Effect of Various Pathological Conditions on Nitric Oxide Level and L-Citrulline Uptake in Motor Neuron-Like (NSC-34) Cell Lines

Abstract

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disorder that causes progressive paralysis. L-Citrulline is a non-essential neutral amino acid produced by L-arginine via nitric oxide synthase (NOS). According to previous studies, the pathogenesis of ALS entails glutamate toxicity, oxidative stress, protein misfolding, and neurofilament disruption. In addition, L-citrulline prevents neuronal cell death in brain ischemia; therefore, we investigated the change in the transport of L-citrulline under various pathological conditions in a cell line model of ALS. We examined the uptake of [¹⁴C]L-citrulline in wild-type (hSOD1wt/WT) and mutant NSC-34/ SOD1^G93A (MT) cell lines. The cell viability was determined via MTT assay. A transport study was performed to determine the uptake of [¹⁴C]L-citrulline. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis was performed to determine the expression levels of rat large neutral amino acid transported 1 (rLAT1) in ALS cell lines. Nitric oxide (NO) assay was performed using Griess reagent. L-Citrulline had a restorative effect on glutamate induced cell death, and increased [¹⁴C]L-citrulline uptake and mRNA levels of the large neutral amino acid transporter (LAT1) in the glutamate-treated ALS disease model (MT). NO levels increased significantly when MT cells were pretreated with glutamate for 24 h and restored by co-treatment with L-citrulline. Co-treatment of MT cells with L-arginine, an NO donor, increased NO levels. NSC-34 cells exposed to high glucose conditions showed a significant increase in [¹⁴C]L-citrulline uptake and LAT1 mRNA expression levels, which were restored to normal levels upon co-treatment with unlabeled L-citrulline. In contrast, exposure of the MT cell line to tumor necrosis factor alpha, lipopolysaccharides, and hypertonic condition decreased the uptake significantly which was restored to the normal level by co-treating with unlabeled L-citrulline. L-Citrulline can restore NO levels and cellular uptake in ALS-affected cells with glutamate cytotoxicity, pro-inflammatory cytokines, or other pathological states, suggesting that L-citrulline supplementation in ALS may play a key role in providing neuroprotection.

Keywords: Amyotrophic lateral sclerosis; Glutamate; Hypoxia-inducible factor; L-citrulline; Large neutral amino acid transporter 1; Nitric oxide.

Fig. 1

Cell viability of MT cell line with glutamate cytotoxicity. (A) Images of the cells were obtained using EVOS XL Core Cell Imaging (100×). (B) MTT assay. MT cell lines were incubated with 1 mM glutamate (Glut) in the presence or absence of 20 mM L-citrulline (Citr) and L-arginine (Arg) for 24 h. Each column represents the mean ± SEM (n=3-4). **p<0.01 vs. the control, ^###p<0.001; vs. glutamate treatment.

Fig. 2

[¹⁴C]L-Citrulline uptake and LAT1 mRNA expression level in MT cell line with glutamate cytotoxicity. MT cells were incubated with 1 mM glutamate (Glut) with/without 20 mM L-citrulline (Citr) and L-arginine (Arg) for 24 h. (A) [¹⁴C]L-Citrulline uptake was performed at pH 7.4 and 37°C for 5 min. (B) The transcription levels of LAT1 mRNA were determined by quantitative real-time PCR analysis and normalized to those of GAPDH. Each column represents the mean ± SEM (n=3-4). **p<0.01 vs. the control, ^##p<0.01 vs. glutamate treatment.

Fig. 3

[¹⁴C]L-Citrulline uptake LAT1 mRNA expression level in SOD1 G93A mutant cell line exposed to various pro-inflammatory conditions. MT cells were incubated with 20 ng/mL of LPS and TNF-α with/without 20 mM L-Citrulline for 24 h. (A) [¹⁴C]L-Citrulline uptake by MT cell lines was performed at pH 7.4 and 37°C for 5 min. (B) The transcription levels of LAT1 mRNA were determined by quantitative real-time PCR analysis and normalized to those of GAPDH. Each value represents the mean ± SEM (n=3-4). **p<0.01, ***p<0.001; significantly different from control. ^###p<0.001; vs. LPS treatment and ⁺⁺⁺p<0.001; vs. TNF-α treatment.

Fig. 4

NO levels in the MT cell line under inflammatory states. (A) NO concentrations were measured using Griess reagent after pre-treatment with glutamate (1 mM) and 20 mM of L-citrulline and L-arginine for 24 h. (B) NO concentrations were measured using Griess reagent after pre-treatment with LPS, TNF-α (20 ng/mL), and 20 mM of L-citrulline for 24 h. Each column represents the mean ± SEM (n=3-4). *p<0.05, **p<0.01, ***p<0.01 vs. the control, ^#p<0.05 vs. glutamate treatment, ^##p<0.01; vs. LPS treatment and ⁺p<0.05 vs. TNF-α treatment.

Fig. 5

Effect of L-citrulline under hypertonic condition. [¹⁴C]L-Citrulline uptake by NSC-34 cell lines (WT; wild type, MT; mutant type) under hypertonic (Hyp) condition and addition of 20 mM L-citrulline (Cit) was performed at pH 7.4 and 37°C for 5 min. (A) LAT1 mRNA expression level on MT cells under hypertonic and high glucose condition. (B) The transcript levels of LAT1 were determined by quantitative real-time PCR analysis and normalized to those of GAPDH Each value represents the mean ± SEM (n=3-4). **p<0.01, significantly different from control. ^##p<0.01, vs hypertonic condition.

Fig. 6

Effect of L-citrulline under high glucose condition. (A) [¹⁴C]L-Citrulline uptake by NSC-34 cell lines was performed at pH 7.4 and 37°C for 5 min under high glucose (HG) condition and addition of 20 mM of L-citrulline (Cit). (B) LAT1 mRNA expression level of NSC-34 cell lines. The transcript levels of LAT1 determined by quantitative real-time PCR analysis and normalized to those of GAPDH. (C) HIF-1α mRNA expression level on high glucose condition. The transcript levels of HIF-1 α determined by quantitative real-time PCR analysis. Each value represents the mean ± SEM (n=3-4). **p<0.01, ***p<0.001; significantly different from control. ^#p<0.05, ^###p<0.001 vs high glucose condition.