Naunyn-Schmiedeberg's Archives of Pharmacology

, Volume 387, Issue 1, pp 67–74 | Cite as

Hydrogen sulfide protects endothelial nitric oxide function under conditions of acute oxidative stress in vitro.

  • Mohammad R. Al-Magableh
  • Barbara K. Kemp-Harper
  • Hooi H. Ng
  • Alyson A. Miller
  • Joanne L. HartEmail author
Original Article


The aim of this study was to examine the ability of H2S, released from NaHS to protect vascular endothelial function under conditions of acute oxidative stress by scavenging superoxide anions (O2 ) and suppressing vascular superoxide anion production. O2 was generated in Krebs' solution by reacting hypoxanthine with xanthine oxidase (Hx-XO) or with the O2 generator pyrogallol to model acute oxidative stress in vitro. O2 generation was measured by lucigenin-enhanced chemiluminescence. Functional responses in mouse aortic rings were assessed using a small vessel myograph. NaHS scavenged O2 in a concentration-dependent manner. Isolated aortic rings exposed to either Hx-XO or pyrogallol displayed significantly attenuated maximum vasorelaxation responses to the endothelium-dependent vasodilator acetylcholine, and significantly reduced NO bioavailability, which was completely reversed if vessels were pre-incubated with NaHS (100 μM). NADPH-stimulated aortic O2 production was significantly attenuated by the NADPH oxidase inhibitor diphenyl iodonium. Prior treatment of vessels with NaHS (100 nM–100 μM; 30 min) inhibited NADPH-stimulated aortic O2 production in a concentration-dependent manner. This effect persisted when NaHS was washed out prior to measuring NADPH-stimulated O2 production. These data show for the first time that NaHS directly scavenges O2 and suppresses vascular NADPH oxidase-derived O2 production in vitro. Furthermore, these properties protect endothelial function and NO bioavailability in an in vitro model of acute oxidative stress. These results suggest that H2S can elicit vasoprotection by both scavenging O2 and by reducing vascular NADPH oxidase-derived O2 production.


Hydrogen sulfide Superoxide Vasoprotection NADPH oxidase 

List of abbreviations


Diphenyl iodonium


Combination of hypoxanthine and xanthine oxidase


Nω-nitro-l-arginine methyl ester hydrochloride


Nicotinamide adenine dinucleotide phosphate




Reactive oxygen species


Superoxide dismutase



Dr Hart was a NHMRC Peter Doherty Fellow and the project was additionally funded by the William Buckland Foundation, ANZ Trustees and the Ramaciotti Foundation. Dr Miller is the recipient of a NHMRC Career Development Fellowship.

Author disclosure statement

No competing financial interests exist.

Supplementary material

210_2013_920_MOESM1_ESM.docx (15 kb)
ESM 1 (DOCX 15 kb)


  1. Al-Magableh MR, Hart JL (2011) Mechanism of vasorelaxation and role of endogenous hydrogen sulfide production in mouse aorta. Naunyn Schmiedebergs Arch Pharmacol 383:403–413PubMedCrossRefGoogle Scholar
  2. Bir SC, Kolluru GK, McCarthy P, Shen X, Pardue S, Pattillo CB, Kevil CG (2012) Hydrogen sulfide stimulates ischemic vascular remodeling through nitric oxide synthase and nitrite reduction activity regulating hypoxia-inducible factor-1alpha and vascular endothelial growth factor-dependent angiogenesis. J Am Heart Assoc 1:e004093PubMedCentralPubMedGoogle Scholar
  3. Brandes RP, Weissmann N, Schroder K (2010) NADPH oxidases in cardiovascular disease. Free Radic Biol Med 49:687–706PubMedCrossRefGoogle Scholar
  4. Bucci M, Papapetropoulos A, Vellecco V, Zhou Z, Pyriochou A, Roussos C, Roviezzo F, Brancaleone V, Cirino G (2010) Hydrogen sulfide is an endogenous inhibitor of phosphodiesterase activity. Arterioscler Thromb Vasc Biol 30:1998–2004PubMedCrossRefGoogle Scholar
  5. Bucci M, Papapetropoulos A, Vellecco V, Zhou Z, Zaid A, Giannogonas P, Cantalupo A, Dhayade S, Karalis KP, Wang R, Feil R, Cirino G (2012) cGMP-dependent protein kinase contributes to hydrogen sulfide-stimulated vasorelaxation. PLoS One 7:e53319PubMedCentralPubMedCrossRefGoogle Scholar
  6. Carballal S, Trujillo M, Cuevasanta E, Bartesaghi S, Moller MN, Folkes LK, Garcia-Bereguiain MA, Gutierrez-Merino C, Wardman P, Denicola A, Radi R, Alvarez B (2011) Reactivity of hydrogen sulfide with peroxynitrite and other oxidants of biological interest. Free Radic Biol Med 50:196–205PubMedCrossRefGoogle Scholar
  7. Chataigneau T, Feletou M, Huang PL, Fishman MC, Duhault J, Vanhoutte PM (1999) Acetylcholine-induced relaxation in blood vessels from endothelial nitric oxide synthase knockout mice. Br J Pharmacol 126:219–226PubMedCrossRefGoogle Scholar
  8. Chen AF, Chen DD, Daiber A, Faraci FM, Li H, Rembold CM, Laher I (2012) Free radical biology of the cardiovascular system. Clin Sci (Lond) 123:73–91CrossRefGoogle Scholar
  9. Cheng Y, Ndisang J, Tang G, Cao K, Wang R (2004) Hydrogen sulfide-induced relaxation of resistance mesenteric artery beds of rats. Am J Physiol Heart Circ Physiol 287:2316–2323CrossRefGoogle Scholar
  10. DeLeon ER, Stoy GF, Olson KR (2012) Passive loss of hydrogen sulfide in biological experiments. Anal Biochem 421:203–207PubMedCrossRefGoogle Scholar
  11. Dombkowski RA, Russell MJ, Olson KR (2004) Hydrogen sulfide as an endogenous regulator of vascular smooth muscle tone in trout. Am J Physiol Regul Integr Comp Physiol 286:R678–R685PubMedCrossRefGoogle Scholar
  12. Dowell FJ, Hamilton CA, McMurray J, Reid JL (1993) Effects of a xanthine oxidase/hypoxanthine free radical and reactive oxygen species generating system on endothelial function in New Zealand white rabbit aortic rings. J Cardiovasc Pharmacol 22:792–797PubMedCrossRefGoogle Scholar
  13. Drummond GR, Selemidis S, Griendling KK, Sobey CG (2011) Combating oxidative stress in vascular disease: NADPH oxidases as therapeutic targets. Nat Rev Drug Discov 10:453–471PubMedCentralPubMedCrossRefGoogle Scholar
  14. Ford A, Al-Magableh M, Gaspari TA, Hart JL (2013) Chronic NaHS treatment is vasoprotective in high fat fed ApoE-/- mice. Int J Vasc Med 2013:915983. doi: 10.1155/2013/915983
  15. Forstermann U (2008) Oxidative stress in vascular disease: causes, defense mechanisms and potential therapies. Nat Clin Pract Cardiovasc Med 5:338–349PubMedCrossRefGoogle Scholar
  16. Fukuto JM, Carrington SJ, Tantillo DJ, Harrison JG, Ignarro LJ, Freeman BA, Chen A, Wink DA (2012) Small molecule signaling agents: the integrated chemistry and biochemistry of nitrogen oxides, oxides of carbon, dioxygen, hydrogen sulfide, and their derived species. Chem Res Toxicol 25:769–793PubMedCrossRefGoogle Scholar
  17. Furne J, Saeed A, Levitt MD (2008) Whole tissue hydrogen sulfide concentrations are orders of magnitude lower than presently accepted values. Am J Physiol Regul Integr Comp Physiol 295:R1479–R1485PubMedCrossRefGoogle Scholar
  18. Gryglewski RJ, Palmer RM, Moncada S (1986) Superoxide anion is involved in the breakdown of endothelium-derived vascular relaxing factor. Nature 320:454–456PubMedCrossRefGoogle Scholar
  19. Kida M, Sugiyama T, Yoshimoto T, Ogawa Y (2013) Hydrogen sulfide increases nitric oxide production with calcium-dependent activation of endothelial nitric oxide synthase in endothelial cells. Eur J Pharm Sci 48:211–215PubMedCrossRefGoogle Scholar
  20. Kietadisorn R, Juni RP, Moens AL (2012) Tackling endothelial dysfunction by modulating NOS uncoupling: new insights into its pathogenesis and therapeutic possibilities. Am J Physiol Endocrinol Metab 302:E481–E495PubMedCrossRefGoogle Scholar
  21. Kimura H, Shibuya N, Kimura Y (2012) Hydrogen sulfide is a signaling molecule and a cytoprotectant. Antioxid Redox Signal 17:45–57PubMedCrossRefGoogle Scholar
  22. Kimura Y, Goto Y, Kimura H (2010) Hydrogen sulfide increases glutathione production and suppresses oxidative stress in mitochondria. Antioxid Redox Signal 12:1–13PubMedCrossRefGoogle Scholar
  23. Kimura Y, Kimura H (2004) Hydrogen sulfide protects neurons from oxidative stress. FASEB J 18:1165–1167PubMedGoogle Scholar
  24. Land WG (2012) Emerging role of innate immunity in organ transplantation: part I: evolution of innate immunity and oxidative allograft injury. Transplant Rev (Orlando) 26:60–72CrossRefGoogle Scholar
  25. Lassegue B, San Martin A, Griendling KK (2012) Biochemistry, physiology, and pathophysiology of NADPH oxidases in the cardiovascular system. Circ Res 110:1364–1390PubMedCentralPubMedCrossRefGoogle Scholar
  26. Li L, Whiteman M, Guan YY, Neo KL, Cheng Y, Lee SW, Zhao Y, Baskar R, Tan CH, Moore PK (2008) Characterization of a novel, water-soluble hydrogen sulfide-releasing molecule (GYY4137): new insights into the biology of hydrogen sulfide. Circulation 117:2351–2360PubMedCrossRefGoogle Scholar
  27. Lu M, Hu LF, Hu G, Bian JS (2008) Hydrogen sulfide protects astrocytes against H2O2 induced neural injury via enhancing glutamate uptake. Free Radic Biol Med 45:1705–1713PubMedCrossRefGoogle Scholar
  28. MacKenzie A, Martin W (1998) Loss of endothelium-derived nitric oxide in rabbit aorta by oxidant stress: restoration by superoxide dismutase mimetics. Br J Pharmacol 124:719–728PubMedCrossRefGoogle Scholar
  29. Mian KB, Martin W (1995) Differential sensitivity of basal and acetylcholine-stimulated activity of nitric oxide to destruction by superoxide anion in rat aorta. Br J Pharmacol 115:993–1000PubMedCrossRefGoogle Scholar
  30. Miller AA, De Silva TM, Judkins CP, Diep H, Drummond GR, Sobey CG (2010) Augmented superoxide production by Nox2-containing NADPH oxidase causes cerebral artery dysfunction during hypercholesterolemia. Stroke 41:784–789PubMedCrossRefGoogle Scholar
  31. Miller AA, Drummond GR, De Silva TM, Mast AE, Hickey H, Williams JP, Broughton BR, Sobey CG (2009) NADPH oxidase activity is higher in cerebral versus systemic arteries of four animal species: role of Nox2. Am J Physiol Heart Circ Physiol 296:H220–H225PubMedCrossRefGoogle Scholar
  32. Miller AA, Drummond GR, Schmidt HH, Sobey CG (2005) NADPH oxidase activity and function are profoundly greater in cerebral versus systemic arteries. Circ Res 97:1055–1062PubMedCrossRefGoogle Scholar
  33. Muzaffar S, Jeremy JY, Sparatore A, Del Soldato P, Angelini GD, Shukla N (2008a) H2S-donating sildenafil (ACS6) inhibits superoxide formation and gp91phox expression in arterial endothelial cells: role of protein kinases A and G. Br J Pharmacol 155:984–994PubMedCrossRefGoogle Scholar
  34. Muzaffar S, Shukla N, Bond M, Newby AC, Angelini GD, Sparatore A, Del Soldato P, Jeremy JY (2008b) Exogenous hydrogen sulfide inhibits superoxide formation, NOX-1 expression and Rac1 activity in human vascular smooth muscle cells. J Vasc Res 45:521–528PubMedCrossRefGoogle Scholar
  35. Olson KR (2012) A practical look at the chemistry and biology of hydrogen sulfide. Antioxid Redox Signal 17:32–44PubMedCrossRefGoogle Scholar
  36. Pacher P, Beckman JS, Liaudet L (2007) Nitric oxide and peroxynitrite in health and disease. Physiol Rev 87:315–424PubMedCentralPubMedCrossRefGoogle Scholar
  37. Predmore BL, Julian D, Cardounel AJ (2011) Hydrogen sulfide increases nitric oxide production from endothelial cells by an akt-dependent mechanism. Front Physiol 2:104PubMedCentralPubMedGoogle Scholar
  38. Predmore BL, Lefer DJ, Gojon G (2012) Hydrogen sulfide in biochemistry and medicine. Antioxid Redox Signal 17:119–140PubMedCrossRefGoogle Scholar
  39. Renga B (2011) Hydrogen sulfide generation in mammals: the molecular biology of cystathionine-beta- synthase (CBS) and cystathionine-gamma-lyase (CSE). Inflamm Allergy Drug Targets 10:85–91PubMedCrossRefGoogle Scholar
  40. Rivera J, Sobey CG, Walduck AK, Drummond GR (2010) Nox isoforms in vascular pathophysiology: insights from transgenic and knockout mouse models. Redox Rep 15:50–63PubMedCrossRefGoogle Scholar
  41. Schramm A, Matusik P, Osmenda G, Guzik TJ (2012) Targeting NADPH oxidases in vascular pharmacology. Vascul Pharmacol 56:216–231PubMedCentralPubMedCrossRefGoogle Scholar
  42. Searcy DG, Whitehead JP, Maroney MJ (1995) Interaction of Cu, Zn superoxide dismutase with hydrogen sulfide. Arch Biochem Biophys 318:251–263PubMedCrossRefGoogle Scholar
  43. Selemidis S, Sobey CG, Wingler K, Schmidt HH, Drummond GR (2008) NADPH oxidases in the vasculature: molecular features, roles in disease and pharmacological inhibition. Pharmacol Ther 120:254–291PubMedCrossRefGoogle Scholar
  44. Shibuya N, Tanaka M, Yoshida M, Ogasawara Y, Togawa T, Ishii K, Kimura H (2009) 3-Mercaptopyruvate sulfurtransferase produces hydrogen sulfide and bound sulfane sulfur in the brain. Antioxid Redox Signal 11:703–714PubMedCrossRefGoogle Scholar
  45. Stasko A, Brezova V, Zalibera M, Biskupic S, Ondrias K (2009) Electron transfer: a primary step in the reactions of sodium hydrosulphide, an H2S/HS(−) donor. Free Radic Res 43:581–593PubMedCrossRefGoogle Scholar
  46. Streeter E, Hart J, Badoer E (2012) An investigation of the mechanisms of hydrogen sulfide-induced vasorelaxation in rat middle cerebral arteries. Naunyn Schmiedebergs Arch Pharmacol 385:991–1002PubMedCrossRefGoogle Scholar
  47. Sun Y, Tang CS, Du JB, Jin HF (2011) Hydrogen sulfide and vascular relaxation. Chin Med J (Engl) 124:3816–3819Google Scholar
  48. Touyz RM, Briones AM, Sedeek M, Burger D, Montezano AC (2011) NOX isoforms and reactive oxygen species in vascular health. Mol Interv 11:27–35PubMedCrossRefGoogle Scholar
  49. Vasquez-Vivar J, Kalyanaraman B, Martasek P (2003) The role of tetrahydrobiopterin in superoxide generation from eNOS: enzymology and physiological implications. Free Radic Res 37:121–127PubMedCrossRefGoogle Scholar
  50. Whiteman M, Armstong J, Chu S, Jia-Ling S, Wong B, Cheung N, Halliwell B, Moore P (2004) The novel neuromodulator hydrogen sulfide: an endogenous peroxynitrite 'scavenger'? J Neurochem 90:765–768PubMedCrossRefGoogle Scholar
  51. Whiteman M, Cheung N, Zhu Y, Chu S, Siau J, Wong B, Armstrong J, Moore P (2005) Hydrogen sulphide: a novel inhibitor of hypochlorous acid-mediated oxidative damage in the brain? Biochem Biophys Res Comm 343:303–310CrossRefGoogle Scholar
  52. Yan S, Chang T, Wang H, Wu L, Wang R, Meng Q (2006) Effects of hydrogen sulfide on homocysteine-induced oxidative stress in vascular smooth muscle cells. Biochem Biophys Res Comm 333:11146–11152Google Scholar
  53. Zhao W, Wang R (2002) H(2)S-induced vasorelaxation and underlying cellular and molecular mechanisms. Am J Physiol Heart Circ Physiol 283:H474–H480PubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Mohammad R. Al-Magableh
    • 1
  • Barbara K. Kemp-Harper
    • 1
  • Hooi H. Ng
    • 2
  • Alyson A. Miller
    • 1
  • Joanne L. Hart
    • 2
    Email author
  1. 1.Department of PharmacologyMonash UniversityClaytonAustralia
  2. 2.School of Medical Sciences and Health Innovations Research Institute (HIRi)RMIT UniversityBundoora WestAustralia

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