Abstract. As a part of the nitroso signalling pathway, nitroso-compounds serve as stores and carriers of NO; as part of the sulphide signalling pathway, bound sulfane-sulphur compounds serve as stores and carriers of H 2 S. Here we hypothesise a coupled sulphide-nitroso signalling pathway, in which H 2 S plays a main role. H 2 S releases NO from the endogenous S-nitroso-compounds nitrosocysteine, nitroso-acetylcysteine and nitroso-albumin. Relaxation of noradrenaline-precontracted aortic rings by H 2 S is also enhanced in the presence of nitroso-albumin, which may implicate the involvement of the nitroso signalling pathway. Pretreatment of albumin, cysteine, N-acetylcysteine and lipids with H 2 S results in binding of sulphur to these compounds creating thus new-modified sulphur compounds that release NO from nitroso-compounds directly and/or through released H 2 S, which suggests sulphide-nitroso signalling pathway participation. This hypothesis is supported by the observation that the pretreatment of noradrenaline-precontracted aortic rings with H 2 S significantly enhanced relaxation induced by nitroso-glutathione in the absence of H 2 S. We assume that the NO release from nitroso-compounds directly by H 2 S or indirectly by the H 2 S-induced sulphur-bound compounds represents coupled sulphide-nitroso signalling, which may explain some of the numerous biological effects of H 2 S that are shared with NO.

Abstract.We studied the involvement of O 2 , pH and low molecular thiols in H 2 S-induced decomposition of S-nitrosoglutathione (GSNO). The GSNO decomposition -• NO release was evaluated by UV-VIS spectroscopy and Griess assay. The H 2 S donor Na 2 S was used. O 2 slightly increased, but was not necessary for the H 2 S-induced GSNO decomposition. The rate of GSNO decomposition depended on pH; the maximum rate was observed at pH 7.4-8.0, and this decreased with lowering pH (6.4-4.5) as well as with increasing pH at 9.0-12.0. H 2 S-induced GSNO decomposition was slowed by the presence of other thiols, such as L-cysteine (Cys), N-acetyl-L-cysteine (NAC) and Lglutathione (GSH), but not in the presence of L-methionine (Met) or oxidized glutathione (GSSG). In sharp contrast, at pH 6.0, H 2 S-induced GSNO decomposition was negligible, yet the presence of Cys, NAC and GSH induced the H 2 S-driven GSNO decomposition (whilst Met and GSSG were inactive). In conclusion we postulate an involvement of low molecular thiols and pH in• NO signaling, by modulating the interactions of H 2 S with nitroso compounds, and hence in part they also appear to control H 2 S-triggered• NO release. The interaction of H 2 S and/or its derivatives with the thiol group may be responsible for the observed effects.

Variants of L1210 leukemia cells-namely, parental P-glycoprotein-negative S cells and R and T cells expressing P-glycoprotein, due to selection with vincristine and transfection with the human p-glycoprotein gene, respectively-were used. The responses of these cell variants to two naturally occurring isothiocyanates-sulforaphane (SFN, from cruciferous vegetables) and allyl isothiocyanate (AITC, from mustard, radish, horseradish and wasabi)-were studied. We obtained conflicting results for the cell death effects induced by isothiocyanates, as measured by i. cell counting, which showed inhibited proliferation, and ii. cell metabolic activity via an MTS assay, which showed an increased MTS signal. These results indicated the hyperactivation of cell metabolism induced by treatment with isothiocyanates. In more detailed study, we found that, depending on the cell variants and the isothiocyanate used in treatment, apoptosis and necrosis (detected by annexin-V cells and propidium iodide staining), as well as autophagy (detected with monodansylcadaverine), were involved in cell death. We also determined the cell levels/expression of Bcl-2 and Bax as representative anti- and pro-apoptotic proteins of the Bcl-2 family, the cell levels/expression of members of the canonical and noncanonical NF-κB pathways, and the cell levels of 16 and 18 kDa fragments of LC3B protein as markers of autophagy.

Garlic, onion and leek have beneficial effects in treatment of numerous health disorders. The aim of the present study was to investigate underlying molecular mechanisms. To test the potency of the aqueous garlic, onion and leek extracts to release NO from GSNO we have measured NO oxidation product, NO(2)-, by the Griess reagent method. Further, we studied the ability of garlic extract to relax noradrenaline-precontracted rat aortic rings in the presence of GSNO and effects of garlic extract on electrical properties of rat heart intracellular chloride channels. We have observed that: i) garlic, onion and leek extracts released NO from GSNO in the order: garlic > onion > leek; ii) the ability of garlic extract to release NO was pH-dependent (8.0 > 7.4 > 6.0) and potentiated by thiols (Cys >> GSH = N-acetyl-cysteine > oxidized glutathione) at concentration 100 µmol/l; iii) the garlic extract (0.045 mg/ml) prolonged relaxation time of aortic rings induced by GSNO (50 nmol/l) and inhibited intracellular chloride channels. We suggest that NO-releasing properties of the garlic, onion and leek extracts and their interaction with Cys and GSH are involved in NO-signalling pathway which contributes to some of its numerous beneficial biological effects.