The Role of the Endocannabinoid System in Opioid-Induced Respiratory

Abstract

The endocannabinoid system (ECS) has emerged as a critical regulator of respiratory function with potential implications for opioid-induced respiratory depression (OIRD), a leading cause of opioid-related mortality. This dissertation examines the multifaceted role of cannabinoid 2 receptor (CB2R) in respiratory control mechanisms and their interaction with opioid effects through a comprehensive series of in vivo experiments using wild-type (WT) and CB2R knockout (CB2RKO) mice.Respiratory parameters assessed via whole-body plethysmography revealed pronounced sexual dimorphism in naïve mice, with females exhibiting significantly higher respiratory rates (RR), tidal volumes (TV), and minute ventilation (MV) than males across all phases of the oestrous cycle. Remarkably, these sex differences were largely abolished in CB2RKO mice, demonstrating CB2R's fundamental role in establishing sex-specific respiratory patterns. Additionally, CB2R deletion significantly altered hypercapnic ventilatory responses, particularly in females in specific phases of the oestrus cycle, suggesting CB2R involvement in central and peripheral chemoreception mechanisms. Pharmacological studies provided further evidence for CB2R's respiratory role, with the CB2R inverse agonist SR144528 significantly decreasing RR in both male and female WT mice. However, contrary to expectations, the CB2R agonists AM2301 and JWH015 failed to attenuate fentanyl or oxycodone-induced respiratory depression, and in some instances exacerbated respiratory suppression, particularly in females under hypercapnic conditions. This indicates complex, context-dependent effects of CB2R modulation that differ from previously reported protective effects against morphine-induced respiratory depression. Molecular analysis of respiratory control centers revealed region- and sex-specific expression patterns of CB1R and CB2R in the pre-Bötzinger complex (pBc) and Kölliker-Fuse nucleus (KF). Similarly, endocannabinoid levels (2-arachidonoylglycerol and anandamide) showed regional and sex-specific variations, with WT females exhibiting higher anandamide levels in the KF compared to males. The μ-opioid receptor (MOR) mRNA was significantly upregulated in both pBc and KF of CB2RKO mice, though protein levels remained unchanged, suggesting post transcriptional modifications. Blood gas analysis demonstrated that CB2R influences oxygen transport and hemoglobin dynamics with sex-specific effects. Female WT mice exhibited higher oxygen saturation (sO2) and fractional oxyhemoglobin of (FO2Hb), differences that were absent in CB2RKO mice. Moreover, acute administration of fentanyl (0.5 mg/kg) and oxycodone (10 mg/kg) significantly increased arterial partial pressure of carbon dioxide (pCO2) levels, particularly in females. Further investigations examined the effects of modulating endocannabinoid levels through enzyme inhibition. The monoacylglycerol lipase inhibitor MJN110 at 10 mg/kg exacerbated morphine-induced respiratory depression, particularly in males under hypercapnic conditions and in females during normoxia. Similarly, the fatty acid amide hydrolase inhibitor JNJ42165279 failed to improve respiratory parameters when co-administered with morphine. These findings suggest that increasing endocannabinoid tone may potentiate rather than alleviate OIRD. This dissertation demonstrates that the ECS, particularly CB2Rs, represents a role in sex differences in respiratory physiology and response to opioids. The complex interplay between CB2R signaling, sex hormones, and respiratory control mechanisms offers new insights into the pathophysiology of OIRD and suggests that ECS-targeted interventions for respiratory disorders must carefully consider sex as a biological variable. These findings advance our understanding of ECS in respiratory control mechanisms and sex-specific involvement of ECS in physiology of the respiratory system.