Enhancement of spinal dorsal horn neuron NMDA receptor phosphorylation as the mechanism of remifentanil induced hyperalgesia: Roles of PKC and CaMKII

Abstract

Experimental studies have demonstrated that modulation of N-Methyl-D-Aspartate (NMDA) receptor subunits 1 and 2 (NR1 and NR2) through phosphorylation can affect remifentanil induced postoperative opioid-induced hyperalgesia (OIH). Also, activations of Protein Kinase C (PKC) and ERK1/2 have been shown to potentiate while activation of CaMKII inhibit opioid induced hyperalgesia (J Neurosurg Anesthesiol. 2016;28(1):44–50). However, the definite mechanism of OIH development, and in particular the potential interplay between NMDA receptors and PKC or CaMKII or ERK1/2 in the development of remifentanil induced hyperalgesia is unclear. The current study was designed to investigate the changes of NMDA function/receptor phosphorylation in the membrane of dorsal horn (DH) neurons following remifentanil application and the impacts of PKC, CaMKII or ERK1/2 on remifentanil induced modulation of NMDA receptors and hyperalgesia. Remifentanil was given intravenously at 1μg/kg/min over a period of 60 minutes (min) in rats, followed immediately by the infusion of either vehicle solution or the respective inhibitors of PKC (chelerythrine, at 0.2mg/kg/min), CaMKII (KN93, at 0.02mg/kg/min) or ERK1/2 (PD98059, 0.2 mg/kg/min) in each group over 10 min. Thereafter, the rat pain behaviors were evaluated by the paw withdrawal mechanical threshold and paw withdrawal thermal latency for a duration of 8 hours (h). In in vitro studies, fetal DH neurons were isolated and primarily cultured in the presence of 4nM remifentanil for 60 min, and then the remifentanil was washed out and replaced immediately by culturing in the absence or presence of chelerythrine (100μM), KN93 (10μM) or PD98059(100μM) respectively in each group for 8 hours. The expression of NMDA receptors subunits and their phosphorylation (NR1, NR2, phospho-NR1, phospho-NR2) were analyzed by Western blotting at 1h, 2h, 4h, 8h after the completion of treatments with protein kinases inhibitors. The functional changes of NMDA receptors were evaluated by electrophysiologic recordings of NMDA current. Remifentanil induced thermal and mechanical hyperalgesia occurred at 2 hours after its administration and lasted for 4 hours. Chelerythrine and KN93 but not PD98059 significantly attenuated remifentanil-induced hyperalgesia compared with control group. The expression of NR1, NR2, phospho-NR1, phospho-NR2 were increased significantly and progressively over time after remifentanil administration as compared to control group, and these increases were all significantly attenuated by either Chelerythrine or KN93 but not by PD98059. Of note, NMDA receptor functional enhancement induced by remifentanil were attenuated by Chelerythrine, KN93, and PD98059 each time points. It is concluded that the enhancements in function and quantity of NMDA receptor via phosphorylation of its subunits through PKC and CaMKII activation may represent the major mechanism whereby remifentanil induced OIH.