High Concentration of L-Arginine Suppresses Nitric Oxide Synthase Activity and Produces Reactive Oxygen Species in NB9 Human Neuroblastoma Cells

Abstract

Hereditary argininemia manifests as neurological disturbance and mental retardation, features not observed in other amino acidemias. The cytotoxic effect of a high concentration of L-arginine (L-Arg) was investigated using NB9 human neuroblastoma cells (NB9), which express neuronal nitric oxide synthase (nNOS). When the concentration of L-Arg in the medium increased from 50 microM to 2 mM after incubation for 48 hr, the intracellular concentration of L-Arg increased from 68.0 +/- 1 pmol/10(6) cells to 1310.0 +/- 5 pmol/10(6) cells and that of L-citrulline (L-Cit) from undetectable levels to 47.1 +/- 0.2 pmol/10(6) cells (mean +/- SD of three independent analyses). This increase in intracellular L-Arg levels caused a decrease in NOS activity by approximately 71%. Flow cytometric analysis showed that reactive oxygen species (ROS) are produced in NB9 exposed to 2 mM L-Arg. The production of ROS was abolished by a NOS inhibitor, NG-nitro-L arginine-methylester. Production of ROS was also observed when NB9 were treated with L-Cit for 48 hr. To investigate the effect of L-Cit on the activity of NOS, a kinetic study on nNOS was conducted using cellular extracts from NB9. The apparent Km value of nNOS for L-Arg was 8.4 microM, with a Vmax value of 8.2 pmol/min/mg protein. L-Cit competitively inhibited NOS activity, as indicated by an apparent Ki value of 65 nM. These results suggest that L-Cit formed by nNOS in L-Arg-loaded neuronal cells inhibits NOS activity and nNOS in these L-Arg-loaded cells functions as a NADPH oxidase to produce ROS, which may cause neurotoxicity in argininemia.

Intracerebroventricular administration of L-arginine improves spatial memory acquisition in triple transgenic mice via reduction of oxidative stress and apoptosis

“…Incubation of the cells with 0.5 mM H 2 O 2 for 24 hours leads to deterioration of viability rate, which can be reversed by an increase in arginine concentration up to one mM. Remarkably, arginine itself demonstrates cytotoxic properties in concentrations greater than one mM ( figure 6c ), which accords with the current literature ( Todoroki et al 1998 ). It was demonstrated that high concentrations (more than one mM) of L-Arginine induce severe DNA damage and arrest at the GI phase.…”

Autophagy-Induced Nanoparticle-Based Clearing of Toxic Huntingtin Protein Aggregates from Neuron Cells

“…30,42 In another, the iron oxide nanoparticle is terminated with arginine with guanidinium groups (iron oxide-arginine) that induces high cell uptake via a nonendocytic approach and excess arginine has the ability to induce ROS by modifying the cellular nitric oxide synthesis pathway and also to deplete intracellular adenosine triphosphate (ATP). 38,43,44 The zinc oxide nanoparticle is selected due to its crystal defectdependent ROS generation property. 32 This nanoparticle is also terminated with primary amines (zinc oxide-amine) to further enhance its ROS generation property.…”

“…This L-citrulline binds to the L-arginine binding site of nitric oxide synthase (NOS), inhibits nitric oxide synthesis, and functions as a NADPH oxidase to produce reactive oxygen species. 44 We further investigated the origin of autophagy induction by carbon-trehalose. Trehalose is known to induce autophagy via the mTOR independent pathway 45 and also known to activate transcription factor EB (TFEB) or lysosome biogenesis factor, which in turn can induce autophagic flux.…”

Gene Expression of Nitric Oxide Synthase and Heme Oxygenase in Placental Villi during Pregnancy with and without Intrauterine Growth Restriction

“…The authentic primer for iNOS was 5'-GTGAGGATCAAAAACTGGGG-3' and the antisense primer was 5'-ACCTGCAGGT TGGACCAC-3'. The authentic primer for eNOS was 5'-CACTGAGATCGGCACGA-GGA-3' and the antisense primer was 5'-GTCACCATCGTGGACCACCA-3' [17]. The authentic sense primer for HO-1 was 5'-GAATTCAGCATGCCCCAGGATTTG-3' and the antisense primer was 5'-TCTAGACTAGCTGGATGTTGAGCAGGA-3' [18].…”