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Here we investigated the effect of the flavonoid galangin in isolated rat thoracic aortic rings. Galangin (0.1-100 microM) induced relaxation in rings pre-contracted with phenylephrine (PE 1 microM) or with KCl (100 mM) or pre-treated with the nitric oxide synthase inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME, 100 microM), the cyclooxygenase inhibitor indomethacin (10 microM) and the adenylate cyclase inhibitor, SQ 22,536 (100 microM). In another set of experiments, rat aortic rings were incubated with galangin (1-100 microM) and the contractile responses to PE (0.001-3 microM) or to KCl (60 mM) were evaluated. We also evaluated the effect of galangin (100 microM) on PE (10 microM)-induced contraction in a Ca2+-free medium. Galangin relaxed aortic rings with or without endothelium. Galangin effect was significantly inhibited by L-NAME. Galangin inhibited the contractile response to PE, either in presence or in absence of external calcium, and to KCl. In the end, we also found that galangin caused nitric oxide (NO) release from aortic rings and abolished the increase in [Ca2+]i triggered by PE or KCl in aortic smooth muscle cells, either in presence and in absence of external Ca2+. Our results suggest that galangin reduces the contractility of rat aortic rings through an endothelium-dependent mechanism, involving NO, and also through an endothelium-independent mechanism, inhibiting calcium movements through cell membranes.

In this article we report an investigation concerning analogues of the thrombin binding aptamer (TBA) in which thymidines have been replaced, one at a time, by the unusual residue 5-hydroxymethyl-2'-deoxyuridine (hmU) that differs from the canonical thymidine only in the presence of an hydroxyl on the 5-methyl group. NMR and CD data clearly indicate that all TBA derivatives preserve the ability to fold in the original ''chair-like'' structure. The presence of the hmU residue doesn't affect notably thermal stability of the modified aptamers compared to the parent one, except the analogue H9 for which a more marked increase of the melting temperature has been observed. Although all TBA analogues have shown decreased affinities to thrombin, derivatives H3, H7 and H9 have proven to possess improved anticoagulant activities in comparison with the parent aptamer. Our data open-up the possibility to enhance the TBA biological properties, just introducing small chemical modifications.

Herein, we report optically pure modified acyclic nucleosides as ideal probes for aptamer modification. These new monomers offer unique advantages in exploring the role played in thrombin inhibition by a single residue modification at key positions of the TBA structure.

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-β-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.

Here we have characterized perthamide C, a cyclopeptide from a Solomon Lithistid sponge Theonella swinhoei, which displays an anti-inflammatory/immunomodulatory activity. The study has been performed using the carragenan-induced mouse paw edema that displays an early (0–6 h) and a late phase (24–96 h). Perthamide C significantly inhibits neutrophils infiltration in tissue both in the early and late phases. This effect was coupled to a reduced expression of the endothelial nitric oxide synthase (eNOS) in the early phase while cyclooxygenase-1 and 2 (COX-1, COX-2), and inducible NOS (iNOS) expression were unaffected. In the late phase perthamide C reduced expression of both NOS isoforms without affecting COXs expression. This peculiar selectivity toward the two enzymes deputed to produce NO lead us to investigate on a possible action of perthamide C on lymphocytes infiltration and activation. We found that perthamide C inhibited the proliferation of peripheral lymphocytes, and that this effect was secondary to its metabolic activation in vivo. Indeed, in vitro perthamide C did not inhibit proliferation as opposite to its metabolite perthamide H. In conclusion, perthamide C selectively interferes with NO generation triggered by either eNOS or iNOS without affecting either COX-1 or COX-2. This in turn leads to modulation of the inflammatory response through a reduction of vascular permeability, neutrophil infiltration as well as lymphocyte proliferation.

DCCC chromatography followed by HPLC purification on the polar extract of marine sponge Theonella swinhoei resulted in the isolation of five new cyclopeptides, perthamides G–K. The new structures, featuring unprecedented amino acid units, were determined by interpretation of extensive spectroscopic and spectrometric data (MS, 1 H and 13 C NMR, COSY, HSQC, HMBC, and ROESY). Pharmacological analysis demonstrated that these natural cyclopeptides are endowed with anti-inflammatory potential as assessed by their ability to reduce carrageenan-induced mouse paw oedema.

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.

Two unprecedented cyclic peptides, solomonamides A and B, were isolated from the marine sponge Theonella swinhoei. The structures were elucidated on the basis of comprehensive 1D and 2D NMR analysis and high-resolution mass spectrometry. A combined approach, involving Marfey's method, QM J based analysis, and DFT J/(13)C calculations, was used for establishing the absolute configuration of the entire molecule. Solomonamide A showed in vivo anti-inflammatory activity.

Hydrogen sulfide (H(2)S), a novel gaseous transmitter, is considered a physiological regulator of vascular homeostasis. Recent evidence suggests H(2)S as an endothelium-hyperpolarizing factor (EDHF) candidate. To address this issue, we evaluated the vascular effect of sodium hydrogen sulfide (NaHS), an H(2)S donor, on the rat mesenteric arterial bed. NaHS concentration-response curve was performed on preconstricted mesenteric arterial bed. To assess the contribution of EDHF, we performed a pharmacologic dissection using indomethacin, N(G)-nitro-l-arginine methyl ester (l-NAME), or apamin and charybdotoxin as cyclooxygenase, nitric-oxide synthase, and calcium-dependent potassium channel inhibitors, respectively. In another set of experiments, we used 4-(4-octadecylphenyl)-4-oxobutenoic acid, baicalein, or proadifen as phospholipase A(2) (PLA(2)), lipoxygenase, and cytochrome P450 inhibitors, respectively. Finally, an immunofluorescence study was performed to support the involvement of PLA(2) in mesenteric artery challenged by NaHS. NaHS promoted a dual vascular effect (i.e., vasoconstriction and vasodilation). l-NAME or baicalein administration affected neither NaHS-mediated vasodilation nor vasoconstriction, whereas apamin and charybdotoxin significantly inhibited NaHS-induced relaxation. Pretreatment with PLA(2) inhibitor abolished both the contracting and the relaxant effect, whereas P450 cytochrome blocker significantly reduced NaHS-mediated relaxation. The immunofluorescence study showed that NaHS caused a migration of cytosolic PLA(2) close to the nucleus, which implicates activation of this enzyme. Our data indicate that H(2)S could activate PLA(2), which in turn releases arachidonic acid leading, initially, to vasoconstriction followed by vasodilation mediated by cytochrome P450-derived metabolites. Because EDHF has been presumed to be a cytochrome P450 derivative of the arachidonic acid, our results suggest that H(2)S acts through EHDF release.

Objective— Carbon monoxide (CO) is a weak soluble guanylyl cyclase stimulator, leading to transient increases in cGMP and vasodilation. The aim of the present work was to measure the effect of CO-releasing molecules (CORMs) on the cGMP/nitric oxide (NO) pathway and to evaluate how selected CORMs affect NO-induced vasorelaxation. Methods and Results— Incubation of smooth muscle cells with some but not all of the CORMs caused a minor increase in cGMP levels. Concentration-response curves were bell-shaped, with higher CORMs concentrations producing lower increases in cGMP levels. Although exposure of cells to CORM-2 enhanced cGMP formation, we observed that the compound inhibited NO-stimulated cGMP accumulation in cells and NO-stimulated soluble guanylyl cyclase activity that could be reversed by superoxide anion scavengers. Reactive oxygen species generation from CORMs was confirmed using luminol-induced chemiluminescence and electron spin resonance. Furthermore, we observed that NO is scavenged by CORM-2. When used alone CORM-2 relaxed vessels through a cGMP-mediated pathway but attenuated NO donor-stimulated vasorelaxation. Conclusion— We conclude that the CORMs examined have context-dependent effects on vessel tone, as they can directly dilate blood vessels, but also block NO-induced vasorelaxation.

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 β-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. Conclusion— 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.

BACKGROUND: Testosterone (T) induces a rapid relaxation in vascular tissues of different species due to a nongenomic effect of this steroid on vessels. Different mechanisms have been proposed to explain T-induced vasodilatation but the effective mechanism(s) and the mediators involved are still a matter of debate. OBJECTIVES: We have evaluated if H(2)S pathway is involved in T vascular effects. DESIGN AND SETTING: Male Wistar rats were sacrificed and thoracic aorta was rapidly dissected and cleaned from fat and connective tissue. Rings of 2-3 mm length were cut and placed in organ baths filled with oxygenated Krebs solution at 37 degrees C and mounted to isometric force transducers. H(2)S determination was performed on thoracic aortic rings incubated with T or vehicle and in presence of inhibitors. H2S concentration was calculated against a calibration curve of NaHS (3-250 microM). Results were expressed as nmoles/mg protein. MEASUREMENTS: Vascular reactivity was evaluated by using isometric transducers. H(2)S determination was performed by using a cystathionine beta-synthetase (CBS) and cystathionine gamma lyase (CSE) activity assay. CSE and CBS protein levels were assessed by Western blot analysis. Statistical analysis was performed by using two-way ANOVA and unpaired Student's t-test where appropriate. RESULTS: T significantly increased conversion of L-cysteine to H(2)S. This effect was significantly reduced by PGG and BCA, two specific inhibitors of CSE. T (10 nM-10 microM) induced a concentration-dependent vasodilatation of rat aortic rings in vitro that was significantly and concentration-dependent inhibited by PGG, BCA, and glybenclamide. Incubation of aorta with T up to 1 h did not change CBS/CSE expression, suggesting that T modulates enzymatic activity. CONCLUSIONS: Here we demonstrate that T vasodilator effect involves H(2)S, a novel gaseous mediator. T modulates H(2)S levels by increasing the enzymatic conversion of L-cysteine to H(2)S.

Two new metabolites, perthamides C and D, have been isolated from the marine sponge Theonella swinhoei. Their structures were determined by interpretation of NMR and ESIMS data. All compounds exhibited in vivo potent anti-inflammatory activity. Biological activity and structural elucidation are reported.

A new modified acyclic nucleoside, namely N(1)-(3-hydroxy-2-hydroxymethyl-2-methylpropyl)-thymidine, was synthesized and transformed into a building block useful for oligonucleotide (ON) automated synthesis. A series of modified thrombin binding aptamers (TBAs) in which the new acyclic nucleoside replaces, one at the time, the thymidine residues were then synthesized and characterized by UV, CD, MS, and (1)H NMR. The biological activity of the resulting TBAs was tested by Prothrombin Time assay (PT assay) and by purified fibrinogen clotting assay. From a structural point of view, nearly all the new TBA analogues show a similar behavior as the unmodified counterpart, being able to fold into a bimolecular or monomolecular quadruplex structure depending on the nature of monovalent cations (sodium or potassium) coordinated in the quadruplex core. From the comparison of structural and biological data, some important structure-activity relationships emerged, particularly when the modification involved the TT loops. In agreement with previous studies we found that the folding ability of TBA analogues is more affected by modifications involving positions 4 and 13, rather than positions 3 and 12. On the other hand, the highest anti-thrombin activities were detected for aptamers containing the modification at T13 or T12 positions, thus indicating that the effects produced by the introduction of the acyclic nucleoside on the biological activity are not tightly connected with structure stabilities. It is noteworthy that the modification at T7 produces an ON being more stable and active than the natural TBA.

Sphingosine-1-phosphate (S1P) has been shown to regulate numerous and diverse cell functions, including smooth muscle contraction. Here we assessed the role of S1P/Sphingosine kinase (SPK) pathway in the regulation of bronchial tone. Our objective was to determine, using an integrated pharmacologic and molecular approach, (1) the role of S1P as endogenous modulator of the bronchial tone, and (2) the linkage between S1P pathway and bronchial hyperresponsiveness. We evaluated S1P effects on isolated bronchi and whole lungs, harvested from Balb/c mice sensitized to ovalbumin (OVA) versus vehicle-treated mice, by measuring bronchial reactivity and lung resistance. We found that S1P administration on nonsensitized mouse bronchi does not cause any direct effect on bronchial tone, while a significant increase in Ach-induced contraction occurs after S1P challenge. Conversely, in OVA-sensitized mice S1P/SPK pathway triggers airway hyperesponsiveness. Indeed, S1P causes a dose-dependent contraction of isolated bronchi. Similarly, in the whole lung system S1P increased airway resistance only in OVA-sensitized mice. The action on bronchi of S1P is coupled to an enhanced expression of SPK(1) and SPK(2) as well as of S1P(2) and S1P(3) receptors. In these experiments the key role for S1P/SPK in hyperreactivity has been confirmed by pharmacologic modulation of SPKs. S1P/SPK pathway does not seem to play a major role in physiologic conditions, while it may become critical in pathologic conditions. These results open new windows for therapeutic strategies in diseases like asthma.

Carrageenan-induced rat paw oedema is a widely used model to investigate the physiopathology of an acute local inflammation. Recently, much attention has been focused on the link between haemostasis and inflammation, and on the impact that inflammation might have on thrombotic events. It is known that the systemic response to inflammation is the “acute phase reaction” that represents a highly complex reaction of the organism to a variety of injuries, aimed to restore homeostasis; one important feature of the acute phase reaction is the hepatic synthesis of proteins involved in the coagulation cascade. Much attention has been focused on the role that systemic inflammation might have on thrombotic events, while there is not much information on the role played by an acute local inflammation on haemostasis, that can lead toward a pro-thrombotic state. The present study was conducted to evaluate the haemostatic balance in the early and the late phase of carrageenan-induced rat paw oedema; i.e. at 3 h, when paw inflammation is maximally expressed, and 24 h following carrageenan injection, when there is an almost complete absence of local inflammatory symptoms. We found that in inflamed animals, 24 h following oedema induction, there was an increase in plasma fibrinogen levels, antithrombin III activity and serum interleukin-6 levels, concomitant to a shortened prothrombin time and to an increased platelet responsiveness to ADP. Furthermore, in inflamed tissues at 3 h there was an increase in antithrombin III proteic expression. Our results demonstrate that a haemostatic imbalance occurs following carrageenan-induced rat paw oedema.

Proteinase activated receptor-2 (PAR2), a seven transmembrane domain G protein coupled receptor, is expressed on airway epithelium and smooth muscle cells and over-expressed in human airways under pathological conditions, such as asthma and chronic obstructive pulmonary disease (CCPD). However, the role of PAR2 in airways has not yet been defined. Aim of the present study, was to evaluate the in vitro rat bronchial response to a synthetic peptide activating PAR2 (PAR2-AP; SLIGRL), following an in vivo treatment with bacterial lipopolysaccharide (LPS). Bronchi from LPS-treated animals showed an increased relaxant response to PAR2-AP, compared to naive animals, the effect was maximum after 20-h pretreatment and reduced by epithelium removal. Western blot analysis showed an increased PAR2 protein expression on bronchi removed 20 h after LPS treatment. PAR2-AP-induced bronchorelaxation was inhibited by ibuprofen, by the selective cyclooxygenase2 (COX-2) inhibitor, diisopropyl fluorophosphate (DFP) and partially by the calcitonin gene related peptide (CGRP) antagonist, rat-CGRP([8-37]). Furthermore, there was a strong immunoreactivity for COX-2 on bronchial epithelium of LPS-treated rats. Prostaglandin E-2 (PGE(2)) tissue release and CGRP tissue content were significantly increased following tissue incubation with PAR2-AP. The in vivo LPS treatment in rats strongly increases the bronchorelaxant effect of PAR2-AP, this effect correlates with an increased tissue protein receptor expression and the COX-2 localization on bronchial epithelium. our work supports a role for PAR(2) as a defence mechanism aimed to preserve bronchial functionality under systemic inflammatory conditions; both COX-2-derived PGE(2) and CGRP are involved in this effect.

Objectives : Protease-activated receptor 2 (PAR2) is a G-protein-coupled receptor proteolytically activated by trypsin, tryptase or factor Xa. Alternatively, PAR2 can be activated by synthetic peptides whose sequence mimics the tethered ligand exposed after receptor cleavage. It is known that PAR2 modulates vascular reactivity, both in vitro and in vivo. The present study was designed to investigate the role of basal nitric oxide and cyclic nucleotides, adenosine 3′5′cyclic monophosphate (cAMP) and guanosine 3′5′ cyclic monophosphate (cGMP), in the vasorelaxation induced by a PAR2-activating peptide (PAR2-AP; SLIGRL-NH2) on rat aorta in vitro. Methods : A concentration–response curve to PAR2-AP was performed on rat thoracic aorta with or without a functional endothelium, and the effect of inhibitors was evaluated. The effect of PAR2-AP (10−7–3 × 10−5 M) on endothelium-denuded aorta was also evaluated after tissue incubation with sodium nitroprusside. Results : PAR2-AP (10−7–3 × 10−5 M) caused an endothelium-dependent relaxation abolished by N ω-nitro-l-arginine methyl ester, L-NAME, and by the guanylyl cyclase inhibitor, 1 H -[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ), but unaffected by geldanamycin. Vasorelaxant effect of PAR2-AP was only partially inhibited by the adenylyl cyclase inhibitor, 9-(tetrahydro-2-furanyl)-9 H -purin-6-amine (SQ 22,536), and it was increased by tissue incubation with the phosphodiesterase inhibitor, rolipram. On tissue without endothelium, PAR2-AP did not cause vasorelaxation, up to a concentration of 10−4 M. However, after tissue incubation with sodium nitroprusside (SNP, 3 × 10−9 M), the vasorelaxant effect of PAR2-AP was restored. Following tissue incubation with PAR2-AP, cAMP levels were significantly increased compared to control values. Conclusions : Our results suggest that vasorelaxation induced by PAR2-AP is modulated by basal nitric oxide with an involvement of both cyclic nucleotides, cGMP and cAMP.

Background Malignant hyperthermia (MH) is a pharmacogenetic disorder of skeletal muscle. The incidence of MH reactions ranges from 1:5000 to 1:50,000 anesthesias, however genetic abnormalities have been estimated as great as one in 3000 subjects [1] . The symptoms of MH include hyperthermia, increase in carbon dioxide production and oxygen consumption, muscle rigidity, rhabdomyolisis, tachycardia and, if untreated, death. Over 90 mutations have been identified in the ryanodine receptor (RYR1) gene and at least 30 are causal for MH. Thegenetic test only detects about 30% of people at riskof MH. Therefore, the “gold standard” for MH diagnosis is the in vitro contracture test. These tests enclose a positive response to caffeine, which is a well-known non specific PDE inhibitor [2] , and to halothane, whose mechanism of action involves KATP channels [3] . Since these actions are among the most accredited molecular mechanisms triggered by H2S [4] , [5] we aimed to evaluate the role of H2S/L-Cys pathway in MH. Methods The procedure is performed accordingly to the “European Group protocol for investigation of malignant hyperthermia susceptibly” Briefly, the muscular biopsy of the vastus group of the quadriceps muscle is harvested under regional anaesthesia. Eight muscular bundles of 15–25 mm length and 2–3 mm thickness are placed in a tissue bath with Krebs solution at 37 °C and connected to an isometric transducer. Electrical stimulus is than applied and the muscle is stretched slowly up to 2 g. After20 min of equilibration caffeine or halothane are added in the tissue bath at progressive concentrations. An increase in resting tension of at least 2 mN allows a MH susceptible (MHS) diagnosis. After diagnosis has been made, muscle bundles from both MHS and MH negative (MHN) subjects have been used for functional studies. Western blot analysis, qRT-PCR for H2S molecular machinery and plasmatic and tissutal H2S content were also performed. Results H2S assay performed on tissues homogenate revealed an significant increase of H2S content in MHS patients compared to MHN. Conversely, no difference was founded in plasmatic levels H2S of both groups of patients. Western blot analysis showed a significant and selective increase of CBS expression in MHS patients compared to MHN, confirmed by qRT-PCR analysis. Functional studies performed on MHN showed that pre-incubation of the tissue with NaHS was able to switch an MHS typical response following challenge with either caffeine or halothane. Conclusion Our data show that in MHS patients there is an increase in CBS expression that accounts for the enhanced concentration of H2S within the skeletal muscle. The involvement of H2S in MH is further confirmed by the finding that incubation of MHN tissues with NaHS prior to the addition of either caffeine or halothane generates a response similar to MHS tissues. In conclusion we demonstrate that L-Cys/CBS/H2S pathway is involved in malygnant hyperthermia. This finding may allows a different diagnostic and/or therapeutic approach.

Background The need for new drugs in melanoma treatment is of great relevance. Indeed, current therapies for the treatment of metastatic melanoma offer a limited clinical benefit and only in recent years there has been an advancement due to the identification of new molecular targets [1] . Hydrogen sulphide (H 2 S) is endogenously produced by the action of three enzymes CBS, CSE and the newly discovered 3-MST [2] . While H 2 S is cytoprotective at physiological concentrations, it seems to have pro-apoptotic actions in cancer cells [3] . However, to date there are not definitive reports on the role played by H 2 S in cancer development. Aim of this study was to determine the possible involvement of H 2 S in human melanoma. Methods The study has been performed by using some relevant human melanoma cell lines such as A375, WM115 and SK-Mel-28. HaCat, a normal human fibroblast cell line, has been used as control. Cellular proliferation was evaluated by the MTT assay. Apoptosis was assayed by flow cytometry analysis by double staining with Annexin V and propidium iodide (PI). NF-kB/DNA-binding activity was evaluated by electrophoretic mobility shift assay. Expression of CBS, CSE, 3-MST was assayed by quantitative real time RT-PCR, expression of Bcl-2, XIAP, c-FLIP, caspase 3, PARP, IKBa and Akt/p-Akt was determined by western blotting. Levels of H 2 S in the supernatant and in total cellular extracts were assayed by colorimetric assay. Results Diallyl trisulfide (DATS) is a garlic-derived polysulfide able to release H 2 S [4] . Our results demonstrate that DATS greatly suppressed, in a time and concentration-dependent manner, proliferation of the three human melanoma cell lines used. The most striking effect was obtained on the A375 cell line whose proliferation was inhibited, following incubation with DATS (10–30–100 μM, 72 h) by 30%, 70%, and 78% respectively ( p 2 S levels found in both supernatants and cellular lysates. Conversely, DATS up to 300 μM did not affect proliferation of HaCat. DATS-induced inhibition of A375 proliferation (10–100 μM, 72 h) was almost completely reversed by haemoglobin (10 μM; p 2 S. Moreover, DATS-induced inhibition of A375 proliferation was due to the induction of apoptosis as demonstrated by FACS analysis with Annexin V/PI staining and further confirmed by the inhibition of Bcl-2, XIAP, FLIP, as well as by the cleavage and consequent activation of caspase-3 and inactivation of poly (ADP ribose) polymerase (PARP-1). Constitutive NF-kB and activated Akt expression have been described in melanoma [5] . We also demonstrated that H 2 S released by DATS suppressed both constitutive NF-kB/DNA-binding activity and Akt phosphorylation suggesting that the apoptotic effect observed following exposure to H 2 S was consistent with the signal transduction pathways activated. Conclusion In conclusion, we demonstrate that H 2 S triggers, in relevant human melanoma cell lines, an apoptotic effect. This effect, in turn, activates downstream a signal pattern that candidates H 2 S as a possible novel therapeutic/target or diagnostic tool.

Background Hydrogen sulphide (H2S) is a novel gaseous mediator enzymatically produced by cystathionine-beta-synthase (CBS) and cystathionine-gamma-lyase (CSE). It is involved in physiological homeostatic processes and several pathological conditions; however, more importantly, H2S has a prominent role in cardiovascular system, where CSE deletion results in hypertension [1] .ACE inhibitors are widely used in controlling blood pressure in hypertensive patients and they represent first line treatment in different cardiovascular diseases, since they also show additional beneficial effects unrelated to ACE inhibition [2] , [3] .In particular, therapeutic use of zofenopril, a sulfhydrylated ACE inhibitor, has raised hypothesis over a potential role of thiol group in such beneficial effects, other than antioxidant activity.Here, we aimed to investigate on vascular effect of zofenoprilat, water-soluble metabolite of zofenopril, with respect to H2S pathway activation. Methods In order to pursue our goal, we performed isolated organ bath experiments by using aorta and carotid arteries harvested from Wistar Kyoto rats, where we tested vascular effect of zofenoprilat, compared to other ACE inhibitors, in presence of CSE inhibitor PAG or after endothelium removal.In addition, we also performed in vivo experiment, where we tested vascular response after two weeks treatment with zofenopril compared to enalapril in spontaneously hypertensive rats.We also determined vascular response to l -cys, precursor for H2S production, and quantified H2S levels in plasma and tissues and CSE and CBS protein levels upon different treatments. Results In vitro data showed that zofenoprilat, but not enalaprilat, was able to relax both aorta and carotid arteries in a concentration dependent fashion (100 nM–1 mM), reaching ∼60% vasodilation in both cases.Endothelium removal significantly reduced zofenoprilat induced vasorelaxation in aorta (EC50:8.5 μM vs 381 μM), but this effect was not observed in carotid.Next, CSE inhibition by PAG nearly abrogated zofenoprilat vasodilation in both aorta and carotid, indicating a role for H2S in this effect.In vivo data, first highlighted a significantly stronger effect of zofenopril vs enalapril treatment in lowering blood pressure (p l -cys was significantly improved in zofenopril vs enalapril treated animals (p Conclusion In conclusion, we show that sulfhydrylated ACE inhibitor zofenopril triggers H2S pathway and regulates different signal transduction cascades, other than inhibition of ACE. We suggest that this action could be, at least partly, explicative of the additional effects reported in the clinical literature about sulfhydrylated ACE inhibitors. Our data also imply the chance to understand, by using a drug already adopted in clinic, the role played by the l -Cys/H2S pathway in human hypertension.