Effects of Reactive Oxygen Species and Nitrogen Species on the Excitability of Spinal Substantia Gelatinosa Neurons

ABSTRACT

Reactive oxygen species (ROS) and nitrogen species (RNS) are both important signaling molecules involved in pain transmission in the dorsal horn of the spinal cord. Xanthine oxidase (XO) is a well-known enzyme for the generation of superoxide anions (O₂˙⁻), while S-nitroso-N-acetyl-DL-penicillamine (SNAP) is a representative nitric oxide (NO) donor. In this study, we used patch clamp recording in spinal slices of rats to investigate the effects of O₂˙⁻ and NO on the excitability of substantia gelatinosa (SG) neurons. We also used confocal scanning laser microscopy to measure XO- and SNAP-induced ROS and RNS production in live slices. We observed that the ROS level increased during the perfusion of xanthine and xanthine oxidase (X/XO) compound and SNAP after the loading of 2',7'-dichlorofluorescin diacetate (H₂DCF-DA), which is an indicator of intracellular ROS and RNS. Application of ROS donors such as X/XO, β-nicotinamide adenine dinucleotide phosphate (NADPH), and 3-morpholinosydnomimine (SIN-1) induced a membrane depolarization and inward currents. SNAP, an RNS donor, also induced membrane depolarization and inward currents. X/XO-induced inward currents were significantly decreased by pretreatment with phenyl N-tert-butylnitrone (PBN; nonspecific ROS and RNS scavenger) and manganese(III) tetrakis(4-benzoic acid) porphyrin (MnTBAP; superoxide dismutase mimetics). Nitro-L-arginine methyl ester (NAME; NO scavenger) also slightly decreased X/XO-induced inward currents, suggesting that X/XO-induced responses can be involved in the generation of peroxynitrite (ONOO⁻). Our data suggest that elevated ROS, especially O₂˙⁻, NO and ONOO⁻, in the spinal cord can increase the excitability of the SG neurons related to pain transmission.