High Turnover Rates for Hydrogen Sulfide Allow for Rapid Regulation of Its Tissue Concentrations

Abstract Aims: Hydrogen sulfide (H2S) is a signaling molecule, which influences many physiological processes. While H2S is produced and degraded in many cell types, the kinetics of its turnover in different tissues has not been reported. In this study, we have assessed the rates of H2S production in murine liver, kidney, and brain homogenates at pH 7.4, 37°C, and at physiologically relevant cysteine concentrations. We have also studied the kinetics of H2S clearance by liver, kidney, and brain homogenates under aerobic and anaerobic conditions. Results: We find that the rate of H2S production by these tissue homogenates is considerably higher than background rates observed in the absence of exogenous substrates. An exponential decay of H2S with time is observed and, as expected, is significantly faster under aerobic conditions. The half-life for H2S under aerobic conditions is 2.0, 2.8, and 10.0?min with liver, kidney, and brain homogenate, respectively. Western-blot analysis of the sulfur dioxygenase, ETHE1, involved in H2S catabolism, demonstrates higher steady-state protein levels in liver and kidney versus brain. Innovation: By combining experimental and simulation approaches, we demonstrate high rates of tissue H2S turnover and provide estimates of steady-state H2S levels. Conclusion: Our study reveals that tissues maintain a high metabolic flux of sulfur through H2S, providing a rationale for how H2S levels can be rapidly regulated. Antioxid. Redox Signal. 17, 22?31.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/98481/1/ars%2E2011%2E4310.pd

Astrocytic Redox Remodeling by Amyloid Beta Peptide

Abstract Astrocytes are critical for neuronal redox homeostasis providing them with cysteine needed for glutathione synthesis. In this study, we demonstrate that the astrocytic redox response signature provoked by amyloid beta (A-) is distinct from that of a general oxidant (tertiary-butylhydroperoxide [t-BuOOH]). Acute A- treatment increased cystathionine --synthase (CBS) levels and enhanced transsulfuration flux in contrast to repeated A- exposure, which decreased CBS and catalase protein levels. Although t-BuOOH also increased transsulfuration flux, CBS levels were unaffected. The net effect of A- treatment was an oxidative shift in the intracellular glutathione/glutathione disulfide redox potential in contrast to a reductive shift in response to peroxide. In the extracellular compartment, A-, but not t-BuOOH, enhanced cystine uptake and cysteine accumulation, and resulted in remodeling of the extracellular cysteine/cystine redox potential in the reductive direction. The redox changes elicited by A- but not peroxide were associated with enhanced DNA synthesis. CBS activity and protein levels tended to be lower in cerebellum from patients with Alzheimer's disease than in age-matched controls. Our study suggests that the alterations in astrocytic redox status could compromise the neuroprotective potential of astrocytes and may be a potential new target for therapeutic intervention in Alzheimer's disease. Antioxid. Redox Signal. 14, 2385-2397.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90483/1/ars-2E2010-2E3681.pd

Na+ and K+ ion imbalances in Alzheimer's disease

AbstractAlzheimer's disease (AD) is associated with impaired glutamate clearance and depressed Na+/K+ ATPase levels in AD brain that might lead to a cellular ion imbalance. To test this hypothesis, [Na+] and [K+] were analyzed in postmortem brain samples of 12 normal and 16 AD individuals, and in cerebrospinal fluid (CSF) from AD patients and matched controls. Statistically significant increases in [Na+] in frontal (25%) and parietal cortex (20%) and in cerebellar [K+] (15%) were observed in AD samples compared to controls. CSF from AD patients and matched controls exhibited no differences, suggesting that tissue ion imbalances reflected changes in the intracellular compartment. Differences in cation concentrations between normal and AD brain samples were modeled by a 2-fold increase in intracellular [Na+] and an 8–15% increase in intracellular [K+]. Since amyloid beta peptide (Aβ) is an important contributor to AD brain pathology, we assessed how Aβ affects ion homeostasis in primary murine astrocytes, the most abundant cells in brain tissue. We demonstrate that treatment of astrocytes with the Aβ 25–35 peptide increases intracellular levels of Na+ (~2–3-fold) and K+ (~1.5-fold), which were associated with reduced levels of Na+/K+ ATPase and the Na+-dependent glutamate transporters, GLAST and GLT-1. Similar increases in astrocytic Na+ and K+ levels were also caused by Aβ 1–40, but not by Aβ 1–42 treatment. Our study suggests a previously unrecognized impairment in AD brain cell ion homeostasis that might be triggered by Aβ and could significantly affect electrophysiological activity of brain cells, contributing to the pathophysiology of AD

The Quantitative Significance of the Transsulfuration Enzymes for H2S Production in Murine Tissues

The enzymes of the transsulfuration pathway, cystathionine --synthase (CBS) and cystathionine --lyase (CSE), are important for the endogenous production of hydrogen sulfide (H2S), a gaseous signaling molecule. The relative contributions of CBS and CSE to H2S generation in different tissues are not known. In this study, we report quantification of CBS and CSE in murine liver and kidney and their contribution to H2S generation in these tissues and in brain at saturating substrate concentrations. We show that CBS protein levels are significantly lower than those of CSE; 60-fold and 20-fold in liver and kidney, respectively. Each enzyme is more abundant in liver compared with kidney, twofold and sixfold for CBS and CSE, respectively. At high substrate concentrations (20-mM each cysteine and homocysteine), the capacity for liver H2S production is approximately equal for CBS and CSE, whereas in kidney and brain, CBS constitutes the major source of H2S, accounting for -80% and -95%, respectively, of the total output. At physiologically relevant concentrations of substrate, and adjusting for the differences in CBS versus CSE levels, we estimate that CBS accounts for only 3% of H2S production by the transsulfuration pathway enzymes in liver. Antioxid. Redox Signal. 15, 363-372.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90506/1/ars-2E2010-2E3781.pd

Structural and Mechanistic Insights into Hemoglobincatalyzed Hydrogen Sulfide Oxidation and the Fate of Polysulfide Products

Hydrogen sulfide is a cardioprotective signaling molecule but is toxic at elevated concentrations. Red blood cells can synthesize H2S but, lacking organelles, cannot dispose of H2S via the mitochondrial sulfide oxidation pathway. We have recently shown that at high sulfide concentrations, ferric hemoglobin oxidizes H2S to a mixture of thiosulfate and iron-bound polysulfides in which the latter species predominates. Here, we report the crystal structure of human hemoglobin containing low spin ferric sulfide, the first intermediate in heme-catalyzed sulfide oxidation. The structure provides molecular insights into why sulfide is susceptible to oxidation in human hemoglobin but is stabilized against it in HbI, a specialized sulfide-carrying hemoglobin from a mollusk adapted to life in a sulfide-rich environment. We have also captured a second sulfide bound at a postulated ligand entry/exit site in the α-subunit of hemoglobin, which, to the best of our knowledge, represents the first direct evidence for this site being used to access the heme iron. Hydrodisulfide, a postulated intermediate at the junction between thiosulfate and polysulfide formation, coordinates ferric hemoglobin and, in the presence of air, generated thiosulfate. At low sulfide/heme iron ratios, the product distribution between thiosulfate and iron-bound polysulfides was approximately equal. The iron-bound polysulfides were unstable at physiological glutathione concentrations and were reduced with concomitant formation of glutathione persulfide, glutathione disulfide, and H2S. Hence, although polysulfides are unlikely to be stable in the reducing intracellular milieu, glutathione persulfide could serve as a persulfide donor for protein persulfidation, a posttranslational modification by which H2S is postulated to signal

Differential Dependence on Cysteine from Transsulfuration versus Transport During T Cell Activation

The synthesis of glutathione, a major cellular antioxidant with a critical role in T cell proliferation, is limited by cysteine. In this study, we evaluated the contributions of the xC- cystine transporter and the transsulfuration pathway to cysteine provision for glutathione synthesis and antioxidant defense in naive versus activated T cells and in the immortalized T lymphocyte cell line, Jurkat. We show that the xC- transporter, although absent in naive T cells, is induced after activation, releasing T cells from their cysteine dependence on antigen-presenting cells. We also demonstrate the existence of an intact transsulfuration pathway in naive and activated T cells and in Jurkat cells. The flux through the transsulfuration pathway increases in primary but not in transformed T cells in response to oxidative challenge by peroxide. Inhibition of the transsulfuration pathway in both primary and transformed T cells decreases cell viability under oxidative-stress conditions. Antioxid. Redox Signal. 15, 39-47.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90472/1/ars-2E2010-2E3496.pd

Structural and Mechanistic Insights into Hemoglobincatalyzed Hydrogen Sulfide Oxidation and the Fate of Polysulfide Products

Hydrogen sulfide is a cardioprotective signaling molecule but is toxic at elevated concentrations. Red blood cells can synthesize H2S but, lacking organelles, cannot dispose of H2S via the mitochondrial sulfide oxidation pathway. We have recently shown that at high sulfide concentrations, ferric hemoglobin oxidizes H2S to a mixture of thiosulfate and iron-bound polysulfides in which the latter species predominates. Here, we report the crystal structure of human hemoglobin containing low spin ferric sulfide, the first intermediate in heme-catalyzed sulfide oxidation. The structure provides molecular insights into why sulfide is susceptible to oxidation in human hemoglobin but is stabilized against it in HbI, a specialized sulfide-carrying hemoglobin from a mollusk adapted to life in a sulfide-rich environment. We have also captured a second sulfide bound at a postulated ligand entry/exit site in the α-subunit of hemoglobin, which, to the best of our knowledge, represents the first direct evidence for this site being used to access the heme iron. Hydrodisulfide, a postulated intermediate at the junction between thiosulfate and polysulfide formation, coordinates ferric hemoglobin and, in the presence of air, generated thiosulfate. At low sulfide/heme iron ratios, the product distribution between thiosulfate and iron-bound polysulfides was approximately equal. The iron-bound polysulfides were unstable at physiological glutathione concentrations and were reduced with concomitant formation of glutathione persulfide, glutathione disulfide, and H2S. Hence, although polysulfides are unlikely to be stable in the reducing intracellular milieu, glutathione persulfide could serve as a persulfide donor for protein persulfidation, a posttranslational modification by which H2S is postulated to signal

Structural and Mechanistic Insights into Hemoglobincatalyzed Hydrogen Sulfide Oxidation and the Fate of Polysulfide Products

Hydrogen sulfide is a cardioprotective signaling molecule but is toxic at elevated concentrations. Red blood cells can synthesize H2S but, lacking organelles, cannot dispose of H2S via the mitochondrial sulfide oxidation pathway. We have recently shown that at high sulfide concentrations, ferric hemoglobin oxidizes H2S to a mixture of thiosulfate and iron-bound polysulfides in which the latter species predominates. Here, we report the crystal structure of human hemoglobin containing low spin ferric sulfide, the first intermediate in heme-catalyzed sulfide oxidation. The structure provides molecular insights into why sulfide is susceptible to oxidation in human hemoglobin but is stabilized against it in HbI, a specialized sulfide-carrying hemoglobin from a mollusk adapted to life in a sulfide-rich environment. We have also captured a second sulfide bound at a postulated ligand entry/exit site in the α-subunit of hemoglobin, which, to the best of our knowledge, represents the first direct evidence for this site being used to access the heme iron. Hydrodisulfide, a postulated intermediate at the junction between thiosulfate and polysulfide formation, coordinates ferric hemoglobin and, in the presence of air, generated thiosulfate. At low sulfide/heme iron ratios, the product distribution between thiosulfate and iron-bound polysulfides was approximately equal. The iron-bound polysulfides were unstable at physiological glutathione concentrations and were reduced with concomitant formation of glutathione persulfide, glutathione disulfide, and H2S. Hence, although polysulfides are unlikely to be stable in the reducing intracellular milieu, glutathione persulfide could serve as a persulfide donor for protein persulfidation, a posttranslational modification by which H2S is postulated to signal

Doxorubicin pharmacokinetics in lymphoma patients treated with doxorubicin-loaded eythrocytes

Doxorubicin-loaded erythrocytes (DLE) were administrated to 15 lymphoma patients. Antibiotic peak concentration in blood decreased by 55%, doxorubicin circulated several times longer, and the area under the concentration-time curve increased 5 times if compared with standard doxorubicin administration. The DLE was well tolerated by patients

An Allosteric Mechanism for Switching between Parallel Tracks in Mammalian Sulfur Metabolism

Methionine (Met) is an essential amino acid that is needed for the synthesis of S-adenosylmethionine (AdoMet), the major biological methylating agent. Methionine used for AdoMet synthesis can be replenished via remethylation of homocysteine. Alternatively, homocysteine can be converted to cysteine via the transsulfuration pathway. Aberrations in methionine metabolism are associated with a number of complex diseases, including cancer, anemia, and neurodegenerative diseases. The concentration of methionine in blood and in organs is tightly regulated. Liver plays a key role in buffering blood methionine levels, and an interesting feature of its metabolism is that parallel tracks exist for the synthesis and utilization of AdoMet. To elucidate the molecular mechanism that controls metabolic fluxes in liver methionine metabolism, we have studied the dependencies of AdoMet concentration and methionine consumption rate on methionine concentration in native murine hepatocytes at physiologically relevant concentrations (40–400 µM). We find that both [AdoMet] and methionine consumption rates do not change gradually with an increase in [Met] but rise sharply (∼10-fold) in the narrow Met interval from 50 to 100 µM. Analysis of our experimental data using a mathematical model reveals that the sharp increase in [AdoMet] and the methionine consumption rate observed within the trigger zone are associated with metabolic switching from methionine conservation to disposal, regulated allosterically by switching between parallel pathways. This regulatory switch is triggered by [Met] and provides a mechanism for stabilization of methionine levels in blood over wide variations in dietary methionine intake