Hydrogen sulfide and nitric oxide metabolites in the blood of free-ranging brown bears and their potential roles in hibernation

During winter hibernation, brown bears (Ursus arctos) lie in dens for half a year without eating while their basal metabolism is largely suppressed. To understand the underlying mechanisms of metabolic depression in hibernation, we measured type and content of blood metabolites of two ubiquitous inhibitors of mitochondrial respiration, hydrogen sulfide (H₂S) and nitric oxide (NO), in winter-hibernating and summer-active free-ranging Scandinavian brown bears. We found that levels of sulfide metabolites were overall similar in summer-active and hibernating bears but their composition in the plasma differed significantly, with a decrease in bound sulfane sulfur in hibernation. High levels of unbound free sulfide correlated with high levels of cysteine (Cys) and with low levels of bound sulfane sulfur, indicating that during hibernation H₂S, in addition to being formed enzymatically from the substrate Cys, may also be regenerated from its oxidation products, including thiosulfate and polysulfides. In the absence of any dietary intake, this shift in the mode of H₂S synthesis would help preserve free Cys for synthesis of glutathione (GSH), a major antioxidant found at high levels in the red blood cells of hibernating bears. In contrast, circulating nitrite and erythrocytic S-nitrosation of glyceraldehyde-3-phosphate dehydrogenase, taken as markers of NO metabolism, did not change appreciably. Our findings reveal that remodeling of H₂S metabolism and enhanced intracellular GSH levels are hallmarks of the aerobic metabolic suppression of hibernating bears.     

Regulation of Sulfur Nutrition in Wild-Type and Transgenic Poplar Over-Expressing γ-Glutamylcysteine Synthetase in the Cytosol as Affected by Atmospheric H2S

This study with poplar (Populus tremula × Populus alba) cuttings was aimed to test the hypothesis that sulfate uptake is regulated by demand-driven control and that this regulation is mediated by phloem-transported glutathione as a shoot-to-root signal. Therefore, sulfur nutrition was investigated at (a) enhanced sulfate demand in transgenic poplar over-expressing γ-glutamylcysteine (γ-EC) synthetase in the cytosol and (b) reduced sulfate demand during short-term exposure to H₂S. H₂S taken up by the leaves increased cysteine, γ-EC, and glutathione concentrations in leaves, xylem sap, phloem exudate, and roots, both in wild-type and transgenic poplar. The observed reduced xylem loading of sulfate after H₂S exposure of wild-type poplar could well be explained by a higher glutathione concentration in the phloem. In transgenic poplar increased concentrations of glutathione and γ-EC were found not only in leaves, xylem sap, and roots but also in phloem exudate irrespective of H₂S exposure. Despite enhanced phloem allocation of glutathione and its accumulation in the roots, sulfate uptake was strongly enhanced. This finding is contradictory to the hypothesis that glutathione allocated in the phloem reduces sulfate uptake and its transport to the shoot. Correlation analysis provided circumstantial evidence that the sulfate to glutathione ratio in the phloem may control sulfate uptake and loading into the xylem, both when the sulfate demand of the shoot is increased and when it is reduced.     

Hydrogen Sulfide Promotes Adipogenesis in 3T3L1 Cells

The effect of hydrogen sulfide (H₂S) on differentiation of 3T3L1-derived adipocytes was examined. Endogenous H₂S was increased after 3T3L1 differentiation. The expression of the H₂S-synthesising enzymes, cystathionine γ-lyase (CSE), cystathionine β-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST), was increased in a time-dependent manner during 3T3L1 differentiation. Expression of genes associated with adipogenesis related genes including fatty acid binding protein 4 (FABP4/aP2), a key regulator of this process, was increased by GYY4137 (a slow-releasing H₂S donor compound) and sodium hydrosulfide (NaHS, a classical H₂S donor) but not by ZYJ1122 or time-expired NaHS. Furthermore expression of these genes were reduced by aminooxyacetic acid (AOAA, CBS inhibitor), DL-propargylglycine (PAG, CSE inhibitor) as well as by CSE small interference RNA (siCSE) and siCBS. The size and number of lipid droplets in mature adipocytes was significantly increased by both GYY4137 and NaHS, which also impaired the ability of CL316,243 (β3-agonist) to promote lipolysis in these cells. In contrast, AOAA and PAG had the opposite effect. Taken together, we show that the H₂S-synthesising enzymes CBS, CSE and 3-MST are endogenously expressed during adipogenesis and that both endogenous and exogenous H₂S modulate adipogenesis and adipocyte maturation.     

Isotope dilution mass spectrometry for the quantification of sulfane sulfurs

Sulfane sulfurs are one type of important reactive sulfur species. These molecules have unique reactivity that allows them to attach reversibly to other sulfur atoms and exhibit regulatory effects in diverse biological systems. Recent studies have suggested that sulfane sulfurs are involved in signal transduction processes regulated by hydrogen sulfide (H₂S). Accurate and reliable measurements of sulfane sulfurs in biological samples are thus needed to reveal their production and mechanisms of actions. Herein we report a convenient and accurate method for the determination of sulfane sulfur concentrations. The method employs a triphenylphosphine derivative (P2) to capture sulfane sulfurs as a stable phosphine sulfide product, PS2. The concentration of PS2 was then determined by isotope dilution mass spectrometry, using a ¹³C₃-labeled phosphine sulfide, PS1, as the internal standard. The specificity and efficiency of the method were proven by model reactions. It was also applied to the measurement of sulfane sulfurs in mouse tissues including brain, kidney, lung, liver, heart, spleen, and blood.     

Regulation of Vascular Tone,Angiogenesis and Cellular Bioenergetics by the 3-Mercaptopyruvate Sulfurtransferase/H2S Pathway: Functional Impairment by Hyperglycemia and Restoration by dl-α-Lipoic Acid

Hydrogen sulfide (H₂S), as a reducing agent and an antioxidant molecule, exerts protective effects against hyperglycemic stress in the vascular endothelium. The mitochondrial enzyme 3-mercaptopyruvate sulfurtransferase (3-MST) is an important biological source of H₂S. We have recently demonstrated that 3-MST activity is inhibited by oxidative stress in vitro and speculated that this may have an adverse effect on cellular homeostasis. In the current study, given the importance of H₂S as a vasorelaxant, angiogenesis stimulator and cellular bioenergetic mediator, we first determined whether the 3-MST/H₂S system plays a physiological regulatory role in endothelial cells. Next, we tested whether a dysfunction of this pathway develops during the development of hyperglycemia and μmol/L to diabetes-associated vascular complications. Intraperitoneal (IP) 3-MP (1 mg/kg) raised plasma H₂S levels in rats. 3-MP (10 1 mmol/L) promoted angiogenesis in vitro in bEnd3 microvascular endothelial cells and in vivo in a Matrigel assay in mice (0.3–1 mg/kg). In vitro studies with bEnd3 cell homogenates demonstrated that the 3-MP-induced increases in H₂S production depended on enzymatic activity, although at higher concentrations (1–3 mmol/L) there was also evidence for an additional nonenzymatic H₂S production by 3-MP. In vivo, 3-MP facilitated wound healing in rats, induced the relaxation of dermal microvessels and increased mitochondrial bioenergetic function. In vitro hyperglycemia or in vivo streptozotocin diabetes impaired angiogenesis, attenuated mitochondrial function and delayed wound healing; all of these responses were associated with an impairment of the proangiogenic and bioenergetic effects of 3-MP. The antioxidants dl-α-lipoic acid (LA) in vivo, or dihydrolipoic acid (DHLA) in vitro restored the ability of 3-MP to stimulate angiogenesis, cellular bioenergetics and wound healing in hyperglycemia and diabetes. We conclude that diabetes leads to an impairment of the 3-MST/H₂S pathway, and speculate that this may contribute to the pathogenesis of hyperglycemic endothelial cell dysfunction. We also suggest that therapy with H₂S donors, or treatment with the combination of 3-MP and lipoic acid may be beneficial in improving angiogenesis and bioenergetics in hyperglycemia.     

Analytical measurement of discrete hydrogen sulfide pools in biological specimens

Hydrogen sulfide (H₂S) is a ubiquitous gaseous signaling molecule that plays a vital role in numerous cellular functions and has become the focus of many research endeavors, including pharmacotherapeutic manipulation. Among the challenges facing the field is the accurate measurement of biologically active H₂S. We have recently reported that the typically used methylene blue method and its associated results are invalid and do not measure bona fide H₂S. The complexity of analytical H₂S measurement reflects the fact that hydrogen sulfide is a volatile gas and exists in the body in various forms, including a free form, an acid-labile pool, and bound as sulfane sulfur. Here we describe a new protocol to discretely measure specific H₂S pools using the monobromobimane method coupled with RP-HPLC. This new protocol involves selective liberation, trapping, and derivatization of H₂S. Acid-labile H₂S is released by incubating the sample in an acidic solution (pH 2.6) of 100 mM phosphate buffer with 0.1 mM diethylenetriaminepentaacetic acid (DTPA), in an enclosed system to contain volatilized H₂S. Volatilized H₂S is then trapped in 100 mM Tris–HCl (pH 9.5, 0.1 mM DTPA) and then reacted with excess monobromobimane. In a separate aliquot, the contribution of the bound sulfane sulfur pool was measured by incubating the sample with 1 mM TCEP (tris(2-carboxyethyl)phosphine hydrochloride), a reducing agent, to reduce disulfide bonds, in 100 mM phosphate buffer (pH 2.6, 0.1 mM DTPA), and H₂S measurement was performed in a manner analogous to the one described above. The acid-labile pool was determined by subtracting the free hydrogen sulfide value from the value obtained by the acid-liberation protocol. The bound sulfane sulfur pool was determined by subtracting the H₂S measurement from the acid-liberation protocol alone compared to that of TCEP plus acidic conditions. In summary, our new method allows very sensitive and accurate measurement of the three primary biological pools of H₂S, including free, acid-labile, and bound sulfane sulfur, in various biological specimens.     

Microbial regulation of host hydrogen sulfide bioavailability and metabolism

Hydrogen sulfide (H₂S), generated through various endogenous enzymatic and nonenzymatic pathways, is emerging as a regulator of physiological and pathological events throughout the body. Bacteria in the gastrointestinal tract also produce significant amounts of H₂S that regulates microflora growth and virulence responses. However, the impact of the microbiota on host global H₂S bioavailability and metabolism remains unknown. To address this question, we examined H₂S bioavailability in its various forms (free, acid labile, or bound sulfane sulfur), cystathionine γ-lyase (CSE) activity, and cysteine levels in tissues from germ-free versus conventionally housed mice. Free H₂S levels were significantly reduced in plasma and gastrointestinal tissues of germ-free mice. Bound sulfane sulfur levels were decreased by 50–80% in germ-free mouse plasma and adipose and lung tissues. Tissue CSE activity was significantly reduced in many organs from germ-free mice, whereas tissue cysteine levels were significantly elevated compared to conventional mice. These data reveal that the microbiota profoundly regulates systemic bioavailability and metabolism of H₂S.     

Serum γ-glutamyl Transferase Levels,Insulin Resistance and Liver Fibrosis in Patients with Chronic Liver Diseases

Background and Aims

Serum levels of γ-glutamyl-transpeptidase(γ-GT) were associated with liver disease severity and metabolic alterations, which in turn are able to affect hepatic damage. In patients with nonalcoholic fatty liver disease (NAFLD), genotype 1 chronic hepatitis C (G1CHC) and chronic hepatitis B (CHB), we assessed the link between liver fibrosis and γ-GT serum levels, and we evaluated if normal or high γ-GT serum levels affect the association between insulin resistance (IR) and severity of liver fibrosis.

Methods

843 consecutive patients with chronic liver disease (CLD)(193 NAFLD, 481 G1CHC, 169 CHB) were evaluated by liver biopsy (Kleiner and Scheuer scores) and clinical and metabolic measurements. IR was diagnosed if HOMA>3. A serum γ-GT concentration of >36 IU/L in females and >61 IU/L in males was considered the threshold value for identifying high levels of γ-GT.

Results

By multivariate logistic regression analysis, abnormal γ-GT serum levels were independently linked to severe liver fibrosis in patients with NAFLD (OR2.711,CI1.120–6.564,p = 0.02), G1CHC (OR3.461,CI2.138–5.603,p<0.001) and CHB (OR2.778,CI1.042–7.414,p = 0.04), together with IR and liver necroinflammation, and with a negative predictive value>80%. Interestingly, among patients with high or normal γ-GT values, even if IR prevalence was significantly higher in patients with severe fibrosis compared to those without, IR remained significantly associated with severe fibrosis in patients with abnormal γ-GT values only (OR4.150,CI1.079–15.970,p = 0.03 for NAFLD; OR2.250,CI1.211–4.181,p = 0.01 for G1CHC; OR3.096,CI2.050–34.220,p = 0.01 for CHB).

Conclusions

In patients with CLD, IR is independently linked to liver fibrosis only in patients with abnormal γ-GT values, without differences according to liver disease etiology, and suggesting a role of γ-GT as a marker of metabolic-induced liver damage. These data could be useful for the clinical and pharmacologic management of patients with CLD.     

Evidence for an intracellular sulfur cycle in cucumber leaves

H₂S emission from cucumber (Cucumis sativus L.) leaf discs supplied with L-cysteine in the dark is inhibited 80–90% by aminooxyacetic acid (AOA), an inhibitor of pyridoxal-phosphate dependent enzymes. Exposure to L-cysteine in the light enhanced the emission of H₂S in response to this sulfur source. Turning off the light reduced the emission of H₂S to the rate observed in continuous dark; turning on the light enhanced the emission of H₂S to the rate observed in continuous light. Therefore, in the light H₂S emission in response to L-cysteine becomes a partially light-dependent process. Treatment with cyanazine, an inhibitor of photosynthetic electron transport, reduced H₂S emission in the light to the rate observed in continuous dark, but did not affect H₂S emission in the dark. In leaf discs pre-exposed to L-cysteine in the light, treatment with cyanazine+ AOA inhibited the emission of H₂S in response to L-cysteine completely. Therefore, only part of the H₂S emitted in response to this sulfur source is derived from a light-independent, but pyridoxal-phosphate-dependent process; the balance of the H₂S emitted is derived from a light-dependent process that can be inhibited by cyanazine. When cucumber leaf discs were supplied with a pulse of L-[^35S]cysteine, radioactively labeled H₂S was emitted in two waves, one during the first hour of exposure to L-cysteine, and a second after 3–4 h; unlabeled H₂S, however, was emitted continuously. The second wave of emission of labeled H₂S was not observed in pulse-chase experiments in which sulfate or cyanazine were added to the treatment solution after 3 h of exposure to L-cysteine, or when the lights was turned off. The labeling pattern of sulfur compounds inside cucumber cells supplied with a pulse of L-[³⁵S]cysteine showed that the labeled H₂S released from L-cysteine partially enters first the sulfite, then the sulfate pool of the cells. The radioactively labeled sulfate, however, is not incorporated into L-cysteine, but enters the H₂S pool of the cells again. These observations are consistent with the idea of an intracellular sulfur cycle in plant cells. The L-cysteine taken up by the leaf discs seems to be desulfhydrated in a light-independent, but pyridoxal-phosphate-dependent process. The H₂S synthesized this way may be partially released into the atmosphere; the other part of the H₂S produced in response to L-cysteine may be oxidized to sulfite, then to sulfate, which is subsequently reduced via the light-depent sulfate assimilation pathway. In the presence of excess L-cysteine, synthesis of additional cysteine may be inhibited, and the sulfide moiety may be split off carrier bound sulfide to enter the H₂S pool of the cells again. It is suggested that the function of this sulfur cycle may be regulation of the free cysteine pool.Abbreviation AOA aminooxyacetic acid     

Role of Hydrogen Sulfide in Early Blood-Brain Barrier Disruption following Transient Focal Cerebral Ischemia

We determined the role of endogenous hydrogen sulfide (H₂S) in cerebral vasodilation/hyperemia and early BBB disruption following ischemic stroke. A cranial window was prepared over the left frontal, parietal and temporal cortex in mice. Transient focal cerebral Ischemia was induced by directly ligating the middle cerebral artery (MCA) for two hours. Regional vascular response and cerebral blood flow (CBF) during ischemia and reperfusion were measured in real time. Early BBB disruption was assessed by Evans Blue (EB) and sodium fluorescein (Na-F) extravasation at 3 hours of reperfusion. Topical treatment with DL-propargylglycine (PAG, an inhibitor for cystathionine γ-lyase (CSE)) and aspartate (ASP, inhibitor for cysteine aminotransferase/3-mercaptopyruvate sulfurtransferase (CAT/3-MST)), but not O-(Carboxymethyl)hydroxylamine hemihydrochloride (CHH, an inhibitor for cystathionine β-synthase (CBS)), abolished postischemic cerebral vasodilation/hyperemia and prevented EB and Na-F extravasation. CSE knockout (CSE^-/-) reduced postischemic cerebral vasodilation/hyperemia but only inhibited Na-F extravasation. An upregulated CBS was found in cerebral cortex of CSE^-/- mice. Topical treatment with CHH didn’t further alter postischemic cerebral vasodilation/hyperemia, but prevented EB extravasation in CSE^-/- mice. In addition, L-cysteine-induced hydrogen sulfide (H₂S) production similarly increased in ischemic side cerebral cortex of control and CSE^-/- mice. Our findings suggest that endogenous production of H₂S by CSE and CAT/3-MST during reperfusion may be involved in postischemic cerebral vasodilation/hyperemia and play an important role in early BBB disruption following transient focal cerebral ischemia.     

The Reg3α (HIP/PAP) Lectin Suppresses Extracellular Oxidative Stress in a Murine Model of Acute Liver Failure

MethodsPrimary hepatocytes stressed with the reactive oxygen species (ROS) inducers TNFα and H₂O₂ were incubated with a recombinant Reg3α protein. ALF was induced in C57BL/6J mice by an anti-CD95 antibody. Livers and primary hepatocytes were harvested for deoxycholate separation of cellular and extracellular fractions, immunostaining, immunoprecipitation and malondialdehyde assays. Fibrin deposition was studied by immunofluorescence in frozen liver explants from patients with ALF.ResultsFibrin deposition occurs during experimental and clinical acute liver injuries. Reg3α bound the resulting transient fibrin network, accumulated in the inflammatory extracellular matrix (ECM), greatly reduced extracellular ROS levels, and improved cell viability. Hepatocyte treatment with ligands of death receptors, e.g. TNFα and Fas, resulted in a twofold increase of malondialdehyde (MDA) level in the deoxycholate-insoluble fractions. Reg3α treatment maintained MDA at a level similar to control cells and thereby increased hepatocyte survival by 35%. No antioxidant effect of Reg3α was noted in the deoxycholate-soluble fractions. Preventing fibrin network formation with heparin suppressed the prosurvival effect of Reg3α.ConclusionsReg3α is an ECM-targeted ROS scavenger that binds the fibrin scaffold resulting from hepatocyte death during ALF. ECM alteration is an important pathogenic factor of ALF and a relevant target for pharmacotherapy.     

RIP3-Dependent Accumulation of Mitochondrial Superoxide Anions in TNF-α-Induced Necroptosis

Excessive production of reactive oxygen species (ROS) is a key phenomenon in tumor necrosis factor (TNF)-α-induced cell death. However, the role of ROS in necroptosis remains mostly elusive. In this study, we show that TNF-α induces the mitochondrial accumulation of superoxide anions, not H₂O₂, in cancer cells undergoing necroptosis. TNF-α-induced mitochondrial superoxide anions production is strictly RIP3 expression-dependent. Unexpectedly, TNF-α stimulates NADPH oxidase (NOX), not mitochondrial energy metabolism, to activate superoxide production in the RIP3-positive cancer cells. In parallel, mitochondrial superoxide-metabolizing enzymes, such as manganese-superoxide dismutase (SOD2) and peroxiredoxin III, are not involved in the superoxide accumulation. Mitochondrial-targeted superoxide scavengers and a NOX inhibitor eliminate the accumulated superoxide without affecting TNF-α-induced necroptosis. Therefore, our study provides the first evidence that mitochondrial superoxide accumulation is a consequence of necroptosis.     

Role of o-acetylserine in hydrogen sulfide emission from pumpkin leaves in response to sulfate

In the presence of excess sulfate, cysteine synthesis in pumpkin (Cucurbita pepo) leaves is not limited by sulfate reduction, but by the availability of O-acetylserine. Feeding of O-acetylserine or its metabolic precursors S-acetyl-coenzyme-A and coenzyme A to leaf discs enhanced the incorportion of [³⁵S]sulfate into reduced sulfur compounds, mainly into cysteine, at the cost of lowered H₂S emission; the uptake and reduction of sulfate is not affected by these treatments. β-Fluoropyruvate, an inhibitor of the generation of S-acetyl-coenzyme A via pyruvate dehydrogenase, stimulated H₂S emission in response to sulfate. This stimulation is overcompensated by addition of O-acetylserine, S-acetyl-coenzyme A, or coenzyme A. These results indicate that, in the presence of high amounts of sulfate, excess sulfur is reduced and emitted as H₂S into the atmosphere. The H₂S emitted seems to be produced by liberation from a precursor of cysteine rather than by cysteine desulfhydration.     

ADH1B and ADH1C Genotype,Alcohol Consumption and Biomarkers of Liver Function: Findings from a Mendelian Randomization Study in 58,313 European Origin Danes

Background

The effect of alcohol consumption on liver function is difficult to determine because of reporting bias and potential residual confounding. Our aim was to determine this effect using genetic variants to proxy for the unbiased effect of alcohol.

Methods

We used variants in ADH1B and ADH1C genes as instrumental variables (IV) to estimate the causal effect of long-term alcohol consumption on alanine aminotransferase (ALT), γ-glutamyl-transferase (γ-GT), alkaline phosphatase (ALP), bilirubin and prothrombin action. Analyses were undertaken on 58,313 Danes (mean age 56).

Results

In both confounder adjusted multivariable and genetic-IV analyses greater alcohol consumption, amongst those who drank any alcohol, was associated with higher ALT [mean difference per doubling of alcohol consumption: 3.4% (95% CI: 3.1, 3.7) from multivariable analyses and 3.7% (−4.5, 11.9) from genetic-IV analyses] and γ-GT [8.2% (7.8, 8.5) and 6.8% (−2.8, 16.5)]. The point estimates from the two methods were very similar and statistically the results from the two methods were consistent with each other for effects with ALT and γ-GT (both p_diff>0.3). Results from the multivariable analyses suggested a weak inverse association of alcohol with ALP [−1.5% (−1.7, −1.3)], which differed from the strong positive effect found in genetic-IV analyses [11.6% (6.8, 16.4)] (p_diff<0.0001). In both multivariable and genetic-IV analyses associations with bilirubin and protrombin action were weak and close to the null.

Conclusions

Our results suggest that greater consumption of alcohol is related to poorer liver function as indicated by higher ALT, γ-GT and ALP, but not to clotting or bilirubin.     

Activation of Swelling-activated Chloride Current by Tumor Necrosis Factor-α Requires ClC-3-dependent Endosomal Reactive Oxygen Production

ClC-3 is a Cl⁻/H⁺ antiporter required for cytokine-induced intraendosomal reactive oxygen species (ROS) generation by Nox1. ClC-3 current is distinct from the swelling-activated chloride current (ICl_swell), but overexpression of ClC-3 can activate currents that resemble ICl_swell. Because H₂O₂ activates ICl_swell directly, we hypothesized that ClC-3-dependent, endosomal ROS production activates ICl_swell. Whole-cell perforated patch clamp methods were used to record Cl⁻ currents in cultured aortic vascular smooth muscle cells from wild type (WT) and ClC-3 null mice. Under isotonic conditions, tumor necrosis factor-α (TNF-α) (10 ng/ml) activated outwardly rectifying Cl⁻ currents with time-dependent inactivation in WT but not ClC-3 null cells. Inhibition by tamoxifen (10 μm) and by hypertonicity (340 mosm) identified them as ICl_swell. ICl_swell was also activated by H₂O₂ (500 μm), and the effect of TNF-α was completely inhibited by polyethylene glycol-catalase. ClC-3 expression induced ICl_swell in ClC-3 null cells in the absence of swelling or TNF-α, and this effect was also blocked by catalase. ICl_swell activation by hypotonicity (240 mosm) was only partially inhibited by catalase, and the size of these currents did not differ between WT and ClC-3 null cells. Disruption of endosome trafficking with either mutant Rab5 (S34N) or Rab11 (S25N) inhibited TNF-α-mediated activation of ICl_swell. Thrombin also activates ROS production by Nox1 but not in endosomes. Thrombin caused H₂O₂-dependent activation of ICl_swell, but this effect was not ClC-3- or Rab5-dependent. Thus, activation of ICl_swell by TNF-α requires ClC-3-dependent endosomal H₂O₂ production. This demonstrates a functional link between two distinct anion currents, ClC-3 and ICl_swell.     

Altered gene expression of hydrogen sulfide-producing enzymes in the liver and muscles tissues of hyperthyroid rats

Thyroid hormones have a role in the regulation of hydrogen sulfide (H₂S) biosynthesis. In this study, we determined the effects of hyperthyroidism on H₂S levels in various tissues and messenger RNA (mRNA) expression of cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MST) in the liver and muscles of the rat. Sixteen male Wistar rats were divided into the hyperthyroid and the control groups. Hyperthyroidism was induced by adding l -thyroxine (12 mg/L) to drinking water for a period of 21 days. H₂S concentrations in serum, liver, aorta, heart, and soleus muscles, as well as mRNA expressions of CBS, CSE, and 3-MST in these tissues were measured at Day 21. Hyperthyroid rats had lower H₂S levels in the serum compared with controls (14.7 ± 1.4 vs. 25.7 ± 1.6 µmol/L, p < 0.001). Compared with controls, hyperthyroid rats had lower levels of H₂S in the aorta (89%), heart (80%), and soleus (103%) muscles, but higher levels in the liver (35%). Hyperthyroidism decreased the ratio of CBS/CSE mRNA expression in the liver and the CSE/CBS mRNA expression in the muscles by decreasing CBS levels in liver (34% cf. controls) and CSE levels in the aorta, heart, and soleus muscles (respectively, 51%, 7%, and 52% cf.). In addition, hyperthyroidism decreased the mRNA expression of 3-MST in the liver (51%) and aorta (33%), and increased it in the heart (300%) and soleus muscle (182%). In conclusion, hyperthyroidism increased H₂S levels in the liver and decreased it in muscles; these effects are at least in part due to increases and decreases in expression of CSE in the liver and muscles, respectively. These data indicate an association between thyroid hormone status and gene expression of the H₂S-producing enzymes in the rat.     

Reactive Oxygen Species Are Induced by Kaposi's Sarcoma-Associated Herpesvirus Early during Primary Infection of Endothelial Cells To Promote Virus Entry

The entry of Kaposi''s sarcoma-associated herpesvirus (KSHV) into human dermal microvascular endothelial cells (HMVEC-d), natural in vivo target cells, via macropinocytosis is initiated through a multistep process involving the binding of KSHV envelope glycoproteins with cell surface α3β1, αVβ3, and αVβ5 integrin molecules and tyrosine kinase ephrin-A2 receptor, followed by the activation of preexisting integrin-associated signaling molecules such as focal adhesion kinase (FAK), Src, c-Cbl, phosphoinositide 3-kinase (PI-3K), and Rho-GTPases. Many viruses, including KSHV, utilize cellular reactive oxygen species (ROS) for viral genomic replication and survival within host cells; however, the role of ROS in early events of viral entry and the induction of signaling has not been elucidated. Here we show that KSHV induced ROS production very early during the infection of HMVEC-d cells and that ROS production was sustained over the observation period (24 h postinfection). ROS induction was dependent on the binding of KSHV to the target cells, since pretreatment of the virus with heparin abolished ROS induction. Pretreatment of HMVEC-d cells with the antioxidant N-acetylcysteine (NAC) significantly inhibited KSHV entry, and consequently gene expression, without affecting virus binding. In contrast, H₂O₂ treatment increased the levels of KSHV entry and infection. In addition, NAC inhibited KSHV infection-induced translocation of αVβ3 integrin into lipid rafts, actin-dependent membrane perturbations, such as blebs, observed during macropinocytosis, and activation of the signal molecules ephrin-A2 receptor, FAK, Src, and Rac1. In contrast, H₂O₂ treatment increased the activation of ephrin-A2, FAK, Src, and Rac1. These studies demonstrate that KSHV infection induces ROS very early during infection to amplify the signaling pathways necessary for its efficient entry into HMVEC-d cells via macropinocytosis.     

The multifaceted roles of sulfane sulfur species in cancer-associated processes

Sulfane sulfur species comprise a variety of biologically relevant hydrogen sulfide (H₂S)-derived species, including per- and poly-sulfidated low molecular weight compounds and proteins. A growing body of evidence suggests that H₂S, currently recognized as a key signaling molecule in human physiology and pathophysiology, plays an important role in cancer biology by modulating cell bioenergetics and contributing to metabolic reprogramming. This is accomplished through functional modulation of target proteins via H₂S binding to heme iron centers or H₂S-mediated reversible per- or poly-sulfidation of specific cysteine residues. Since sulfane sulfur species are increasingly viewed not only as a major source of H₂S but also as key mediators of some of the biological effects commonly attributed to H₂S, the multifaceted role of these species in cancer biology is reviewed here with reference to H₂S, focusing on their metabolism, signaling function, impact on cell bioenergetics and anti-tumoral properties.