Ca²⁺/calmodulin‐dependent protein kinase II in spinal dorsal horn contributes to the pain hypersensitivity induced by γ‐aminobutyric acid type a receptor inhibition

Abstract

The fast inhibitory synaptic transmission mediated by the γ-aminobutyric acid type A receptor (GABAA R) within spinal dorsal horn exerts a gating control over the synaptic conveyance of nociceptive information from the periphery to higher brain regions. Although a large body of evidence has demonstrated that the impairment of GABAergic inhibition alone is sufficient to elicit pain hypersensitivity in intact animals, the underlying mechanisms remain to be characterized. The present study shows that Ca(2+) /calmodulin-dependent protein kinase II (CaMKII) is an important signaling protein downstream of reduced GABAergic inhibition. We found that pharmacological removal of inhibition by intrathecal application of the GABAA R antagonist bicuculline significantly enhanced the autophosphorylation of CaMKII at Thr286 in spinal dorsal horn of mice. In addition to increased CaMKII activity, bicuculline also promoted CaMKII interaction with N-methyl-D-aspartate (NMDA)-subtype glutamate receptors and induced the translocation of CaMKII from cytosolic compartments to the synaptosomal membrane fraction. Immunoblotting analysis revealed that the phosphorylation levels of NMDA receptor NR2B subunit at Ser1303 and of AMPA-subtype glutamate receptor GluR1 subunit at Ser831, two important CaMKII phosphorylation sites, were substantially enhanced after bicuculline application. Behavioral tests illustrated that intrathecal administration of the CaMKII inhibitor KN-93, NMDA receptor antagonist D-APV, or AMPA receptor antagonist GYKI 52466 effectively ameliorated the mechanical allodynia evoked by bicuculline. These data thus indicate that CaMKII signaling is critical for the reduced inhibition to evoke spinal sensitization.

Peripheral inflammation activated focal adhesion kinase signaling in spinal dorsal horn of mice

“…The P3 pellet was homogenized in sodium dodecyl sulfate (SDS) sample buffer and boiled at 95°C for 5 min before processing. To assay extracellular signal‐regulated kinase1/2 (ERK1/2) phosphorylation (Suo et al, ), the spinal dorsal horn was homogenized in radioimmunoprecipitation assay buffer (50 mM Tris‐HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% NP‐40, 0.1% SDS, 0.5% sodium deoxycholate, and protease/phosphatase inhibitors). After centrifugation at 14,000 g for 10 min, the supernatant was harvested, and the protein concentration was measured with a BCA protein assay kit (Pierce, Rockford, IL).…”

“…For Western blot analysis, scanned digital images were quantified in ImageJ (NIH). The relative band density of NMDA receptor was calculated by the ratio of its signal to β‐actin signal, whereas ERK phosphorylation level was determined by the ratio of phosphorylation signal to ERK signal (Suo et al, ). Statistical significance was set at P < 0.05 by Student t ‐test or one‐way ANOVA, followed by post hoc Tukey's honestly significant difference test.…”

Resolvins: Potent Pain Inhibiting Lipid Mediators via Transient Receptor Potential Regulation

“…PKA phosphorylates Ser890 and Ser897 on NR1 and Ser845 on GluR1. CaMKII phosphorylates Ser1303 on NR2B and Ser831 on GluR1 (Suo et al, 2013;Ji et al, 2018). These processes correspond to the early, phosphorylation-dependent phase of central sensitization in which extracellular signalregulated protein kinase (ERK) is important to sustain central sensitization.…”