Article

Activation and inhibition of soluble guanylyl cyclase by S-nitrosocysteine: Involvement of amino acid transport system L

Medical College of Wisconsin, Milwaukee, Wisconsin, United States
Free Radical Biology and Medicine (Impact Factor: 5.74). 06/2009; 47(3):269-74. DOI: 10.1016/j.freeradbiomed.2009.04.027
Source: PubMed

ABSTRACT

In this study the mechanism by which S-nitrosocysteine (CysNO) activates soluble guanylyl cyclase (sGC) has been investigated. CysNO is the S-nitrosated derivative of the amino acid cysteine and has previously been shown to be transported into various cell types by amino acid transport system L. Here we show, using both neuroblastoma and pulmonary artery smooth muscle cells, that CysNO stimulates cGMP formation at low concentrations, but this effect is lost at higher concentrations. Stimulation of cGMP accumulation occurs only after its transport into the cell and subsequent flavoprotein reductase-mediated metabolism to form nitric oxide (NO). Consequently, CysNO can be regarded as a cell-targeted NO-releasing agent. However, CysNO also functions as an NO-independent thiol-modifying agent and can compromise cellular antioxidant defenses in a concentration-dependent manner. The observed biphasic nature of CysNO-dependent cGMP accumulation seems to be due to these two competing mechanisms. At higher concentrations, CysNO probably inactivates guanylyl cyclase through modification of an essential thiol group on the enzyme, either directly or as a result of a more generalized oxidative stress. We show here that higher concentrations of CysNO can increase cellular S-nitrosothiol content to nonphysiological levels, deplete cellular glutathione, and inhibit cGMP formation in parallel. Although the inhibition of sGC by S-nitrosation has been suggested as a mechanism of nitrovasodilator tolerance, in the case of CysNO, it seems to be more a reflection of a generalized oxidative stress placed upon the cell by the nonphysiological levels of intracellular S-nitrosothiol generated upon CysNO exposure.

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Available from: Joseph Riego, Aug 12, 2014
    • "Before infusion, endogenous SNO concentrations were below the limit of detection in both ipsilateral femoral vein and brachial artery plasma. At the end of the 0.8 ml/min infusion period, plasma SNO concentrations in the ipsilateral femoral vein (assumed to closely approximate concentrations in the ipsilateral artery) were 30 μmol/L (lower than 40 μmol/L, at which L-cysNO might inhibit rather than activate sGC [10]), over 90% of which were in the low molecular weight fraction. In contrast, plasma SNO concentrations in the brachial artery (assumed to closely approximate the concentrations in the mesenteric and contralateral femoral arteries) were 0.6 μmol/L and were all in the high molecular weight fraction, suggesting virtually complete S-transnitrosylation from low to high molecular weight thiols during systemic recirculation. "
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    ABSTRACT: S-nitrosothiols (SNOs) such as S-nitroso-L-cysteine (L-cysNO) are endogenous compounds with potent vasodilatory activity. During circulation in the blood, the NO moiety can be exchanged among various thiol-containing compounds by S-transnitrosylation, resulting in SNOs with differing capacities to enter the cell (membrane permeability). To determine whether the vasodilating potency of SNOs is dependent upon membrane permeability, membrane-permeable L-cysNO and impermeable S-nitroso-D-cysteine (D-cysNO) and S-nitroso-glutathione (GSNO) were infused into one femoral artery of anesthetized adult sheep while measuring bilateral femoral and systemic vascular conductances. L-cysNO induced vasodilation in the infused hind limb, whereas D-cysNO and GSNO did not. L-cysNO also increased intracellular NO in isolated arterial smooth muscle cells, whereas GSNO did not. The infused SNOs remained predominantly in a low molecular weight form during first-passage through the hind limb vasculature, but were converted into high molecular weight SNOs upon systemic recirculation. At systemic concentrations of ~0.6μmol/L, all three SNOs reduced mean arterial blood pressure by ~50%, with pronounced vasodilation in the mesenteric bed. Pharmacokinetics of L-cysNO and GSNO were measured in vitro and in vivo and correlated with their hemodynamic effects, membrane permeability, and S-transnitrosylation. These results suggest local vasodilation by SNOs in the hind limb requires membrane permeation, whereas systemic vasodilation does not. The systemic hemodynamic effects of SNOs occur after equilibration of the NO moiety amongst the plasma thiols via S-transnitrosylation.
    No preview · Article · Dec 2015 · Free Radical Biology and Medicine
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    • "CysNO is widely used as an “NO donor” to S-nitrosate cells [5, 6, 14, 15], although S-nitrosation was not associated with the action of NO under our assay conditions. Among various the mechanisms for S-nitrosation by extracellular S-nitrosothiols [17, 18, 20–23, 28, 38, 41], Hogg et al. [17, 18, 41] and Whorton et al. [28, 38] have shown that in several cell types, including erythrocytes, endothelial cells, smooth muscle cells, and epithelial cells, S-nitrosation of cellular proteins involves LAT-mediated CysNO uptake. Through this study, we add embryonic fibroblast 3Y1 cells to this growing cell inventory. "
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    ABSTRACT: The mechanism of protein S-nitrosation in cells is not fully understood. Using rat 3Y1 cells, we addressed this issue. Among S-nitrosothiols and NO donors tested, only S-nitrosocysteine (CysNO) induced S-nitrosation when exposed in Hanks' balanced salt solution (HBSS) and not in serum-containing general culture medium. In HBSS, NO release from CysNO was almost completely abolished by sequestering metal ions with a metal chelator without affecting cellular S-nitrosation. In contrast, L-leucine, a substrate of L-type amino acid transporters (LATs), significantly inhibited S-nitrosation. The absence of S-nitrosation with CysNO in general culture medium resulted not only from a competition with amino acids in the medium for LATs but also from transnitrosation of cysteine residues in serum albumin. Collectively, these results suggest that in simple buffered saline, CysNO-dependent S-nitrosation occurs through a cellular incorporation-dependent mechanism, but if it occurs in general culture media, it may be through an NO-dependent mechanism.
    Full-text · Article · Aug 2011 · Oxidative Medicine and Cellular Longevity
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    • "An interesting feature to emerge from these studies is that in general, cellular effects mediated by cysNO/L-AT are insensitive to the presence in the extracellular medium of NO scavengers, such as oxyhaemoglobin, thus excluding NO release from the mechanism (Zhang and Hogg, 2004; Zhu et al., 2008). An exception is when cysNO-mediated stimulation of sGC is considered – this process is inhibited by oxyhaemoglobin, but only because intracellular reduction of cysNO to NO is required before a cyclic GMP response can occur (Riego et al., 2009). "
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    ABSTRACT: S-nitrosothiols have a number of potential clinical applications, among which their use as antithrombotic agents has been emphasized. This is largely because of their well-documented platelet inhibitory effects, which show a degree of platelet selectivity, although the mechanism of this remains undefined. Recent progress in understanding how nitric oxide (NO)-related signalling is delivered into cells from stable S-nitrosothiol compounds has revealed a variety of pathways, in particular denitrosation by enzymes located at the cell surface, and transport of intact S-nitrosocysteine via the amino acid transporter system-L (L-AT). Differences in the role of these pathways in platelets and vascular cells may in part explain the reported platelet-selective action. In addition, emerging evidence that S-nitrosothiols regulate key targets on the exofacial surfaces of cells involved in the thrombotic process (for example, protein disulphide isomerase, integrins and tissue factor) suggests novel antithrombotic actions, which may not even require transmembrane delivery of NO.
    Preview · Article · Mar 2010 · British Journal of Pharmacology
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