Neuronal NOS Activates Spinal NADPH Oxidase 2 Contributing to Central Sigma-1 Receptor-Induced Pain Hypersensitivity in Mice

We recently demonstrated that activation of spinal sigma-1 receptors (Sig-1Rs) induces pain hypersensitivity via the activation of neuronal nitric oxide synthase (nNOS) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (Nox2). However, the potential direct interaction between nNOS-derived nitric oxide (NO) and Nox2-derived reactive oxygen species (ROS) is poorly understood, particularly with respect to the potentiation of N-methyl-D-aspartate (NMDA) receptor activity in the spinal cord associated with the development of central sensitization. Thus, the main purpose of this study was to investigate whether Sig-1R-induced and nNOS-derived NO modulates spinal Nox2 activation leading to an increase in ROS production and ultimately to the potentiation of NMDA receptor activity and pain hypersensitivity. Intrathecal pretreatment with the nNOS inhibitor, 7-nitroindazole or with the Nox inhibitor, apocynin significantly inhibited the mechanical and thermal hypersensitivity induced by intrathecal administration of the Sig-1R agonist, 2-(4-morpholinethyl) 1-phenylcyclohexanecarboxylate hydrochloride (PRE084). Conversely, pretreatment with 5,10,15,20-tetrakis-(4-sulphonatophenyl)-porphyrinato iron(III) (FeTPPS; a scavenger of peroxynitrite, a toxic reaction product of NO and superoxide) had no effect on the PRE084-induced pain hypersensitivity. Pretreatment with 7-nitroindazole significantly reduced the PRE084-induced increase in Nox2 activity and concomitant ROS production in the lumbar spinal cord dorsal horn, whereas apocynin did not alter the PRE084-induced changes in nNOS phosphorylation. On the other hand pretreatment with apocynin suppressed the PRE084-induced increase in the protein kinase C (PKC)-dependent phosphorylation of NMDA receptor GluN1 subunit (pGluN1) at Ser896 site in the dorsal horn. These findings demonstrate that spinal Sig-1R-induced pain hypersensitivity is mediated by nNOS activation, which leads to an increase in Nox2 activity ultimately resulting in a ROS-induced increase in PKC-dependent pGluN1 expression.