Int J Oral Biol.  2016 Sep;41(3):141-147. 10.11620/IJOB.2016.41.3.141.

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

Affiliations
  • 1Department of Oral Physiology, College of Dentistry, Institute of Wonkwang Biomaterial and Implant, Wonkwang University, Iksan 570-749, Korea. physio1@wonkwang.ac.kr

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.

Keyword

substantia gelatinosa neuron; superoxide; nitric oxide; excitation; patch clamp

MeSH Terms

Adenine
Animals
Humans
Membranes
Microscopy, Confocal
Neurons*
Nitric Oxide
Nitrogen*
Perfusion
Peroxynitrous Acid
Rats
Reactive Oxygen Species*
Spinal Cord
Spinal Cord Dorsal Horn
Substantia Gelatinosa*
Superoxide Dismutase
Superoxides
Tissue Donors
Xanthine
Xanthine Oxidase
Adenine
Nitric Oxide
Nitrogen
Peroxynitrous Acid
Reactive Oxygen Species
Superoxide Dismutase
Superoxides
Xanthine
Xanthine Oxidase
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