ABSTRACT
The abnormal regulation of inducible nitric oxide synthase (iNOS) and neuronal nitric oxide synthase (nNOS) is associated with neurodegenerative disorders. Recombinant arginine deiminase (rADI) is a selective NO modulator of iNOS and eNOS in endothelial cells, and it also exhibits neuroprotective activity in an iNOS-induced neuron-microglia coculture system. However, the effect of rADI on nNOS remains unknown. Addressing this issue is important for evaluating the potential application of rADI in neurodegenerative diseases. SH-SY5Y cells were treated with N-methyl-D-aspartic acid (NMDA) to activate nNOS. NMDA increased NO production by 39.7 ± 3.9% via nNOS under arginine-containing conditions, but there was no significant increase in both arginine-free and rADI pretreated arginine-containing (citrulline) buffer. Subsequently, neither NMDA nor rADI alone caused cytotoxicity, whereas cotreatment with NMDA and rADI resulted in dissipation of the cell mitochondrial membrane potential and decreased cell viability. The mechanism of rADI cytotoxicity in the presence of NMDA is caused by the inhibition of NO production via nNOS mediated by the NMDA receptor, which was abolished when extracellular arginine was absent, even in the presence of citrulline. rADI not only reduced NO production but also caused cellular toxicity in nNOS-activated SH-SY5Y cells, suggesting a dual role for rADI in NOS-mediated neurotoxicity.
Subject(s)
Hydrolases/metabolism , Neurodegenerative Diseases/enzymology , Nitric Oxide Synthase Type I/biosynthesis , Nitric Oxide/biosynthesis , Recombinant Proteins/metabolism , Arginine/metabolism , Cell Culture Techniques , Cell Survival , Humans , Hydrolases/administration & dosage , Hydrolases/genetics , Membrane Potential, Mitochondrial/genetics , N-Methylaspartate/administration & dosage , Neurodegenerative Diseases/etiology , Neurodegenerative Diseases/pathology , Neurons/drug effects , Neurons/metabolism , Neurons/pathology , Nitric Oxide Synthase Type I/genetics , Nitric Oxide Synthase Type I/metabolism , Nitric Oxide Synthase Type II/genetics , Nitric Oxide Synthase Type II/metabolism , Recombinant Proteins/administration & dosage , Recombinant Proteins/geneticsABSTRACT
Modulation of nitric oxide (NO) production is considered a promising approach to therapy of diseases involving excessive inducible nitric oxide synthase (iNOS) expression, such as certain neuronal diseases. Recombinant arginine deiminase (rADI, EC3.5.3.6) catalyzes the conversion of L-arginine (L-arg), the sole substrate of NOS for NO production, to L-citrulline (L-cit) and ammonia. To understand the effect of the depletion of L-arg by rADI on NO concentration and neuroprotection, a direct coculture of neuron SHSY5Y cells and microglia BV2 cells treated with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) was used as a model of iNOS induction. The results showed that rADI preserved cell viability (4-fold higher compared with the cells treated with LPS/IFN-gamma only) by the MTT assay, corresponding with the results of neuronal viability by neuron-specific immunostaining assay. NO production (mean +/- SD) decreased from 67.0 +/- 1.3 to 19.5 +/- 5.5 microM after a 2-day treatment of rADI by the Griess assay; meanwhile, induction of iNOS protein expression by rADI was observed. In addition, rADI substantially preserved the neuronal function of dopamine uptake in the coculture. The replenishment of L-arg in the coculture eliminated the neuroprotective and NO-suppressive effects of rADI in the coculture, indicating that L-arg played a crucial role in the effects of rADI. These results highlight the important role of L-arg in the neuron-microglia coculture in excessive induction of iNOS. Regulation of L-arg by ADI demonstrated that rADI has a potentially therapeutic role in iNOS-related neuronal diseases.
