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

Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
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


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
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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.
    Oxidative Medicine and Cellular Longevity 08/2011; 2011(5):450317. DOI:10.1155/2011/450317 · 3.36 Impact Factor
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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.
    British Journal of Pharmacology 03/2010; 159(8):1572-80. DOI:10.1111/j.1476-5381.2010.00670.x · 4.84 Impact Factor
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    ABSTRACT: The pharmacological effects of nitric oxide (NO) administered as a gas are dependent on the conversion to S-nitrosocysteine, and as such are largely mediated by the L-type amino-acid transporters (LATs) in several cell types. The dipeptide transporter PEPT2 has been proposed as a second route for S-nitrosothiol (SNO) transport, but this has never been demonstrated. Because NO governs impor-tant immune functions in alveolar macrophages, we exposed rat alveolar macrophages (primary and NR8383 cells) to NO gas at the air–liquid interface 6 LPS stimulation in the presence of PEPT2 sub-strate Cys-Gly (or the LAT substrate L-Cys) 6 transporter competi-tors. We found that SNO uptake and NO-dependent actions, such as the activation of soluble guanylyl cyclase (sGC), the augmentation of sGC-dependent filamentous actin (F-actin) polymerization, phagocytosis, and the inhibition of NF-kB activation, were signifi-cantly augmented by the addition of Cys-Gly in a manner dependent on PEPT2 transport. We found parallel (and greater) effects that were dependent on LAT transport. The contribution of cystine/ cysteine shuttling via system x cystine transporter (xCT) to SNO uptake was relatively minor. The observed effects were unaf-fected by NO synthase inhibition. The NO gas treatment of alve-olar macrophages increased SNO uptake, the activation of sGC, F-actin polymerization, and phagocytosis, and inhibited NF-kB activation, in a manner dependent on SNO transport via PEPT2, as well as via LAT. Inhaled nitric oxide (NO) gas therapy has been widely consid-ered to exert its pharmacologic effects by diffusion to the target molecules. However, we recently showed that the pharmacologic effects of NO gas treatment of alveolar epithelium in vitro (1) and on pulmonary vascular resistance ex vivo (2) are dependent on S-nitrosocysteine (CSNO) uptake through the L-type amino-acid transporter (LAT). This suggests that the pharmacologic effects depend in part on the formation of extracellular S-nitrosothiols (SNO), notably CSNO that is taken up via LAT. A second potential route for SNO uptake involves the formation of S-nitrosocysteinyl glycine (SNO-Cys-Gly) that could be taken up via the dipeptide transporter PEPT2. PEPT2 is a member of the solute carrier family (SLC15A2), a proton-dependent dipeptide/ tripeptide transporter (3), that we have previously shown is expressed in rat alveolar epithelium (4), and has been shown to be expressed in murine macrophages (5). Although SNO-Cys-Gly has been proposed to mediate SNO-dependent effects (6), to our knowledge, this has never been demonstrated directly. The potential for the formation of SNO-Cys-Gly in the alveolus is significant, because glutathione is relatively abundant in the alveolar lining fluid, and the metab-olism of glutathione to Cys-Gly or S-nitroso-glutathione to SNO-Cys-Gly (7) could be mediated by g-glutamyltranspepti-dase (gGT), which is expressed in alveolar epithelium (8) and alveolar macrophages (9). We sought to determine the potential route for SNO uptake in alveolar macrophages during NO therapy, because the alve-olar macrophage takes part in the pulmonary immune response (10) and would receive exposure to relatively high concentra-tions of exogenous NO during inhaled NO treatment. The al-veolar macrophage can also generate substantial endogenous NO via nitric oxide synthase 2 (NOS2) (11). Because SNO modifications can affect the immune responses of the alveolar macrophage (12), we sought to determine the mechanisms gov-erning SNO transport during NO gas treatment. No studies, to our knowledge, have addressed SNO effects in alveolar macro-phages in the context of NO gas treatment. To test the contributions of SNO transport pathways in alve-olar macrophages under basal and LPS-stimulated conditions in the context of NO gas treatment, we exposed freshly isolated rat alveolar macrophages or rat alveolar macrophage–derived (NR8383) cells (16) to NO gas at an air–liquid interface in the presence of PEPT2 and LAT substrates and their corresponding competitors, to determine the relative contributions of the two transport pathways to SNO uptake, activation of soluble gua-nylyl cyclase (sGC), and downstream-target filamentous actin (F-actin) polymerization (13). We also examined the effects of the two transport pathways on the ability of NO treatment to inhibit LPS-stimulated NF-kB activation. NF-kB is a transcrip-tion factor that regulates inflammatory cytokine production, and is known to be inhibited by S-nitrosylation in vivo under lung injury conditions for which NO gas would be considered for therapy (14, 15). We found that PEPT2 and LAT1 were expressed in macro-phages and NR8383 cells, and that exposure to NO gas induced in-tracellular SNO accumulations that were dependent on PEPT2 and LAT. Accumulations of SNO were dependent on PEPT2 and LAT transport under basal and LPS-stimulated conditions. Likewise, the activation of sGC and the inhibition of NF-kB in macrophages by NO gas treatment under basal and LPS-stimulated conditions were also dependent on PEPT2 and LAT transport, with LAT transport being predominant. These transporter-dependent effects of NO on sGC activation and the downstream effects of sGC on F-actin polymerization, or on the inhibition of NF-kB activation in alveo-lar macrophages, were not completely independent of endogenous NO production. SNO accumulation and sGC activation after NO gas exposure were induced by prolonged exposure to LPS in a nitric oxide synthase (NOS)-dependent manner, but NO
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