Fan G H, Zhao J, Wu Y L, Lou L G, Zhang Z, Jing Q, Ma L, Pei G
Shanghai Institute of Cell Biology, Chinese Academy of Sciences, People's Republic of China.
Mol Pharmacol. 1998 Apr;53(4):684-90. doi: 10.1124/mol.53.4.684.
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 delta-opioid receptor agonist. The effects of NMDA were dose and time dependent with an IC50 value of 5 nM and could be blocked by NMDA receptor antagonists. After NMDA treatment, the DPDPE dose-response curve shifted to the right (EC50 value increased approximately 7-fold, from 6 to 40 nM), and the maximal response induced by DPDPE was reduced by approximately 60%. The effects of NMDA were reversible, and the DPDPE response could recover within 60 min. The functional responses of delta-, mu-, and kappa-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 Gialpha2 phosphorylation. Transient transfection into NG108-15 cells of the wild-type Gialpha2 and a mutated Gialpha2 (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 Gialpha2-transfected cells but much less affected in the mutant Gialpha2-transfected cells. In summary, NMDA attenuates opioid receptor/G protein coupling, and this process requires activation of PKC.
在神经母细胞瘤X胶质瘤杂交（NG108-15）细胞中研究了N-甲基-D-天冬氨酸（NMDA）对阿片受体介导的G蛋白激活的影响。用NMDA处理细胞导致[D-青霉胺2，D-青霉胺5]-脑啡肽（DPDPE，一种δ-阿片受体激动剂）刺激的[35S]鸟苷-5'-O-（3-硫代）三磷酸结合显著减弱。NMDA的作用具有剂量和时间依赖性，IC50值为5 nM，并且可被NMDA受体拮抗剂阻断。NMDA处理后，DPDPE剂量反应曲线向右移动（EC50值增加约7倍，从6 nM增加到40 nM），并且DPDPE诱导的最大反应降低了约60%。NMDA的作用是可逆的，DPDPE反应可在60分钟内恢复。NMDA处理也显著减弱了原代培养神经元中δ-、μ-和κ-阿片受体的功能反应。NMDA对阿片受体介导的G蛋白激活的抑制作用可通过共同给予蛋白激酶C（PKC）抑制剂或通过消除细胞外Ca2+来阻断。相应地，NMDA处理NG108细胞显著提高了细胞PKC活性并刺激了Gialpha2磷酸化。将野生型Gialpha2和突变型Gialpha2（Ser144Ala）瞬时转染到NG108-15细胞中导致DPDPE刺激的G蛋白激活增加2倍。在野生型Gialpha2转染的细胞中，NMDA处理极大地抑制了DPDPE反应，但在突变型Gialpha2转染的细胞中受影响较小。总之，NMDA减弱阿片受体/G蛋白偶联，并且该过程需要PKC的激活。
