Recombinant arginine deiminase reduces inducible nitric oxide synthase iNOS‐mediated neurotoxicity in a coculture of neurons and microglia

Abstract

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.