Direct inhibition of endothelial nitric oxide synthase by hydrogen sulfide: Contribution to dual modulation of vascular tension
Introduction
Hydrogen sulfide (H2S) has traditionally been known for its toxic effects on living organisms. However, H2S is now considered a gasotransmitter in the mammalian body including the central nervous system and circulatory system (Wang, 2002). Endogenous H2S is formed from l-cysteine mainly by two pyridoxal-5′-phosphate-dependent enzymes, cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) (Stipanuk and Beck, 1982, Swaroop et al., 1992). CBS is highly expressed in the brain, modulating neuronal functions (Abe and Kimura, 1996, Kimura, 2000). CSE has been demonstrated to be expressed in the vascular systems including the aorta, tail artery, mesenteric artery, pulmonary artery and portal vein, while CBS is not detectable in those blood vessels (Hosoki et al., 1997, Zhao et al., 2001). According to in situ hybridization analysis of rat aorta, CSE mRNA appears to be present in the smooth muscle, but not endothelial, layer (Zhao et al., 2001). Under physiological conditions, the concentration of H2S is 1–160 μM in the mammalian tissues and blood (Abe and Kimura, 1996, Bhatia et al., 2005, Fiorucci et al., 2006, Li et al., 2005, Zhao et al., 2001).
Vascular actions of H2S are extremely complex, showing great species and strain differences (Cheng et al., 2004, Dombkowski et al., 2005, Hosoki et al., 1997, Olson, 2005, Zhao and Wang, 2002, Zhao et al., 2001). Olson's group (Dombkowski et al., 2005, Olson, 2005) has shown that H2S at high concentrations contracts rat aorta, while H2S exhibits both contractile and relaxant activities in rat pulmonary artery precontracted with norepinephrine. In contrast, some other groups have reported that H2S induces relaxation responses in isolated rat vascular tissues including the aorta and the mesenteric artery in vitro (Cheng et al., 2004, Hosoki et al., 1997, Zhao and Wang, 2002, Zhao et al., 2001). Although the mechanisms for the contractile activity of H2S have yet to be clarified, there is some information about the mechanisms for the relaxant activity of H2S in blood vessels. The H2S-evoked vasorelaxation appears to involve both endothelium-dependent and -independent mechanisms. Most importantly, the vasodilation induced by H2S is attributable, at least in part, to activation of ATP-sensitive potassium (K+ATP) channels (Cheng et al., 2004, Zhao et al., 2001). Nonetheless, the H2S-induced vasorelaxation is only partially blocked by glibenclamide, a K+ATP channel inhibitor at 10 μM, an appropriate specific concentration (Cheng et al., 2004, Zhao et al., 2001), implying involvement of additional mechanisms independent of K+ATP channels. Functions of H2S also appear to be closely related to nitric oxide (NO). Interestingly, NO donors up-regulate expression and activity of CSE in vascular tissues and cultured aortic smooth muscle cells (Leffler et al., 2006, Zhao et al., 2001). H2S potentiates expression of inducible NO synthase (iNOS) following stimulation with interleukin-1β in cultured rat vascular smooth muscle cells (Jeong et al., 2006), while H2S inhibits expression of iNOS in RAW264.7 macrophages stimulated with lipopolysaccharide (Oh et al., 2006). However, the direct effect of H2S on activity of NO synthase remains to be studied.
In the present study, we carefully investigated characteristics of the vascular actions of H2S in aortic rings isolated from rats and mice, and then examined direct effects of H2S on activity of recombinant endothelial NO synthase (eNOS). Here, our results, for the first time to our best knowledge, indicate that H2S directly inhibits eNOS activity, leading to enhancement of vascular tension.
Section snippets
Major chemicals
We used sodium hydrosulfide (NaHS) as a H2S donor, since the use of NaHS enables us to define actual concentrations of H2S in the solution more accurately and reproducibly than bubbling H2S gas itself (Distrutti et al., 2006). In the neutral solution, one-third of NaHS exists as H2S and the remaining two-thirds are present as HS− (Beauhamp et al., 1984). NaHS, NG-nitro-l-arginine methyl ester hydrochloride (l-NAME) and glibenclamide were purchased from Sigma–Aldrich (St. Louis, MO).
Contractile and relaxant activity of the H2S donor NaHS in the aortic rings isolated from rats and mice
In the rat (Fig. 1a and c) and mouse (Fig. 1b and d) aortic rings precontracted with PE, the H2S donor NaHS, when applied cumulatively, caused small increase in vascular tension at low concentrations (100–300 μM in rat aorta and 30–100 μM in mouse aorta) and relaxation at high concentrations (1000–3000 μM in rat aorta and 300–3000 μM in mouse aorta). Pretreatment with glibenclamide, a K+ATP channel inhibitor, at 10 μM dramatically enhanced the contractile activity of NaHS at low concentrations and
Discussion
The present study employing NaHS as the H2S donor shows that H2S plays a dual role in vascular tension modulation, causing contraction and relaxation in both rat and mouse aortae. Together with the findings from the tissue bioassay, our study employing recombinant bovine eNOS reveals, for the first time to our best knowledge, that H2S directly inhibits eNOS, possibly leading to enhancement of the tension in the precontracted aortic tissues. Our data also demonstrate that the relaxant effect of H
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