Olianas Maria C, Dedoni Simona, Ambu Rossano, Onali Pierluigi
Department of Neuroscience, Section of Biochemical Pharmacology, University of Cagliari, 09042 Monserrato, Ca, Italy.
Eur J Pharmacol. 2009 Apr 1;607(1-3):96-101. doi: 10.1016/j.ejphar.2009.02.025. Epub 2009 Feb 23.
The clozapine metabolite N-desmethylclozapine (NDMC) has been recently shown to act at different neurotransmitter receptors and to display both antagonist and agonist activities. We have previously reported that in cells over-expressing the recombinant delta-opioid receptor NDMC behaved as partial agonist with high intrinsic activity, but its action at the receptors naturally expressed in human brain remained to be investigated. In the present study, we examined whether NDMC was able to bind to and activate delta-opioid receptors in membranes of post-mortem human frontal cortex. In radioligand binding assays, NDMC competition curves displayed high- (K(i)=26 nM) and low-affinity (K(i)=3 microM) components, whose proportion was regulated by guanine nucleotides in an agonist-like fashion. In functional assays, NDMC stimulated [(35)S]GTPgammaS binding (EC(50)=905 nM) and inhibited cyclic AMP formation (EC(50)=590 nM) as effectively as delta-opioid agonists, whereas clozapine was much less potent and efficacious and clozapine N-oxide was completely inactive. The NDMC agonist activity was potently antagonized by the delta-opioid antagonist naltrindole, but not by the micro-opioid receptor antagonist CTAP (D-phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH(2)) or the kappa-opioid antagonist nor-binaltorphimine. Moreover, blockade of either acetylcholine muscarinic, dopamine D(2) or serotonin 5HT(1A) receptors failed to affect NDMC agonist activity. These data demonstrate that at clinically relevant concentrations NDMC behaves as an efficacious agonist at delta-opioid receptors of human frontal cortex.
氯氮平代谢物N-去甲基氯氮平（NDMC）最近被证明可作用于不同的神经递质受体，并表现出拮抗剂和激动剂活性。我们之前报道过，在过表达重组δ-阿片受体的细胞中，NDMC表现为具有高内在活性的部分激动剂，但其在人脑中天然表达的受体上的作用仍有待研究。在本研究中，我们检测了NDMC是否能够结合并激活死后人类额叶皮质膜中的δ-阿片受体。在放射性配体结合试验中，NDMC竞争曲线显示出高亲和力（K(i)=26 nM）和低亲和力（K(i)=3 μM）成分，其比例受鸟嘌呤核苷酸以激动剂样方式调节。在功能试验中，NDMC刺激[(35)S]GTPγS结合（EC(50)=905 nM）并抑制环磷酸腺苷形成（EC(50)=590 nM），其效果与δ-阿片激动剂一样有效，而氯氮平的效力和效能要低得多，氯氮平N-氧化物则完全无活性。NDMC的激动剂活性被δ-阿片拮抗剂纳曲吲哚强烈拮抗，但未被μ-阿片受体拮抗剂CTAP（D-苯丙氨酸-半胱氨酸-酪氨酸-D-色氨酸-精氨酸-苏氨酸-青霉胺-苏氨酸-NH(2)）或κ-阿片拮抗剂去甲二氢吗啡酮拮抗。此外，阻断乙酰胆碱毒蕈碱、多巴胺D(2)或5-羟色胺5HT(1A)受体均未能影响NDMC的激动剂活性。这些数据表明，在临床相关浓度下，NDMC在人类额叶皮质的δ-阿片受体上表现为有效的激动剂。
