N-Methyl-d-Aspartate Attenuates Opioid Receptor-Mediated G Protein Activation and This Process Involves Protein Kinase C

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Abstract
The effects of N-methyl-d-aspartate (NMDA) on opioid receptor-mediated G protein activation were explored in neuroblastoma X glioma hybrid (NG108–15) cells. Treatment of the cells with NMDA resulted in a remarkable attenuation of [35S]guanosine-5′-O-(3-thio)triphosphate binding stimulated by [d-Pen2,d-Pen5]-enkephalin (DPDPE), a δ-opioid receptor agonist. The effects of NMDA were dose and time dependent with an IC50 value of 5 nmand could be blocked by NMDA receptor antagonists. After NMDA treatment, the DPDPE dose-response curve shifted to the right (EC50 value increased ∼7-fold, from 6 to 40 nm), and the maximal response induced by DPDPE was reduced by ∼60%. The effects of NMDA were reversible, and the DPDPE response could recover within 60 min. The functional responses of δ-, μ-, and κ-opioid receptors in primarily cultured neurons also were attenuated significantly by NMDA treatment. The inhibitory effects of NMDA on opioid receptor-mediated G protein activation could be blocked by coadministration of the protein kinase C (PKC) inhibitors or by elimination of the extracellular Ca2+. Correspondingly, NMDA treatment of NG108 cells significantly elevated cellular PKC activity and stimulated Giα2 phosphorylation. Transient transfection into NG108–15 cells of the wild-type Giα2and a mutated Giα2 (Ser144Ala) resulted in a 2-fold increase in DPDPE-stimulated G protein activation. The DPDPE responses were greatly inhibited by NMDA treatment in the wild-type Giα2-transfected cells but much less affected in the mutant Giα2-transfected cells. In summary, NMDA attenuates opioid receptor/G protein coupling, and this process requires activation of PKC.