Hydrogen sulfide increases nitric oxide production with calcium-dependent activation of endothelial nitric oxide synthase in endothelial cells

doi:10.1016/j.ejps.2012.11.001

European Journal of Pharmaceutical Sciences

Volume 48, Issues 1–2, 23 January 2013, Pages 211-215

https://doi.org/10.1016/j.ejps.2012.11.001 Get rights and content

Abstract

Hydrogen sulfide (H₂S) was recently discovered to be synthesized in mammalian tissues by several different enzymes. Numerous studies have shown that H₂S has vasodilator and antihypertensive effects in the cardiovascular system. However, intracellular mechanisms of the H₂S-induced vasodilation and its interactions with other endothelium-derived relaxing factors, such as nitric oxide (NO), remain unclear. We investigated whether H₂S directly regulates endothelial NO synthase (eNOS) activity and NO production in endothelial cells. NaHS, a H₂S donor, dose-dependently increased NO production in cultured endothelial cells. This effect was abolished by a calcium chelator (BAPTA-AM), but not by the absence of extracellular calcium. The NaHS-induced NO production was partially blocked by inhibitors of ryanodine receptor (dantrolene) or inositol 1,4,5-triphosphate receptor (xestospongin C). NaHS significantly increased intracellular calcium concentrations, and this effect was attenuated by dantrolene or xestospongin C. NaHS induced phosphorylation of eNOS at the activating phosphoserine residue 1179. The NaHS-induced eNOS phosphorylation and NO production were not affected by a PI3K/Akt inhibitor (wortmannin). The data of this study suggest that H₂S directly acts on endothelial cells to induce eNOS activation and NO production by releasing calcium from the intracellular store in endoplasmic reticulum, which may explain one of mechanisms of its vasodilator function.

Graphical abstract

Introduction

Hydrogen sulfide (H₂S) is a pungent toxic gas, but recently it was discovered that H₂S is synthesized from L-cysteine in various mammalian tissues by several different enzymes, such as cystathionine gamma-lyase (CSE), cystathionine beta-synthase (CBS), 3-mercaptopyruvate sulfurtransferase (3-MST) (Kimura, 2010).

H₂S has various physiological functions in many tissues, such as controlling synaptic activities and cognitive functions in the brain, protection of neurons and myocardial cells from oxidative stress (Kimura, 2011), and protection of islet cells from glucose toxicity (Kaneko et al., 2009). H₂S also has anti-inflammatory effect in various tissues and organs by reduction of mitochondrial oxygen consumption (Kimura, 2011).

In the cardiovascular system, H₂S induces myocardial preconditioning and reduces ischemia-reperfusion injury in the heart by reduction of neutrophil adhesion (Szabó, 2007). Moreover, H₂S has vasodilator functions and antihypertensive effects by smooth muscle relaxation, drawing attentions as a candidate for a component of endothelium-derived relaxing factors (EDRFs) (Kimura, 2011). H₂S relaxes vascular smooth muscle cells by opening of K_ATP channels (Zhao et al., 2001) and by enhancing the effects of nitric oxide (NO) (Hosoki et al., 1997). Indeed, mice lacking CSE display pronounced hypertension caused by reduced production of H₂S and diminished endothelium-dependent vasorelaxation (Yang et al., 2008).

It has been well known that the most potent endogenous vasorelaxant is NO, which is produced in vascular endothelial cells by endothelial nitric oxide synthase (eNOS). eNOS is a calcium/calmodulin (Ca²⁺/CaM) dependent enzyme that is activated in response to the stimulation of a variety of Ca²⁺-mobilizing cell surface receptors in vascular endothelium. CaM is activated by increase in intracellular free calcium concentration ([Ca²⁺]_i) (Dudzinski et al., 2006), which is induced either by influx of extracellular calcium via channels such as voltage-dependent and receptor-operated calcium channels, or by release from intracellular stores in endoplasmic reticulum (ER) via receptors such as inositol 1,4,5-triphosphate receptors (IP₃Rs) and ryanodine receptors (RyRs). CaM activates eNOS by binding at the canonical binding site located at residues 493–510, which induces a conformational change to facilitate electron transfer. CaM also activates calcium/calmodulin-dependent protein kinase (CaMKII), which phosphorylates eNOS at the activating phosphoserine residue 1177 (human eNOS) or 1179 (bovine eNOS). In addition to regulatory pathway involving Ca²⁺/CaM, eNOS phosphorylation modulating its enzymatic activity is regulated by various pathways. Diverse agonists, such as VEGF and insulin, activate phosphatidylinositol 3-kinase (PI3K) isoforms, which in turn activate kinase Akt and ultimately phosphorylate eNOS at serine 1179 (Dimmeler et al., 1999). Cross-talk between the various agonist-mediated signaling pathways would represent an important mechanism of eNOS regulation, as for example, VEGF induces both the calcium- and phosphorylation-dependent pathways of eNOS activity.

Although recent reports have shown the molecular cross-talk between H₂S and NO, H₂S has been reported to either enhance or attenuate NO synthesis and/or NO mediated vasorelaxation in different studies (Kimura, 2010, Skovgaard et al., 2011). In this study, we investigated whether H₂S directly regulates eNOS activity and NO production in cultured endothelial cells.

Section snippets

Materials

Fetal bovine serum, Dulbecco’s modified Eagle’s medium, Hanks’ balanced salt solution, wortmannin, and bradykinin acetate salt were purchased from Sigma–Aldrich (St. Louis, MO). NO₂/NO₃ Assay Kit-FX (Fluorometric), 2,3-diaminonaphthalene Kit and BAPTA-AM were from Dojindo Chemicals (Japan). Xestospongin C, dantrolene, and fura 2-AM were from Wako (Japan). Polyclonal antibodies against phospho-Akt (Ser-473), Akt and phospho-eNOS (Ser-1179) were from Cell Signaling Technology (Beverly, MA).

The NaHS-induced increase in NO production in BAECs

We first examined the effect of a H₂S donor, NaHS, on NO production in BAECs. Treatment with NaHS for 30 min increased NO production from BAECs in a dose-dependent manner (25–200 μM); a significant increase was induced at a concentration as low as 25 μM by 27 ± 2% (n = 8, P < 0.05) and the peak at 200 μM by 164 ± 7% (n = 8, P < 0.05) (Fig. 1).

The involvement of intracellular and extracellular calcium in the NaHS-induced NO production

We next examined the effect of an intracellular calcium chelator (BAPTA-AM). Pretreatment with BAPTA-AM (20 μM) abolished the NaHS-induced NO production (n = 4, P < 0.05) (

Discussion

This study has demonstrated the effects of H₂S on calcium-dependent activation of eNOS and NO production. It has been shown that endogenous H₂S enhanced the NO-induced smooth muscle relaxation in the thoracic aorta (Hosoki et al., 1997), and that the H₂S-induced vasorelaxation is partially but significantly attenuated either by removal of the endothelium, or by blockade of NO synthase, suggesting that H₂S induces the release of NO or EDRFs from endothelial cells (Zhao and Wang, 2002, Cheng et

Conclusion

In conclusion, these findings strongly suggest that H₂S directly acts on endothelial cells to induce eNOS activation and NO production by mainly releasing calcium from the intracellular store in ER, which may explain one of mechanisms of its vasodilator function as an EDRF. NO has already been clinically applied for its vasodilating and antihypertensive effects. Therefore, further elucidation of mechanisms of H₂S as an EDRF will contribute to therapeutic application of H₂S.

Acknowledgements

This study was supported in part by the Grant-in-aid from the Ministry of Education, Science, Sports and Culture, and the Ministry of Health, Welfare and Labor of Japan. This study was also supported by a grant from the Takeda Science Foundation (to T.S.).

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Citation Excerpt :
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Hydrogen sulfide increases nitric oxide production with calcium-dependent activation of endothelial nitric oxide synthase in endothelial cells

Abstract

Graphical abstract

Introduction

Section snippets

Materials

The NaHS-induced increase in NO production in BAECs

The involvement of intracellular and extracellular calcium in the NaHS-induced NO production

Discussion

Conclusion

Acknowledgements

Atherosclerosis

Vascul. Pharmacol.

FEBS Lett.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

FEBS Lett.

J. Biol. Chem.

Hydrogen sulfide-induced relaxation of resistance mesenteric artery beds of rats

Am. J. Physiol. Heart Circ. Physiol.

Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation

Nature

The regulation and pharmacology of endothelial nitric oxide synthase

Annu. Rev. Pharmacol. Toxicol.

Phosphorylation of Thr(495) regulates Ca(2+)/calmodulin-dependent endothelial nitric oxide synthase activity

Circ. Res.

Regulation of endothelium-derived nitric oxide production by the protein kinase Akt

Nature

Endothelin receptor subtype B mediates synthesis of nitric oxide by cultured bovine endothelial cells

J. Clin. Invest.