Effects of facilitated desensitization of µ-opioid receptors on analgesic and respiratory depressive tolerance development in vivo

阿片类药物是治疗严重疼痛最有效的药物，但其长期临床应用受到镇痛耐受性和依赖性发展的限制。奇怪的是，其他阿片类药物的作用，如呼吸抑制或便秘，很少或根本没有耐受性。耐受性发展的第一个也是最关键的生化步骤是激动剂诱导的μ-阿片受体（MOP）C-末端Ser/Thr残基的磷酸化，导致受体脱敏。使用一种新开发的技术来可视化大脑中磷酸化的MOP受体，我们意外地观察到MOP磷酸化在阿片类药物暴露后并不普遍发生。事实上，发现MOP表达和磷酸化之间存在显著的不匹配，在一些特别富含MOP表达的脑区域（如丘脑核和纹状体）中，可检测到的MOP磷酸化显著低。这导致了在这些大脑区域中“缺乏激酶”的假设，特别是G蛋白偶联受体激酶（GRK）。我们推测，呼吸抑制或便秘的耐受性发展有限可能是由于缺乏GRKs的共表达而导致MOP脱敏失败。为了验证这一假设，我们建议生成共表达内源性MOP受体与GRK 2或GRK 5的小鼠模型，已知GRK 2或GRK 5以不同的方式促进由高效或低效阿片类药物诱导的MOP磷酸化。我们假设，在这些小鼠中，所有MOP受体将经历激动剂诱导的磷酸化，从而脱敏，导致耐受性增加，不仅镇痛作用，而且呼吸抑制和便秘后，反复阿片类药物暴露。也有可能促进MOP脱敏降低了这些小鼠中阿片类药物的成瘾性。对阿片类药物镇痛和呼吸抑制作用的耐受性增加在临床上是可取的，因为阿片类药物剂量递增不会受到潜在致命性呼吸骤停的限制。拟议的项目旨在提供重要的数据，以支持这样的临床策略，并可能刺激药物的探索，调节GRK的表达或活性作为一种新的治疗概念。

Opioids are the most effective drugs for treating severe pain, but their long-term clinical use is limited by the development of analgesic tolerance and dependence. Curiously, other opioid effects such as respiratory depression or constipation develop little or no tolerance. The first and most critical biochemical step in tolerance development is agonist-induced phosphorylation of the C-terminal Ser/Thr residues of the µ-opioid receptor (MOP), leading to desensitization of the receptor. Using a newly developed technique to visualize phosphorylated MOP receptors in the brain, we made the unexpected observation that MOP phosphorylation does not occur ubiquitously after opioid exposure. In fact, a significant mismatch between MOP expression and phosphorylation was found, with detectable MOP phosphorylation strikingly low in some brain regions particularly rich in MOP expression, such as the nucleus accumbens and striatum. This led to the hypothesis of "lack of kinases" in these brain regions, particularly G protein-coupled receptor kinases (GRK). We hypothesize that limited tolerance development in respiratory depression or constipation may be due to a failure of MOP desensitization because of the lack of co-expression of GRKs. To test this hypothesis, we propose to generate mouse models that co-express endogenous MOP receptors with either GRK2 or GRK5, which are known to contribute in different ways to MOP phosphorylation induced by high- or low-efficacy opioids. We hypothesize that in these mice, all MOP receptors will undergo agonist-induced phosphorylation and thus desensitization, leading to increased tolerance not only to analgesic effects but also to respiratory depression and constipation after repeated opioid exposure. It is also possible that facilitated MOP desensitization reduces the addictive properties of opioids in these mice. Increased tolerance to analgesic and respiratory depressant effects of opioids would be clinically desirable, as opioid dose escalation would not be limited by potentially fatal respiratory arrest. The proposed project aims to provide important data to support such a clinical strategy and may stimulate the exploration of drugs that modulate GRK expression or activity as a novel therapeutic concept.