Elsevier

Nitric Oxide

Volume 78, 1 August 2018, Pages 32-40
Nitric Oxide

Cystine/glutamate transporter, system xc, is involved in nitric oxide production in mouse peritoneal macrophages

https://doi.org/10.1016/j.niox.2018.05.005Get rights and content

Highlights

  • xCT deficiency causes a decrease in NO production of macrophages.

  • Cysteine supplied via xCT is involved in NO production in macrophages.

  • Decrease in intracellular glutathione does not influence NO production in macrophages.

  • ROS levels in xCT-deficient macrophages were higher than those of wild-type cells.

Abstract

The amino acid transport system xc is important for maintaining intracellular glutathione levels and extracellular redox balance. The main component of system xc, xCT, is strongly induced by various stimuli, including oxidative stress and bacterial lipopolysaccharides (LPS) in macrophages. In the present study, we investigated the production of nitric oxide by LPS-stimulated mouse peritoneal macrophages isolated from both xCT-deficient and wild-type mice. After culturing macrophages in the presence of LPS for 24–48 h, nitrite levels in the medium of xCT-deficient macrophages were significantly decreased compared to that of wild-type cells. However, the transport activity of arginine, a precursor of nitric oxide, and the expression of nitric oxide synthase 2 in xCT-deficient macrophages were similar to those of wild-type cells. When wild-type macrophages were cultured in the medium that contained no cystine, nitric oxide production was decreased to the level similar to that of the xCT-deficient macrophages. When xCT-deficient macrophages were cultured with 2-mercaptoethanol, intracellular cysteine levels were increased and nitrite accumulation in the medium was significantly increased. On the other hand, when these cells were cultured with buthionine sulfoximine, an inhibitor of glutathione synthesis, nitrite accumulation in the medium was essentially unchanged, although intracellular glutathione levels were very low. Reactive oxygen species levels in xCT-deficient macrophages were higher than those of wild-type cells, and treatment with LPS caused an increase in oxidative stress in both cells. These results suggest that intracellular cysteine supplied by xCT contributes to nitric oxide production and the reduction of oxidative stress in macrophages.

Introduction

Macrophages produce large amounts of reactive oxygen species (ROS) and nitric oxide (NO) in response to inflammatory stimuli such as bacterial lipopolysaccharides (LPS) and interferon γ, and function to protect the host from bacterial infection [1,2]. In macrophages, NO synthase 2 (NOS2) is induced by various stimuli, including LPS and interferon γ, which cause the production of large amounts of NO. Recent studies have revealed that NO produced by macrophages functions, not only as an antimicrobial, tumoricidal, and tissue-damaging effector molecule operating in the innate immune system, but also as an effector of adaptive immune responses and cyto-protection [3]. Although NO itself plays multiple physiological roles, it also reacts with other gaseous molecules and is converted to reactive nitrogen oxide species (RNOS). Among the known RNOS, peroxynitrite (ONOO), which is formed by the reaction of NO with superoxide, exerts oxidative damage to many biological molecules [4]. Acting in coordination, antioxidative and redox molecules protect cells against oxidative and nitrosative stress by eliminating ROS and RNOS.

Glutathione is the most abundant non-protein thiol that is produced in cells and plays pleiotropic roles, such as antioxidation and the detoxification of toxicants. The antioxidative functions of glutathione are effectively expressed by donating electrons to peroxides via glutathione peroxidase (GPX) [5]. Thus, an insufficiency of glutathione triggers the redox imbalance and makes cells more vulnerable to oxidative insults, leading to cell death.

Intracellular glutathione levels are maintained by de novo synthesis from constituent amino acids, glutamate, glycine, and cysteine that is catalyzed by the action of γ-glutamylcysteine synthetase (γGCS) and glutathione synthetase (GSS) as well as by recycling oxidized glutathione by glutathione reductase (GSR) in a NADPH-dependent manner [6]. A redox-sensitive transcriptional regulatory factor, Nrf2, regulates the expression of the enzymes that are involved in both the de novo synthesis and reductive recycling of the oxidized glutathione [7,8]. Among three substrate amino acids, cysteine is generally maintained at low levels in many types of cells and thus constitutes a rate-limiting factor for the synthesis of glutathione [9].

Several systems, including amino acid transporters and metabolic protein degradation, function to supply cysteine to cells [10]. In some types of cells, cysteine can be supplied via the transsulfuration pathway in association with methionine metabolism [11]. A reduced form of extracellular cysteine is transported into cells via neutral amino acid transporters, e.g., ASCTs and LAT2 [12,13]. The oxidized form of cysteine, i.e., cystine, is taken up by cells via system xc, which is composed of two protein components; xCT (SLC7A11) and 4F2hc (SLC3A2) [14]. In many cultured cells, xCT is induced by various stimuli such as electrophilic agents [15], amino acid deprivation [16], bacterial lipopolysaccharide [17], and contributes to the maintenance of intracellular glutathione level. xCT is the main component of the system xc and mediates the transport of cystine and cystathionine into cells in exchange with glutamate [14,18].

xCT is constitutively expressed in a limited number of organs, such as the thymus, spleen, and cerebral meninges [19]. Upon oxidative stress, xCT is induced in alveolar macrophages, the kidney, and liver, and hence a xCT deficiency aggravates the oxidative damage in these organs [[20], [21], [22], [23]]. xCT-deficient (KO) mice show healthy phenotypes except for a redox imbalance in the blood plasma [24]. Due to the oxidative conversion of cysteine to cystine in the culture medium, intracellular glutathione levels depend on the extent of cystine uptake via xCT in many types of cultured cells under routine culture conditions. Mouse embryonic fibroblasts (MEF) isolated from KO mice are unable to survive under routine culture conditions due to decreased glutathione levels [18,24].

We previously reported that the activities of system xc and system y+, which mediates the transport of cationic amino acids, are strongly induced by a very small amount of LPS in macrophages [17,25]. Because arginine is a substrate of system y+ and a precursor of NO, it seems rational that system y+ and NOS2 are simultaneously induced by LPS. The question arises as to whether the simultaneous induction of system xc and system y+ has any physiological and/or pathophysiological significance. In the present study, we compared NO production by peritoneal macrophages isolated from both KO and wild-type (WT) mice under various culture conditions.

Section snippets

Materials

All chemicals and agents were purchased from Sigma (St. Louis, MO, USA) or Wako Pure Chemicals Industries (Osaka, Japan), unless otherwise stated.

Mice

KO mice were generated, as described in a previous study using a gene-targeting technique [24]. The genotype was determined by PCR, as reported previously. After backcrossing the C57BL/6N mice more than 10 times, the resulting WT and KO mice were used in the experiments reported here. All of the mice had free access to standard rodent chow and water.

Production of nitric oxide in xCT-deficient macrophages

To investigate a role of xCT in NO production in macrophages, we measured the levels of nitrite, a stable metabolite of NO, in culture medium of LPS-stimulated macrophages isolated from xCT-deficient (KO) and wild-type (WT) mice. Nitrite levels were increased in the culture medium of both KO and WT macrophages in a time-dependent manner. However, nitrite levels in medium of KO macrophages were significantly lower than that of WT cells (Fig. 1A). In both macrophages, arginine uptake activity was

Discussion

The findings reported in this study show that NO production in xCT-deficient (KO) macrophages is significantly decreased compared to that in WT cells. xCT primarily functions as an amino acid transporter which mediates an exchange of cystine and glutamate on the plasma membrane. There are several possible explanations for how such a deficiency in a transporter could cause a decrease in NO production. One possibility is the inactivation of post-translational modification of the NOS2 protein.

Author contribution

SK performed most of the experiments, SH, TH, MS, RK assisted in some of the experiments, such as western blot of proteins and detection of oxidative stress of macrophages. SB, JF advised the design of some experiments and helped writing manuscript. SK, JF and HS designed the experiments and wrote the paper.

Conflicts of interest

The authors declared no conflicts of interest.

Acknowledgement

This work was supported by the JSPS KAKENHI Grant-in-Aid for Research Activity Start-up (16H06648) to SK.

References (39)

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