Regulation of Eukaryotic Elongation Factor 2 Kinase in Mouse Sensory Neurons and Anti-nociceptive Effects of a Selective Inhibitor

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      Eukaryotic elongation factor 2 (eEF2) is an essential component of the translation machinery that catalyzes translocation of the ribosome along the mRNA transcript. Phosphorylation of eEF2 causes a transient suspension of ongoing translation. eEF2 kinase, the only enzyme known to phosphorylate eEF2, is activated by calcium/calmodulin and AMP-activated kinase (AMP kinase), a key sensor of ATP availability, among other mechanisms. Using an antibody selective for phosphorylated eEF2 (peEF2), we examined eEF2 regulation in sensory neurons of the mouse dorsal root ganglion (DRG). Immunohistochemistry for peEF2 revealed tonically high levels of peEF2 in a subset of primarily small-diameter DRG neurons, particularly putative nociceptors positive for either TRPV1 or IB4. In dissociated DRG neurons, application of 50 mM K+ for 1 or 10 minutes induced intense peEF2 staining in virtually all neurons; this staining remained elevated above baseline 60 minutes after stimulation and was prevented by a selective eEF2 kinase inhibitor, A484954 (30 micromolar). Both peEF2 and eEF2K levels in DRG increased in response to hindpaw inflammatory insult (complete Freud's adjuvant injection), as measured by western blot. Therefore, A484954 bioavailability and pharmacokinetics were evaluated by mass spectrometry in plasma and brain 30, 60 and 90 minutes after oral administration of 1 or 7.5 mg/kg by gavage. Peak plasma and brain concentrations were observed 60 minutes after administration. Daily oral administration of A484954 in the carrageenan model of inflammatory pain significantly reduced hyperalgesia to radiant heat throughout the behavioral response to carrageenan, suggesting that suppression of eEF2 phosphorylation is anti-nociceptive. L3-5 DRG, lumbar spinal dorsal horn and whole brain were analyzed to confirm inhibition of eEF2 phosphorylation by oral A484954. These results indicate that peEF2 is rapidly induced in response to sensory neuron activation and sustained during inflammatory hyperalgesia. Suppression of eEF2 phosphorylation is anti-nociceptive, possibly through a direct action on DRG nociceptors. Grant support from NIH NIGMS R01102346 and P20GM103643.
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