- Email: [email protected]
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Abstracts / Nitric Oxide 39 (2014) S16–S49
RIN14B cells, which was also inhibited by HC030031 and DTT. These results indicates that H2S has both inhibitory and excitatory effects via opening KATP and TRPA1 channels, respectively, suggesting the possibility of bidirectional modulation by H2S in secretory cells.
not proceed further. However, under hypoxic conditions the reaction between H2S and 8-NO2-cGMP goes to completion and yields the amino derivative 8-NH2-cGMP, a reaction which may be relevant to the first step in the cellular recycling of 8-NO2-cGMP into cGMP.
http://dx.doi.org/10.1016/j.niox.2014.03.122
http://dx.doi.org/10.1016/j.niox.2014.03.124
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Spontaneous oxidation of hydrogen sulfide to persulfide (s) allows signaling via sulfhydration
H2S induced S-nitrosoglutathione decomposition – Effect of thiols, ph and oxygen
Eric R. DeLeon a,b, Kenneth R. Olson a,b, Evelyn Huang a, Yan Gao b a University of Notre Dame, Notre Dame, USA b Indiana University School of Medicine South Bend, South Bend, USA
Marian Grman a,b, Anton Misak b, Karol Ondrias b a Faculty of Mathematics, Physics and Informatics, Comenius University, Bratislava, Slovakia b Institute of Molecular Physiology and Genetics SAS, Bratislava, Slovakia
‘‘Sulfhydration’’ has been proposed to be a means of hydrogen sulfide (H2S) signaling in which the H2S reacts with the sulfur of a cysteine residue in proteins, thereby forming a polysulfide and changing the function of the latter, as described by the equation RSH + H2S ? RSSH. However, this requires oxidation of the cysteine RSH, which cannot be accomplished by H2S, as the sulfur is in its most reduced form (oxidation state of 2). Hydropersulfides (H2Sn, n = 2–8, oxidation number = 0), can oxidize RSH and have been suggested to be a contaminant of salts commonly used to produce H2S (NaSH, Na2S) and account for much of the activity originally attributed to H2S. Here, we use the green fluorescent protein, roGFP2, that contains two reactive cysteine residues (Cys147, Cys204) and is a specific reporter of redox status, being fully oxidized by 30 mM tert-butyl hydroperoxide (t-BuOOH), and fully reduced by dithiothreitol (DTT, 30 mM), to determine if H2S can spontaneously form hydro-persulfides in normoxic (21% O2) laboratory conditions. We show that all concentrations ranging from 1lM-300uM H2S have very little reductive effect on roGFP, but that H2S > 300 uM slowly reoxidizes roGFP in a closed system given an hour to react. We attribute this to spontaneous oxidation of H2S to hydropersulfides. Addition of mixed persulfides (K2Sn, n = 1–8) oxidizes roGFP at low concentrations (1uM-300uM), supporting our hypothesis. These results show that H2Sn can be formed from H2S and thereby signal via sulfhydration. Support, NSF IOS-1051627 and NSF DGE-1313583.
Hydrogen sulfide (H2S) and nitric oxide (NO) have some similar biological properties. Interaction of NO with sulfhydryl group of thiols leads to the formation of Snitrosothiols, the biological reservoirs of NO. Snitrosoglutathione (GSNO) is the most abundant and relatively stable S-nitrosothiol. We studied the effect of low molecular thiols, pH and oxygen on the reaction of H2S donor Na2S (0.2 mM) with GSNO (0.2 mM) by time resolved UV–vis absorption spectroscopy. We found that this decomposition is pH dependent, the maximum rate is at physiological pH 7.4, higher or lower pH decelerated the reaction (7.4 > 8.0 6.0). Next we studied the effect of low molecular thiols - L-cysteine (Cys), N-acetyl-L-cysteine (NAC) and reduced glutathione (GSH) on this reaction. At physiological and alkaline pH (7.4 and 8.0), they had a deceleration effect (NAC > GSH > Cys). At acidic pH (6.0), they had the reverse effect – acceleration of GSNO decomposition (Cys > GSH > NAC). Oxidized thiols (oxidized glutathione or cystine) had no effect at any studied pH values. Measurements in nitrogen deaerated solutions revealed that oxygen is not necessary for H2S induced GSNO decomposition, although it slightly accelerates the reaction. Presented results can contribute to understanding of the involvement of H2S and low molecular thiols in NO signaling pathways. This work was supported by VEGA 2/0050/13 and APVV-0074-11. http://dx.doi.org/10.1016/j.niox.2014.03.125
http://dx.doi.org/10.1016/j.niox.2014.03.123
P76 P74 The reaction between H2S and 8-nitro-cGMP: Substitution or reduction? Erwan Galardon, Vida Terzic, Isabelle Artaud UMR 8601, LCBPT, CNRS-Université Paris Descartes, PRES Sorbonne Paris Cité, 45 rue des Sts Pères, 75006 Paris, France Amongst the molecular reactions of H2S accounting for its role as gaseous transmitter is the reaction between hydrogen sulfide and electrophiles. The reaction between H2S and 8-NO2-guanosine 30 ; 50 -cyclic monophosphate (8-NO2-cGMP) leading to the thiol derivative 8-SH-cGMP by ‘‘sulfhydration’’ was proposed to take place in cells, but the chemical reaction is in fact sluggish and requires the catalysts cysteine and transition metals or metalloproteins (Nishida et al., Nat. Chem. Biol. 8 (2012) 714–724). In this work, we show that the direct reaction between H2S and 8-NO2-guanosine (8-NO2-Gua, used as a model of 8-NO2-cGMP) or 8-NO2-cGMP does not lead to the substitution of the nitro group by HS, but to a redox reaction yielding the radical [8-NO2-Gua]-. Because of futile redox cycling with dioxygen, the reaction does
Vasorelaxation induced by naturally occurring triterpenes involves NO and H2S releasing: Ex vivo and in silico studies César Ibarra-Alvarado, Fabian López-Vallejo, Mariana Solís-Gutiérrez, Francisco J. Luna-Vázquez, Alejandra Rojas-Molina Laboratorio de Investigación Química y Farmacológica de Productos Naturales, Facultad de Química, Universidad Autónoma de Querétaro. Querétaro, Qro., Mexico Cardiovascular diseases are the main cause of death in the western world. Altered endothelial signal transduction, which involves reduced bioavailability of NO, CO, and H2S, is associated with several of this kind of diseases. The aim of this study was to investigate the vasorelaxant effect of six naturally occurring triterpenes and to explore the involvement of gasotransmitters in their mechanism of action. The vasodilator activity of ursolic acid, uvaol, betulinic acid, friedelin, oleanolic acid, and lupeol was assessed by using the isolated rat aorta. All compounds displayed a significant relaxant effect on endothelium-intact vessels, in a concentration-dependent manner. This effect was significantly reduced by L-NAME. Ursolic acid displayed the highest maximal vasodilator effect, which was endothelium-dependent. Furthermore, propargyl glycine (PAG), a cysta-
Abstracts / Nitric Oxide 39 (2014) S16–S49
RIN14B cells, which was also inhibited by HC030031 and DTT. These results indicates that H2S has both inhibitory and excitatory effects via opening KATP and TRPA1 channels, respectively, suggesting the possibility of bidirectional modulation by H2S in secretory cells.
not proceed further. However, under hypoxic conditions the reaction between H2S and 8-NO2-cGMP goes to completion and yields the amino derivative 8-NH2-cGMP, a reaction which may be relevant to the first step in the cellular recycling of 8-NO2-cGMP into cGMP.
http://dx.doi.org/10.1016/j.niox.2014.03.122
http://dx.doi.org/10.1016/j.niox.2014.03.124
P73
P75
Spontaneous oxidation of hydrogen sulfide to persulfide (s) allows signaling via sulfhydration
H2S induced S-nitrosoglutathione decomposition – Effect of thiols, ph and oxygen
Eric R. DeLeon a,b, Kenneth R. Olson a,b, Evelyn Huang a, Yan Gao b a University of Notre Dame, Notre Dame, USA b Indiana University School of Medicine South Bend, South Bend, USA
Marian Grman a,b, Anton Misak b, Karol Ondrias b a Faculty of Mathematics, Physics and Informatics, Comenius University, Bratislava, Slovakia b Institute of Molecular Physiology and Genetics SAS, Bratislava, Slovakia
‘‘Sulfhydration’’ has been proposed to be a means of hydrogen sulfide (H2S) signaling in which the H2S reacts with the sulfur of a cysteine residue in proteins, thereby forming a polysulfide and changing the function of the latter, as described by the equation RSH + H2S ? RSSH. However, this requires oxidation of the cysteine RSH, which cannot be accomplished by H2S, as the sulfur is in its most reduced form (oxidation state of 2). Hydropersulfides (H2Sn, n = 2–8, oxidation number = 0), can oxidize RSH and have been suggested to be a contaminant of salts commonly used to produce H2S (NaSH, Na2S) and account for much of the activity originally attributed to H2S. Here, we use the green fluorescent protein, roGFP2, that contains two reactive cysteine residues (Cys147, Cys204) and is a specific reporter of redox status, being fully oxidized by 30 mM tert-butyl hydroperoxide (t-BuOOH), and fully reduced by dithiothreitol (DTT, 30 mM), to determine if H2S can spontaneously form hydro-persulfides in normoxic (21% O2) laboratory conditions. We show that all concentrations ranging from 1lM-300uM H2S have very little reductive effect on roGFP, but that H2S > 300 uM slowly reoxidizes roGFP in a closed system given an hour to react. We attribute this to spontaneous oxidation of H2S to hydropersulfides. Addition of mixed persulfides (K2Sn, n = 1–8) oxidizes roGFP at low concentrations (1uM-300uM), supporting our hypothesis. These results show that H2Sn can be formed from H2S and thereby signal via sulfhydration. Support, NSF IOS-1051627 and NSF DGE-1313583.
Hydrogen sulfide (H2S) and nitric oxide (NO) have some similar biological properties. Interaction of NO with sulfhydryl group of thiols leads to the formation of Snitrosothiols, the biological reservoirs of NO. Snitrosoglutathione (GSNO) is the most abundant and relatively stable S-nitrosothiol. We studied the effect of low molecular thiols, pH and oxygen on the reaction of H2S donor Na2S (0.2 mM) with GSNO (0.2 mM) by time resolved UV–vis absorption spectroscopy. We found that this decomposition is pH dependent, the maximum rate is at physiological pH 7.4, higher or lower pH decelerated the reaction (7.4 > 8.0 6.0). Next we studied the effect of low molecular thiols - L-cysteine (Cys), N-acetyl-L-cysteine (NAC) and reduced glutathione (GSH) on this reaction. At physiological and alkaline pH (7.4 and 8.0), they had a deceleration effect (NAC > GSH > Cys). At acidic pH (6.0), they had the reverse effect – acceleration of GSNO decomposition (Cys > GSH > NAC). Oxidized thiols (oxidized glutathione or cystine) had no effect at any studied pH values. Measurements in nitrogen deaerated solutions revealed that oxygen is not necessary for H2S induced GSNO decomposition, although it slightly accelerates the reaction. Presented results can contribute to understanding of the involvement of H2S and low molecular thiols in NO signaling pathways. This work was supported by VEGA 2/0050/13 and APVV-0074-11. http://dx.doi.org/10.1016/j.niox.2014.03.125
http://dx.doi.org/10.1016/j.niox.2014.03.123
P76 P74 The reaction between H2S and 8-nitro-cGMP: Substitution or reduction? Erwan Galardon, Vida Terzic, Isabelle Artaud UMR 8601, LCBPT, CNRS-Université Paris Descartes, PRES Sorbonne Paris Cité, 45 rue des Sts Pères, 75006 Paris, France Amongst the molecular reactions of H2S accounting for its role as gaseous transmitter is the reaction between hydrogen sulfide and electrophiles. The reaction between H2S and 8-NO2-guanosine 30 ; 50 -cyclic monophosphate (8-NO2-cGMP) leading to the thiol derivative 8-SH-cGMP by ‘‘sulfhydration’’ was proposed to take place in cells, but the chemical reaction is in fact sluggish and requires the catalysts cysteine and transition metals or metalloproteins (Nishida et al., Nat. Chem. Biol. 8 (2012) 714–724). In this work, we show that the direct reaction between H2S and 8-NO2-guanosine (8-NO2-Gua, used as a model of 8-NO2-cGMP) or 8-NO2-cGMP does not lead to the substitution of the nitro group by HS, but to a redox reaction yielding the radical [8-NO2-Gua]-. Because of futile redox cycling with dioxygen, the reaction does
Vasorelaxation induced by naturally occurring triterpenes involves NO and H2S releasing: Ex vivo and in silico studies César Ibarra-Alvarado, Fabian López-Vallejo, Mariana Solís-Gutiérrez, Francisco J. Luna-Vázquez, Alejandra Rojas-Molina Laboratorio de Investigación Química y Farmacológica de Productos Naturales, Facultad de Química, Universidad Autónoma de Querétaro. Querétaro, Qro., Mexico Cardiovascular diseases are the main cause of death in the western world. Altered endothelial signal transduction, which involves reduced bioavailability of NO, CO, and H2S, is associated with several of this kind of diseases. The aim of this study was to investigate the vasorelaxant effect of six naturally occurring triterpenes and to explore the involvement of gasotransmitters in their mechanism of action. The vasodilator activity of ursolic acid, uvaol, betulinic acid, friedelin, oleanolic acid, and lupeol was assessed by using the isolated rat aorta. All compounds displayed a significant relaxant effect on endothelium-intact vessels, in a concentration-dependent manner. This effect was significantly reduced by L-NAME. Ursolic acid displayed the highest maximal vasodilator effect, which was endothelium-dependent. Furthermore, propargyl glycine (PAG), a cysta-