N-METHYL-D-ASPARTATE ATTENUATES OPIOID RECEPTOR-MEDIATED G-PROTEIN ACTIVATION AND THIS PROCESS INVOLVES PROTEIN-KINASE-C

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 remark able attenuation of [S-35]guanosine-5'-O-(3-thio)triphosphate binding stimulated by [D-Pen(2),D-Pen(5)]-enkephalin (DPDPE), a delta-opioid r eceptor agonist. The effects of NMDA were dose and time dependent with an IC50 value of 5 nM and could be blocked by NMDA receptor antagonis ts. After NMDA treatment, the DPDPE dose-response curve shifted to the right (EC50 value increased similar to 7-fold, from 6 to 40 nM), and the maximal response induced by DPDPE was reduced by similar to 60%. T he effects of NMDA were reversible, and the DPDPE response could recov er within 60 min. The functional responses of delta-, mu-, and kappa-o pioid receptors in primarily cultured neurons also were attenuated sig nificantly by NMDA treatment. The inhibitory effects of NMDA on opioid receptor-mediated G protein activation could be blocked by coadminist ration of the protein kinase C (PKC) inhibitors or by elimination of t he extracellular Ca2+. Correspondingly, NMDA treatment of NG108 cells significantly elevated cellular PKC activity and stimulated G(i alpha 2) phosphorylation. Transient transfection into NG108-15 cells of the wild-type G(i alpha 2) and a mutated G(i alpha 2) (Ser144Ala) resulted in a 2-fold increase in DPDPE-stimulated G protein activation. The DP DPE responses were greatly inhibited by NMDA treatment in the wild-typ e G(i alpha 2)-transfected cells but much less affected in the mutant G(i alpha 2)-transfected cells. In summary, NMDA attenuates opioid rec eptor/G protein coupling, and this process requires activation of PKC.