Inhibition of arterial contraction by dinitrosyl–iron complexes: critical role of the thiol ligand in determining rate of nitric oxide (NO) release and formation of releasable NO stores by S-nitrosation

doi:10.1016/j.bcp.2003.07.017

Biochemical Pharmacology

Volume 66, Issue 12, 15 December 2003, Pages 2365-2374

https://doi.org/10.1016/j.bcp.2003.07.017 Get rights and content

Abstract

The inhibition of arterial tone produced by two nitric oxide (NO) derivatives of biological relevance, dinitrosyl–iron complexes with cysteine (DNIC-CYS) or with glutathione (DNIC-GSH), was compared. Both compounds induced vasorelaxation within the same concentration range (3–300 nM) in endothelium-denuded rat aortic rings. Consistent with a faster rate of NO release from DNIC-CYS than from DNIC-GSH, the relaxant effect of DNIC-CYS was rapid in onset and tended to recover with time, whereas the one of DNIC-GSH developed slowly and was sustained. In addition, DNIC-GSH (0.3 and 1 μM) but not DNIC-CYS (1 μM) induced, even after washout of the drug, a persistent hyporesponsiveness to vasoconstrictors and a relaxant effect of low molecular weight thiols like N-acetylcysteine (NAC, which can displace NO from preformed NO stores). Both effects of DNIC-GSH were associated with elevation of cyclic GMP content and were attenuated by NO scavengers or a cyclic GMP-dependent protein kinases inhibitor. In rings previously exposed to DNIC-GSH, addition of mercuric chloride (which can cleave the cysteineNO bond of S-nitrosothiols) elicited relaxation, completely blunted the one of NAC and also abolished the persistent elevation of NO content. In conclusion, this study shows that whereas both DNIC-CYS and DNIC-GSH elicited a NO release-associated relaxant effect in isolated arteries, only DNIC-GSH induced an inhibition of contraction which persisted after drug removal. The persistent effect of DNIC-GSH was attributed to the formation of releasable NO stores in arterial tissue, most probably as S-nitrosothiols. Thus, the nature of the thiol ligand plays a critical role in determining the mechanisms and duration of the effect of LMW-DNIC in arteries.

Introduction

DNIC are NO derivatives of biological relevance. They are formed in various cells and tissues, especially during NO overproduction by the inducible NOS (NOS-2) (for reviews, see [1], [2]). High molecular weight and LMW-DNIC exist, with cysteine residues of proteins or with LMW thiols like cysteine or glutathione as ligand, respectively. LMW-DNIC are much more instable than the protein-bound species [2], [3] and, as demonstrated in acellular systems, easily release NO or transfer it, alone or together with iron, to various targets [2], [4], [5], [6], [7], [8], [9]. In cells or tissues, LMW-DNIC exert cyclic GMP-dependent [4], [10] and -independent effects [11], [12], [13]. The latter have been attributed to nitrosative modification of proteins via the transfer of the NO or Fe(NO)₂ group, forming protein S-nitrosothiols or protein-bound DNIC, respectively [2], [12], [13]. Besides being responsible for post-translational modification of various proteins, formation of S-nitrosothiols or DNIC on proteins are also involved in NO transport and storage in blood and tissues [14], [15], [16], [17].

In arteries expressing NOS-2, protein-bound DNIC are formed and may contribute to vascular hyporesponsiveness to vasoconstrictors [18], [19]. LMW-DNIC exert vasorelaxant effects which are likely due to the activation of the cyclic GMP pathway [10], [20] and have been postulated as the ‘endothelium-derived relaxing factor’ released by endothelium-dependent agonists [21]. LMW-DNIC can also enhance the release of NE from perivascular nerves by cyclic GMP-independent mechanisms [13]. The transfer of the Fe(NO)₂ group of LMW-DNIC, resulting in the formation of NO stores as protein-bound DNIC, has been also demonstrated in isolated arteries [4]. However, the potential role of S-nitrosation of cysteine residues in the effects of LMW-DNIC on vascular tone is not documented. As recently demonstrated [22], persistent S-nitrosation of protein is another mechanism of formation of releasable NO stores in arteries. It accounts for the long-lasting inhibition of arterial tone induced by GSNO, another LMW NO derivative of biological significance [23].

The aim of the present study was to compare the effect of two LMW-DNIC of biological relevance, DNIC-CYS and DNIC-GSH, on vascular tone. Their ability to induce or not persistent inhibition of contraction by S-nitrosation reactions was especially investigated. For this purpose, responses to contractile agonists were assessed in rat aortic rings previously exposed to the LMW-DNIC, and then carefully washed out. The potential role of S-nitrosation of tissue thiol was investigated using thiol-modifying reagents, LMW thiols and mercuric chloride. LMW thiols can displace NO bioactivity from protein S-nitrosothiols [15], [22], [24] and/or from protein-bound DNIC [4], [18], [25], whereas mercuric chloride is known to cleave the cysteine-NO bond of S-nitrosothiols [26].

Section snippets

Rate of NO release from DNIC

Conversion of oxyHb (10 μM) to methemoglobin (metHb) in 100 mM phosphate buffer (NaH₂PO₄/Na₂HPO₄ 100 mM, pH 7.4, 37°) was applied to study the release of NO from LMW-DNIC. MetHb formation was continuously monitored in a diode array spectrophotometer (Hewlett-Packard 8453) by recording the absorbance changes at 401 nm (isosbestic point at 411 nm) and calculated from the absorbance changes (ε_401–411=38 mM⁻¹ cm⁻¹) [27]. The initial rate of metHb formation was determined between 0.5 and 5 min after the

Rate of NO release from LMW-DNIC

The rate of release of NO from DNIC-CYS and DNIC-GSH was compared using the oxyHb assay in buffer solution. As illustrated in Fig. 1, DNIC-CYS released NO at significant higher rates (about 3.5 times) than DNIC-GSH.

Vasorelaxant effect of LMW-DNIC

In rat aortic rings precontracted with NE, DNIC-CYS and DNIC-GSH produced concentration-dependent relaxant effect, with similar potencies (Fig. 2A). The ec₅₀ values of DNIC-CYS and DNIC-GSH were 31±4 nM (N=8) and 32±8 nM (N=8), respectively. The relaxant effect of DNIC-CYS was rapid in

Discussion

The major finding of the present study is that the LMW-DNIC of biological relevance, DNIC-CYS and DNIC-GSH, differ in their ability to induce or not an inhibition of arterial tone which persists after washout of the drug. The persistent inhibition of contraction obtained with DNIC-GSH is attributed to the formation of releasable NO stores on tissue thiols, most probably as S-nitrosothiols.

The vasorelaxant properties of LMW-DNIC have been previously reported [4], [10], [13], [20] and attributed

Acknowledgements

The authors thank Véronique Freund for helpful contribution in contractile experiments. This work was partially supported by a grant from Fondation de France. J.L.A. is recipient of a fellowship from CAPES-Brazil.

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Endothelial dysfunction predisposing to cardiovascular diseases is defined as an imbalance in the production of vasodilating factors, such as nitric oxide (NO), and vasoconstrictive factors. To insure its physiological role, NO, a radical with very short half-life, requires to be stored and transported to its action site. S-nitrosothiols (RSNOs) like S-nitrosoglutathione (GSNO) represent the main form of NO storage within the vasculature. The NO store formed by RSNOs is still bioavailable to trigger vasorelaxation. In this way, RSNOs are an emerging class of NO donors with a potential to restore NO bioavailability within cardiovascular disorders.
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All the studied RSNOs were able to generate a NO store materialized by a three to five times increase in NOx species inside aortae. NACNO was the most potent RSNO to produce a vascular NO store bioavailable for vasorelaxation and the most efficient to induce vascular hyporeactivity to PHE in endothelium-removed aortae. GSNO and SNAP were equivalent and more efficient than SNP. In endothelium-intact aortae, the NO store was also formed whereas it seemed less available for vasorelaxation and did not influence PHE-induced vasoconstriction. In conclusion, RSNOs – NACNO in a better extent – are able to restore NO bioavailability as a functional NO store within the vessel wall, especially when the endothelium is removed. This was associated with a hyporeactivity to the vasoconstrictive agent phenylephrine. Treatment with RSNOs could present a benefit to restore NO-dependent functions in pathological states associated with injured endothelium.
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Nitrogen monoxide (NO) is vital for many essential biological processes as a messenger and effector molecule. The physiological importance of NO is the result of its high affinity for iron in the active sites of proteins such as guanylate cyclase. Indeed, NO possesses a rich coordination chemistry with iron and the formation of dinitrosyl–dithiolato iron complexes (DNICs) is well documented. In mammals, NO generated by cytotoxic activated macrophages has been reported to play a role as a cytotoxic effector against tumor cells by binding and releasing intracellular iron. Studies from our laboratory have shown that two proteins traditionally involved in drug resistance, namely multidrug-resistance protein 1 and glutathione S-transferase, play critical roles in intracellular NO transport and storage through their interaction with DNICs (R.N. Watts et al., Proc. Natl. Acad. Sci. USA 103:7670–7675, 2006; H. Lok et al., J. Biol. Chem. 287:607–618, 2012). Notably, DNICs are present at high concentrations in cells and are biologically available. These complexes have a markedly longer half-life than free NO, making them an ideal “common currency” for this messenger molecule. Considering the many critical roles NO plays in health and disease, a better understanding of its intracellular trafficking mechanisms will be vital for the development of new therapeutics.
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The resulting ground-state Fe–N–O moiety may be linear or bent [1]. The dinitrosyl Fe(NO)2 moiety has been suggested as a biologically relevant entity [2–7]. Exogenous and endogenous NO, upon exposure to and contact with Fe-containing biomolecules, generates a species exhibiting a strong EPR signal at g = 2.03 attributed to an “Fe(NO)2”-containing species [3].
A series of iron dinitrosyl complexes of the form Fe(NO)₂(P(C₆H₄X)₃)₂ (X = p-OMe (1), p-Me (2), m-Me (3), p-H (4), p-F (5), p-Cl (6), p-CF₃ (7)) has been prepared from the reactions of Fe(NO)₂(CO)₂ and the respective triarylphosphines. Complexes 1–7 have been characterized by IR and ³¹P NMR spectroscopy, and by X-ray crystallography for 1 and 7. In general, the compounds with the more basic phosphines display lower υ_NO stretches in the IR spectra than those with the less basic phosphines, and the trends in υ_NO as a function of Hammett parameter and solvent donor/acceptor number were analyzed. The redox behavior of compounds 1–7 in CH₂Cl₂ were studied by cyclic voltammetry at a Pt electrode. In general, the compounds undergo one-electron oxidations. Infrared spectroelectrochemistry revealed that the oxidations generate the derivatives with υ_NOs that are ∼100 cm⁻¹ higher in energy indicative of Fe(NO)₂-centered oxidations.
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Indeed, by incubating aortic segments with DNICs, Mülsch et al. (90) reported relaxation of pre-contracted femoral artery segments. Similar observations were also made by Alencar et al. (59). The generation of NO by eNOS and the formation of DNICs must now be assessed in endothelial cells, with emphasis on the role of GSTs and MRP1 in NO storage and transport.
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Inhibition of arterial contraction by dinitrosyl–iron complexes: critical role of the thiol ligand in determining rate of nitric oxide (NO) release and formation of releasable NO stores by S-nitrosation

Abstract

Introduction

Section snippets

Rate of NO release from DNIC

Rate of NO release from LMW-DNIC

Vasorelaxant effect of LMW-DNIC

Discussion

Acknowledgements

FEBS Lett.

J. Biol. Chem.

Biochem. Pharmacol.

Biochem. Pharmacol.

FEBS Lett.

Eur. J. Pharmacol.

FEBS Lett.

J. Biol. Chem.

Methods Enzymol.

Anal. Biochem.

Biochim. Biophys. Acta

Gastroenterology

EPR characterization of molecular targets for NO in mammalian cells and organelles

FASEB J.

Dinitrosyl complexes of iron with thiol-containing ligands and their reverse conversion into nitrosothiols

Biokhimiia

S-Nitrosation of serum albumin by dinitrosyl–iron complex

J. Biol. Chem.

Molecular mechanisms underlying the role of nitric oxide in the cardiovascular system

Exp. Opin. Invest. Drugs

Nitric oxide-related cyclic GMP-independent effect of N-acetylcysteine in lipopolysaccharide treated rat aorta

Br. J. Pharmacol.

Novel activation of non-selective cationic channels by dinitrosyl iron-thiosulfate in PC12 cells

J. Physiol.