continuous infusion of the SSNO⁻ mix (0.16 µmol/kg per min) significantly decreases MAP compared with baseline, and its effect is more rapid in onset compared with that of NaHS (2.9 µmol/kg per min). Infusion of SULFI/NO equimolar to SSNO⁻ (0.16 µmol/kg per min) has only a mild, nonsignificant effect on blood pressure compared with vehicle control (SI Appendix, Table S6) (n = 3 per group; RM two-way ANOVA treatments, P = 0.00471). *Sidak’s P < 0.05 vs. baseline; **Sidak’s P < 0.01 vs. baseline; ^§P < 0.01 vs. NaHS. (D) Continuous infusion of SSNO⁻ mix (0.16 µmol/kg per minute) increases cardiac contractility (VTI, CO, and PFV) after 60 and 120 min of infusion (n = 3), whereas NaHS (2.9 µmol/kg per minute; shown in Inset) does not have any effect. SULFI/NO markedly increases cardiac contractility already 10 min after the start of infusion (n = 3; RM two-way ANOVA, P < 0.0001). *Sidak’s P < 0.05 vs. vehicle; **Sidak’s P < 0.01 vs. vehicle; ***Sidak’s P < 0.001 vs. vehicle; ^#two-way ANOVA SULFI/NO vs. SSNO, P = 0.0046. RM, repeated measurements.

Fig. 6.

Chemical reaction cascade depicting pathways of formation and decomposition of SSNO⁻, HS_n⁻, and SULFI/NO. Reactions are not mass balanced; numbers in red refer to the reactions described in the text. A more detailed discussion of the reaction mechanisms can be found in SI Appendix. The pK_a values of HS• and HSS• are unknown but likely exceed 7; therefore, at pH 7.4, these species are radical anions.