Mu-opioid receptors transiently activate the Akt-nNOS pathway to produce sustained potentiation of PKC-mediated NMDAR-CaMKII signaling.

Pilar Sánchez-Blázquez,Javier Garzón,María Rodríguez-Muñoz,Rafael Linden

doi:10.1371/journal.pone.0011278

Abstract

BackgroundIn periaqueductal grey (PAG) matter, cross-talk between the Mu-opioid receptor (MOR) and the glutamate N-methyl-D-Aspartate receptor (NMDAR)-CaMKII pathway supports the development of analgesic tolerance to morphine. In neurons, histidine triad nucleotide binding protein 1 (HINT1) connects the regulators of G protein signaling RGSZ1 and RGSZ2 to the C terminus of the MOR. In response to morphine, this HINT1-RGSZ complex binds PKCγ, and afterwards, the interplay between PKCγ, Src and Gz/Gi proteins leads to sustained potentiation of NMDAR-mediated glutamate responses.Methodology/Principal FindingsFollowing an intracerebroventricular (icv) injection of 10 nmol morphine, Akt was recruited to the synaptosomal membrane and activated by Thr308 and Ser473 phosphorylation. The Akt activation was immediately transferred to neural Nitric Oxide Synthase (nNOS) Ser1417. Afterwards, nitric oxide (NO)-released zinc ions recruited PKCγ to the MOR to promote the Src-mediated phosphorylation of the Tyr1325 NMDAR2A subunit. This action increased NMDAR calcium flux and CaMKII was activated in a calcium-calmodulin dependent manner. CaMKII then acted on nNOS Ser847 to produce a sustained reduction in NO levels. The activation of the Akt-nNOS pathway was also reduced by the binding of these proteins to the MOR-HINT1 complex where they remained inactive. Tolerance to acute morphine developed as a result of phosphorylation of MOR cytosolic residues, uncoupling from the regulated G proteins which are transferred to RGSZ2 proteins. The diminished effect of morphine was prevented by LNNA, an inhibitor of nNOS function, and naltrindole, a delta-opioid receptor antagonist that also inhibits Akt.Conclusions/SignificanceAnalysis of the regulatory phosphorylation of the proteins included in the study indicated that morphine produces a transient activation of the Akt/PKB-nNOS pathway. This activation occurs upstream of PKCγ and Src mediated potentiation of NMDAR activity, ultimately leading to morphine tolerance. In summary, the Akt-nNOS pathway acts as a primer for morphine-triggered events which leads to the sustained potentiation of the NMDAR-CaMKII pathway and MOR inhibition.

Highlights

The coexistence of ligand-regulated metabotropic and ionotropic receptors in the postsynapse raises the possibility of mutual interactions triggered by the relative abundance of their presynaptic mediators
This kinase enhances the activity of Src [5]; protein kinase C (PKC) promotes the regulatory phosphorylation of serine and threonine residues in the cytosolic regions of NR1 and NR2 subunits [6,7,8]; it directly elicits the potentiation of Nmethyl-D-Aspartate receptor (NMDAR) responses by removing the inhibitory binding of Ca-Calmodulin (Ca2+-CaM) on NR1 subunits [9]
Because the histidine triad nucleotide binding protein 1 (HINT1) protein is required for morphine to recruit PKCc to the Mu-opioid receptor (MOR) environment, we evaluated in HINT1 (2/2) mice this possibility but with Akt and neural Nitric Oxide Synthase (nNOS)

Summary

Introduction

The coexistence of ligand-regulated metabotropic and ionotropic receptors in the postsynapse raises the possibility of mutual interactions triggered by the relative abundance of their presynaptic mediators. G-protein coupled receptors (GPCRs) regulate NMDAR activity through the action of nonreceptor tyrosine kinases including Src, Fyn and serine/threonine kinases such as protein kinase C (PKC) [3]. Non-receptor tyrosine kinases phosphorylate specific tyrosine residues in the cytosolic tail of NR2 and, potentially, NR3 subunits [4], producing an increase in the permeation of Ca2+ ions towards the cytosolic side of the postsynapse. This HINT1-RGSZ complex binds PKCc, and afterwards, the interplay between PKCc, Src and Gz/Gi proteins leads to sustained potentiation of NMDAR-mediated glutamate responses

Methods

Results

Conclusion