Mark E. Wood

A novel hydrogen sulfide donor causes stomatal opening and reduces nitric oxide accumulation

Effects of hydrogen sulfide (H(2)S) on plant physiology have been previously studied, but such studies have relied on the use of NaSH as a method for supplying H(2)S to tissues. Now new compounds which give a less severe H(2)S shock and a more prolonged exposure to H(2)S have been developed. Here the effects of one such compound, GYY4137, has been investigated to determine its effects on stomatal closure in Arabidopsis thaliana. It was found that both NaSH and GYY4137 caused stomatal opening in the light and prevented stomatal closure in the dark. Nitric oxide (NO) has been well established as a mediator of stomatal movements and here it was found that both NaSH and GYY4137 reduced the accumulation of NO in guard cells, perhaps suggesting a mode of action for H(2)S in this system. GYY4137, and future related compounds, will be important tools to unravel the effects of plant exposure to H(2)S and to determine how H(2)S may fit into plant cell signalling pathways.

AP39, a novel mitochondria-targeted hydrogen sulfide donor, stimulates cellular bioenergetics, exerts cytoprotective effects and protects against the loss of mitochondrial DNA integrity in oxidatively stressed endothelial cells in vitro

The purpose of the current study was to investigate the effect of the recently synthesized mitochondrially-targeted H2S donor, AP39 [(10-oxo-10-(4-(3-thioxo-3H-1,2-dithiol-5yl)phenoxy)decyl) triphenylphosphonium bromide], on bioenergetics, viability, and mitochondrial DNA integrity in bEnd.3 murine microvascular endothelial cells in vitro, under normal conditions, and during oxidative stress. Intracellular H2S was assessed by the fluorescent dye 7-azido-4-methylcoumarin. For the measurement of bioenergetic function, the XF24 Extracellular Flux Analyzer was used. Cell viability was estimated by the combination of the MTT and LDH methods. Oxidative protein modifications were measured by the Oxyblot method. Reactive oxygen species production was monitored by the MitoSOX method. Mitochondrial and nuclear DNA integrity were assayed by the Long Amplicon PCR method. Oxidative stress was induced by addition of glucose oxidase. Addition of AP39 (30-300 nM) to bEnd.3 cells increased intracellular H2S levels, with a preferential response in the mitochondrial regions. AP39 exerted a concentration-dependent effect on mitochondrial activity, which consisted of a stimulation of mitochondrial electron transport and cellular bioenergetic function at lower concentrations (30-100 nM) and an inhibitory effect at the higher concentration of 300 nM. Under oxidative stress conditions induced by glucose oxidase, an increase in oxidative protein modification and an enhancement in MitoSOX oxidation was noted, coupled with an inhibition of cellular bioenergetic function and a reduction in cell viability. AP39 pretreatment attenuated these responses. Glucose oxidase induced a preferential damage to the mitochondrial DNA; AP39 (100 nM) pretreatment protected against it. In conclusion, the current paper documents antioxidant and cytoprotective effects of AP39 under oxidative stress conditions, including a protection against oxidative mitochondrial DNA damage.

The complex effects of the slow-releasing hydrogen sulfide donor GYY4137 in a model of acute joint inflammation and in human cartilage cells

The role of hydrogen sulfide (H2S) in inflammation remains unclear with both pro‐ and anti‐inflammatory actions of this gas described. We have now assessed the effect of GYY4137 (a slow‐releasing H2S donor) on lipopolysaccharide (LPS)‐evoked release of inflammatory mediators from human synoviocytes (HFLS) and articular chondrocytes (HAC) in vitro. We have also examined the effect of GYY4137 in a complete Freunds adjuvant (CFA) model of acute joint inflammation in the mouse. GYY4137 (0.1–0.5 mM) decreased LPS‐induced production of nitrite (NO2−), PGE2, TNF‐α and IL‐6 from HFLS and HAC, reduced the levels and catalytic activity of inducible nitric oxide synthase (iNOS) and cyclooxygenase‐2 (COX‐2) and reduced LPS‐induced NF‐κB activation in vitro. Using recombinant human enzymes, GYY4137 inhibited the activity of COX‐2, iNOS and TNF‐α converting enzyme (TACE). In the CFA‐treated mouse, GYY4137 (50 mg/kg, i.p.) injected 1 hr prior to CFA increased knee joint swelling while an anti‐inflammatory effect, as demonstrated by reduced synovial fluid myeloperoxidase (MPO) and N‐acetyl‐β‐D‐glucosaminidase (NAG) activity and decreased TNF‐α, IL‐1β, IL‐6 and IL‐8 concentration, was apparent when GYY4137 was injected 6 hrs after CFA. GYY4137 was also anti‐inflammatory when given 18 hrs after CFA. Thus, although GYY4137 consistently reduced the generation of pro‐inflammatory mediators from human joint cells in vitro, its effect on acute joint inflammation in vivo depended on the timing of administration.

Inducible hydrogen sulfide synthesis in chondrocytes and mesenchymal progenitor cells: is H2S a novel cytoprotective mediator in the inflamed joint?

Hydrogen sulfide (H2S) has recently been proposed as an endogenous mediator of inflammation and is present in human synovial fluid. This study determined whether primary human articular chondrocytes (HACs) and mesenchymal progenitor cells (MPCs) could synthesize H2S in response to pro‐inflammatory cytokines relevant to human arthropathies, and to determine the cellular responses to endogenous and pharmacological H2S. HACs and MPCs were exposed to IL‐1β, IL‐6, TNF‐α and lipopolysaccharide (LPS). The expression and enzymatic activity of the H2S synthesizing enzymes cystathionine‐β‐synthase (CBS) and cystathionine‐γ‐lyase (CSE) were determined by Western blot and zinc‐trap spectrophotometry, respectively. Cellular oxidative stress was induced by H2O2, the peroxynitrite donor SIN‐1 and 4‐hydroxynonenal (4‐HNE). Cell death was assessed by 3‐(4,5‐dimethyl‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. Mitochondrial membrane potential (DCm) was determined in situ by flow cytometry. Endogenous H2S synthesis was inhibited by siRNA‐mediated knockdown of CSE and CBS and pharmacological inhibitors D,L‐propargylglycine and aminoxyacetate, respectively. Exogenous H2S was generated using GYY4137. Under basal conditions HACs and MPCs expressed CBS and CSE and synthesized H2S in a CBS‐dependent manner, whereas CSE expression and activity was induced by treatment of cells with IL‐1β, TNF‐α, IL‐6 or LPS. Oxidative stress‐induced cell death was significantly inhibited by GYY4137 treatment but increased by pharmacological inhibition of H2S synthesis or by CBS/CSE‐siRNA treatment. These data suggest CSE is an inducible source of H2S in cultured HACs and MPCs. H2S may represent a novel endogenous mechanism of cytoprotection in the inflamed joint, suggesting a potential opportunity for therapeutic intervention.

Regulation of Heart Function by Endogenous Gaseous Mediators—Crosstalk Between Nitric Oxide and Hydrogen Sulfide

Both nitric oxide (NO) and hydrogen sulfide (H(2)S) are two important gaseous mediators regulating heart function. The present study examined the interaction between these two biological gases and its role in the heart. We found that l-arginine, a substrate of NO synthase, decreased the amplitudes of myocyte contraction and electrically induced calcium transients. Sodium hydrogen sulfide (an H(2)S donor), which alone had minor effect, reversed the negative inotropic effects of l-arginine. The effect of l-arginine + sodium hydrogen sulfide was abolished by three thiols (l-cysteine, N-acetyl-cysteine, and glutathione), suggesting that the effect of H(2)S + NO is thiol sensitive. The stimulatory effect on heart contractility was also induced by GYY4137, a slow-releasing H(2)S donor, when used together with sodium nitroprusside, an NO-releasing donor. More importantly, enzymatic generation of H(2)S from recombinant cystathionine-γ-lyase protein also interacted with endogenous NO generated from l-arginine to stimulate heart contraction. In summary, our data suggest that endogenous NO may interact with H(2)S to produce a new biological mediator that produces positive inotropic effect. The crosstalk between H(2)S and NO also suggests an intriguing potential for the endogenous formation of a thiol-sensitive molecule, which may be of physiological significance in the heart.

The synthesis and functional evaluation of a mitochondria-targeted hydrogen sulfide donor, (10-oxo-10-(4-(3-thioxo-3H-1,2-dithiol-5-yl)phenoxy)decyl) triphenylphosphonium bromide (AP39)

Synthesis and bioavailability of the endogenous gasomediator hydrogen sulfide (H2S) is perturbed in many disease states, including those involving mitochondrial dysfunction. There is intense interest in developing pharmacological agents to generate H2S. We have synthesised a novel H2S donor molecule coupled to a mitochondria-targeting moiety (triphenylphosphonium; TPP+) and compared the effectiveness of the compound against a standard non-TPP+ containing H2S donor (GYY4137) in the inhibition of oxidative stress-induced endothelial cell death. Our study suggests mitochondria-targeted H2S donors are useful pharmacological tools to study the mitochondrial physiology of H2S in health and disease.

Hydrogen sulfide effects on stomatal apertures.

Hydrogen sulfide (H2S) has recently been reported to be a signaling molecule in plants. It has been well established that is has such roles in animals and it has been suggested that it is included into the group of gasotransmitters. We have recently shown that hydrogen sulfide causes stomatal opening in the model plant Arabidopsis thaliana. H2S can be supplied to the plant tissues from donors such as sodium hydrosulfide (NaSH) or more recently from slow release H2S donor molecules such as GYY4137. Both give similar effects, that is, they cause stomatal opening. Furthermore both H2S donors reduced the accumulation of nitric oxide (NO) induced by abscisic acid (ABA) treatment of leaf tissues. Here similar work has been repeated in a crop plant, Capsium anuum, and similar data has been obtained, suggesting that such effects of hydrogen sulfide on plants is not confined to model species.

Leptin-Induced Endothelium-Dependent Vasorelaxation of Peripheral Arteries in Lean and Obese Rats: Role of Nitric Oxide and Hydrogen Sulfide

Adipose tissue hormone leptin induces endothelium-dependent vasorelaxation mediated by nitric oxide (NO) and endothelium-derived hyperpolarizing factors (EDHF). Previously it has been demonstrated that in short-term obesity the NO-dependent and the EDHF-dependent components of vascular effect of leptin are impaired and up-regulated, respectively. Herein we examined the mechanism of the EDHF-dependent vasodilatory effect of leptin and tested the hypothesis that alterations of acute vascular effects of leptin in obesity are accounted for by chronic hyperleptinemia. The study was performed in 5 groups of rats: (1) control, (2) treated with exogenous leptin for 1 week to induce hyperleptinemia, (3) obese, fed highly-palatable diet for 4 weeks, (4) obese treated with pegylated superactive rat leptin receptor antagonist (PEG-SRLA) for 1 week, (5) fed standard chow and treated with PEG-SRLA. Acute effect of leptin on isometric tension of mesenteric artery segments was measured ex vivo. Leptin relaxed phenylephrine-preconstricted vascular segments in NO- and EDHF-dependent manner. The NO-dependent component was impaired and the EDHF-dependent component was increased in the leptin-treated and obese groups and in the latter group both these effects were abolished by PEG-SRLA. The EDHF-dependent vasodilatory effect of leptin was blocked by either the inhibitor of cystathionine γ-lyase, propargylglycine, or a hydrogen sulfide (H2S) scavenger, bismuth (III) subsalicylate. The results indicate that NO deficiency is compensated by the up-regulation of EDHF in obese rats and both effects are accounted for by chronic hyperleptinemia. The EDHF-dependent component of leptin-induced vasorelaxation is mediated, at least partially, by H2S.

Primary intraocular lens implantation for penetrating lens trauma in Africa

OBJECTIVE This study aimed to audit the surgical strategy of primary posterior chamber intraocular lens implantation for cases of recent penetrating trauma involving the lens in an African population. DESIGN Retrospective, noncomparative case series. PARTICIPANTS Seventy-two cases are reported, including all patients who underwent primary intraocular lens implantation for traumatic cataract extraction performed within 1 month of injury between 1988 and 1996. MAIN OUTCOME MEASURES Demographic characteristics and follow-up attendance rates are analyzed. Surgical technique and the occurrence of intraoperative and postoperative complications are reported. Visual outcomes are reported with detailed analysis for cases of poor visual outcome. RESULTS Mean age was 14.3 years (standard deviation = 11.1), 57 (79%) were male and 15 (21%) were female (chi-square = 23.66, P < 0.01). Fifty-eight patients (80%) attended for follow-up with a mean follow-up duration of 14.3 months (standard deviation = 17.3). No demographic or surgical differences were identified between attendees and nonattendees. The posterior capsule had been breached by the trauma in 27 (38%) cases, and 15 of these required anterior vitrectomy. Capsular fixation of the implant was achieved in 49% of patients, the remainder having sulcus fixation. Intraoperative rupture of the posterior capsule occurred in four cases. The only common postoperative complication was acute fibrinous anterior uveitis, which occurred in 29 (40%) patients, and 32% of patients followed up for at least 6 months required secondary posterior capsulotomy. This was more common in younger patients (chi-square = 4.2, P < 0.05). Corrected postoperative visual acuities were available for 51 patients, of which 71% achieved 20/60 or better visual acuity. Patients 6 years of age or younger were less likely to achieve 20/60 (chi-square = 6.61, P = 0.01). CONCLUSIONS This surgical strategy has proved successful, producing good visual results and causing no sight-threatening complications. Primary posterior capsulotomy may be appropriate for younger patients.

AP39, A mitochondrially targeted hydrogen sulfide donor, exerts protective effects in renal epithelial cells subjected to oxidative stress in vitro and in acute renal injury in vivo

ABSTRACT This study evaluated the effects of AP39 [(10-oxo-10-(4-(3-thioxo-3H-1,2-dithiol-5yl) phenoxy)decyl) triphenyl phosphonium bromide], a mitochondrially targeted donor of hydrogen sulfide (H2S) in an in vitro model of hypoxia/oxidative stress injury in NRK-49F rat kidney epithelial cells (NRK cells) and in a rat model of renal ischemia-reperfusion injury. Renal oxidative stress was induced by the addition of glucose oxidase, which generates hydrogen peroxide in the culture medium at a constant rate. Glucose oxidase (GOx)-induced oxidative stress led to mitochondrial dysfunction, decreased intracellular ATP content, and, at higher concentrations, increased intracellular oxidant formation (estimated by the fluorescent probe 2, 7-dichlorofluorescein, DCF) and promoted necrosis (estimated by the measurement of lactate dehydrogenase release into the medium) of the NRK cells in vitro. Pretreatment with AP39 (30–300 nM) exerted a concentration-dependent protective effect against all of the above effects of GOx. Most of the effects of AP39 followed a bell-shaped concentration–response curve; at the highest concentration of GOx tested, AP39 was no longer able to afford cytoprotective effects. Rats subjected to renal ischemia/reperfusion responded with a marked increase (over four-fold over sham control baseline) blood urea nitrogen and creatinine levels in blood, indicative of significant renal damage. This was associated with increased neutrophil infiltration into the kidneys (assessed by the myeloperoxidase assay in kidney homogenates), increased oxidative stress (assessed by the malondialdehyde assay in kidney homogenates), and an increase in plasma levels of IL-12. Pretreatment with AP39 (0.1, 0.2, and 0.3 mg/kg) provided a dose-dependent protection against these pathophysiological alterations; the most pronounced protective effect was observed at the 0.3 mg/kg dose of the H2S donor; nevertheless, AP39 failed to achieve a complete normalization of any of the injury markers measured. The partial protective effects of AP39 correlated with a partial improvement of kidney histological scores and reduced TUNEL staining (an indicator of DNA damage and apoptosis). In summary, the mitochondria-targeted H2S donor AP39 exerted dose-dependent protective effects against renal epithelial cell injury in vitro and renal ischemia-reperfusion injury in vivo. We hypothesize that the beneficial actions of AP39 are related to the reduction of cellular oxidative stress, and subsequent attenuation of various positive feed-forward cycles of inflammatory and oxidative processes.