A Chemical Characterisation Of Hydrogen Sulfide-Mediated Production Of Nitric Oxide From Nitrite And S-nitrosoalbumin

Maria Letizia Lo Faro1, John More2, Paul Winyard1, Matthew Whiteman1. 1Peninsula Medical School, University of Exeter, Exeter, UK, 2BioProducts Laboratory, Elstree, UK.

Until recently nitrite (NO2-) has been considered just an inert metabolite of nitric oxide (·NO). In fact it is an important biological reservoir of ·NO. NO2- alters hypoxic signalling and vasodilation, and protects the vasculature after ischaemia-reperfusion injury.

S-nitrosothiols (RSNOs), another example of ·NO-derived species, have similarly gained great attention for their vasodilatory properties and vascular protective effects after ischaemic events. The mechanism suggested to explain both NO2-- and RSNO-mediated effects is ·NO production. NO2- can be reduced to ·NO at low pH and low O2 tension by xanthine oxidase, deoxyhaemoglobin, mitochondrial complexes and ascorbate.

Hydrogen sulfide (H2S) is a recently discovered gasotransmitter which exerts many physiological effects similar to those of ·NO (e.g. vasodilation, promotion or inhibition of inflammation etc…)

The aim of this work was to test whether H2S could promote ·NO production from NO2- and RSNOs. We also examined whether H2S was able to interact with NO2- and RSNOs to affect cGMP production from human aortic smooth muscle cells (HASMC) and protect endothelial cells from oxidative stress-induced cytotoxicity.

·NO production from NO2- and S-nitrosoalbumin (SNOA) was chemically characterised by electron paramagnetic resonance (EPR) spin trapping and the spin trap used for the experiments was the Fe2+ complex of N-methyl-D-glucamine dithiocarbamate (MGD). ·NO was also assessed by gas phase ozone-based chemiluminescence. In the EPR experiments H2S was generated using the sulfide salt NaSH. On addition of H2S to NO2-, in the presence of the spin trap, a triplet EPR signal attributable to the NO-Fe2+-(MGD)2 spin adduct was detected. Peak integration of the EPR spectra of spin-trapped •NO showed that 5 min incubation (at room temperature) of 1 mM NO2- with 1 mM NaSH increased •NO formation compared to NO2- alone, with increases of 25.8±7.7% (mean± SEM) and 22.7±7.0% at pH 4 and pH 7.4, respectively (n = 4) (final ·NO concentrations in the presence of NaSH were 579 ± 67 µM and 6.5 ± 1.9 µM at pH 4 and 7.4 respectively) . Chemiluminescence confirmed that H2S rapidly reduced NO2- directly to ·NO, at pH 7.4 and that the reaction was thiol-dependent. Chemiluminescence also showed that, in the presence of NaSH, RSNO levels in SNOA significantly decreased (n = 3, p<0.01, data analysed by 2-way ANOVA) almost as soon as the reagents were mixed together.

This work sets the basis to assess whether H2S mediated-reduction of NO2- and RSNOs can be considered a physiologically relevant alternative pathway for ·NO production.