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Hydrogen sulfide: a new EDRF

2009; Elsevier BV; Volume: 76; Issue: 7 Linguagem: Inglês

10.1038/ki.2009.221

1523-1755

Rui Wang,

Eicosanoids and Hypertension Pharmacology

The first endothelium-derived relaxing factor (EDRF) ever identified is a gasotransmitter, nitric oxide (NO). Recent studies have provided several lines of evidence to support the premise that hydrogen sulfide (H2S), another gasotransmitter, is a new EDRF. H2S production is catalyzed in mammalian cells by cystathionine β-synthase (CBS) and/or cystathionine γ-lyase (CSE). The expression of CSE proteins and the activity of CBS have been observed in vascular endothelial cells. A measurable amount of H2S is produced from endothelium upon muscarinic cholinergic stimulation. The endothelium-dependent vasorelaxation induced by H2S shares many common mechanistic traits with those of endothelium-derived hyperpolarizing factor (EDHF). Deficiency in CSE expression increases blood pressure in CSE knockout mice and significantly diminishes endothelium-dependent relaxation of resistance arteries. More extensive and mechanistic studies in the future will help to determine whether H2S is a new EDRF or the very EDHF. The first endothelium-derived relaxing factor (EDRF) ever identified is a gasotransmitter, nitric oxide (NO). Recent studies have provided several lines of evidence to support the premise that hydrogen sulfide (H2S), another gasotransmitter, is a new EDRF. H2S production is catalyzed in mammalian cells by cystathionine β-synthase (CBS) and/or cystathionine γ-lyase (CSE). The expression of CSE proteins and the activity of CBS have been observed in vascular endothelial cells. A measurable amount of H2S is produced from endothelium upon muscarinic cholinergic stimulation. The endothelium-dependent vasorelaxation induced by H2S shares many common mechanistic traits with those of endothelium-derived hyperpolarizing factor (EDHF). Deficiency in CSE expression increases blood pressure in CSE knockout mice and significantly diminishes endothelium-dependent relaxation of resistance arteries. More extensive and mechanistic studies in the future will help to determine whether H2S is a new EDRF or the very EDHF. Dilation of blood vessels can be regulated by a group of labile humoral substances released from the endothelium, namely endothelium-derived relaxing factors (EDRFs). The paradigm-shaking discovery of nitric oxide (NO) as an EDRF decades ago1.Furchgott R.F. Zawadzki J.V. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine.Nature. 1980; 288: 373-376Crossref PubMed Scopus (9605) Google Scholar marked the beginning of a new era in cardiovascular research. Upon activation by different stimuli, such as acetylcholine and bradykinin, endothelial cells have their intracellular calcium level increased, which subsequently activates the endothelial NO synthase (eNOS). This leads to the production of NO and citrulline from the substrates L-arginine and molecular O2. NO is released from endothelial cells and stimulates guanylyl cyclase in vascular smooth muscle cells (SMCs). Consequently, cGMP levels in vascular SMCs are elevated and the cells relax (Figure 1). The initial excitement of NO discovery has transformed into many new explorations and triggered wave after wave of aftershocks. It is generally agreed upon that there are more than one EDRF, which are produced by the endothelium and cause blood vessel dilation. Prostacyclin (PGI2) is produced from the endothelium through cyclooxygenase-1, and binds to specific receptors in SMCs and activates adenylate cyclase. Thus, increased cAMP levels in SMCs relax the cells (Figure 1). In most cases, NO-mediated endothelium-dependent vasorelaxation occurs in large conduit arteries whereas the endothelium-dependent relaxation of peripheral resistance arteries does not depend on NO production. After eliminating the endothelial production of NO and PGI2 by knocking out the expression of eNOS and cyclooxygenase-1, there is still residual endothelium-dependent relaxation.2.Scotland R.S. Madhani M. Chauhan S. et al.Investigation of vascular responses in endothelial nitric oxide synthase /cyclooxygenase-1 double-knockout mice: key role for endothelium-derived hyperpolarizing factor in the regulation of blood pressure in vivo.Circulation. 2005; 111: 796-803Crossref PubMed Scopus (169) Google Scholar Even considering only non-prostanoid EDRF, NO may not be alone. The long-standing mystery surrounding the endothelium-derived hyperpolarizing factor (EDHF) has not been solved, and the roles of other gasotransmitters3.Wang R. Two's company, three's a crowd – Can H2S be the third endogenous gaseous transmitter?.FASEB J. 2002; 16: 1792-1798Crossref PubMed Scopus (1425) Google Scholar in endothelium-dependent vasorelaxation have been questioned in recent years. The chemical profile of hydrogen sulfide (H2S) is similar to that of NO. Both are small molecules of gas, generated in mammalian cells by enzymatic catalyzation, and freely permeable to lipid bilayer. H2S has been traditionally viewed as a toxic gas, and is infamous for its of 'rotten egg' smell. Its toxicological effect is mainly manifested as acute intoxication and loss of central respiratory drive. Only in the last decade, biological and physiological importance of this gas molecule has been exposed under limelight. In mammalian cells, H2S is produced from L-cysteine, catalyzed by one of two pyridoxal-5′-phosphate-dependent enzymes, cystathionine β-synthase (CBS) and/or cystathionine γ-lyase (CSE).3.Wang R. Two's company, three's a crowd – Can H2S be the third endogenous gaseous transmitter?.FASEB J. 2002; 16: 1792-1798Crossref PubMed Scopus (1425) Google Scholar In addition to H2S, L-cysteine catabolism also generates ammonium and pyruvate (Figure 2). The expressions of CBS and CSE are tissue-type specific. CBS is reported to be the predominant H2S-generating enzyme in the brain and nervous system and CSE is the predominant H2S-generating enzyme in pancreatic β-islets, different vascular tissues, and the heart.3.Wang R. Two's company, three's a crowd – Can H2S be the third endogenous gaseous transmitter?.FASEB J. 2002; 16: 1792-1798Crossref PubMed Scopus (1425) Google Scholar Physiological range of H2S in circulation is 10–100 μM in fish, rats, and human. Endogenous level of H2S in brain tissue is approximately 50–160 μM. Our knowledge on the cardiovascular effects of H2S has amounted in recent years, and the momentum in searching the physiological and pathophysiological roles of H2S has been built up. H2S induces relaxation of different vascular tissues (for example, rat aorta and mesenteric arteries), and protects the heart from ischemia/reperfusion damage. H2S can induce vasorelaxation by directly opening KATP channels in vascular SMCs. KATP channel blocker, glibenclamide, inhibited H2S-induced dilation of aorta,4.Zhao W. Zhang J. Lu Y. et al.The vasorelaxant effect of H2S as a novel endogenous gaseous KATP channel opener.EMBO J. 2001; 20: 6008-6016Crossref PubMed Scopus (1547) Google Scholar mesenteric artery beds,5.Cheng Y. Ndisang J.F. Tang G. et al.Hydrogen sulfide-induced relaxation of resistance mesenteric artery beds of rats.Am J Physiol. 2004; 287: H2316-H2323Crossref PubMed Scopus (367) Google Scholar or hepatic microcirculation6.Fiorucci S. Antonelli E. Mencarelli A. et al.The third gas: H2S regulates perfusion pressure in both the isolated and perfused normal rat liver and in cirrhosis.Hepatology. 2005; 42: 539-548Crossref PubMed Scopus (438) Google Scholar and abolished H2S-evoked blood pressure decrease in vivo.4.Zhao W. Zhang J. Lu Y. et al.The vasorelaxant effect of H2S as a novel endogenous gaseous KATP channel opener.EMBO J. 2001; 20: 6008-6016Crossref PubMed Scopus (1547) Google Scholar Using the whole-cell and single-channel patch-clamp technique, direct evidence was obtained that exogenous H2S activated KATP channels and hyperpolarized cell membrane of rat aorta and mesenteric artery SMCs.4.Zhao W. Zhang J. Lu Y. et al.The vasorelaxant effect of H2S as a novel endogenous gaseous KATP channel opener.EMBO J. 2001; 20: 6008-6016Crossref PubMed Scopus (1547) Google Scholar Glibenclamide also abolished H2S-facilitated carotid sinus baroreflex.7.Xiao L. Wu Y.M. Zhang H. et al.Hydrogen sulfide facilitates carotid sinus baroreflex in anesthetized rats.Acta Pharmacol Sin. 2006; 27: 294-298Crossref PubMed Scopus (26) Google Scholar Inhibition of endogenous H2S production with D,L-propargylglycine, a membrane permeable and irreversible inhibitor of CSE, reduced whole-cell KATP currents.4.Zhao W. Zhang J. Lu Y. et al.The vasorelaxant effect of H2S as a novel endogenous gaseous KATP channel opener.EMBO J. 2001; 20: 6008-6016Crossref PubMed Scopus (1547) Google Scholar In myocardium, a mitochondrial KATP channel blocker (5-hydroxydecanoate) or a plasma membrane KATP channel blocker (glibenclamide) abolished H2S-offered cardioprotection against myocardiac infarction damage.8.Johansen D. Ytrehus K. Baxter G.F. et al.Exogenous hydrogen sulfide (H2S) protects against regional myocardial ischemia-reperfusion injury – evidence for a role of KATP channels.Basic Res Cardiol. 2006; 101: 53-60Crossref PubMed Scopus (240) Google Scholar With a broader concept to encompass the origination from endothelium and downstream target of SMCs, we have more than one EDRF. Certainly, EDHF can be categorized into the EDRF class and the idea of H2S being a new EDRF is both intriguing and appealing. Elucidation of the source of H2S production in the blood vessel wall and the physiological role of this gas in regulating cardiovascular function may pave the way for new therapeutic approach, either pharmacologically or genetically, to treat and prevent many different cardiovascular diseases, including hypertension and heart damage. In an earlier study, CSE mRNA expression was detected in smooth muscles of rat aorta wall, using an in situ hybridization method.4.Zhao W. Zhang J. Lu Y. et al.The vasorelaxant effect of H2S as a novel endogenous gaseous KATP channel opener.EMBO J. 2001; 20: 6008-6016Crossref PubMed Scopus (1547) Google Scholar In cultured rat aortic endothelial cells, CSE mRNA was not detected using reverse transcription-PCR.4.Zhao W. Zhang J. Lu Y. et al.The vasorelaxant effect of H2S as a novel endogenous gaseous KATP channel opener.EMBO J. 2001; 20: 6008-6016Crossref PubMed Scopus (1547) Google Scholar CSE protein expression was not conducted at that time due to the lack of CSE-specific antibody.4.Zhao W. Zhang J. Lu Y. et al.The vasorelaxant effect of H2S as a novel endogenous gaseous KATP channel opener.EMBO J. 2001; 20: 6008-6016Crossref PubMed Scopus (1547) Google Scholar In the liver, CSE was found in hepatocytes and hepatic stellate cells, but not in sinusoidal endothelial cells.6.Fiorucci S. Antonelli E. Mencarelli A. et al.The third gas: H2S regulates perfusion pressure in both the isolated and perfused normal rat liver and in cirrhosis.Hepatology. 2005; 42: 539-548Crossref PubMed Scopus (438) Google Scholar It appeared that the vascular endothelium per se was not equipped with H2S-generating enzymes. This notion has been recently challenged and it no longer holds true. CSE protein has been detected in cultured bovine aortic endothelial cells and human umbilical vein endothelial cells. Yang et al.9.Yang G. Wu L. Jiang B. et al.H2S as a physiologic vasorelaxant: Hypertension in mice with deletion of cystathionine gamma-lyase.Science. 2008; 322: 587-590Crossref PubMed Scopus (1733) Google Scholar provided immunohistological evidence for a predominant localization of CSE protein in the endothelial layer of vascular tissues of wild-type mice. This newly defined endothelial localization of CSE can be largely ascribed to the availability of newly developed selective and specific anti-CSE antibody. Direct evidence for the endothelium production of H2S was also obtained. Cultured vascular endothelial cells produce measurable H2S and silencing CSE in these cells significantly decreases H2S production.9.Yang G. Wu L. Jiang B. et al.H2S as a physiologic vasorelaxant: Hypertension in mice with deletion of cystathionine gamma-lyase.Science. 2008; 322: 587-590Crossref PubMed Scopus (1733) Google Scholar Similar to NO, acute production of H2S from vascular endothelial cells is triggered by muscarinic cholinergic activation. Methacholine, a cholinergic receptor agonist, increased H2S levels in cultured endothelial cells by more than twofold. Atropine, a cholinergic antagonist, abolished methacholine effect. In the presence of the calcium ionophore A23187, endothelial production of H2S is significantly increased. Chelating of intracellular-free calcium with BAPTA abolished the effect of A23187 on H2S production as well as lowering the basal level of H2S in endothelial cells. Furthermore, co-immunoprecipitation study demonstrated the binding of calmodulin to recombinant CSE in vitro. This binding is contingent on the presence of calcium, and abolished by calmodulin antagonist W7. These results demonstrated that a calcium–calmodulin system is the prerequisite for muscarinic activation of CSE in endothelial cells. Once CSE is knocked down from endothelial cells, the effects of methacholine and A23187 on H2S production were abolished, indicating CSE activation is the key link in calcium–calmodulin-dependent H2S production.9.Yang G. Wu L. Jiang B. et al.H2S as a physiologic vasorelaxant: Hypertension in mice with deletion of cystathionine gamma-lyase.Science. 2008; 322: 587-590Crossref PubMed Scopus (1733) Google Scholar The vasorelaxant effect of H2S is also endothelium-dependent. The removal of endothelium attenuated the relaxation of rat aortic tissues induced by H2S and shifted H2S concentration–response curve to the right with EC50 changed from 136 to 273 μM.10.Zhao W. Wang R. H2S-induced vasorelaxation and underlying cellular and molecular mechanisms.Am J Physiol. 2002; 283: H474-H480Crossref PubMed Scopus (433) Google Scholar Although this observation did not address whether H2S is endothelium derived or not, it shows that the presence of an intact endothelium does affect H2S-induced vasorelaxation. The endothelium dependence of H2S effect was more pronounced in isolated and perfused rat mesenteric artery bed.5.Cheng Y. Ndisang J.F. Tang G. et al.Hydrogen sulfide-induced relaxation of resistance mesenteric artery beds of rats.Am J Physiol. 2004; 287: H2316-H2323Crossref PubMed Scopus (367) Google Scholar The removal of the functional endothelium significantly reduced H2S-induced relaxation of rat mesenteric artery bed by about sevenfold with EC50 of H2S changed from 25 to 161 μM (P<0.05). This tissue type-selective endothelium-dependent effect of H2S is similar to that of EDHF. The EC50 of H2S in inducing vasorelaxation is quite close to the reported endogenous level of H2S in plasma, which suggests that under physiological in vivo conditions the vascular tone of resistance arteries is likely regulated by endogenous H2S. Long-term effect of H2S on the endothelium is observed as the increased proliferation of vascular endothelial cells, although H2S inhibits proliferation of many types of cells, such as vascular SMCs.11.Yang G. Sun X. Wang R. Hydrogen sulfide-induced apoptosis of human aorta smooth muscle cells via the activation of MAP kinases and caspase-3.FASEB J. 2004; 18: 1782-1784Crossref PubMed Scopus (251) Google Scholar NaHS (10–20 μM), an H2S donor, increased cell growth, migration, scratched wound healing, and tube-like structure formation in cultured human umbilical vein endothelial cells. These effects of NaHS were mediated by PI3K activation and Akt phosphorylation. Furthermore, NaHS treatment (10 and 50 μmol/kg/day, intraperitoneally) of mice significantly promoted neovascularization in vivo.12.Cai W.J. Wang M.J. Moore P.K. et al.The novel proangiogenic effect of hydrogen sulfide is dependent on Akt phosphorylation.Cardiovasc Res. 2007; 76: 29-40Crossref PubMed Scopus (315) Google Scholar Deficiency in the expression of CSE significantly lowers the H2S level in cardiovascular tissues, especially in endothelial cells. Methacholine-induced endothelium-dependent relaxation of resistance mesenteric arteries was significantly diminished in CSE-deficient mice.9.Yang G. Wu L. Jiang B. et al.H2S as a physiologic vasorelaxant: Hypertension in mice with deletion of cystathionine gamma-lyase.Science. 2008; 322: 587-590Crossref PubMed Scopus (1733) Google Scholar The half-life of NO in blood is counted in seconds and it can be scavenged by oxyhemoglobin. H2S seems to be more stable in protein-free solution.13.Zhao W. Ndisang J.F. Wang R. Modulation of endogenous production of H2S in rat tissues.Can J Physiol Pharmacol. 2003; 81: 848-853Crossref PubMed Scopus (195) Google Scholar However, H2S can also be scavenged by methemoglobin or disulfide-containing molecules such as oxidized glutathione.3.Wang R. Two's company, three's a crowd – Can H2S be the third endogenous gaseous transmitter?.FASEB J. 2002; 16: 1792-1798Crossref PubMed Scopus (1425) Google Scholar Therefore, the half-life of free H2S in blood may also be short. The unique property of EDHF, different from other putative EDRFs, is its specific action of hyperpolarizing vascular SMCs so as to close voltage-dependent calcium channels. The effect of EDHF is mainly mediated by small-conductance KCa channels and aided by intermediate-conductance KCa channels, which can be blocked by the co-application of apamin and charybdotoxin. It has been debated, and the case has not yet been settled, whether EDHF is a diffusible EDRF or a simple electrotonic phenomenon that relays membrane potential change in endothelium to the underneath SMCs through low-electrical resistance myoendothelial gap junctions. Among diffusible chemical candidates for EDHF are K+ ions, endothelium-derived C-type natriuretic peptide, H2O2, and P450 metabolites/epoxyeicosatrienoic acids. H2S possesses many common features of EDHF.(1)Some studies have shown that the vasorelaxing effect of H2S appears to be more potent on small-resistance mesenteric arteries than on large aortic vascular tissues (Figure 3). It is known that the contribution of EDHF to endothelium-dependent vasorelaxation is much greater in smaller arteries, including mesenteric artery and coronary artery.(2)Zhao et al.4.Zhao W. Zhang J. Lu Y. et al.The vasorelaxant effect of H2S as a novel endogenous gaseous KATP channel opener.EMBO J. 2001; 20: 6008-6016Crossref PubMed Scopus (1547) Google Scholar has shown that the co-application of apamin (50 nM) and charybdotoxin (50 nM) significantly weakened H2S-induced relaxation of endothelium-intact rat aortic tissues. The removal of endothelium similarly reduced H2S effect as apamin/charybdotoxin did. The same inhibitory effect of apamin/charybdotoxin on H2S-induced relaxation was also observed in rat mesenteric arteries.5.Cheng Y. Ndisang J.F. Tang G. et al.Hydrogen sulfide-induced relaxation of resistance mesenteric artery beds of rats.Am J Physiol. 2004; 287: H2316-H2323Crossref PubMed Scopus (367) Google Scholar On the other hand, whether treatment of the whole animal with apamin/charybdotoxin can affect H2S-induced hypotension has not been reported. These results suggest that either H2S itself is an EDHF or that H2S releases EDHF from the endothelium. As both H2S and NO are bioactive gas molecules, interaction between these two gasotransmitters has long been suspected. Pre-treating rat aortic tissues with 60 μM H2S inhibited the vasorelaxant effect of sodium nitroprusside, an NO donor. Incubation of NaHS with a range of NO donors or NO gas in vitro was reported to form a nitrosothiol molecule. In this way, H2S acts similar to an NO scavenger.14.Whiteman M. Li L. Kostetski I. et al.Evidence for the formation of a novel nitrosothiol from the gaseous mediators nitric oxide and hydrogen sulphide.Biochem Biophys Res Commun. 2006; 343: 303-310Crossref PubMed Scopus (293) Google Scholar The formation of this nitrosothiol molecule inhibited the vasorelaxant effect of NO both in vitro and in vivo. Low concentrations of NaHS or H2S gas in solution reversed the relaxant effects of acetylcholine and histamine on rat aortae. NaHS also inhibited the conversion of [3H]-arginine into [3H]-citrulline by recombinant eNOS.15.Kubo S. Doe I. Kurokawa Y. et al.Direct inhibition of endothelial nitric oxide synthase by hydrogen sulfide: contribution to dual modulation of vascular tension.Toxicology. 2007; 232: 138-146Crossref PubMed Scopus (157) Google Scholar In another study on isolated rat aortas and cultured human umbilical vein endothelial cells, NaHS incubation reduced eNOS activity, eNOS transcript abundance, and L-arginine transport. H2S treatment reduced protein expression of eNOS, but not of nNOS and iNOS. The downregulation of the vascular L-arginine/eNOS/NO pathway was observed after intraperitoneal injection of NaHS (14 μmol/kg) into rats.16.Geng B. Cui Y. Zhao J. et al.Hydrogen sulfide downregulates the aortic L-arginine/nitric oxide pathway in rats.Am J Physiol Regul Integr Comp Physiol. 2007; 293: R1608-R1618Crossref PubMed Scopus (103) Google Scholar In contrast, H2S by itself had no effect on NO production from the rat vascular SMCs, but it augmented interleukin-β-induced NO production and this effect was associated with increased expression of iNOS.17.Jeong S.O. Pae H.O. Oh G.S. et al.Hydrogen sulfide potentiates interleukin-1beta-induced nitric oxide production via enhancement of extracellular signal-regulated kinase activation in rat vascular smooth muscle cells.Biochem Biophys Res Commun. 2006; 345: 938-944Crossref PubMed Scopus (73) Google Scholar Whether H2S and NO at low concentrations have a synergistic effect on vascular relaxation has been controversial. To be equipped with both NO and H2S as EDRFs will enable blood vessels with heterogeneity to relax with selective and defined stimuli. Multiple EDRFs may help to add layers of control or backup mechanisms should one EDRF fail under pathological conditions. Moreover, the interaction between these two EDRFs will either compensate the loss or deficit, or buffer the overproduction or overaction, of one of the two. Accumulating data on the cardiovascular effects of endogenous H2S has stimulated mounting speculation that H2S might be another EDRF or the very EDHF. Whereas the score for the home team is up and all fans are celebrating, it is still too early to announce the winner before hitting a home run. Many challenges should be appropriately addressed regarding the viewpoint of H2S being a new EDRF. The premise that H2S is an EDRF for small resistance arteries that NO is an EDRF for large arteries, NO-mediated endothelium-dependent relaxation of large arteries should be investigated in CSE knockout mice so that the relative contribution of NO and H2S to endothelium-dependent vasorelaxation can be deciphered. As mentioned earlier, endogenous H2S can be generated by another enzyme, CBS. Wang et al.18.Wang J. Dudman N.P. Wilcken D.E. et al.Homocysteine catabolism: levels of 3 enzymes in cultured human vascular endothelium and their relevance to vascular disease.Atherosclerosis. 1992; 97: 97-106Abstract Full Text PDF PubMed Scopus (44) Google Scholar reported the activity of CBS as reflected by the production of cystathionine in cultured human umbilical venous endothelial cells. However, no attempt was taken then to detect CBS mRNA or protein in these cells, which had been cultured for 14 days with the addition of 100 μM L-homocysteine. This observation hints that CBS may function as an inducible H2S-generating enzyme in the vascular endothelial cells, which merits further investigation. The upregulation of CBS may occur when homocysteine level or other links of trans-sulfuration pathway are altered. As such, not only the sulfur metabolism is affected, endothelium-dependent production of H2S and vasorelaxation would also be regulated under different physiological and pathophysiological conditions. It is of note that EDHF plays a critical role in regulating endothelium-dependent vasorelaxation in female mice, whereas NO and PGI2 are the predominant EDRFs in male mice.2.Scotland R.S. Madhani M. Chauhan S. et al.Investigation of vascular responses in endothelial nitric oxide synthase /cyclooxygenase-1 double-knockout mice: key role for endothelium-derived hyperpolarizing factor in the regulation of blood pressure in vivo.Circulation. 2005; 111: 796-803Crossref PubMed Scopus (169) Google Scholar Whether heterogeneity of gender affects the role of H2S as an EDRF should be examined. The qualification of H2S as an EDHF needs electrophysiological recording of membrane potential changes. H2O2 has been suggested as the EDHF.19.Shimokawa H. Morikawa K. Hydrogen peroxide is an endothelium-derived hyperpolarizing factor in animals and humans.J Mol Cell Cardiol. 2005; 39: 725-732Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar In cultured rat aortic A-10 cells, low level of NaHS decreased the production of H2O2, ONOO−, and O2− in the presence of homocysteine, and improved cell viability.20.Yan S.K. Chang T. Wang H. et al.Effects of hydrogen sulfide on homocysteine-induced oxidative stress in vascular smooth muscle cells.Biochem Biophys Res Commun. 2006; 351: 485-491Crossref PubMed Scopus (159) Google Scholar Whether the endothelium-dependent vasorelaxant effect of H2S is mediated by altered H2O2 production or altered redox status in endothelial cells remains unclear. Relative contributions of KATP channels and small/intermediate KCa channels to the vascular effect of H2S need to be further clarified in different vascular tissues in order to determine whether H2S assumes the role of EDRF or EDHF depending on vascular tissue types. In conclusion, H2S is an accredited candidate for EDRF and shares common features with the putative EDHF. It is produced both in vascular endothelial cells and SMCs. Upon muscarinic cholinergic stimulation, endothelium releases H2S, which has two different but complementary vascular actions. The activation of KATP channels in vascular SMCs and of charybdotoxin/apamin sensitive KCa channels by H2S would compound to hyperpolarize SMCs, leading to vasorelaxation (Figure 1). Being an EDRF, H2S plays an important role in regulating endothelium-dependent vasoactive activities. Once endothelium-derived H2S is diminished, selective resistance blood vessels will over-constrict and blood pressure increases.9.Yang G. Wu L. Jiang B. et al.H2S as a physiologic vasorelaxant: Hypertension in mice with deletion of cystathionine gamma-lyase.Science. 2008; 322: 587-590Crossref PubMed Scopus (1733) Google Scholar The H2S deficiency will also likely affect the pathogenesis progress of vascular complications of diabetes, coronary vascular diseases, atherosclerosis, and cardiac ischemic damage, to name a few. More extensive and mechanistic studies in the future are expected to further stimulate and intensify the debate on the candidacy of H2S as a new EDRF/EDHF. No matter what the verdict of this debate, the importance of H2S in regulating cardiovascular function will be certainly better appreciated. The author declared no competing interests. This work was supported by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada (NSERC).