Eric DeLeon - Academia.edu (original) (raw)
Papers by Eric DeLeon
The FASEB Journal, 2016
The mechanism(s) by which cells detect hypoxia and transduce this into appropriate homeostatic re... more The mechanism(s) by which cells detect hypoxia and transduce this into appropriate homeostatic responses is a contentious issue. Vascular oxygen sensing is generally attributed to hypoxic generatio...
The FASEB Journal, 2016
The mechanism(s) by which cells detect hypoxia and transduce this into appropriate homeostatic re... more The mechanism(s) by which cells detect hypoxia and transduce this into appropriate homeostatic responses is a contentious issue. Vascular oxygen sensing is generally attributed to hypoxic generatio...
Acta Physiologica
Numerous studies have shown that H2S serves as an acute oxygen sensor in a variety of cells. We h... more Numerous studies have shown that H2S serves as an acute oxygen sensor in a variety of cells. We hypothesize that H2S also serves in extended oxygen sensing.
American journal of physiology. Regulatory, integrative and comparative physiology, Jan 23, 2017
Fluorescence spectroscopy and microscopy has been used extensively to monitor biomolecules, espec... more Fluorescence spectroscopy and microscopy has been used extensively to monitor biomolecules, especially reactive oxygen species (ROS) and more recently reactive sulfide (RSS) species. Nearly all fluorophores are either excited by or emit light between 450 and 550 nm which is similar to the absorbance of heme proteins and metal-centered porphyrins. Here we examined the effects of catalase (Cat), reduced and oxidized hemoglobin (Hb and metHb), albumin (alb), manganese (III) Tetrakis (4-Benzoic Acid) porphyrin chloride (MnTBAP), iron proto-porphyrin IX (hemin) and copper proto-porphyrin IX (CuPPIX) on the fluorescence properties of fluorescein. We also examined the effects of catalase and MnTBAP on fluorophores for ROS (dichlorofluorescein, DCF), polysulfides (3',6'-Di(O-thiosalicyl)fluorescein, SSP4) and H2S (7-azido-4-methylcoumarin, AzMC) previously activated by H2O2, a mixed polysulfide (H2Sn, n=1-7) and H2S, respectively. All except albumin concentration-dependently inhibit...
Shock (Augusta, Ga.), Jan 11, 2017
Hydrogen sulfide (H2S) is a novel signaling molecule most recently found to be of fundamental imp... more Hydrogen sulfide (H2S) is a novel signaling molecule most recently found to be of fundamental importance in cellular function as a regulator of apoptosis, inflammation, and perfusion. Mechanisms of endogenous H2S signaling are poorly understood, however, signal transmission is thought to occur via persulfidation at reactive cysteine residues on proteins. Although much has been discovered about how H2S is synthesized in the body, less is known about how it is metabolized. Recent studies have discovered a multitude of different targets for H2S therapy, including those related to protein modification, intracellular signaling, and ion channel depolarization. The most difficult part of studying hydrogen sulfide has been finding a way to accurately and reproducibly measure it. The purpose of this review is to: 1) elaborate on the biosynthesis and catabolism of H2S in the human body, 2) review current knowledge of the mechanisms of action of this gas in relation to ischemic injury, 3) defi...
American journal of physiology. Regulatory, integrative and comparative physiology, Sep 20, 2016
In lung epithelial cells, hypoxia decreases the expression and activity of sodium transporting mo... more In lung epithelial cells, hypoxia decreases the expression and activity of sodium transporting molecules, thereby reducing the rate of transepithelial sodium absorption. The mechanisms underlying the sensing of hypoxia and subsequent coupling to sodium transporting molecules remain unclear. Hydrogen sulfide (H2S) has recently been recognized as a cellular signaling molecule whose intracellular concentrations critically depend on oxygen levels. Therefore it was questioned whether endogenously produced H2S contributes to hypoxic inhibition of sodium transport. In electrophysiological Ussing chamber experiments, hypoxia was established by decreasing oxygen concentrations in the chambers. Hypoxia concentration-dependently and reversibly decreased amiloride-sensitive sodium absorption by cultured H441 monolayers and freshly dissected porcine tracheal epithelia due to inhibition of basolateral Na(+)/K(+)-ATPase. Exogenous application of H2S by the sulfur salt Na2S mimicked the effect of h...
American journal of physiology. Regulatory, integrative and comparative physiology, Jan 13, 2016
Stepwise one-electron reduction of oxygen to water produces reactive oxygen species (ROS) that ar... more Stepwise one-electron reduction of oxygen to water produces reactive oxygen species (ROS) that are chemically and biochemically similar to reactive sulfide species (RSS) derived from one-electron oxidation of hydrogen sulfide to elemental sulfur. Both ROS and RSS are endogenously generated and signal via protein thiols. Given the similarities between ROS and RSS we wondered if extant methods for measuring the former would also detect the latter. Here we compared ROS to RSS sensitivity of five common ROS methods, redox-sensitive green fluorescent protein (roGFP), 2', 7'-dihydrodichlorofluorescein, MitoSox Red, Amplex Red and amperometric electrodes. All methods detected RSS and were as or more sensitive to RSS than to ROS. roGFP, arguably the "gold standard" for ROS measurement, was over two-hundred fold more sensitive to the mixed polysulfide H2Sn (n=1-8) than to H2O2. These findings suggest that RSS may be far more prevalent in intracellular signaling than previou...
Nitric Oxide
“Sulfhydration” has been proposed to be a means of hydrogen sulfide (H 2 S) signaling in which th... more “Sulfhydration” has been proposed to be a means of hydrogen sulfide (H 2 S) signaling in which the H 2 S 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 + H 2 S → RSSH. However, this requires oxidation of the cysteine RSH, which cannot be accomplished by H 2 S, as the sulfur is in its most reduced form (oxidation state of −2). Hydropersulfides (H 2 S n , n = 2–8, oxidation number = 0), can oxidize RSH and have been suggested to be a contaminant of salts commonly used to produce H 2 S (NaSH, Na 2 S) and account for much of the activity originally attributed to H 2 S. 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 H 2 S can spontaneously form hydro-persulfides in normoxic (21% O 2 ) laboratory conditions. We show that all concentrations ranging from 1 μ M-300uM H 2 S have very little reductive effect on roGFP, but that H 2 S > 300 uM slowly reoxidizes roGFP in a closed system given an hour to react. We attribute this to spontaneous oxidation of H 2 S to hydropersulfides. Addition of mixed persulfides (K 2 S n , n = 1–8) oxidizes roGFP at low concentrations (1uM-300uM), supporting our hypothesis. These results show that H 2 S n can be formed from H 2 S and thereby signal via sulfhydration. Support, NSF IOS-1051627 and NSF DGE-1313583.
Nitric Oxide, 2014
Hydrogen sulfide (H2S) signaling has been implicated in physiological processes in practically al... more Hydrogen sulfide (H2S) signaling has been implicated in physiological processes in practically all organ systems studied to date. At times the excitement of this new field has outpaced the technical expertise or practical knowledge with which to accurately assess these advancements. Recently, the myriad of proposed H2S actions has spawned interest in using indicators of H2S metabolism, especially plasma H2S concentrations, as a means of identifying a variety of pathophysiological conditions or to predict clinical outcomes. While this is a noteworthy endeavor, there are a number of contraindications to this practice at this time. First, there is little consensus regarding normal, i.e., "physiological" concentrations of H2S in either plasma or tissue. In fact, it has been shown that the methods most often employed for these measurements are associated with substantial artifact. Second, interactions, or presumed lack thereof, of H2S with other biomolecules (e.g., O2, H2O2, pH, etc.) or analytical reagents (e.g., reducing reagents, N-ethylmaleimide, phenylarsine, etc.) are often assumed but not evaluated. Third, the experimental design and/or statistical analyses may not be sufficient to justify using H2S concentration in tissue or blood as a predictive biomarker of pathophysiology. In this study, we first briefly review the problems associated with plasma and tissue H2S measurements and the associated errors and we provide some simple methods to evaluate whether the data obtained is physiologically relevant. Second we provide a brief analysis of H2S interactions with the above biomolecules. Third, we provide a statistical tool with which to determine the clinical applicability of H2S measurements. It is hoped that these points will provide a rational background for future work.
CLEO: 2013, 2013
ABSTRACT We present work looking at the two-photon optical characterization of water soluble chem... more ABSTRACT We present work looking at the two-photon optical characterization of water soluble chemical probes based on surfactant nanomicelles encapsulation and ormosil PEBBLEs as well as in-vitro two-photon imaging of oxygen and hydrogen sulfide.
Analytical Biochemistry, 2012
Hydrogen sulfide (H(2)S) is a volatile gas of considerable interest as a physiologically relevant... more Hydrogen sulfide (H(2)S) is a volatile gas of considerable interest as a physiologically relevant signaling molecule, but this volatility has typically been overlooked in the context of biological experiments. We examined volatility of 10 and 100 μM H(2)S (Na(2)S·9H(2)O) in real time with polarographic electrodes in three commonly employed experimental apparatuses: 24-well tissue culture plates (WP), muscle myograph baths (MB), and the Langendorff perfused heart apparatus (LPH). H(2)S loss from all apparatuses was rapid and exponential, with half-times (t(1/2)) of 5 min (WP), less than 4 min (MB), and less than 0.5 min (LPH). The t(1/2) for H(2)S loss from MB bubbled with 100% oxygen was slightly longer than that for MB bubbled with 100% nitrogen; both were significantly shorter than stirred but unbubbled MB (>9 min). Therefore, even without tissue, H(2)S rapidly disappears from buffer under a variety of experimental conditions, and this is due to volatilization, not oxidation. The inability to maintain H(2)S concentration, even briefly, questions the accuracy of dose-response studies and the relevance of long-term (>10 min) exposure to a single treatment of H(2)S. These results also help to explain the discrepancy between low H(2)S concentrations in blood and tissues versus high concentrations of exogenous H(2)S required to produce physiological responses.
Redox Biology, 2017
Catalase is well-known as an antioxidant dismutating H 2 O 2 to O 2 and H 2 O. However, catalases... more Catalase is well-known as an antioxidant dismutating H 2 O 2 to O 2 and H 2 O. However, catalases evolved when metabolism was largely sulfur-based, long before O 2 and reactive oxygen species (ROS) became abundant, suggesting catalase metabolizes reactive sulfide species (RSS). Here we examine catalase metabolism of H 2 S n , the sulfur analog of H 2 O 2 , hydrogen sulfide (H 2 S) and other sulfur-bearing molecules using H 2 S-specific amperometric electrodes and fluorophores to measure polysulfides (H 2 S n ; SSP4) and ROS (dichlorofluorescein, DCF). Catalase eliminated H 2 S n , but did not anaerobically generate H 2 S, the expected product of dismutation. Instead, catalase concentration-and oxygen-dependently metabolized H 2 S and in so doing acted as a sulfide oxidase with a P 50 of 20 mmHg. H 2 O 2 had little effect on catalase-mediated H 2 S metabolism but in the presence of the catalase inhibitor, sodium azide (Az), H 2 O 2 rapidly and efficiently expedited H 2 S metabolism in both normoxia and hypoxia suggesting H 2 O 2 is an effective electron acceptor in this reaction. Unexpectedly, catalase concentration-dependently generated H 2 S from dithiothreitol (DTT) in both normoxia and hypoxia, concomitantly oxidizing H 2 S in the presence of O 2. H 2 S production from DTT was inhibited by carbon monoxide and augmented by NADPH suggesting that catalase heme-iron is the catalytic site and that NADPH provides reducing equivalents. Catalase also generated H 2 S from garlic oil, diallyltrisulfide, thioredoxin and sulfur dioxide, but not from sulfite, metabisulfite, carbonyl sulfide, cysteine, cystine, glutathione or oxidized glutathione. Oxidase activity was also present in catalase from Aspergillus niger. These results show that catalase can act as either a sulfide oxidase or sulfur reductase and they suggest that these activities likely played a prominent role in sulfur metabolism during evolution and may continue do so in modern cells as well. This also appears to be the first observation of catalase reductase activity independent of peroxide dismutation.
Hydrogen sulfide (H 2 S) has been shown to affect gastrointestinal (GI) motility and signaling in... more Hydrogen sulfide (H 2 S) has been shown to affect gastrointestinal (GI) motility and signaling in mammals and O 2-dependent H 2 S metabolism has been proposed to serve as an O 2 ʻsensorʼ that couples hypoxic stimuli to effector responses in a variety of other O 2-sensing tissues. The low P O2 values and high H 2 S concentrations routinely encountered in the GI tract suggest that H 2 S might also be involved in hypoxic responses in these tissues. In the present study we examined the effect of H 2 S on stomach, esophagus, gallbladder and intestinal motility in the rainbow trout (Oncorhynchus mykiss) and coho salmon (Oncorhynchus kisutch) and we evaluated the potential for H 2 S in oxygen sensing by examining GI responses to hypoxia in the presence of known inhibitors of H 2 S biosynthesis and by adding the sulfide donor cysteine (Cys). We also measured H 2 S production by intestinal tissue in real time and in the presence and absence of oxygen. In tissues exhibiting spontaneous contractions, H 2 S inhibited contraction magnitude (area under the curve and amplitude) and frequency, and in all tissues it reduced baseline tension in a concentration-dependent relationship. Longitudinal intestinal smooth muscle was significantly more sensitive to H 2 S than other tissues, exhibiting significant inhibitory responses at 1-10mmoll-1 H 2 S. The effects of hypoxia were essentially identical to those of H 2 S in longitudinal and circular intestinal smooth muscle; of special note was a unique transient stimulatory effect upon application of both hypoxia and H 2 S. Inhibitors of enzymes implicated in H 2 S biosynthesis (cystathionine -synthase and cystathionine -lyase) partially inhibited the effects of hypoxia whereas the hypoxic effects were augmented by the sulfide donor Cys. Furthermore, tissue production of H 2 S was inversely related to O 2 ; addition of Cys to intestinal tissue homogenate stimulated H 2 S production when the tissue was gassed with 100% nitrogen (~0% O 2), whereas addition of oxygen (~10% O 2) reversed this to net H 2 S consumption. This study shows that the inhibitory effects of H 2 S on the GI tract of a non-mammalian vertebrate are identical to those reported in mammals and they provide further evidence that H 2 S is a key mediator of the hypoxic response in a variety of O 2-sensitive tissues.
AJP: Regulatory, Integrative and Comparative Physiology, 2013
H2S derived from organic thiol metabolism has been proposed serve as an oxygen sensor in a variet... more H2S derived from organic thiol metabolism has been proposed serve as an oxygen sensor in a variety of systems because of its susceptibility to oxidation and its ability to mimic hypoxic responses in numerous oxygen-sensing tissues. Thiosulfate, an intermediate in oxidative H2S metabolism can alternatively be reduced and regenerate H2S. We propose that this contributes to the H2S-mediated oxygen-sensing mechanism. H2S formation from thiosulfate in buffers and in a variety of mammalian tissues and in lamprey dorsal aorta was examined in real time using a polarographic H2S sensor. Inferences of intracellular H2S production were made by examining hypoxic pulmonary vasoconstriction (HPV) in bovine pulmonary arteries under conditions in which increased H2S production would be expected and in mouse and rat aortas, where reducing conditions should mediate vasorelaxation. In Krebs-Henseleit (mammalian) and Cortland (lamprey) buffers, H2S was generated from thiosulfate in the presence of the ...
Redox biology, May 20, 2017
Reactive sulfur species (RSS) such as HS, HS, HS, (n = 2-7) and HSare chemically similar to HO an... more Reactive sulfur species (RSS) such as HS, HS, HS, (n = 2-7) and HSare chemically similar to HO and the reactive oxygen species (ROS) HO, HO, Oand act on common biological effectors. RSS were present in evolution long before ROS, and because both are metabolized by catalase it has been suggested that "antioxidant" enzymes originally evolved to regulate RSS and may continue to do so today. Here we examined RSS metabolism by Cu/Zn superoxide dismutase (SOD) using amperometric electrodes for dissolved HS, a polysulfide-specific fluorescent probe (SSP4), and mass spectrometry to identify specific polysulfides (HS-HS). HS was concentration- and oxygen-dependently oxidized by 1μM SOD to polysulfides (mainly HS, and to a lesser extent HSand HS) with an ECof approximately 380μM HS. HS concentrations > 750μM inhibited SOD oxidation (IC= 1.25mM) with complete inhibition when HS > 1.75mM. Polysulfides were not metabolized by SOD. SOD oxidation preferred dissolved HS over hydrosu...
American journal of physiology. Regulatory, integrative and comparative physiology, Jun 13, 2016
The health benefits of garlic and other organosulfur-containing foods are well recognized and hav... more The health benefits of garlic and other organosulfur-containing foods are well recognized and have been attributed to both pro-oxidant and antioxidant activities. The effects of garlic are surprisingly similar to those of hydrogen sulfide (H2S) which is also known to be released from garlic under certain conditions. However, recent evidence suggests that polysulfides, not H2S, may be the actual mediator of physiological signaling. In this study we monitored formation of H2S and polysulfides from garlic oil in buffer and in HEK293 cells with fluorescent dyes, AzMC and SSP4, respectively and redox activity with two redox indicators roGFP and DCF. Our results show that H2S release from garlic oil in buffer requires other low molecular weight thiols such as cysteine (Cys) or glutathione (GSH), whereas polysulfides are readily detected in garlic oil alone. Administration of garlic oil to cells rapidly increases intracellular polysulfide but has minimal effects on H2S unless Cys or GSH ar...
The FASEB Journal, 2016
The mechanism(s) by which cells detect hypoxia and transduce this into appropriate homeostatic re... more The mechanism(s) by which cells detect hypoxia and transduce this into appropriate homeostatic responses is a contentious issue. Vascular oxygen sensing is generally attributed to hypoxic generatio...
The FASEB Journal, 2016
The mechanism(s) by which cells detect hypoxia and transduce this into appropriate homeostatic re... more The mechanism(s) by which cells detect hypoxia and transduce this into appropriate homeostatic responses is a contentious issue. Vascular oxygen sensing is generally attributed to hypoxic generatio...
Acta Physiologica
Numerous studies have shown that H2S serves as an acute oxygen sensor in a variety of cells. We h... more Numerous studies have shown that H2S serves as an acute oxygen sensor in a variety of cells. We hypothesize that H2S also serves in extended oxygen sensing.
American journal of physiology. Regulatory, integrative and comparative physiology, Jan 23, 2017
Fluorescence spectroscopy and microscopy has been used extensively to monitor biomolecules, espec... more Fluorescence spectroscopy and microscopy has been used extensively to monitor biomolecules, especially reactive oxygen species (ROS) and more recently reactive sulfide (RSS) species. Nearly all fluorophores are either excited by or emit light between 450 and 550 nm which is similar to the absorbance of heme proteins and metal-centered porphyrins. Here we examined the effects of catalase (Cat), reduced and oxidized hemoglobin (Hb and metHb), albumin (alb), manganese (III) Tetrakis (4-Benzoic Acid) porphyrin chloride (MnTBAP), iron proto-porphyrin IX (hemin) and copper proto-porphyrin IX (CuPPIX) on the fluorescence properties of fluorescein. We also examined the effects of catalase and MnTBAP on fluorophores for ROS (dichlorofluorescein, DCF), polysulfides (3',6'-Di(O-thiosalicyl)fluorescein, SSP4) and H2S (7-azido-4-methylcoumarin, AzMC) previously activated by H2O2, a mixed polysulfide (H2Sn, n=1-7) and H2S, respectively. All except albumin concentration-dependently inhibit...
Shock (Augusta, Ga.), Jan 11, 2017
Hydrogen sulfide (H2S) is a novel signaling molecule most recently found to be of fundamental imp... more Hydrogen sulfide (H2S) is a novel signaling molecule most recently found to be of fundamental importance in cellular function as a regulator of apoptosis, inflammation, and perfusion. Mechanisms of endogenous H2S signaling are poorly understood, however, signal transmission is thought to occur via persulfidation at reactive cysteine residues on proteins. Although much has been discovered about how H2S is synthesized in the body, less is known about how it is metabolized. Recent studies have discovered a multitude of different targets for H2S therapy, including those related to protein modification, intracellular signaling, and ion channel depolarization. The most difficult part of studying hydrogen sulfide has been finding a way to accurately and reproducibly measure it. The purpose of this review is to: 1) elaborate on the biosynthesis and catabolism of H2S in the human body, 2) review current knowledge of the mechanisms of action of this gas in relation to ischemic injury, 3) defi...
American journal of physiology. Regulatory, integrative and comparative physiology, Sep 20, 2016
In lung epithelial cells, hypoxia decreases the expression and activity of sodium transporting mo... more In lung epithelial cells, hypoxia decreases the expression and activity of sodium transporting molecules, thereby reducing the rate of transepithelial sodium absorption. The mechanisms underlying the sensing of hypoxia and subsequent coupling to sodium transporting molecules remain unclear. Hydrogen sulfide (H2S) has recently been recognized as a cellular signaling molecule whose intracellular concentrations critically depend on oxygen levels. Therefore it was questioned whether endogenously produced H2S contributes to hypoxic inhibition of sodium transport. In electrophysiological Ussing chamber experiments, hypoxia was established by decreasing oxygen concentrations in the chambers. Hypoxia concentration-dependently and reversibly decreased amiloride-sensitive sodium absorption by cultured H441 monolayers and freshly dissected porcine tracheal epithelia due to inhibition of basolateral Na(+)/K(+)-ATPase. Exogenous application of H2S by the sulfur salt Na2S mimicked the effect of h...
American journal of physiology. Regulatory, integrative and comparative physiology, Jan 13, 2016
Stepwise one-electron reduction of oxygen to water produces reactive oxygen species (ROS) that ar... more Stepwise one-electron reduction of oxygen to water produces reactive oxygen species (ROS) that are chemically and biochemically similar to reactive sulfide species (RSS) derived from one-electron oxidation of hydrogen sulfide to elemental sulfur. Both ROS and RSS are endogenously generated and signal via protein thiols. Given the similarities between ROS and RSS we wondered if extant methods for measuring the former would also detect the latter. Here we compared ROS to RSS sensitivity of five common ROS methods, redox-sensitive green fluorescent protein (roGFP), 2', 7'-dihydrodichlorofluorescein, MitoSox Red, Amplex Red and amperometric electrodes. All methods detected RSS and were as or more sensitive to RSS than to ROS. roGFP, arguably the "gold standard" for ROS measurement, was over two-hundred fold more sensitive to the mixed polysulfide H2Sn (n=1-8) than to H2O2. These findings suggest that RSS may be far more prevalent in intracellular signaling than previou...
Nitric Oxide
“Sulfhydration” has been proposed to be a means of hydrogen sulfide (H 2 S) signaling in which th... more “Sulfhydration” has been proposed to be a means of hydrogen sulfide (H 2 S) signaling in which the H 2 S 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 + H 2 S → RSSH. However, this requires oxidation of the cysteine RSH, which cannot be accomplished by H 2 S, as the sulfur is in its most reduced form (oxidation state of −2). Hydropersulfides (H 2 S n , n = 2–8, oxidation number = 0), can oxidize RSH and have been suggested to be a contaminant of salts commonly used to produce H 2 S (NaSH, Na 2 S) and account for much of the activity originally attributed to H 2 S. 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 H 2 S can spontaneously form hydro-persulfides in normoxic (21% O 2 ) laboratory conditions. We show that all concentrations ranging from 1 μ M-300uM H 2 S have very little reductive effect on roGFP, but that H 2 S > 300 uM slowly reoxidizes roGFP in a closed system given an hour to react. We attribute this to spontaneous oxidation of H 2 S to hydropersulfides. Addition of mixed persulfides (K 2 S n , n = 1–8) oxidizes roGFP at low concentrations (1uM-300uM), supporting our hypothesis. These results show that H 2 S n can be formed from H 2 S and thereby signal via sulfhydration. Support, NSF IOS-1051627 and NSF DGE-1313583.
Nitric Oxide, 2014
Hydrogen sulfide (H2S) signaling has been implicated in physiological processes in practically al... more Hydrogen sulfide (H2S) signaling has been implicated in physiological processes in practically all organ systems studied to date. At times the excitement of this new field has outpaced the technical expertise or practical knowledge with which to accurately assess these advancements. Recently, the myriad of proposed H2S actions has spawned interest in using indicators of H2S metabolism, especially plasma H2S concentrations, as a means of identifying a variety of pathophysiological conditions or to predict clinical outcomes. While this is a noteworthy endeavor, there are a number of contraindications to this practice at this time. First, there is little consensus regarding normal, i.e., "physiological" concentrations of H2S in either plasma or tissue. In fact, it has been shown that the methods most often employed for these measurements are associated with substantial artifact. Second, interactions, or presumed lack thereof, of H2S with other biomolecules (e.g., O2, H2O2, pH, etc.) or analytical reagents (e.g., reducing reagents, N-ethylmaleimide, phenylarsine, etc.) are often assumed but not evaluated. Third, the experimental design and/or statistical analyses may not be sufficient to justify using H2S concentration in tissue or blood as a predictive biomarker of pathophysiology. In this study, we first briefly review the problems associated with plasma and tissue H2S measurements and the associated errors and we provide some simple methods to evaluate whether the data obtained is physiologically relevant. Second we provide a brief analysis of H2S interactions with the above biomolecules. Third, we provide a statistical tool with which to determine the clinical applicability of H2S measurements. It is hoped that these points will provide a rational background for future work.
CLEO: 2013, 2013
ABSTRACT We present work looking at the two-photon optical characterization of water soluble chem... more ABSTRACT We present work looking at the two-photon optical characterization of water soluble chemical probes based on surfactant nanomicelles encapsulation and ormosil PEBBLEs as well as in-vitro two-photon imaging of oxygen and hydrogen sulfide.
Analytical Biochemistry, 2012
Hydrogen sulfide (H(2)S) is a volatile gas of considerable interest as a physiologically relevant... more Hydrogen sulfide (H(2)S) is a volatile gas of considerable interest as a physiologically relevant signaling molecule, but this volatility has typically been overlooked in the context of biological experiments. We examined volatility of 10 and 100 μM H(2)S (Na(2)S·9H(2)O) in real time with polarographic electrodes in three commonly employed experimental apparatuses: 24-well tissue culture plates (WP), muscle myograph baths (MB), and the Langendorff perfused heart apparatus (LPH). H(2)S loss from all apparatuses was rapid and exponential, with half-times (t(1/2)) of 5 min (WP), less than 4 min (MB), and less than 0.5 min (LPH). The t(1/2) for H(2)S loss from MB bubbled with 100% oxygen was slightly longer than that for MB bubbled with 100% nitrogen; both were significantly shorter than stirred but unbubbled MB (>9 min). Therefore, even without tissue, H(2)S rapidly disappears from buffer under a variety of experimental conditions, and this is due to volatilization, not oxidation. The inability to maintain H(2)S concentration, even briefly, questions the accuracy of dose-response studies and the relevance of long-term (>10 min) exposure to a single treatment of H(2)S. These results also help to explain the discrepancy between low H(2)S concentrations in blood and tissues versus high concentrations of exogenous H(2)S required to produce physiological responses.
Redox Biology, 2017
Catalase is well-known as an antioxidant dismutating H 2 O 2 to O 2 and H 2 O. However, catalases... more Catalase is well-known as an antioxidant dismutating H 2 O 2 to O 2 and H 2 O. However, catalases evolved when metabolism was largely sulfur-based, long before O 2 and reactive oxygen species (ROS) became abundant, suggesting catalase metabolizes reactive sulfide species (RSS). Here we examine catalase metabolism of H 2 S n , the sulfur analog of H 2 O 2 , hydrogen sulfide (H 2 S) and other sulfur-bearing molecules using H 2 S-specific amperometric electrodes and fluorophores to measure polysulfides (H 2 S n ; SSP4) and ROS (dichlorofluorescein, DCF). Catalase eliminated H 2 S n , but did not anaerobically generate H 2 S, the expected product of dismutation. Instead, catalase concentration-and oxygen-dependently metabolized H 2 S and in so doing acted as a sulfide oxidase with a P 50 of 20 mmHg. H 2 O 2 had little effect on catalase-mediated H 2 S metabolism but in the presence of the catalase inhibitor, sodium azide (Az), H 2 O 2 rapidly and efficiently expedited H 2 S metabolism in both normoxia and hypoxia suggesting H 2 O 2 is an effective electron acceptor in this reaction. Unexpectedly, catalase concentration-dependently generated H 2 S from dithiothreitol (DTT) in both normoxia and hypoxia, concomitantly oxidizing H 2 S in the presence of O 2. H 2 S production from DTT was inhibited by carbon monoxide and augmented by NADPH suggesting that catalase heme-iron is the catalytic site and that NADPH provides reducing equivalents. Catalase also generated H 2 S from garlic oil, diallyltrisulfide, thioredoxin and sulfur dioxide, but not from sulfite, metabisulfite, carbonyl sulfide, cysteine, cystine, glutathione or oxidized glutathione. Oxidase activity was also present in catalase from Aspergillus niger. These results show that catalase can act as either a sulfide oxidase or sulfur reductase and they suggest that these activities likely played a prominent role in sulfur metabolism during evolution and may continue do so in modern cells as well. This also appears to be the first observation of catalase reductase activity independent of peroxide dismutation.
Hydrogen sulfide (H 2 S) has been shown to affect gastrointestinal (GI) motility and signaling in... more Hydrogen sulfide (H 2 S) has been shown to affect gastrointestinal (GI) motility and signaling in mammals and O 2-dependent H 2 S metabolism has been proposed to serve as an O 2 ʻsensorʼ that couples hypoxic stimuli to effector responses in a variety of other O 2-sensing tissues. The low P O2 values and high H 2 S concentrations routinely encountered in the GI tract suggest that H 2 S might also be involved in hypoxic responses in these tissues. In the present study we examined the effect of H 2 S on stomach, esophagus, gallbladder and intestinal motility in the rainbow trout (Oncorhynchus mykiss) and coho salmon (Oncorhynchus kisutch) and we evaluated the potential for H 2 S in oxygen sensing by examining GI responses to hypoxia in the presence of known inhibitors of H 2 S biosynthesis and by adding the sulfide donor cysteine (Cys). We also measured H 2 S production by intestinal tissue in real time and in the presence and absence of oxygen. In tissues exhibiting spontaneous contractions, H 2 S inhibited contraction magnitude (area under the curve and amplitude) and frequency, and in all tissues it reduced baseline tension in a concentration-dependent relationship. Longitudinal intestinal smooth muscle was significantly more sensitive to H 2 S than other tissues, exhibiting significant inhibitory responses at 1-10mmoll-1 H 2 S. The effects of hypoxia were essentially identical to those of H 2 S in longitudinal and circular intestinal smooth muscle; of special note was a unique transient stimulatory effect upon application of both hypoxia and H 2 S. Inhibitors of enzymes implicated in H 2 S biosynthesis (cystathionine -synthase and cystathionine -lyase) partially inhibited the effects of hypoxia whereas the hypoxic effects were augmented by the sulfide donor Cys. Furthermore, tissue production of H 2 S was inversely related to O 2 ; addition of Cys to intestinal tissue homogenate stimulated H 2 S production when the tissue was gassed with 100% nitrogen (~0% O 2), whereas addition of oxygen (~10% O 2) reversed this to net H 2 S consumption. This study shows that the inhibitory effects of H 2 S on the GI tract of a non-mammalian vertebrate are identical to those reported in mammals and they provide further evidence that H 2 S is a key mediator of the hypoxic response in a variety of O 2-sensitive tissues.
AJP: Regulatory, Integrative and Comparative Physiology, 2013
H2S derived from organic thiol metabolism has been proposed serve as an oxygen sensor in a variet... more H2S derived from organic thiol metabolism has been proposed serve as an oxygen sensor in a variety of systems because of its susceptibility to oxidation and its ability to mimic hypoxic responses in numerous oxygen-sensing tissues. Thiosulfate, an intermediate in oxidative H2S metabolism can alternatively be reduced and regenerate H2S. We propose that this contributes to the H2S-mediated oxygen-sensing mechanism. H2S formation from thiosulfate in buffers and in a variety of mammalian tissues and in lamprey dorsal aorta was examined in real time using a polarographic H2S sensor. Inferences of intracellular H2S production were made by examining hypoxic pulmonary vasoconstriction (HPV) in bovine pulmonary arteries under conditions in which increased H2S production would be expected and in mouse and rat aortas, where reducing conditions should mediate vasorelaxation. In Krebs-Henseleit (mammalian) and Cortland (lamprey) buffers, H2S was generated from thiosulfate in the presence of the ...
Redox biology, May 20, 2017
Reactive sulfur species (RSS) such as HS, HS, HS, (n = 2-7) and HSare chemically similar to HO an... more Reactive sulfur species (RSS) such as HS, HS, HS, (n = 2-7) and HSare chemically similar to HO and the reactive oxygen species (ROS) HO, HO, Oand act on common biological effectors. RSS were present in evolution long before ROS, and because both are metabolized by catalase it has been suggested that "antioxidant" enzymes originally evolved to regulate RSS and may continue to do so today. Here we examined RSS metabolism by Cu/Zn superoxide dismutase (SOD) using amperometric electrodes for dissolved HS, a polysulfide-specific fluorescent probe (SSP4), and mass spectrometry to identify specific polysulfides (HS-HS). HS was concentration- and oxygen-dependently oxidized by 1μM SOD to polysulfides (mainly HS, and to a lesser extent HSand HS) with an ECof approximately 380μM HS. HS concentrations > 750μM inhibited SOD oxidation (IC= 1.25mM) with complete inhibition when HS > 1.75mM. Polysulfides were not metabolized by SOD. SOD oxidation preferred dissolved HS over hydrosu...
American journal of physiology. Regulatory, integrative and comparative physiology, Jun 13, 2016
The health benefits of garlic and other organosulfur-containing foods are well recognized and hav... more The health benefits of garlic and other organosulfur-containing foods are well recognized and have been attributed to both pro-oxidant and antioxidant activities. The effects of garlic are surprisingly similar to those of hydrogen sulfide (H2S) which is also known to be released from garlic under certain conditions. However, recent evidence suggests that polysulfides, not H2S, may be the actual mediator of physiological signaling. In this study we monitored formation of H2S and polysulfides from garlic oil in buffer and in HEK293 cells with fluorescent dyes, AzMC and SSP4, respectively and redox activity with two redox indicators roGFP and DCF. Our results show that H2S release from garlic oil in buffer requires other low molecular weight thiols such as cysteine (Cys) or glutathione (GSH), whereas polysulfides are readily detected in garlic oil alone. Administration of garlic oil to cells rapidly increases intracellular polysulfide but has minimal effects on H2S unless Cys or GSH ar...