Nitric Oxide 31 (2013) S11–S65
Contents lists available at SciVerse ScienceDirect
Nitric Oxide journal homepage: www.elsevier.com/locate/yniox
Abstracts of the 2nd European Conference on the Biology of Hydrogen Sulfide, September 8–11th 2013, Exeter, UK Opening Address and Public Lecture Physiological function of hydrogen sulfide and beyond
polysulfides is much greater than H2S. The production of H2S and the physiological function of H2S and polysulfides will be discussed. http://dx.doi.org/10.1016/j.niox.2013.06.010
Hideo Kimura National Institute of Neuroscience, Kodaira, Tokyo, Japan The relatively high concentrations of endogenous sulfide in the mammalian brain were measured in 1989, suggesting that hydrogen sulfide (H2S) might have a physiological function. In 1996 we demonstrated that cystathionine b-synthase (CBS) is a H2S producing enzyme in the brain and that H2S facilitates the induction of hippocampal long-term potentiation (LTP), a synaptic model of memory, by enhancing the activity of N-methyl D-aspartate (NMDA) receptors. The following year we demonstrated that another H2S producing enzyme, cystathionine c-lyase (CSE) is found in tissues including vasculature and that H2S relaxes them. Based on these observations we proposed that H2S is a neuromodulator and a smooth muscle relaxant. In addition to the function as a signaling molecule, we and others found a cytoprotective effect of this molecule; H2S protects neurons from oxidative stress. This finding led to the identification of the protection of various organs including the heart, pancreas, retina, and the kidney against ischemia–reperfusion injury. From our finding that the brains of CBS knockout mice was still able to produce H2S, we found another pathway; 3-mercaptopyruvate sulfur transferase (3MST) along with cysteine aminotransferase (CAT). 3MST produces H2S in the presence of thioredoxin or dihydrolipoic acid (DHLA). We recently found a novel pathway to produce H2S from D-cysteine, a negative control. D-Amino acid oxidase (DAO) metabolizes D-cysteine to an achiral a–keto acid, 3-mercaptopyruvate (3MP), which is further metabolized to H2S by 3MST. This pathway is mainly localized in the cerebellum and the kidney. The production of H2S from D-cysteine is 80 times more efficient than that from L-cysteine in the kidney, and the administration of Dcysteine to mice ameliorates renal ischemia–reperfusion injury more effectively than L-cysteine. These results show a therapeutic potential of D-cysteine to the renal diseases and even to the kidney transplantation. Our additional contribution to this field is the discovery of H2S-derived polysulfides, which exist in the brain and activate transient receptor potential ankyrin-1 (TRPA1) channels 300 times more potently than H2S. TRPA1 channels mediate the sensory transduction and respond to a variety of stimuli, including cold temperature, pungent compounds and environmental irritants, but its endogenous ligand has not been identified. The sulfane sulfur of polysulfides is reactive electrophile and readily transferred to a nucleophilic protein thiolate, to generate the protein persulfide (sulfhydration). The sulfhydration activity of
Plenary Speakers PL01 H2S: Controversies and conundrums Kenneth R. Olson Indiana University School of Medicine – South Bend, South Bend, IN 46617, USA Research into the biological effects of hydrogen sulfide (H2S) have at times outpaced an understanding of the physical and chemical properties of this signaling molecule. In this talk some of the more outstanding ‘‘controversies and conundrums’’ of H2S will be examined. Perhaps the most pressing issue is what are ‘‘physiological’’ concentrations of H2S in blood and tissues? Can many of the values reported in the literature, especially those in blood in excess of 20 lmol/l be realistic, even when they can’t be detected by the nose, or when they should be theoretically fatal? A brief overview of the more commonly used methods for these analyses such as methylene blue, ion selective electrodes, monobromobimane, headspace gas, fluorescent dyes and polarographic electrodes will be examined along with their advantages and shortcomings. A number of these methods especially the methylene blue and ion selective methods are associated with obvious artifacts and these will be described. Strong support will be given to the polarographic electrode as to date this is the only method available for analyzing H2S concentration in unadulterated, living tissues and in real time. Tissue concentrations of H2S, often reported in nmoles or lmoles per mg protein often lead to excessive intracellular concentrations when expressed per volume of cytosol and these calculations will be examined critically. Volatility of H2S is another problem, the extent of which is seldom appreciated. In tissue culture wells the half-life may be less than 5 min, it is even shorter in preparations that are aerated with either oxygen or nitrogen and in the Langendorff heart preparation little H2S makes a single complete circuit through the system. How these problems affect long-range studies will be examined. Diffusion of H2S into and within tissues will be examined from a theoretical perspective and these studies will illustrate potential mechanisms for regulating H2S concentration as well as provide some perspective regarding potential pitfalls associated with exogenous H2S applications. Other problems that will be addressed include persulfides as contaminants
S12
Abstracts / Nitric Oxide 31 (2013) S11–S65
of commonly used sulfide salts, H2S as a contaminant of other sulfides, notably dithiothreitol (DTT) and whether or not we should be concerned about chemical interactions of H2S with O2, H2O2. Other analytic compounds that are often used to modify protein sulfhydryl groups or inhibit unrelated enzyme pathways such as Nmethylmalemide, phenyl arsine and zinc protoporphyrin IX also may react with H2S and these will be examined. Recently there has been an increased interest and emphasis on correlating plasma H2S concentrations with a broad spectrum of pathophysiological conditions including cancer, obesity, diabetes, hypertension, coronary heart disease, COPD, asthma and even survival probability in patients in septic shock in intensive care units. The utility of such endeavors will be examined from a variety of perspectives including methods and procedures used to measure H2S, the validity of values obtained and statistical approaches regarding trends and differences and the probability of making false positive and false negative errors. Hopefully this talk will initiate constructive dialogue and positively impact the field. Supported: National Science Foundation, IOS 1051627.
the levels of several metabolites and intermediates in the transulfuration pathway. For example, CBS inhibition leads to increased homocysteine levels. To avoid this unwanted side effect when long term inhibition of CBS or CSE is desired (such as in conditions where H2S production is up regulated contributing to disease progression), scavenging of H2S would be the preferred approach. Our efforts to develop H2S scavengers will be presented during the meeting. We conclude that design and development of new inhibitors for each of the H2S-synthesizing enzymes, as well as H2S scavengers is an unmet need for the field, so that the role of H2S-regulated pathways in the development and treatment of disease can be evaluated.
Acknowledgements This work has been co-financed by the European Union (European Social Fund – ESF) and Greek national funds through the Operational Program ’’Education and Lifelong Learning’’ of the National Strategic Reference Framework (NSRF) – Research Funding Program: Thalis; Investing in knowledge society through the European Social Fund (MIS380259) and Aristeia 2011 (1436).
http://dx.doi.org/10.1016/j.niox.2013.06.011 http://dx.doi.org/10.1016/j.niox.2013.06.012
PL02 Modulation of H2S levels in cells and tissues using pharmacological agents: Advances and pitfalls Andreas Papapetropoulos Dept. of Pharmacy, Lab. of Molecular Pharmacology, University of Patras, Greece The biological roles of endogenous H2S are multiple and rapidly expanding. Hydrogen sulfide exerts its effects in most organ systems including the central and peripheral nervous system, the cardiovascular, gastrointestinal and respiratory systems; H2S also participates in the regulation of cellular metabolism and immunological/inflammatory responses. Much of the enzymatically generated H2S is derived from two pyridoxal-50 -phosphate (PLP)-dependent enzymes responsible for the metabolism of L-cysteine: cystathionine beta synthase (CBS) and cystathionine gamma lyase (CSE); a third pathway that catalyzes the production of H2S from L-cysteine via the combined action of 3-mercaptopyruvate sulphurtransferase and cysteine aminotransferase (3MST/CAT) has also been described. CSE and CBS catalyze several H2S-generating reactions using cysteine and/or homocysteine as substrates. CBS is the predominant H2S-generating enzyme in the brain and nervous system and is highly expressed in liver and kidney, while CSE is mainly expressed in the liver and in vascular and non-vascular smooth muscle. To study the role of this novel gasotransmitter, investigators have relied on genetic models, knock down of H2S-producing enzymes by siRNA, and on pharmacological inhibitors. As genetic and siRNA approaches require additional expertise and access to specialized facilities, most researchers prefer to use pharmacological inhibitors. The most commonly used agents to inhibit H2S biosynthesis include propargylglycine (PAG), b-cyanoalanine (BCA), aminooxyacetic acid (AOAA), trifluoroalanine and hydroxylamine. Using recombinant human H2S-producing enzymes we have found that PAG exhibits selectivity towards CSE vs. CBS, but its potency is lower than that of L-aminoethoxyvinylglycine (AVG). BCA, although selective for CSE, it also inhibits CBS when used at high concentrations. In contrast, none of the compounds tested exhibited significant selectivity towards CBS. In addition, the above-mentioned compounds did not inhibit 3MST. It should also be stressed that even the inhibitors that were found to be selective for CSE have been shown to inhibit other PLP-dependent enzymes. Inhibiting CSE or CBS activity will affect
PL03 Expression of H2S-catalyzing enzymes during ‘‘acute chronic disease’’ Peter Radermacher a, Oscar McCook a, Pierre Asfar b, Enrico Calzia a, Mark E. Wood d, Csaba Szabó c, Matthew Whiteman d, Rui Wang e a Klinik für Anästhesiologie, Universitätsklinikum, Ulm, Germany b Département de Réanimation Médicale, Centre Hospitalier Universitaire, Angers, France c Department of Anesthesiology, University of Texas Medical Branch, Galveston, TX, USA d University of Exeter Medical School, Exeter, UK e Department of Biology, Lakehead University, Thunder Bay, Ontario, Canada Disturbed endogenous H2S production as a result down-regulation of the H2S-catalyzing enzymes cysthathionine-c-lyase (CSE) and cysthathione-b-synthase (CBS) is associated with chronic cardiovascular pathology, e.g. hypertension, atherosclerosis, and chronic kidney disease [1–3]. CSE and CBS up-regulation was found during acute hyperinflammatory states resulting from circulatory shock [4–11], but little is known on the effect of acute stress states on CSE and CBS expression during chronic disease. Therefore we measured CBS and CSE expression before and after porcine kidney ischemia/reperfusion (I/R) injury comparing otherwise healthy animals and swine with ubiquitous atherosclerosis resulting from a mutation of the LDL receptor together with a cholesterol-enriched diet [12,13]. CBS expression was not present in native biopsies, and minimal only post I/R, i.e. in necrotic tubules and cells only. Atherosclerotic swine had lower CSE expression in native biopsies. I/R injury caused a downregulation of the CSE enzyme in both groups, which was more pronounced in the atherosclerotic animals. In the atherosclerotic swine fecal peritonitis-induced septic shock also caused marked down-regulation of kidney CSE expression when compared to sham-operated animals. Therefore, the effect of H2S-supplementation using the slow-releasing H2S donor GYY4137 [14] in atherosclerotic swine with septic shock will be shown. GYY4137 is used to avoid the short and high and therefore potentially deleterious peak sulfide concentrations associated with NaSH or Na2S administration. Equivocal data are available on H2S-catalyzing enzymes during cigarette smoke-induced lung disease: both reduced [15,16] and increased
Contents lists available at SciVerse ScienceDirect
Nitric Oxide journal homepage: www.elsevier.com/locate/yniox
Abstracts of the 2nd European Conference on the Biology of Hydrogen Sulfide, September 8–11th 2013, Exeter, UK Opening Address and Public Lecture Physiological function of hydrogen sulfide and beyond
polysulfides is much greater than H2S. The production of H2S and the physiological function of H2S and polysulfides will be discussed. http://dx.doi.org/10.1016/j.niox.2013.06.010
Hideo Kimura National Institute of Neuroscience, Kodaira, Tokyo, Japan The relatively high concentrations of endogenous sulfide in the mammalian brain were measured in 1989, suggesting that hydrogen sulfide (H2S) might have a physiological function. In 1996 we demonstrated that cystathionine b-synthase (CBS) is a H2S producing enzyme in the brain and that H2S facilitates the induction of hippocampal long-term potentiation (LTP), a synaptic model of memory, by enhancing the activity of N-methyl D-aspartate (NMDA) receptors. The following year we demonstrated that another H2S producing enzyme, cystathionine c-lyase (CSE) is found in tissues including vasculature and that H2S relaxes them. Based on these observations we proposed that H2S is a neuromodulator and a smooth muscle relaxant. In addition to the function as a signaling molecule, we and others found a cytoprotective effect of this molecule; H2S protects neurons from oxidative stress. This finding led to the identification of the protection of various organs including the heart, pancreas, retina, and the kidney against ischemia–reperfusion injury. From our finding that the brains of CBS knockout mice was still able to produce H2S, we found another pathway; 3-mercaptopyruvate sulfur transferase (3MST) along with cysteine aminotransferase (CAT). 3MST produces H2S in the presence of thioredoxin or dihydrolipoic acid (DHLA). We recently found a novel pathway to produce H2S from D-cysteine, a negative control. D-Amino acid oxidase (DAO) metabolizes D-cysteine to an achiral a–keto acid, 3-mercaptopyruvate (3MP), which is further metabolized to H2S by 3MST. This pathway is mainly localized in the cerebellum and the kidney. The production of H2S from D-cysteine is 80 times more efficient than that from L-cysteine in the kidney, and the administration of Dcysteine to mice ameliorates renal ischemia–reperfusion injury more effectively than L-cysteine. These results show a therapeutic potential of D-cysteine to the renal diseases and even to the kidney transplantation. Our additional contribution to this field is the discovery of H2S-derived polysulfides, which exist in the brain and activate transient receptor potential ankyrin-1 (TRPA1) channels 300 times more potently than H2S. TRPA1 channels mediate the sensory transduction and respond to a variety of stimuli, including cold temperature, pungent compounds and environmental irritants, but its endogenous ligand has not been identified. The sulfane sulfur of polysulfides is reactive electrophile and readily transferred to a nucleophilic protein thiolate, to generate the protein persulfide (sulfhydration). The sulfhydration activity of
Plenary Speakers PL01 H2S: Controversies and conundrums Kenneth R. Olson Indiana University School of Medicine – South Bend, South Bend, IN 46617, USA Research into the biological effects of hydrogen sulfide (H2S) have at times outpaced an understanding of the physical and chemical properties of this signaling molecule. In this talk some of the more outstanding ‘‘controversies and conundrums’’ of H2S will be examined. Perhaps the most pressing issue is what are ‘‘physiological’’ concentrations of H2S in blood and tissues? Can many of the values reported in the literature, especially those in blood in excess of 20 lmol/l be realistic, even when they can’t be detected by the nose, or when they should be theoretically fatal? A brief overview of the more commonly used methods for these analyses such as methylene blue, ion selective electrodes, monobromobimane, headspace gas, fluorescent dyes and polarographic electrodes will be examined along with their advantages and shortcomings. A number of these methods especially the methylene blue and ion selective methods are associated with obvious artifacts and these will be described. Strong support will be given to the polarographic electrode as to date this is the only method available for analyzing H2S concentration in unadulterated, living tissues and in real time. Tissue concentrations of H2S, often reported in nmoles or lmoles per mg protein often lead to excessive intracellular concentrations when expressed per volume of cytosol and these calculations will be examined critically. Volatility of H2S is another problem, the extent of which is seldom appreciated. In tissue culture wells the half-life may be less than 5 min, it is even shorter in preparations that are aerated with either oxygen or nitrogen and in the Langendorff heart preparation little H2S makes a single complete circuit through the system. How these problems affect long-range studies will be examined. Diffusion of H2S into and within tissues will be examined from a theoretical perspective and these studies will illustrate potential mechanisms for regulating H2S concentration as well as provide some perspective regarding potential pitfalls associated with exogenous H2S applications. Other problems that will be addressed include persulfides as contaminants
S12
Abstracts / Nitric Oxide 31 (2013) S11–S65
of commonly used sulfide salts, H2S as a contaminant of other sulfides, notably dithiothreitol (DTT) and whether or not we should be concerned about chemical interactions of H2S with O2, H2O2. Other analytic compounds that are often used to modify protein sulfhydryl groups or inhibit unrelated enzyme pathways such as Nmethylmalemide, phenyl arsine and zinc protoporphyrin IX also may react with H2S and these will be examined. Recently there has been an increased interest and emphasis on correlating plasma H2S concentrations with a broad spectrum of pathophysiological conditions including cancer, obesity, diabetes, hypertension, coronary heart disease, COPD, asthma and even survival probability in patients in septic shock in intensive care units. The utility of such endeavors will be examined from a variety of perspectives including methods and procedures used to measure H2S, the validity of values obtained and statistical approaches regarding trends and differences and the probability of making false positive and false negative errors. Hopefully this talk will initiate constructive dialogue and positively impact the field. Supported: National Science Foundation, IOS 1051627.
the levels of several metabolites and intermediates in the transulfuration pathway. For example, CBS inhibition leads to increased homocysteine levels. To avoid this unwanted side effect when long term inhibition of CBS or CSE is desired (such as in conditions where H2S production is up regulated contributing to disease progression), scavenging of H2S would be the preferred approach. Our efforts to develop H2S scavengers will be presented during the meeting. We conclude that design and development of new inhibitors for each of the H2S-synthesizing enzymes, as well as H2S scavengers is an unmet need for the field, so that the role of H2S-regulated pathways in the development and treatment of disease can be evaluated.
Acknowledgements This work has been co-financed by the European Union (European Social Fund – ESF) and Greek national funds through the Operational Program ’’Education and Lifelong Learning’’ of the National Strategic Reference Framework (NSRF) – Research Funding Program: Thalis; Investing in knowledge society through the European Social Fund (MIS380259) and Aristeia 2011 (1436).
http://dx.doi.org/10.1016/j.niox.2013.06.011 http://dx.doi.org/10.1016/j.niox.2013.06.012
PL02 Modulation of H2S levels in cells and tissues using pharmacological agents: Advances and pitfalls Andreas Papapetropoulos Dept. of Pharmacy, Lab. of Molecular Pharmacology, University of Patras, Greece The biological roles of endogenous H2S are multiple and rapidly expanding. Hydrogen sulfide exerts its effects in most organ systems including the central and peripheral nervous system, the cardiovascular, gastrointestinal and respiratory systems; H2S also participates in the regulation of cellular metabolism and immunological/inflammatory responses. Much of the enzymatically generated H2S is derived from two pyridoxal-50 -phosphate (PLP)-dependent enzymes responsible for the metabolism of L-cysteine: cystathionine beta synthase (CBS) and cystathionine gamma lyase (CSE); a third pathway that catalyzes the production of H2S from L-cysteine via the combined action of 3-mercaptopyruvate sulphurtransferase and cysteine aminotransferase (3MST/CAT) has also been described. CSE and CBS catalyze several H2S-generating reactions using cysteine and/or homocysteine as substrates. CBS is the predominant H2S-generating enzyme in the brain and nervous system and is highly expressed in liver and kidney, while CSE is mainly expressed in the liver and in vascular and non-vascular smooth muscle. To study the role of this novel gasotransmitter, investigators have relied on genetic models, knock down of H2S-producing enzymes by siRNA, and on pharmacological inhibitors. As genetic and siRNA approaches require additional expertise and access to specialized facilities, most researchers prefer to use pharmacological inhibitors. The most commonly used agents to inhibit H2S biosynthesis include propargylglycine (PAG), b-cyanoalanine (BCA), aminooxyacetic acid (AOAA), trifluoroalanine and hydroxylamine. Using recombinant human H2S-producing enzymes we have found that PAG exhibits selectivity towards CSE vs. CBS, but its potency is lower than that of L-aminoethoxyvinylglycine (AVG). BCA, although selective for CSE, it also inhibits CBS when used at high concentrations. In contrast, none of the compounds tested exhibited significant selectivity towards CBS. In addition, the above-mentioned compounds did not inhibit 3MST. It should also be stressed that even the inhibitors that were found to be selective for CSE have been shown to inhibit other PLP-dependent enzymes. Inhibiting CSE or CBS activity will affect
PL03 Expression of H2S-catalyzing enzymes during ‘‘acute chronic disease’’ Peter Radermacher a, Oscar McCook a, Pierre Asfar b, Enrico Calzia a, Mark E. Wood d, Csaba Szabó c, Matthew Whiteman d, Rui Wang e a Klinik für Anästhesiologie, Universitätsklinikum, Ulm, Germany b Département de Réanimation Médicale, Centre Hospitalier Universitaire, Angers, France c Department of Anesthesiology, University of Texas Medical Branch, Galveston, TX, USA d University of Exeter Medical School, Exeter, UK e Department of Biology, Lakehead University, Thunder Bay, Ontario, Canada Disturbed endogenous H2S production as a result down-regulation of the H2S-catalyzing enzymes cysthathionine-c-lyase (CSE) and cysthathione-b-synthase (CBS) is associated with chronic cardiovascular pathology, e.g. hypertension, atherosclerosis, and chronic kidney disease [1–3]. CSE and CBS up-regulation was found during acute hyperinflammatory states resulting from circulatory shock [4–11], but little is known on the effect of acute stress states on CSE and CBS expression during chronic disease. Therefore we measured CBS and CSE expression before and after porcine kidney ischemia/reperfusion (I/R) injury comparing otherwise healthy animals and swine with ubiquitous atherosclerosis resulting from a mutation of the LDL receptor together with a cholesterol-enriched diet [12,13]. CBS expression was not present in native biopsies, and minimal only post I/R, i.e. in necrotic tubules and cells only. Atherosclerotic swine had lower CSE expression in native biopsies. I/R injury caused a downregulation of the CSE enzyme in both groups, which was more pronounced in the atherosclerotic animals. In the atherosclerotic swine fecal peritonitis-induced septic shock also caused marked down-regulation of kidney CSE expression when compared to sham-operated animals. Therefore, the effect of H2S-supplementation using the slow-releasing H2S donor GYY4137 [14] in atherosclerotic swine with septic shock will be shown. GYY4137 is used to avoid the short and high and therefore potentially deleterious peak sulfide concentrations associated with NaSH or Na2S administration. Equivocal data are available on H2S-catalyzing enzymes during cigarette smoke-induced lung disease: both reduced [15,16] and increased
