We want to thank Pappagallo et al. [1] for their commentary about our recent article [2] which delved into the pharmacodynamic divergence of (R)- and (S)-methadone. Based on our experimental findings, we concluded that (S)-methadone, like (R,S)-methadone and (R)-methadone, is an agonist at the μ opioid receptor (MOR) but with much lower potency. Importantly, (S)-methadone exhibits a unique pharmacodynamic effect by acting as a MOR antagonist when MOR is complexed with the galanin 1 receptor (Gal₁R). Since our previous studies showed that MOR-Gal₁R heteromers mediate the dopaminergic effects of opioids [3], this specific antagonist property of (S)-methadone explains the low efficacy of (R,S)-methadone at activating the dopaminergic system and at eliciting euphoric effects. Accordingly, at sufficient doses/concentrations, (S)-methadone counteracts the locomotor activating and dopamine releasing effects of (R)-methadone. While (S)-methadone may act as an antagonist at the N-methyl-D-aspartate receptor (NMDAR), the drug concentration needed to achieve this effect is higher than what is needed to interact with MORs in vivo. As such, we stated “The currently assumed role of NMDAR blockade in the purported antidepressant effects of (S)-MTD should be reframed in the context of its MOR agonistic properties.” Pappagallo et al. [1] disagreed with this statement. We address their comments below.

Our hot plate findings are a key component of the MOR agonist effects of (S)-methadone, but they are not the only evidence we provided. (S)-methadone also produces hypothermia and partial catalepsy [2], two prototypical effects of MOR agonists in rats. We also used [³⁵S]GTPγS binding in rat brain sections to show that (S)-methadone increases GPCR activity in the striatum at a concentration that does not interact with NMDARs, and that this response was blocked by pretreatment with the preferential MOR antagonist naltrexone. Since the [³⁵S]GTPγS assay is specific to GPCRs and does not involve ion channel activation or inhibition, we conclude that this activation is mediated by opioid receptors, most likely MORs. We also used BRET in transfected cells to show that (S)-methadone acts as an agonist at MOR. Finally, we found that 30 mg/kg (S)-methadone, a dose used to produce antidepressant-like effects in rats (10–40 mg/kg) [4, 5], occupies nearly 80% of striatal MORs without significant occupancy of NMDARs, using an assay which has previously shown occupancy by ketamine at these receptors [6]. Thus, we used diverse and independent approaches to confirm that (S)-methadone binds to and activates MORs at doses/concentrations that do not bind NMDARs in vivo.

我们要感谢 Pappagallo 等人 [1] 对我们最近的文章 [2] 的评论，该文章深入探讨了 （R）- 和 （S）-美沙酮的药效学差异。根据我们的实验结果，我们得出结论，（S）-美沙酮与 （R，S）-美沙酮和 （R）-美沙酮一样，是μ阿片受体 （MOR） 的激动剂，但效力要低得多。重要的是，当 MOR 与甘丙肽 1 受体 （Gal₁R） 复合时，（S）-美沙酮通过充当 MOR 拮抗剂而表现出独特的药效学效应。由于我们之前的研究表明 MOR-Gal₁R 异聚体介导阿片类药物的多巴胺能作用 [3]，因此 （S）-美沙酮的这种特异性拮抗剂特性解释了 （R，S）-美沙酮在激活多巴胺能系统和引发欣快作用方面的低疗效。因此，在足够的剂量/浓度下，（S）-美沙酮抵消 （R）-美沙酮的运动激活和多巴胺释放作用。虽然 （S）-美沙酮可以作为 N-甲基-D-天冬氨酸受体 （NMDAR） 的拮抗剂，但达到这种效果所需的药物浓度高于在体内与 MORs 相互作用所需的药物浓度。因此，我们说“目前假设的 NMDAR 阻断在 （S）-MTD 的所谓抗抑郁作用中的作用应该在其 MOR 激动特性的背景下重新构建。Pappagallo 等人 [1] 不同意这种说法。我们在下面讨论他们的评论。

我们的热板研究结果是 （S）-美沙酮的 MOR 激动剂作用的关键组成部分，但它们并不是我们提供的唯一证据。（S）-美沙酮还会产生体温过低和部分僵硬 [2]，这是 MOR 激动剂在大鼠中的两种典型作用。我们还在大鼠脑切片中使用 [³⁵S] GTPγS 结合来表明 （S）-美沙酮在不与 NMDAR 相互作用的浓度下增加纹状体中的 GPCR 活性，并且这种反应被优先的 MOR 拮抗剂纳曲酮预处理阻断。由于 [³⁵S]GTPγS 测定对 GPCR 具有特异性，不涉及离子通道激活或抑制，因此我们得出结论，这种激活是由阿片受体介导的，很可能是 MOR。我们还在转染细胞中使用 BRET 来证明 （S）-美沙酮在 MOR 中起激动剂的作用。最后，我们发现 30 mg/kg （S）-美沙酮，一种用于在大鼠 （10-40 mg/kg） 中产生抗抑郁药样作用的剂量 [4， 5]，占据了纹状体 MOR 的近 80%，而 NMDAR 没有显着占据，使用先前显示氯胺酮对这些受体的占据率的测定 [6]。因此，我们使用了多种独立方法来确认 （S）-美沙酮以体内不结合 NMDAR 的剂量/浓度结合并激活 MOR。