Hydrogen sulfide donors alleviate itch secondary to the activation of type-2 protease activated receptors (PAR-2) in mice.

Published onlineJOURNAL ARTICLEHydrogen sulfide (H2S) has been highlighted as an endogenous signaling molecule and we have previously found that it can inhibit histamine-mediated itching. Pruritus is the most common symptom of cutaneous diseases and anti-histamines are the usual treatment; however, anti-histamine-resistant pruritus is common in some clinical settings. In this way, the involvement of mediators other than histamine in the context of pruritus requires new therapeutic targets. Considering that the activation of proteinase-activated receptor 2 (PAR-2) is involved in pruritus both in rodents and humans, in this study we investigated the effect of H2S donors on the acute scratching behavior mediated by PAR-2 activation in mice, as well as some of the possible pharmacological mechanisms involved. The intradermal injection of the PAR-2 peptide agonist SLIGRL-NH2 (8-80nmol) caused a dose-dependent scratching that was unaffected by intraperitoneal pre-treatment with the histamine H1 antagonist pyrilamine (30mg/kg). Co-injection of SLIGRL-NH2 (40nmol) with either the slow-release H2S donor GYY4137 (1 and 3nmol) or the spontaneous donor NaHS (1 and 0.3nmol) significantly reduced pruritus. Co-treatment with the KATP channel blocker glibenclamide (200nmol) or the nitric oxide (NO) donor sodium nitroprusside (10nmol) abolished the antipruritic effects of NaHS; however, the specific soluble guanylyl cyclase inhibitor ODQ (30μg) had no significant effects. The transient receptor potential ankyrin type 1 (TRPA1) antagonist HC-030031 (20μg) significantly reduced SLIGRL-NH2-induced pruritus; however pruritus induced by the TRPA1 agonist AITC (1000nmol) was unaffected by NaHS. Based on these data, we conclude that pruritus secondary to PAR-2 activation can be reduced by H2S, which acts through KATP channel opening and involves NO in a cyclic guanosine monophosphate (cGMP)-independent manner. Furthermore, TRPA1 receptors mediate the pruritus induced by activation of PAR-2, but H2S does not interfere with this pathway. These results provide additional support for the development of new therapeutical alternatives, mainly intended for treatment of pruritus in patients unresponsive to anti-histamines.MNM and SKPC are recipients of fellowships from the National Council for Scientific and Technological Development (CNPq) and grants from the Sao Paulo Research Foundation (FAPESP). RT, MW and MEW would like to thank the Brian Ridge Scholarship for its support (RT)

Protective effects of exogenous and endogenous hydrogen sulfide in mast cell-mediated pruritus and cutaneous acute inflammation in mice.

Published onlineJournal ArticleThis is the author accepted manuscript. The final version is available from Elsevier via the DOI in this record.The recently described 'gasomediator' hydrogen sulfide (H2S) has been involved in pain mechanisms, but its effect on pruritus, a sensory modality that similarly to pain acts as a protective mechanism, is poorly known and controversial. The effects of the slow-releasing (GYY4137) and spontaneous H2S donors (Na2S and Lawesson's reagent, LR) were evaluated in histamine and compound 48/80 (C48/80)-dependent dorsal skin pruritus and inflammation in male BALB/c mice. Animals were intradermally (i.d.) injected with C48/80 (3μg/site) or histamine (1μmol/site) alone or co-injected with Na2S, LR or GYY4137 (within the 0.3-100nmol range). The involvement of endogenous H2S and KATP channel-dependent mechanism were also evaluated. Pruritus was assessed by the number of scratching bouts, whilst skin inflammation was evaluated by the extravascular accumulation of intravenously injected (125)I-albumin (plasma extravasation) and myeloperoxidase (MPO) activity (neutrophil recruitment). Histamine or C48/80 significantly evoked itching behavior paralleled by plasma extravasation and increased MPO activity. Na2S and LR significantly ameliorated histamine or C48/80-induced pruritus and inflammation, although these effects were less pronounced or absent with GYY4137. Inhibition of endogenous H2S synthesis exacerbated C48/80-induced responses, whereas the blockade of KATP channels by glibenclamide did not. High-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) revealed that Na2S and LR, but not GYY4137, significantly attenuated C48/80-induced histamine release from rat peritoneal mast cell in vitro. We provide first evidences that H2S exerted protective effect against acute pruritus mediated via histaminergic pathways in murine skin, thus making of H2S donors a potential alternative/complementary therapy for treatment of acute pruritus.Sao Paulo Research Foundation (Fapesp grant numbers: 2013/04.151-3, 2014/15.576-8, 2014/24.518-1) and CNPq (grant number: 163278/2012-1). GDN, MNM and SKPC are recipients of fellowships from the National Council for Scientific and Technological Development (CNPq). We thank Irene M Gouvea, Flávia B de Lira and Mauro Sucupira for their techinical support

Vasorelaxant Activity of AP39, a Mitochondria-Targeted H2S Donor, on Mouse Mesenteric Artery Rings In Vitro

This is the final version. Available from MDPI via the DOI in this record. The data presented in this study are available on request from the corresponding author.Mitochondria-targeted hydrogen sulfide (H2S) donor compounds, such as compound AP39, supply H2S into the mitochondrial environment and have shown several beneficial in vitro and in vivo effects in cardiovascular conditions such as diabetes and hypertension. However, the study of their direct vascular effects has not been addressed to date. Thus, the objective of the present study was to analyze the effects and describe the mechanisms of action of AP39 on the in vitro vascular reactivity of mouse mesenteric artery. Protein and gene expressions of the H2S-producing enzymes (CBS, CSE, and 3MPST) were respectively analyzed by Western blot and qualitative RT-PCR, as well the in vitro production of H2S by mesenteric artery homogenates. Gene expression of CSE and 3MPST in the vessels has been evidenced by RT-PCR experiments, whereas the protein expression of all the three enzymes was demonstrated by Western blotting experiments. Nonselective inhibition of H2S-producing enzymes by AOAA abolished H2S production, whereas it was partially inhibited by PAG (a CSE selective inhibitor). Vasorelaxation promoted by AP39 and its H2S-releasing moiety (ADT-OH) were significantly reduced after endothelium removal, specifically dependent on NO-cGMP signaling and SKCa channel opening. Endogenous H2S seems to participate in the mechanism of action of AP39, and glibenclamide-induced KATP blockade did not affect the vasorelaxant response. Considering the results of the present study and the previously demonstrated antioxidant and bioenergetic effects of AP39, we conclude that mitochondria-targeted H2S donors may offer a new promising perspective in cardiovascular disease therapeutics.The Royal SocietySao Paulo Research Foundation (FAPESP)Sao Paulo Research Foundation (FAPESP)Brazilian National Council for Scientific and Technological Development (CNPq)Brazilian National Council for Scientific and Technological Development (CNPq)Coordination for the Improvement of Higher Education Personnel (CAPES

Antihypertensive properties of a nitric oxide-releasing naproxen derivative in two-kidney, one-clip rats.

Nonsteroidal anti-inflammatory drugs have been reported to exacerbate hypertension. In this study, we tested the hypothesis that a nitric oxide-releasing derivative of naproxen would ameliorate hypertension in the rat. Hypertension was induced by partially occluding one renal artery (the "2K,1C" model), and 2 wk later the rats started receiving naproxen, the nitric oxide-releasing derivative HCT-3012, or vehicle each day for 2 wk. Naproxen significantly exacerbated the hypertension. HCT-3012 significantly reduced blood pressure relative to both the naproxen- and vehicle-treated groups. Both naproxen and HCT-3012 markedly suppressed whole blood thromboxane B(2) synthesis. In studies of anesthetized rats, naproxen significantly enhanced the late hypertensive response to endothelin-1 and significantly blunted the early hypotensive response. In contrast, HCT-3102 did not affect either response to endothelin-1. In vitro, HCT-3012 significantly reduced the responsiveness of aortic rings to the contractile effects of phenylephrine. These studies suggest that HCT-3012 reduces blood pressure in hypertensive rats, not simply through the vasodilatory actions of the nitric oxide it releases, but through alterations in the responsiveness of the vasculature to endogenous pressor agents

Plasma hydroxy metronidazole/metronidazole ratio in anti-HCV carriers with and without apparent liver disease

Aims: To evaluate plasma hydroxy-metronidazole/metronidazole ratio as a dynamic liver function test in HCV-infected individuals with/without liver disease, in the absence of liver cirrhosis. Methods: Metronidazole was administered intravenously in healthy volunteers, asymptomatic anti-HCV-positive blood donors, and in chronic hepatitis C patients. Serology to HCV was determined by a second generation assay and confirmed by gelatin particle agglutination test using recombinant antigens C22-3 and C200. Plasma concentration of metronidazole and hydroxy-metronidazole was measured by high performance liquid chromatography in samples collected 5, 10, 20 and 30 min following the end of metronidazole infusion. Results: Chronic hepatitis C patients had abnormal liver enzymes, while healthy volunteers and anti-HCV-positive blood donors had normal liver biochemistry tests. Plasma metronidazole concentration was similar,in all groups studied. 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