Variants of Neural Nitric Oxide Synthase in the Spinal Cord of Neuropathic Rats and Their Effects on Nuclear Factor-κB (NF-κB) Activity in PC12 Cells

Abstract

Neuropathic pain due to nerve injury is associated with overactivity of spinal N-methyl-D-aspartate (NMDA) receptors and nitric oxide synthases (NOS). Spinal NOS and NMDA receptors could act in a concerted manner to excite each other in nociceptive signaling. Among the 3 major NOS isoforms, neuronal NOS (nNOS) has the most functional relationship with NMDA receptors through a PDZ-PDZ (PSD-95, Dlg, ZO-1 homology) postsynapse interaction. However, some nNOS variants lack the PDZ domain, which may result in the changes in the interaction with the NMDA receptor and subsequent localization and enzymatic activity. The aim of this study was to determine which nNOS variants are expressed in the spinal cord in neuropathic rats and deduce their role in neuropathic pain by testing the effects of these kinds of nNOS on nuclear factor-kappaB (NF-kappaB) activity in PC12 cells. Western blot analysis revealed that there were at least 3 bands of nNOS (155, 135, and 125 kDa) in the spinal cord and, moreover, that nNOS at 135 kDa decreased significantly after development of neuropathic pain. 5'-RACE-PCR and Southern blots determined that the nNOS at 155 and 135 kDa corresponded to nNOSalpha and nNOSbeta, respectively, which was confirmed by RT-PCR. PC12 cells transfected with the nNOSalpha gene had no effect on NF-kappaB activity, but nNOSbeta without the PDZ domain significantly decreased that in PC12 cells. Considering the importance of spinal NF-kappaB signaling in neuropathic rat, it could be concluded that changes in spinal nNOS variants and quantity after peripheral nerve injury implicate nNOS in the generation of neuropathic pain. This article presents data demonstrating that nNOS variants change in the spinal cord of the rats after neuropathic pain and result in differential effects on NF-kappaB activity in PC12 cells. These changes in nNOS variants and their different characteristics may account for the spinal NO paradox role in neuropathic pain. Furthermore, these data suggest that nNOSbeta may represent a new therapeutic target for the treatment of chronic neuropathic pain.