Regulation of soluble guanylyl cyclase redox state by hydrogen sulfide.

Soluble guanylate cyclase (sGC) is a receptor for nitric oxide (NO). Binding of NO to ferrous (Fe(2+)) heme increases its catalytic activity, leading to the production of cGMP from GTP. Hydrogen sulfide (H2S) is a signaling molecule that exerts both direct and indirect anti-oxidant effects. In the present, study we aimed to determine whether H2S could regulate sGC redox state and affect its responsiveness to NO-releasing agents and sGC activators. Using cultured rat aortic smooth muscle cells, we observed that treatment with H2S augmented the response to the NO donor DEA/NO, while attenuating the response to the heme-independent activator BAY58-2667 that targets oxidized sGC. Similarly, overexpression of H2S-synthesizing enzyme cystathionine-γ lyase reduced the ability of BAY58-2667 to promote cGMP accumulation. In experiments with phenylephrine-constricted mouse aortic rings, treatment with rotenone (a compound that increases ROS production), caused a rightward shift of the DEA/NO concentration-response curve, an effect partially restored by H2S. When rings were pre-treated with H2S, the concentration-response curve to BAY 58-2667 shifted to the right. Using purified recombinant human sGC, we observed that treatment with H2S converted ferric to ferrous sGC enhancing NO-donor-stimulated sGC activity and reducing BAY 58-2667-triggered cGMP formation. The present study identified an additional mechanism of cross-talk between the NO and H2S pathways at the level of redox regulation of sGC. Our results provide evidence that H2S reduces sGC heme Fe, thus, facilitating NO-mediated cellular signaling events.

Guanylyl cyclase activation reverses resistive breathing-induced lung injury and inflammation.

Inspiratory resistive breathing (RB), encountered in obstructive lung diseases, induces lung injury. The soluble guanylyl cyclase (sGC)/cyclic guanosine monophosphate (cGMP) pathway is down-regulated in chronic and acute animal models of RB, such as asthma, chronic obstructive pulmonary disease, and in endotoxin-induced acute lung injury. Our objectives were to: (1) characterize the effects of increased concurrent inspiratory and expiratory resistance in mice via tracheal banding; and (2) investigate the contribution of the sGC/cGMP pathway in RB-induced lung injury. Anesthetized C57BL/6 mice underwent RB achieved by restricting tracheal surface area to 50% (tracheal banding). RB for 24 hours resulted in increased bronchoalveolar lavage fluid cellularity and protein content, marked leukocyte infiltration in the lungs, and perturbed respiratory mechanics (increased tissue resistance and elasticity, shifted static pressure-volume curve right and downwards, decreased static compliance), consistent with the presence of acute lung injury. RB down-regulated sGC expression in the lung. All manifestations of lung injury caused by RB were exacerbated by the administration of the sGC inhibitor, 1H-[1,2,4]oxodiazolo[4,3-]quinoxalin-l-one, or when RB was performed using sGCα1 knockout mice. Conversely, restoration of sGC signaling by prior administration of the sGC activator BAY 58-2667 (Bayer, Leverkusen, Germany) prevented RB-induced lung injury. Strikingly, direct pharmacological activation of sGC with BAY 58-2667 24 hours after RB reversed, within 6 hours, the established lung injury. These findings raise the possibility that pharmacological targeting of the sGC-cGMP axis could be used to ameliorate lung dysfunction in obstructive lung diseases.

Inspiratory resistive breathing induces MMP-9 and MMP-12 expression in the lung.

Inspiratory resistive breathing (IRB) is characterized by large negative intrathoracic pressures and was shown to induce pulmonary inflammation in previously healthy rats. Matrix metalloproteinases (MMP)-9 and -12 are induced by inflammation and mechanical stress in the lung. We hypothesized that IRB induces MMP-9 and -12 in the lung. Anesthetized, tracheostomized rats breathed spontaneously through a two-way valve, connected to an inspiratory resistance, with the tidal inspiratory tracheal pressure set at 50% of the maximum. Quietly breathing animals served as controls. After 3 and 6 h of IRB, respiratory mechanics were measured, bronchoalveolar lavage (BAL) was performed, lung injury score was estimated, and lung MMP-9 was estimated by zymography and ELISA. MMP-9 and MMP-12 immunohistochemistry was performed. Isolated normal alveolar macrophages were incubated with BAL from rats that underwent IRB. After 18 h, MMP-9 and -12 levels were measured in supernatants, and immunocytochemistry was performed. Macrophages were treated with IL-1ß, IL-6, or TNF-α, and MMP-9 in supernatants was measured. After 6 h of IRB, leukocytes in BAL increased, and IL-1ß and IL-6 levels were elevated. Elasticity and injury score were increased after 3 and 6 h of IRB. Lung MMP-9 levels increased after 6 h of IRB. MMP-9 and MMP-12 were detected in alveolar macrophages and epithelial (bronchial/alveolar) cells after 3 and 6 h of IRB. MMP-9 and MMP-12 were found in supernatants after treatment with 6 h of IRB BAL. Cytosolic immunostaining was detected after treatment with 3 and 6 h of IRB BAL. All cytokines induced MMP-9 in culture supernatants. In conclusion, IRB induces MMP-9 and -12 in the lung of previously healthy rats.

Role of cGMP in hydrogen sulfide signaling.

The importance of hydrogen sulfide (H2S) in physiology and disease is being increasingly recognized in recent years. Unlike nitric oxide (NO) that signals mainly through soluble guanyl cyclase (sGC)/cGMP, H2S is more promiscuous, affecting multiple pathways. It interacts with ion channels, enzymes, transcription factors and receptors. It was originally reported that H2S does not alter the levels of cyclic nucleotides. More recent publications, however, have shown increases in intracellular cGMP following exposure of cells or tissues to exogenously administered or endogenously produced H2S. Herein, we discuss the evidence for the participation of cGMP in H2S signaling and reconcile the seemingly divergent results presented in the literature on the role of this cyclic nucleotide in the biological actions of H2S.

Phosphinodithioate and Phosphoramidodithioate Hydrogen Sulfide Donors.

Hydrogen sulfide is rapidly emerging as a key physiological mediator and potential therapeutic tool in numerous areas such as acute and chronic inflammation, neurodegenerative and cardiovascular disease, diabetes, obesity and cancer. However, the vast majority of the published studies have employed crude sulfide salts such as sodium hydrosulfide (NaSH) and sodium sulfide (Na2S) as H2S "donors" to generate H2S. Although these salts are cheap, readily available and easy to use, H2S generated from them occurs as an instantaneous and pH-dependent dissociation, whereas endogenous H2S synthesis from the enzymes cystathionine γ-lyase, cystathionine-ß-synthase and 3-mercaptopyruvate sulfurtransferase is a slow and sustained process. Furthermore, sulfide salts are frequently used at concentrations (e.g. 100 µM to 10 mM) far in excess of the levels of H2S reported in vivo (nM to low µM). For the therapeutic potential of H2S is to be properly harnessed, pharmacological agents which generate H2S in a physiological manner and deliver physiologically relevant concentrations are needed. The phosphorodithioate GYY4137 has been proposed as "slow-release" H2S donors and has shown promising efficacy in cellular and animal model diseases such as hypertension, sepsis, atherosclerosis, neonatal lung injury and cancer. However, H2S generation from GYY4137 is inefficient necessitating its use at high concentrations/doses. However, structural modification of the phosphorodithioate core has led to compounds (e.g. AP67 and AP105) with accelerated rates of H2S generation and enhanced biological activity. In this review, the therapeutic potential and limitations of GYY4137 and related phosphorodithioate derivatives are discussed.

Hydrogen sulfide replacement therapy protects the vascular endothelium in hyperglycemia by preserving mitochondrial function.

The goal of the present studies was to investigate the role of changes in hydrogen sulfide (H(2)S) homeostasis in the pathogenesis of hyperglycemic endothelial dysfunction. Exposure of bEnd3 microvascular endothelial cells to elevated extracellular glucose (in vitro "hyperglycemia") induced the mitochondrial formation of reactive oxygen species (ROS), which resulted in an increased consumption of endogenous and exogenous H(2)S. Replacement of H(2)S or overexpression of the H(2)S-producing enzyme cystathionine-γ-lyase (CSE) attenuated the hyperglycemia-induced enhancement of ROS formation, attenuated nuclear DNA injury, reduced the activation of the nuclear enzyme poly(ADP-ribose) polymerase, and improved cellular viability. In vitro hyperglycemia resulted in a switch from oxidative phosphorylation to glycolysis, an effect that was partially corrected by H(2)S supplementation. Exposure of isolated vascular rings to high glucose in vitro induced an impairment of endothelium-dependent relaxations, which was prevented by CSE overexpression or H(2)S supplementation. siRNA silencing of CSE exacerbated ROS production in hyperglycemic endothelial cells. Vascular rings from CSE(-/-) mice exhibited an accelerated impairment of endothelium-dependent relaxations in response to in vitro hyperglycemia, compared with wild-type controls. Streptozotocin-induced diabetes in rats resulted in a decrease in the circulating level of H(2)S; replacement of H(2)S protected from the development of endothelial dysfunction ex vivo. In conclusion, endogenously produced H(2)S protects against the development of hyperglycemia-induced endothelial dysfunction. We hypothesize that, in hyperglycemic endothelial cells, mitochondrial ROS production and increased H(2)S catabolism form a positive feed-forward cycle. H(2)S replacement protects against these alterations, resulting in reduced ROS formation, improved endothelial metabolic state, and maintenance of normal endothelial function.

Soluble guanylyl cyclase is a target of angiotensin II-induced nitrosative stress in a hypertensive rat model.

Nitric oxide (NO) by activating soluble guanylyl cyclase (sGC) is involved in vascular homeostasis via induction of smooth muscle relaxation. In cardiovascular diseases (CVDs), endothelial dysfunction with altered vascular reactivity is mostly attributed to decreased NO bioavailability via oxidative stress. However, in several studies, relaxation to NO is only partially restored by exogenous NO donors, suggesting sGC impairment. Conflicting results have been reported regarding the nature of this impairment, ranging from decreased expression of one or both subunits of sGC to heme oxidation. We showed that sGC activity is impaired by thiol S-nitrosation. Recently, angiotensin II (ANG II) chronic treatment, which induces hypertension, was shown to generate nitrosative stress in addition to oxidative stress. We hypothesized that S-nitrosation of sGC occurs in ANG II-induced hypertension, thereby leading to desensitization of sGC to NO hence vascular dysfunction. As expected, ANG II infusion increases blood pressure, aorta remodeling, and protein S-nitrosation. Intravital microscopy indicated that cremaster arterioles are resistant to NO-induced vasodilation in vivo in anesthetized ANG II-treated rats. Concomitantly, NO-induced cGMP production decreases, which correlated with S-nitrosation of sGC in hypertensive rats. This study suggests that S-nitrosation of sGC by ANG II contributes to vascular dysfunction. This was confirmed in vitro by using A7r5 smooth muscle cells infected with adenoviruses expressing sGC or cysteine mutants: ANG II decreases NO-stimulated activity in the wild-type but not in one mutant, C516A. This result indicates that cysteine 516 of sGC mediates ANG II-induced desensitization to NO in cells.

Thioglycine and L-thiovaline: biologically active H2S-donors.

Thioglycine and l-thiovaline are stable under acidic and basic conditions but in the presence of bicarbonate they liberate the gasotransmitter H(2)S. In cells both thioamino acids were proven to enhance cGMP formation and promote vasorelaxation in mouse aortic rings. Given that H(2)S is known to lower arterial hypertension, reduce oxidative stress and exhibit cardioprotective effects in preclinical models, H(2)S donors hold promise as novel treatments for cardiovascular diseases.

Hydrogen sulfide is an endogenous stimulator of angiogenesis.

The goal of the current study was to investigate the role of exogenous and endogenous hydrogen sulfide (H(2)S) on neovascularization and wound healing in vitro and in vivo. Incubation of endothelial cells (ECs) with H(2)S enhanced their angiogenic potential, evidenced by accelerated cell growth, migration, and capillary morphogenesis on Matrigel. Treatment of chicken chorioallantoic membranes (CAMS) with H(2)S increased vascular length. Exposure of ECs to H(2)S resulted in increased phosphorylation of Akt, ERK, and p38. The K(ATP) channel blocker glibenclamide or the p38 inhibitor SB203580 abolished H(2)S-induced EC motility. Since glibenclamide inhibited H(2)S-triggered p38 phosphorylation, we propose that K(ATP) channels lay upstream of p38 in this process. When CAMs were treated with H(2)S biosynthesis inhibitors dl-propylargylglycine or beta-cyano-L-alanine, a reduction in vessel length and branching was observed, indicating that H(2)S serves as an endogenous stimulator of the angiogenic response. Stimulation of ECs with vascular endothelial growth factor (VEGF) increased H(2)S release, while pharmacological inhibition of H(2)S production or K(ATP) channels or silencing of cystathionine gamma-lyase (CSE) attenuated VEGF signaling and migration of ECs. These results implicate endothelial H(2)S synthesis in the pro-angiogenic action of VEGF. Aortic rings isolated from CSE knockout mice exhibited markedly reduced microvessel formation in response to VEGF when compared to wild-type littermates. Finally, in vivo, topical administration of H(2)S enhanced wound healing in a rat model, while wound healing was delayed in CSE(-/-) mice. We conclude that endogenous and exogenous H(2)S stimulates EC-related angiogenic properties through a K(ATP) channel/MAPK pathway.

Hydrogen sulfide is an endogenous inhibitor of phosphodiesterase activity.

OBJECTIVE: Recent studies have demonstrated that hydrogen sulfide (H(2)S) is produced within the vessel wall from L-cysteine regulating several aspects of vascular homeostasis. H(2)S generated from cystathione γ-lyase (CSE) contributes to vascular tone; however, the molecular mechanisms underlying the vasorelaxing effects of H(2)S are still under investigation. METHODS AND RESULTS: Using isolated aortic rings, we observed that addition of L-cysteine led to a concentration-dependent relaxation that was prevented by the CSE inhibitors DL-propargylglyicine (PAG) and ß-cyano-l-alanine (BCA). Moreover, incubation with PAG or BCA resulted in a rightward shift in sodium nitroprusside-and isoproterenol-induced relaxation. Aortic tissues exposed to PAG or BCA contained lower levels of cGMP, exposure of cells to exogenous H(2)S or overexpression of CSE raised cGMP concentration. RNA silencing of CSE expression reduced intracellular cGMP levels confirming a positive role for endogenous H(2)S on cGMP accumulation. The ability of H(2)S to enhance cGMP levels was greatly reduced by the nonselective phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. Finally, addition of H(2)S to a cell-free system inhibited both cGMP and cAMP breakdown. CONCLUSIONS: These findings provide direct evidence that H(2)S acts as an endogenous inhibitor of phosphodiesterase activity and reinforce the notion that this gasotransmitter could be therapeutically exploited.

The soluble guanylyl cyclase inhibitor NS-2028 reduces vascular endothelial growth factor-induced angiogenesis and permeability.

Nitric oxide (NO) is known to promote vascular endothelial growth factor (VEGF)-stimulated permeability and angiogenesis. However, effector molecules that operate downstream of NO in this pathway remain poorly characterized. Herein, we determined the effect of soluble guanylyl cyclase (sGC) inhibition on VEGF responses in vitro and in vivo. Treatment of endothelial cells (EC) with VEGF stimulated eNOS phosphorylation and cGMP accumulation; pretreatment with the sGC inhibitor 4H-8-bromo-1,2,4-oxadiazolo(3,4-d)benz(b)(1,4)oxazin-1-one (NS-2028) blunted cGMP levels without affecting VEGF-receptor phosphorylation. Incubation of cells with NS-2028 blocked the mitogenic effects of VEGF. In addition, cells in which sGC was inhibited exhibited no migration and sprouting in response to VEGF. To study the mechanisms through which NS-2028 inhibits EC migration, we determined the effects of alterations in cGMP levels on p38 MAPK. Initially, we observed that inhibition of sGC attenuated VEGF-stimulated activation of p38. In contrast, the addition of 8-Br-cGMP to EC stimulated p38 phosphorylation. The addition of cGMP elevating agents (BAY 41-2272, DETA NO and YC-1) enhanced EC migration. To test whether sGC also mediated the angiogenic effects of VEGF in vivo, we used the rabbit cornea assay. Animals receiving NS-2028 orally displayed a reduced angiogenic response to VEGF. As increased vascular permeability occurs prior to new blood vessel formation, we determined the effect of NS-2028 in vascular leakage. Using a modified Miles assay, we observed that NS-2028 attenuated VEGF-induced permeability. Overall, we provide evidence that sGC mediates the angiogenic and permeability-promoting activities of VEGF, indicating the significance of sGC as a downstream effector of VEGF-triggered responses.

Protein kinase G phosphorylates soluble guanylyl cyclase on serine 64 and inhibits its activity.

OBJECTIVE: Binding of nitric oxide (NO) to soluble guanylyl cyclase (sGC) leads to increased cGMP synthesis that activates cGMP-dependent protein kinase (PKG). Herein, we tested whether sGC activity is regulated by PKG. METHODS AND RESULTS: Overexpression of a constitutively active form of PKG (DeltaPKG) stimulated (32)P incorporation into the alpha1 subunit. Serine to alanine mutation of putative sites revealed that Ser64 is the main phosphorylation site for PKG. Using a phospho-specific antibody we observed that endogenous sGC phosphorylation on Ser 64 increases in cells and tissues exposed to NO, in a PKG-inhibitable manner. Wild-type (wt) sGC coexpressed with DeltaPKG exhibited lower basal and NO-stimulated cGMP accumulation, whereas the S64A alpha1/beta1 sGC was resistant to the PKG-induced reduction in activity. Using purified sGC we observed that the S64D alpha1 phosphomimetic /beta1 dimer exhibited lower Vmax; moreover, the decrease in Km after NO stimulation was less pronounced in S64D alpha1/beta1 compared to wild-type sGC. Expression of a phosphorylation-deficient sGC showed enhanced responsiveness to endothelium-derived NO, reduced desensitization to acute NO exposure, and allowed for greater VASP phosphorylation. CONCLUSIONS: We conclude that PKG phosphorylates sGC on Ser64 of the alpha1 subunit and that phosphorylation inhibits sGC activity, establishing a negative feedback loop.

Zoledronic acid is effective against experimental malignant pleural effusion.

RATIONALE: Aminobiphosphonates, such as zoledronic acid (ZA), exert potent indirect antitumor effects and are currently being tested against human solid tumors. The antitumor actions of aminobiphosphonates, including angiostasis, are relevant to the pathogenesis of malignant pleural effusion (MPE), but no study has addressed the efficacy of these compounds against malignant pleural disease. OBJECTIVES: Here we hypothesized that treatment of immunocompetent mice with ZA would halt tumor progression in a mouse model of adenocarcinoma-induced MPE. METHODS: To induce MPE in mice, Lewis lung carcinoma cells were delivered directly into the pleural space. Subsequently, animals were treated with ZA in both a prevention and a regression protocol. MEASUREMENTS AND MAIN RESULTS: ZA treatment resulted in significant reductions in pleural fluid accumulation and tumor dissemination, while it significantly prolonged survival. These effects of ZA were linked to enhanced apoptosis of pleural tumor cells, decreased formation of new vessels in pleural tumors, and reduced pleural vascular permeability. In addition, ZA was able to inhibit the recruitment of mononuclear cells to pleural tumors, with concomitant reductions in matrix metalloproteinase-9 release into the pleural space. Finally, ZA limited the expression of proinflammatory and angiogenic mediators, as well as the activity of small GTP proteins Ras and RhoA, in tumor cells in vivo and in vitro. CONCLUSIONS: ZA is effective against experimental MPE, suggesting that this intervention should be considered for testing in clinical trials.

cGMP-dependent and -independent angiogenesis-related properties of nitric oxide.

Nitric oxide exerts a stimulatory role during postnatal angiogenesis. Although soluble guanylyl cyclase (sGC) mediates many of the effects of nitric oxide (NO) in the vascular system, the contribution of cGMP-dependent vs cGMP-independent pathways in NO-induced angiogenesis remains unclear. Herein, we determined the effects of a NO donor (sodium nitroprusside; SNP) and a NO-independent sGC activator (BAY 41-2272) in the growth and migration of rat aortic endothelial cells (RAEC). RAEC lack enzymatically active sGC as suggested by their inability to accumulate cGMP upon exposure to SNP. However, treatment of RAEC with SNP promoted a modest increase in their proliferation and migration that was dependent on extracellular signal regulated kinase1/2 activation. Moreover, when RAEC were exposed to vascular endothelial growth factor we observed an increase in migration that was inhibited by NO synthase, but not sGC, inhibition. Infection of cells with adenoviruses containing sGC greatly increased the efficacy of SNP as a mitogenic and migratory stimulus. We conclude that NO is capable of stimulating EC proliferation and mobility in the absence of sGC; however, increased intracellular levels of cGMP following sGC activation greatly amplify the angiogenic potential of NO.

Tyrosine phosphorylation of eNOS regulates myocardial survival after an ischaemic insult: role of PYK2.

AIMS: Endothelial nitric oxide (NO) synthase (eNOS) is known to play a cardioprotective protective. However, the molecular mechanisms regulating eNOS activity during ischaemia/reperfusion (I/R) injury are incompletely understood. eNOS is a substrate for several kinases that positively or negatively affect its enzymatic activity. Herein, we sought to correlate eNOS phosphorylation status with cardiomyocyte survival and we investigated the contribution of the proline-rich tyrosine kinase 2 (PYK2)/eNOS axis to the regulation of myocardial infarct size in vivo. METHODS AND RESULTS: Exposure of H9c2 cardiomyocytes to H2O2 lead to PYK2 phosphorylation on its activator site (Y402) and eNOS phosphorylation on the inhibitor site Y656 and the activator site S1176. Both H2O2-induced eNOS phosphorylation events were abolished by PYK2 pharmacological inhibition or gene knockdown. Activity assays demonstrated that phosphorylation of the tyrosine inhibitory site exerts a dominant effect over S1176. In cardiomyocytes subjected to oxidative stress or oxygen-glucose deprivation, inhibition of PYK2 limited cell injury; this effect was prevented by inhibition of NO production. In vivo, ischaemia-reperfusion induced an early activation of PYK2, leading to eNOS phosphorylation on Y656, which, in turn, reduced NO output, as judged by the low tissue levels of its downstream effector cGMP. Moreover, pharmacological blockade of PYK2 alleviated eNOS inhibition and prevented cardiac damage following I/R injury in wild-type, but not in eNOS KO mice. CONCLUSION: The current studies demonstrate that PYK2 is a pivotal regulator of eNOS function in myocardial infarction and identify PYK2 as a novel therapeutic target for cardioprotection.

Angiopoietin-1 inhibits endothelial permeability, neutrophil adherence and IL-8 production.

1 Angiopoietin-1 (Ang1) is an angiogenic growth factor that binds to the Tie2 receptor on vascular endothelium, promoting blood vessel maturation and integrity. In the present study, we have investigated whether Ang1 also possesses anti-inflammatory properties by determining its effects on endothelial barrier function, neutrophil (PMN) adherence to endothelial cells (EC) and production of the PMN chemotactic factor interleukin-8 (IL-8). 2 Pretreatment of endothelial monolayers with Ang1 attenuated the permeability increase induced by thrombin in both lung microvascular cells and a human endothelial cell line. Similarly, Ang1 prevented the permeability-inducing effects of platelet-activating factor, bradykinin and histamine. 3 Pretreatment of EC with Ang1 also reduced the adherence of PMN to EC stimulated by thrombin. In contrast to its ability to counteract the increase in monolayer permeability brought about by various inflammatory agents, Ang1 did not affect the ability of histamine, PAF, or tumor necrosis factor-alpha to stimulate PMN adherence to EC. 4 In addition to its ability to inhibit PMN adherence, Ang1 diminished IL-8 production from EC challenged with thrombin in a concentration-dependent manner. 5 When EC were preincubated with the specific Rho kinase (ROCK) inhibitor Y-27632, we observed a reduction in PMN adherence in response to thrombin, as well as a decrease in thrombin-stimulated IL-8 production. Coincubation of monolayers with Y-27632 and Ang1 did not further attenuate the above-mentioned responses. However, Ang-1 failed to inhibit the activation of RhoA in response to thrombin, suggesting that inhibition of EC adhesiveness for PMN and IL-8 production by Ang1 does not result from reduced ROCK activation. 6 We conclude that Ang1 can counteract several aspects of the inflammatory response, including endothelial permeability, PMN adherence to EC as well as inhibition of IL-8 production by EC.

Hydrogen sulfide accounts for the peripheral vascular effects of zofenopril independently of ACE inhibition.

AIMS: Therapeutic use of sulfhydrylated inhibitor S-zofenopril has raised different hypotheses regarding the role played by its thiol group in the beneficial clinical effects exerted compared with other angiotensin-converting enzyme (ACE) inhibitors. Here, we investigated hydrogen sulfide (H2S) pathway as accountable for extra-beneficial effects in vascular function. METHODS AND RESULTS: Spontaneously hypertensive rat (SHRs) and control Wistar Kyoto (WKY) rats were treated with either S-zofenopril or enalapril in vivo. Aorta and carotid were harvested and ex vivo vascular reactivity to acetylcholine (Ach) and L-cysteine (L-cys) assessed. Cystathionine-ß-synthase (CBS), cystathionine-γ-lyase (CSE), and 3-mercaptosulfur-transferase (3MST) expression, as well as H2S levels, were evaluated in both vascular tissues. The vascular response to Ach in both carotid and aorta was impaired in SHR (~30%, P < 0.001). S-zofenopril, but not enalapril, restored this response, while L-cys-induced relaxation was enhanced. CSE expression in vessels and tissue/plasma H2S levels were restored to WKY values in SHRs receiving S-zofenopril. In contrast, CBS and 3MST expression were not modified by treatments. S-zofenoprilat, an active metabolite of S-zofenopril, releases H2S in a 'cell-free' assay and it directly relaxed vessels in vitro in a concentration-dependent manner (P < 0.001). In vivo administration of R-zofenoprilat diasteroisomer, which does not inhibit ACE, did not modify blood pressure; nonetheless, it retained the beneficial effect on SHR vascular function as well as restored plasma/tissue H2S levels. CONCLUSION: Our findings establish that S-zofenopril improves vascular function by potentiating the H2S pathway in a model of spontaneous hypertension. This novel mechanism, unrelated to ACE inhibition and based on H2S release, could explain the beneficial effects of sulfhydrylated ACE inhibitors reported in the clinical literature.

Selectivity of commonly used pharmacological inhibitors for cystathionine ß synthase (CBS) and cystathionine γ lyase (CSE).

BACKGROUND AND PURPOSE: Hydrogen sulfide (H2S) is a signalling molecule that belongs to the gasotransmitter family. Two major sources for endogenous enzymatic production of H2S are cystathionine ß synthase (CBS) and cystathionine γ lyase (CSE). In the present study, we examined the selectivity of commonly used pharmacological inhibitors of H2S biosynthesis towards CSE and CBS. EXPERIMENTAL APPROACH: To address this question, human CSE or CBS enzymes were expressed and purified from Escherichia coli as fusion proteins with GSH-S-transferase. After purification, the activity of the recombinant enzymes was tested using the methylene blue method. KEY RESULTS: ß-Cyanoalanine (BCA) was more potent in inhibiting CSE than propargylglycine (PAG) (IC50 14 ± 0.2 µM vs. 40 ± 8 µM respectively). Similar to PAG, L-aminoethoxyvinylglycine (AVG) only inhibited CSE, but did so at much lower concentrations. On the other hand, aminooxyacetic acid (AOAA), a frequently used CBS inhibitor, was more potent in inhibiting CSE compared with BCA and PAG (IC50 1.1 ± 0.1 µM); the IC50 for AOAA for inhibiting CBS was 8.5 ± 0.7 µM. In line with our biochemical observations, relaxation to L-cysteine was blocked by AOAA in aortic rings that lacked CBS expression. Trifluoroalanine and hydroxylamine, two compounds that have also been used to block H2S biosynthesis, blocked the activity of CBS and CSE. Trifluoroalanine had a fourfold lower IC50 for CBS versus CSE, while hydroxylamine was 60-fold more selective against CSE. CONCLUSIONS AND IMPLICATIONS: In conclusion, although PAG, AVG and BCA exhibit selectivity in inhibiting CSE versus CBS, no selective pharmacological CBS inhibitor is currently available.

Insights into soluble guanylyl cyclase activation derived from improved heme-mimetics.

Recently, the structure of BAY 58-2667 bound to the Nostoc sp. H-NOX domain was published. On the basis of this structural information, we designed BAY 58-2667 derivatives and tested their effects on soluble guanylyl cyclase (sGC) activity. Derivative 20 activated sGC 4.8-fold more than BAY 58-2667. Co-crystallization of 20 with the Ns H-NOX domain revealed that the increased conformational distortion at the C-terminal region of αF helix containing 110-114 residues contributes to the higher activation triggered by 20.

cGMP-dependent protein kinase contributes to hydrogen sulfide-stimulated vasorelaxation.

A growing body of evidence suggests that hydrogen sulfide (H2S) is a signaling molecule in mammalian cells. In the cardiovascular system, H2S enhances vasodilation and angiogenesis. H2S-induced vasodilation is hypothesized to occur through ATP-sensitive potassium channels (K(ATP)); however, we recently demonstrated that it also increases cGMP levels in tissues. Herein, we studied the involvement of cGMP-dependent protein kinase-I in H2S-induced vasorelaxation. The effect of H2S on vessel tone was studied in phenylephrine-contracted aortic rings with or without endothelium. cGMP levels were determined in cultured cells or isolated vessel by enzyme immunoassay. Pretreatment of aortic rings with sildenafil attenuated NaHS-induced relaxation, confirming previous findings that H2S is a phosphodiesterase inhibitor. In addition, vascular tissue levels of cGMP in cystathionine gamma lyase knockouts were lower than those in wild-type control mice. Treatment of aortic rings with NaHS, a fast releasing H2S donor, enhanced phosphorylation of vasodilator-stimulated phosphoprotein in a time-dependent manner, suggesting that cGMP-dependent protein kinase (PKG) is activated after exposure to H2S. Incubation of aortic rings with a PKG-I inhibitor (DT-2) attenuated NaHS-stimulated relaxation. Interestingly, vasodilatory responses to a slowly releasing H2S donor (GYY 4137) were unaffected by DT-2, suggesting that this donor dilates mouse aorta through PKG-independent pathways. Dilatory responses to NaHS and L-cysteine (a substrate for H2S production) were reduced in vessels of PKG-I knockout mice (PKG-I⁻/⁻). Moreover, glibenclamide inhibited NaHS-induced vasorelaxation in vessels from wild-type animals, but not PKG-I⁻/⁻, suggesting that there is a cross-talk between K(ATP) and PKG. Our results confirm the role of cGMP in the vascular responses to NaHS and demonstrate that genetic deletion of PKG-I attenuates NaHS and L-cysteine-stimulated vasodilation.