Spinal neuronal NOS activation mediates sigma‐1 receptor‐induced mechanical and thermal hypersensitivity in mice: involvement of PKC‐dependent GluN1 phosphorylation

Abstract

BACKGROUND AND PURPOSE We recently demonstrated that activation of the spinal sigma‐1 receptor induces mechanical and thermal hypersensitivity via calcium‐dependent second messenger cascades and phosphorylation of the spinal NMDA receptor GluN1 subunit (pGluN1). Here we examined the role of NO in this process, as it plays a critical role in PKC‐mediated calcium signalling and the potentiation of NMDA receptor function. EXPERIMENTAL APPROACH The effects of intrathecal (i.t.) pretreatment with nNOS inhibitors on PRE084 (sigma‐1 receptor agonist)‐induced pain were assessed in mice by use of mechanical allodynia and thermal hyperalgesia tests. Western blot analysis, immunoprecipitation and immunohistochemical techniques were used to determine effects of these treatments on spinal pGluN1‐immunoreactive (ir) cells, whether PRE084 induces a time‐dependent modification of nNOS activity in the dorsal horn, and if any changes in nNOS activity can be blocked by sigma‐1 receptor, calcineurin or soluble guanylyl cyclase (sGC) inhibitors. KEY RESULTS PRE084, injected i.t., induced mechanical and thermal hypersensitivity, and increased the number of PKC‐ and PKA‐dependent pGluN1‐ir cells in spinal cord. This PRE084‐induced hypersensitivity and increase in PKC‐dependent pGluN1 expression were blocked by pretreatment with NG‐nitro‐L‐arginine methyl ester (L‐NAME) or 7‐nitroindazole (7‐NI). PRE084 also time‐dependently decreased the ratio of phosphorylated nNOS (pnNOS) to nNOS expression and the number of spinal pnNOS‐ir cells. This decrease in pnNOS was prevented by BD1047, a sigma‐1 receptor antagonist and cyclosporin A, a calcineurin inhibitor, but not by a sGC inhibitor. CONCLUSIONS AND IMPLICATIONS Spinal sigma‐1 receptor‐induced sensitization is mediated by an increase in nNOS activity, which is associated with an NO‐induced increase in PKC‐dependent pGluN1 expression.

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

“…6,14) As previously described, Sig-1R-induced pain is closely associated with nNOS activation as well as Nox2-derived ROS production and both ROS and NO participate in NMDA receptor signaling. 9) However, the hypothetical interaction between these two signaling pathways is poorly understood, particularly as it relates to the potentiation of NMDA receptor activity in the spinal cord.…”

“…The doses of all drugs were selected based on or slightly modified from established doses previously used in the literature including those used in previous studies from our laboratories. 6,13,[16][17][18][19] PRE, L-NIL and FeTPPS were dissolved in physiological saline. 7-NI was dissolved in 5% dimethyl sulfoxide (DMSO) in Corn oil.…”

“…Sensitization to innocuous mechanical stimulation was examined using a von Frey filament (North Coast Medical, Morgan Hill, CA, U.S.A.) as previously described. 6) Based on this previous study, the 0.16 g von Frey filament was selected for testing and was applied 10 times to each hind paw with each application separated by 10 s intervals. Then, the number of paw withdrawal responses was counted.…”

“…Since phosphorylation of nNOS (pnNOS) at serine 847 (Ser847) inhibits nNOS activity, 5,6) a decrease in the pnNOS expression is considered to reflect an increase in the active forms of nNOS. The changes in the expression of pnNOS in the spinal cord dorsal horn were measured by using an immmunohistochemistry.…”

“…administration of the nNOS inhibitor, 7-nitroindazole, reduces sigma-1 receptor (Sig-1R)-induced pain hypersensitivity and suppresses the Sig-1R-induced increase in protein kinase C (PKC)-dependent phosphorylation of GluN1 subunit (pGluN1) resulting in the potentiation of NMDA receptor function. 6) These findings raise the possibility that the action of NO on nociceptive transmission at spinal synapses could be closely linked to Sig-1R-induced functional activation of NMDA receptors.…”