Levodopa modulating effects of inducible nitric oxide synthase and reactive oxygen species in glioma cells

Abstract

Neurological injury and Parkinson disease (PD) are often associated with the increase of nitric oxide (NO) and free radicals from resident glial cells in the brain. In vitro, exposure to L-3-4-dihydroxyphenylalanine (L-DOPA), one of the main therapeutic agents for the treatment of PD, can lead to neurotoxicity. In this study, lipopolysaccharide (LPS) and interferon-gamma (IFN-g) were used to stimulate C6 glioma cells in the presence of varying concentrations of L-DOPA (1 μM-1 mM). The results indicated a slight augmentation of NO 2 − production at low concentrations of L-DOPA (<100 μM) and complete inhibition of NO 2 − at higher concentrations (500 μM, 1 mM), (p < 0.001). Western blot analysis corroborated that L-DOPA effects on iNOS was at the level of its protein expression. Total reactive oxygen species (ROS) were detected using 2′, 7′-dichlorofluorescein diacetate fluorescence dye (2′, 7′-DCFC) and there was an increase of intensity with the increasing concentrations of L-DOPA. Furthermore, large amounts of superoxide (O 2 −) and hydrogen peroxide (H 2O 2) were generated from the autoxidation of L-DOPA. C6 cells contain high levels of catalase, with inadequate levels of superoxide dismutase (SOD); therefore, there was an accumulation of O 2 −, tantamount to elevation in 2′7′-DCFC intensity. Simultaneous accumulation of O 2 − and NO 2 − would propel formation of peroxynitrite (ONOO-). SOD completely attenuated the autoxidation of L-DOPA and significantly reversed the inhibitory effects on iNOS at high concentrations. The data obtained confirmed that the observed effects on iNOS were not due to the activation of the D 1 or β1 adrenergic receptors by L-DOPA. It was concluded from this study that L-DOPA contributed to the modulation of iNOS and to the increase of O 2 − production in the stimulated glioma cells in vitro.