Glutamine Cooperatively Upregulates Lipopolysaccharide-Induced Nitric Oxide Production in BV2 Microglial Cells through the ERK and Nrf-2/HO-1 Signaling Pathway

• Main Authors: Rajapaksha Gedara Prasad Tharanga Jayasooriya, Ilandarage Menu Neelaka Molagoda, Matharage Gayani Dilshara, Yung Hyun Choi, Gi-Young Kim
• Format: Article
• Language: English
• Published: MDPI AG 2020-06-01
• Series: Antioxidants
• Subjects: glutamine; nitric oxide; inducible nitric oxide synthase; nuclear factor-erythroid 2-related factor 2; heme oxygenase-1
• Online Access: https://www.mdpi.com/2076-3921/9/6/536

Glutamine (Gln) is a nonessential α-amino acid for protein biosynthesis. However, the mechanism through which Gln regulates NO production in microglial cells is still unclear. In this study, we investigated whether the presence or absence of Gln affects NO production in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. Our data revealed that Gln depletion decreased cell viability accompanied by mild cytotoxicity, and blocked LPS-induced NO production concomitant with a significant decrease in inducible NO synthase (iNOS) expression. Additionally, Gln depletion for 24 h blocked the restoration of LPS-mediated NO production in the presence of Gln, suggesting that Gln depletion caused long-term immune deprivation. In particular, sodium-coupled amino acid transporter 1 and 2 (<i>SNAT1</i> and <i>SNAT2</i>), which are the main Gln transporters, were highly upregulated in LPS-stimulated BV2 microglial cells, in the presence of Gln accompanied by NO production. Regardless of the presence of Gln, LPS positively stimulated nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) expression, and transient <i>Nrf2</i> knockdown and HO-1 inhibition stimulated LPS-induced NO production and <i>iNOS</i> expression; however, transient <i>Nrf2</i> knockdown did not affect <i>SNAT1</i> and <i>SNAT2</i> expression, indicating that Gln transporters, <i>SNAT1</i> and <i>SNAT2</i>, were not regulated by Nrf2, which downregulated the HO-1-mediated NO production. Moreover, Gln depletion significantly reduced LPS-induced extracellular signal-regulated kinase (ERK) phosphorylation; furthermore, a specific ERK inhibitor, PD98059, and transient <i>ERK</i> knockdown attenuated LPS-stimulated NO production and <i>iNOS</i> expression, in the presence of Gln, accompanied by downregulation of <i>SNAT1</i> and <i>SNAT2</i>, suggesting that the ERK signaling pathway was related to LPS-mediated NO production via SNAT1 and SNAT2. Altogether, our data indicated that extracellular Gln is vital for NO production from microglia in inflammatory conditions.