David W. Kraus

Hydrogen sulfide attenuates myocardial ischemia-reperfusion injury by preservation of mitochondrial function

The recent discovery that hydrogen sulfide (H2S) is an endogenously produced gaseous second messenger capable of modulating many physiological processes, much like nitric oxide, prompted us to investigate the potential of H2S as a cardioprotective agent. In the current study, we demonstrate that the delivery of H2S at the time of reperfusion limits infarct size and preserves left ventricular (LV) function in an in vivo model of myocardial ischemia-reperfusion (MI-R). This observed cytoprotection is associated with an inhibition of myocardial inflammation and a preservation of both mitochondrial structure and function after I-R injury. Additionally, we show that modulation of endogenously produced H2S by cardiac-specific overexpression of cystathionine γ-lyase (α-MHC-CGL-Tg mouse) significantly limits the extent of injury. These findings demonstrate that H2S may be of value in cytoprotection during the evolution of myocardial infarction and that either administration of H2S or the modulation of endogenous production may be of clinical benefit in ischemic disorders.

Hydrogen sulfide mediates the vasoactivity of garlic

The consumption of garlic is inversely correlated with the progression of cardiovascular disease, although the responsible mechanisms remain unclear. Here we show that human RBCs convert garlic-derived organic polysulfides into hydrogen sulfide (H2S), an endogenous cardioprotective vascular cell signaling molecule. This H2S production, measured in real time by a novel polarographic H2S sensor, is supported by glucose-maintained cytosolic glutathione levels and is to a large extent reliant on reduced thiols in or on the RBC membrane. H2S production from organic polysulfides is facilitated by allyl substituents and by increasing numbers of tethering sulfur atoms. Allyl-substituted polysulfides undergo nucleophilic substitution at the α carbon of the allyl substituent, thereby forming a hydropolysulfide (RSnH), a key intermediate during the formation of H2S. Organic polysulfides (R-Sn-R′; n > 2) also undergo nucleophilic substitution at a sulfur atom, yielding RSnH and H2S. Intact aorta rings, under physiologically relevant oxygen levels, also metabolize garlic-derived organic polysulfides to liberate H2S. The vasoactivity of garlic compounds is synchronous with H2S production, and their potency to mediate relaxation increases with H2S yield, strongly supporting our hypothesis that H2S mediates the vasoactivity of garlic. Our results also suggest that the capacity to produce H2S can be used to standardize garlic dietary supplements.

High fat diet induces dysregulation of hepatic oxygen gradients and mitochondrial function in vivo.

NAFLD (non-alcoholic fatty liver disease), associated with obesity and the cardiometabolic syndrome, is an important medical problem affecting up to 20% of western populations. Evidence indicates that mitochondrial dysfunction plays a critical role in NAFLD initiation and progression to the more serious condition of NASH (non-alcoholic steatohepatitis). Herein we hypothesize that mitochondrial defects induced by exposure to a HFD (high fat diet) contribute to a hypoxic state in liver and this is associated with increased protein modification by RNS (reactive nitrogen species). To test this concept, C57BL/6 mice were pair-fed a control diet and HFD containing 35% and 71% total calories (1 cal≈4.184 J) from fat respectively, for 8 or 16 weeks and liver hypoxia, mitochondrial bioenergetics, NO (nitric oxide)-dependent control of respiration, and 3-NT (3-nitrotyrosine), a marker of protein modification by RNS, were examined. Feeding a HFD for 16 weeks induced NASH-like pathology accompanied by elevated triacylglycerols, increased CYP2E1 (cytochrome P450 2E1) and iNOS (inducible nitric oxide synthase) protein, and significantly enhanced hypoxia in the pericentral region of the liver. Mitochondria from the HFD group showed increased sensitivity to NO-dependent inhibition of respiration compared with controls. In addition, accumulation of 3-NT paralleled the hypoxia gradient in vivo and 3-NT levels were increased in mitochondrial proteins. Liver mitochondria from mice fed the HFD for 16 weeks exhibited depressed state 3 respiration, uncoupled respiration, cytochrome c oxidase activity, and mitochondrial membrane potential. These findings indicate that chronic exposure to a HFD negatively affects the bioenergetics of liver mitochondria and this probably contributes to hypoxic stress and deleterious NO-dependent modification of mitochondrial proteins.

Sulfide consumption by mussel gill mitochondria is not strictly tied to oxygen reduction: measurements using a novel polarographic sulfide sensor.

SUMMARY Some organisms that survive in environments rich in hydrogen sulfide possess specific metabolic pathways for sulfide oxidation and subsequent use of reducing equivalents in oxidative phosphorylation, a process called chemolithoheterotrophy. This process is dependent on ambient oxygen partial pressure and environmental sulfide exposure. To define accurately the kinetics of sulfide metabolism and its dependence on cellular conditions, we have developed a polarographic sulfide sensor (PSS) to measure sulfide concentrations directly and continuously under physiological conditions. The ribbed mussel Geukensia demissa, an inhabitant of sulfide-rich coastal sediments, consumes sulfide in a chemolithoheterotrophic metabolic strategy. Gill mitochondria use sulfide as respiratory substrate for ATP production, and sulfide consumption is sufficiently rapid and so kinetically complex that only continuous real-time detection captures these events. Under normoxic conditions, oxygen and sulfide consumption are matched. Under hypoxic to anoxic conditions, however, sulfide consumption continues without commensurate oxygen consumption, and these results can be duplicated at higher oxygen conditions by selective blockade of terminal oxidases. These metabolic capabilities depend on prior environmental sulfide exposure, which suggests substantial mitochondrial metabolic plasticity. The recent finding that endogenous sulfide is a critical cell signaling molecule in all organisms suggests that the metabolic pathways that tightly control cellular sulfide levels are widespread. Sensors that accurately report sulfide concentrations under physiologically relevant conditions are valuable tools with which to explore the expanding role of sulfide in biological systems.

Novel Method for Measuring S-Nitrosothiols Using Hydrogen Sulfide

Recent advances in techniques that allow sensitive and specific measurement of S-nitrosothiols (RSNOs) have provided evidence for a role for these compounds in various aspects of nitric oxide (NO) biology. The most widely used approach is to couple reaction chemistry that selectively reduces RSNOs by one electron to produce NO, with the sensitive detection of the latter under anaerobic conditions using ozone based chemiluminescence in NO analyzers. Herein, we report a novel reaction that is readily adaptable for commercial NO analyzers that utilizes hydrogen sulfide (H2S), a gas that can reduce RSNO to NO and, analogous to NO, is produced by endogenous metabolism and has effects on diverse biological functions. We discuss factors that affect H2S based methods for RSNO measurement and discuss the potential of H2S as an experimental tool to measure RSNO.

Sulfide-Stimulation of Oxygen Consumption Rate and Cytochrome Reduction in Gills of the Estuarine Mussel Geukensia demissa

Organisms, such as the mussel Geukensia demissa, that inhabit high-sulfide sediments have mechanisms that impede sulfide poisoning of aerobic respiration. Oxygen consumption rates (nO2) of excised ciliated gills from freshly collected G. demissa were stimulated 3-fold at sulfide concentrations between 200 and 500 μM and remained stimulated at 1000 μM. Maintenance of mussels in sulfide-free conditions resulted in less stimulation of gill nO2 at <500 μM sulfide and inhibition between 500 and 1000 {mu}M sulfide. Gills of Mytilus galloprovincialis from a sulfide-free environment were inhibited by {ge}200 μM sulfide. These results indicate that sulfide stimulation of nO2 may be correlated to environmental exposure to sulfide. Serotonin, a neurohormonal stimulant of ciliary beating, further increased sulfide-stimulated nO2, possibly in support of energy demand. Sulfide-stimulated nO2 was negligible in boiled gills and was 61% inhibited by cyanide, implicating the participation of mitochondrial electron flux. Mitochondrial cytochromes c and oxidase oxidation/ reduction state changed little at <500 μM sulfide, but reduction occurred at 500-2000 μM sulfide, suggesting that although cytochrome oxidation/reduction state may be regulated in the face of increased electron flux, regulation may fail at inhibitory sulfide levels. Sulfide-stimulated nO2 may represent a detoxification mechanism in G. demissa.

A Physiological Comparison of Bivalve Mollusc Cerebro-visceral Connectives With and Without Neurohemoglobin. II. Neurohemoglobin Characteristics

Several bivalve mollusc species possess hemoglobin in their nervous systems whereas most species do not. The function of this neurohemoglobin was investigated in situ in cerebro-visceral connectives of Tellina alternata and Spisula solidissima. Both neurohemoglobins, located in glial cells, exhibit high oxygen affinities and relatively high Hill numbers. The rate of oxygen diffusion into the connective begins to fall below the consumption rate near the PO2 at which each neurohemoglobin begins to unload oxygen, assuming the perineural sheath presents an effective barrier to oxygen diffusion. The neurohemoglobin could thus act as an oxygen store during periods of low PO2. Oxygen unloading from the neurohemoglobin proceeds for a considerable length of time at a constant rate. The long duration may be attributed to the geometry of the connective and to the perineural sheath, whose primary function may be to retain oxygen within the connective during anoxic conditions. The constant unloading rate may be attributed to neurohemoglobin cooperativity in situ because the driving force for unloading remains nearly constant at the P50 of each neurohemoglobin. An oxygen supply at a constant rate for an extended period of time would be useful to an animal requiring aerobic nervous function during anoxic conditions.

Assessing NO-dependent vasodilatation using vessel bioassays at defined oxygen tensions.

Results from vessel bioassays have provided the foundation for much of our understanding of the mechanisms that control vascular homeostasis and blood flow. The seminal observations that led to the discovery that nitric oxide (NO) is a critical mediator of vascular relaxation were made with the use of such methodology, and many studies have used NO-dependent vessel relaxation as an experimental readout for understanding mechanisms that regulate vascular NO function. Studies have coupled controlling oxygen tensions within vessel bioassay chambers to begin to understand how oxygen-specifically hypoxia-regulate NO function, and this context has identified red cells-specifically hemoglobin within-as critical modulators. Alone, vessel bioassays or measuring oxygen partial pressures (pO2) is relatively straightforward, but the combination necessitates consideration of several factors. We use the example of deoxygenated red cells/hemoglobin-dependent potentiation of nitrite-dependent dilation to illustrate the salient factors that are critical to consider in designing and interpreting experiments aimed at understanding the interplay between oxygen and NO function in the vasculature.

Metabolic and cardiac signaling effects of inhaled hydrogen sulfide and low oxygen in male rats

Low concentrations of inhaled hydrogen sulfide (H(2)S) induce hypometabolism in mice. Biological effects of H(2)S in in vitro systems are augmented by lowering O(2) tension. Based on this, we hypothesized that reduced O(2) tension would increase H(2)S-mediated hypometabolism in vivo. To test this, male Sprague-Dawley rats were exposed to 80 ppm H(2)S at 21% O(2) or 10.5% O(2) for 6 h followed by 1 h recovery at room air. Rats exposed to H(2)S in 10.5% O(2) had significantly decreased body temperature and respiration compared with preexposure levels. Heart rate was decreased by H(2)S administered under both O(2) levels and did not return to preexposure levels after 1 h recovery. Inhaled H(2)S caused epithelial exfoliation in the lungs and increased plasma creatine kinase-MB activity. The effect of inhaled H(2)S on prosurvival signaling was also measured in heart and liver. H(2)S in 21% O(2) increased Akt-P(Ser473) and GSK-3β-P(Ser9) in the heart whereas phosphorylation was decreased by H(2)S in 10.5% O(2), indicating O(2) dependence in regulating cardiac signaling pathways. Inhaled H(2)S and low O(2) had no effect on liver Akt. In summary, we found that lower O(2) was needed for H(2)S-dependent hypometabolism in rats compared with previous findings in mice. This highlights the possibility of species differences in physiological responses to H(2)S. Inhaled H(2)S exposure also caused tissue injury to the lung and heart, which raises concerns about the therapeutic safety of inhaled H(2)S. In conclusion, these findings demonstrate the importance of O(2) in influencing physiological and signaling effects of H(2)S in mammalian systems.

A Physiological Comparison of Bivalve Mollusc Cerebro-visceral Connectives With and Without Neurohemoglobin. III. Oxygen Demand

Several bivalve mollusc species possess neurohemoglobin in their nervous systems whereas most species do not. The cerebro-visceral connectives of Tellina alternata and Spisula solidissima with neurohemoglobin and Tagelus plebeius and Geukensia demissa without neurohemoglobin exhibit similar electrical characteristics dictated mostly by axon size (0.3-0.4 µm mean axon diameter, Kraus et al., 1988). Action potential conduction is sensitive to a depletion of both ambient and neurohemoglobin-bound oxygen. Connectives without neurohemoglobin and connectives with carbon monoxide neurohemoglobin ceased to conduct action potentials within 5-10 minutes after exposure to anoxic conditions. Connectives with neurohemoglobin conducted action potentials throughout the time course of neurohemoglobin deoxygenation, lasting 20-30 mintues.Connectives without neurohemoglobin exhibited an approximate five-fold elevation in oxygen consumption rate during action potential conduction, as predicted by axon diameter. However, con...