Background: L-glutamine (Gln) suppresses inflammation via rapid up-regulation of MAPK phosphatase (MKP)-1, deactivating p38 and JNK mitogen-activated protein kinases (MAPKs). However, the high dosage required for this may cause serious side effects. Objective: To facilitate reduced Gln intake, we developed a less-hydrolysable Gln derivative, α, δ-N-acetyl-glutamine (α, δ-NAG), which is resistant to the hydrolytic action of glutaminase. Methods: We developed α, δ-NAG by substituting the NH 2 group in α-chain and δ-amide group of Gln with acetyl groups. We employed the ovalbumin model, previously developed by us, to examine sequential asthmatic events, including neutrophilia/Th1 and eosinophilia/Th2 responses. MKP-1 was knocked down using small-interfering RNA (siRNA). Gln levels and intracellular calcium concentration ([Ca 2+] i) were analysed using multiple reaction monitoring chromatograms and confocal laser scanning microscopy, respectively. Results: Oral administration of α, δ-NAG and Gln suppressed all the parameters at 0.2 and 2 g/kg body weight, respectively. MKP-1 siRNA abrogated the beneficial effects of α, δ-NAG. α, δ-NAG up-regulated MKP-1 in an ERK MAPK-dependent manner. α, δ-NAG transiently increased [Ca 2+] I, resulting in increased Ras activity. Inhibition of Gα q, a G-protein subfamily, abrogated the effects of α, δ-NAG on [Ca 2+] I and Ras activity. Inhibition of Gα q, Ca 2+, and Ras abrogated the effects of α, δ-NAG, such as signalling pathways (ERK phosphorylation and MKP-1 up-regulation) and clinical signs (neutrophilia/Th1 responses) in asthmatic mice. Conclusion: α, δ-NAG exhibits strong anti-inflammatory activity (~ 10,000-fold stronger than that of Gln), likely attributable to its up-regulation of MKP-1 by activating pathways involving the G protein-coupled receptor (GPCR)/Gα q/Ca 2+/Ras/ERK cascade.

Background: The administration of L-glutamine (Gln) suppresses allergic airway inflammation via the rapid upregulation of MAPK phosphatase (MKP)-1, which functions as a negative regulator of inflammation by deactivating p38 and JNK mitogen-activated protein kinases (MAPKs). However, the role of endogenous Gln remains to be elucidated. Therefore, we investigated the mechanism by which endogenous Gln regulates MKP-1 induction and allergic airway inflammation in an ovalbumin-based murine asthma model. Methods: We depleted endogenous Gln levels using l-γ-glutamyl- p-nitroanilide (GPNA), an inhibitor of the Gln transporter ASCT2, and glutamine synthetase small interfering (si)RNA. Lentivirus expressing MKP-1 was injected to achieve overexpression of MKP-1. Asthmatic phenotypes were assessed using our previously developed ovalbumin-based murine model, which is suitable for examining sequential asthmatic events, including neutrophil infiltration. Gln levels were analyzed using a Gln assay kit. Results: GPNA or glutamine synthetase siRNA successfully depleted endogenous Gln levels. Importantly, homeostatic MKP-1 induction did not occur at all, which resulted in prolonged p38 MAPK and cytosolic phospholipase A 2 (cPLA 2) phosphorylation in Gln-deficient mice. Gln deficiency augmented all examined asthmatic reactions, but it exhibited a strong bias toward increasing the neutrophil count, which was not observed in MKP-1-overexpressing lungs. This neutrophilia was inhibited by a cPLA 2 inhibitor and a leukotriene B4 inhibitor, but not by dexamethasone. Conclusion: Gln deficiency leads to the impairment of MKP-1 induction and activation of p38 MAPK and cPLA 2, resulting in the augmentation of neutrophilic, more so than eosinophilic, airway inflammation.