Impact of Morphine withdrawal on genes related to the TLR2 pathway expressed in the Prefrontal Cortex.

Millions of people suffer from opioid use disorder, because of the ongoing opioid epidemic. The aversive symptoms of withdrawal are a leading factor for drug relapses, yet there are limited therapeutic options to minimize or prevent withdrawal symptoms. The mechanism behind opioid withdrawal is still not fully understood, thus preventing the development of new therapeutics. This study is an extension of our previously proposed mechanism of a toll-like receptor 2 (TLR2) mediated withdrawal response as a result of morphine induced microbial change that occurs during morphine withdrawal. Transcriptome analysis of the pre-frontal cortex indicated that there was increased expression of genes related to TLR2 signaling in morphine withdrawal treated animals compared to placebo controls. Antibiotic treatment further altered TLR2 related genes, recovering some of the morphine induced effect and leading to additional suppression of some genes related to the TLR2 pathway. Morphine withdrawal induced gene expression was attenuated in a whole body TLR2 knockout model. These results provide more support that TLR2 plays an integral role in morphine withdrawal mechanisms and could be a potential therapeutic target to minimize opioid withdrawal associated co-morbidities.

YTHDF1 in periaqueductal gray inhibitory neurons contributes to morphine withdrawal responses in mice.

BACKGROUND: Physical symptoms and aversion induced by opioid withdrawal strongly affect the management of opioid addiction. YTH N6-methyladenosine (m6A) RNA binding protein 1 (YTHDF1), an m6A-binding protein, from the periaqueductal gray (PAG) reportedly contributes to morphine tolerance and hyperalgesia. However, the role of YTHDF1 in morphine withdrawal remains unclear. METHODS: A naloxone-precipitated morphine withdrawal model was established in C57/BL6 mice or transgenic mice. YTHDF1 was knocked down via adeno-associated virus transfection. Combined with the results of the single-cell RNA sequencing analysis, the changes in morphine withdrawal somatic signs and conditioned place aversion (CPA) scores were compared when YTHDF1 originating from different neurons in the ventrolateral periaqueductal gray (vlPAG) was knocked down. We further explored the role of inflammatory factors and transcription factors related to inflammatory response in morphine withdrawal. RESULTS: Our results revealed that YTHDF1 expression was upregulated in the vlPAG of mice with morphine withdrawal and that the knockdown of vlPAG YTHDF1 attenuated morphine withdrawal-related somatic signs and aversion. The levels of NF-κB and p-NF-κB were reduced after the inhibition of YTHDF1 in the vlPAG. YTHDF1 from vlPAG inhibitory neurons, rather than excitatory neurons, facilitated morphine withdrawal responses. The inhibition of YTHDF1 in vlPAG somatostatin (Sst)-expressing neurons relieved somatic signs of morphine withdrawal and aversion, whereas the knockdown of YTHDF1 in cholecystokinin (Cck)-expressing or parvalbumin (PV)-expressing neurons did not change morphine withdrawal-induced responses. The activity of c-fos + neurons, the intensity of the calcium signal, the density of dendritic spines, and the frequency of mIPSCs in the vlPAG, which were increased in mice with morphine withdrawal, were decreased with the inhibition of YTHDF1 from vlPAG inhibitory neurons or Sst-expressing neurons. Knockdown of NF-κB in Sst-expressing neurons also alleviated morphine withdrawal-induced responses. CONCLUSIONS: YTHDF1 originating from Sst-expressing neurons in the vlPAG is crucial for the modulation of morphine withdrawal responses, and the underlying mechanism might be related to the regulation of the expression and phosphorylation of NF-κB.

Non-invasive temporal interference brain stimulation reduces preference on morphine-induced conditioned place preference in rats.

Long-term use of opioid drugs such as morphine can induce addiction in the central nervous system through dysregulation of the reward system of the brain. Deep brain stimulation (DBS) is a non-pharmacological technique capable of attenuating behavioral responses associated with opioid drug consumption and possesses the capability to selectively activate and target localized brain regions with a high spatial resolution. However, long-term implantation of electrodes in brain tissue may limit the effectiveness of DBS due to changes in impedance, position, and shape of the tip of the stimulation electrode and the risk of infection of nerve tissue around the implanted electrode. The main objective of the current study is to evaluate the effect of temporal interference (TI) brain stimulation on addictive behaviors of morphine-induced conditioned place preference (CPP) in rats. TI stimulation is a non-invasive technique used transcranially to modulate neural activity within targeted brain regions. It involves applying two high-frequency currents with slightly different frequencies, resulting in interference and targeted stimulation of different brain areas with the desired spatial resolution. The results indicated that TI stimulation with the amplitude of I 1 = I 2 = 0.5 mA, carrier frequency of 2 kHz, frequency difference of 25 Hz, ON-OFF stimulation frequency of 0.25 Hz, and total duration of 10 min in three consecutive days resulted in a significant reduction of morphine preference in the morphine-stimulation group in comparison with the morphine group (p < 0.001). These findings highlight the potential of TI stimulation as a modulatory intervention in mitigating the addictive properties of morphine and provide valuable insights into the therapeutic implications of this stimulation paradigm for treatment of opioid drugs in human subjects.

Effects of anterior quadratus lumborum block versus erector spinae plane block on postoperative acute pain in percutaneous nephrolithotomy: a prospective, observational study.

BACKGROUND: The study aimed to compare the pain-relieving effectiveness of anterior quadratus lumborum block (QLB3) and erector spinae plane block (ESPB), both of which have been documented to provide relief during abdominal surgery. METHODS: This prospective observational study, conducted between February and July 2023, included 96 patients who had undergone percutaneous nephrolithotomy (PCNL). Patients were divided into three groups: QLB3, ESPB, and control (no block) and received the corresponding nerve block in the preanesthetic room for regional block. Cumulative morphine consumption during the initial 24 h after PCNL, numerical rating scale resting/movement scores, intraoperative remifentanil usage, rescue analgesic requirements, time when the first analgesic was requested, and postoperative nausea and vomiting scores were documented and compared between the groups. RESULTS: Total median morphine consumption in the first 24 h postoperatively was similar in the QLB3 and ESPB groups but higher in the control group (QLB3, 7 mg [(Q1-Q3) 7-8.5]; ESPB, 8 mg [6.5-9]; control, 12.5 [10-17]; P < 0.001). Similarly, median intraoperative remifentanil consumption did not differ between the block groups but was higher in the control group (QLB3, 1082 µg [IQR 805.5-1292.7]; ESPB, 1278 µg [940.2-1297.5]; control, 1561 µg [1315-2068]; P < 0.001). The number of patients receiving rescue analgesic medication was similar in the block groups but higher in the control group (QLB3, n = 9 [30%]; ESPB, n = 14 [46.7%]; control, n = 21 [70%]; P = 0.008). CONCLUSIONS: QLB3 and ESPB were adequate and comparable in providing postoperative analgesia as part of multimodal analgesia after PCNL. TRIAL REGISTRATION: The study was registered on ClinicalTrials.gov (Identifier: NCT05822492).

Intrathecal Morphine Enhances Postoperative Analgesia and Recovery in Robotic-Assisted Laparoscopic Partial Nephrectomy: A Retrospective Study of 272 Patients.

BACKGROUND Robot-assisted laparoscopic partial nephrectomy (RAPN) has been increasingly used for treating renal tumors due to its advantages over other approaches. However, RAPN can induce acute incisional, peritoneal, visceral, and referred pain. Therefore, acute pain control in robotic surgery is a concern. This retrospective study aimed to evaluate the efficacy of intrathecal morphine (ITM) for postoperative analgesia and recovery after RAPN. MATERIAL AND METHODS We retrospectively investigated consecutive patients who underwent RAPN at our institute between 2020 and 2021. Among the 272 patients who met the inclusion criteria, 135 patients were administered 200 µg of ITM preoperatively (ITM group), while 137 patients were not (control group). Postoperative pain assessments using the numeric rating scale (NRS), opioid requirements, and recovery profiles during the first postoperative 24 h were compared between the 2 groups. RESULTS As the primary endpoint, the incidence of moderate-to-severe pain (24-h average NRS pain score ≥4) was significantly lower in the ITM group than in the control group (36.3% vs 61.3%, P<0.001). Pain scores and cumulative opioid requirements were also significantly lower in the ITM group for all assessments (P<0.001). Moreover, the ITM group had a higher score on the Quality of Recovery-15 questionnaire on the first postoperative day (129 vs 120, P=0.003) despite an increased rate of postoperative nausea/vomiting (27.4% vs 13.1%, P=0.003). CONCLUSIONS Our findings indicate that ITM provided superior pain control during the early period following RAPN, with reduced postoperative opioid requirements. Moreover, ITM improved patient satisfaction with recovery.

Morphine induces inflammatory responses via both TLR4 and cGAS-STING signaling pathways.

BACKGROUND: Opioid activation of the microglia or macrophage Toll-like receptor 4 (TLR4) and associated inflammatory cytokine release are implicated in opioid-induced hyperalgesia and tolerance. The cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS-STING) signaling pathway, activated by double-stranded DNA including mitochondrial DNA (mtDNA), has emerged as another key mediator of inflammatory responses. This study tested the hypothesis that morphine induces immune inflammatory responses in microglia and macrophages involving TLR4 and cGAS-STING pathway. METHODS: BV2 microglia and Raw 264.7 (Raw) macrophage cells were exposed to morphine with and without a STING inhibitor (C176) for 6 h or TLR 4 inhibitor (TAK242) for 24 h. Western blotting and RT-qPCR analyses assessed TLR4, cGAS, STING, nuclear factor-kappa B (NF-κB), and pro-inflammatory cytokine expression. Morphine-induced mitochondria dysfunction was quantified by reactive oxygen species (ROS) release using MitoSOX, mtDNA release by immunofluorescence, and RT-qPCR. Polarization of BV2 and Raw cells was assessed by inducible nitric oxide (iNOS) and CD86 expression. The role of mtDNA on morphine-related inflammation was investigated by mtDNA depletion of the cells with ethidium bromide (EtBr) or cell transfection of mtDNA extracted from morphine-treated cells. RESULTS: Morphine significantly increased the expression of TLR4, cGAS, STING, p65 NF-κB, and cytokines (IL-6 and TNF-α) in BV2 and Raw cells. Morphine-induced mitochondrial dysfunction by increased ROS and mtDNA release; the increased iNOS and CD86 evidenced inflammatory M1-like phenotype polarization. TLR4 and STING inhibitors reduced morphine-induced cytokine release in both cell types. The transfection of mtDNA activated inflammatory signaling proteins, cytokine release, and polarization. Conversely, mtDNA depletion led to the reversal of these effects. CONCLUSION: Morphine activates the cGAS-STING pathway in macrophage cell types. Inhibition of the STING pathway can be an additional method to overcome immune cell inflammation-related morphine tolerance and opioid-induced hyperalgesia.

Intrathecal administration of MCRT produced potent antinociception in chronic inflammatory pain models via µ-δ heterodimer with limited side effects.

An important goal in the opioid field is to discover effective analgesic drugs with minimal side effects. MCRT demonstrated potent antinociceptive effects with limited side effects, making it a promising candidate. However, its pharmacological properties and how it minimizes side effects remain unknown. Various mouse pain and opioid side effect models were used to evaluate the antinociceptive properties and safety at the spinal level. The targets of MCRT were identified through cAMP measurement, isolated tissue assays, and pharmacological experiments. Immunofluorescence was employed to visualize protein expression. MCRT displayed distinct antinociceptive effects between acute and chronic inflammatory pain models due to its multifunctional properties at the µ opioid receptor (MOR), µ-δ heterodimer (MDOR), and neuropeptide FF receptor 2 (NPFFR2). Activation of NPFFR2 reduced MOR-mediated antinociception, leading to bell-shaped response curves in acute pain models. However, activation of MDOR produced more effective antinociception in chronic inflammatory pain models. MCRT showed limited tolerance and opioid-induced hyperalgesia in both acute and chronic pain models and did not develop cross-tolerance to morphine. Additionally, MCRT did not exhibit addictive properties, gastrointestinal inhibition, and effects on motor coordination. Mechanistically, peripheral chronic inflammation or repeated administration of morphine and MCRT induced an increase in MDOR in the spinal cord. Chronic administration of MCRT had no apparent effect on microglial activation in the spinal cord. These findings suggest that MCRT is a versatile compound that provides potent antinociception with minimal opioid-related side effects. MDOR could be a promising target for managing chronic inflammatory pain and addressing the opioid crisis.

Immunological effects of post-operative epidural analgesia versus oral opioids in VATS.

INTRODUCTION: Anaesthetic choices in cancer surgery, including the use of epidural analgesia, may affect immune function during the perioperative period and might play an important role in subsequent cancer spread and recurrence. METHODS: This was a prospective, randomised, controlled, double-blinded, single-centre study allocating patients scheduled for video-assisted thoracoscopic surgery (VATS) lobectomy to post-operative pain management using either thoracic epidural analgesia or oral morphine. We compared pre-, per-, and post-operative plasma levels of interleukin (IL)-6, IL-10, IL-12, and interferon (IFN)-γ using regression analysis, and conducted a two-year survival follow-up. RESULTS: A total of 66 patients were randomised. Fifty-six received the allocated treatment and were analysed. None of the investigated cytokines exhibited significant between-group differences in plasma concentrations when adjusted for the chosen covariates (p ≥ 0.204). A two-year follow-up showed no difference in survival between the two groups (p = 0.5). CONCLUSION: Our study found no differences in the impact on the innate, non-specific immune system related to epidural analgesia for pain management in VATS. FUNDING: The Danish Cancer Society (R150-A10139). Oberstinde Kirsten Jensa la Cours Mindelegat (JSP-25076). University of Southern Denmark, Region of Southern Denmark and Department of Anaesthesia and Intensive Care, Odense University Hospital. TRIAL REGISTRATION: NCT02359175 (ClinicalTrials.gov).

The effectiveness and outcomes of epidural analgesia in patients undergoing open liver resection: a propensity score matching analysis.

BACKGROUND: Open liver resection necessitates a substantial upper abdominal inverted-L incision, resulting in severe pain and compromising patient recovery. Despite the efficacy of epidural analgesia in providing adequate postoperative analgesia, the potential epidural-related adverse effects should be carefully considered. This study aims to compare the efficacy and safety of continuous epidural analgesia and intravenous analgesia in open liver resection. METHODS: A retrospective study was conducted, collecting data from patients who underwent open liver resection between 2007 and 2017. Propensity score matching was implemented to mitigate confounding variables, with patients being matched in a 1:1 ratio based on propensity scores. The primary outcome was the comparison of postoperative morphine consumption at 24, 48, and 72 hours between the two groups. Secondary outcomes included pain scores, postoperative outcomes, and epidural-related adverse effects. RESULTS: A total of 612 patients were included, and after matching, there were 204 patients in each group. Opioid consumption at 24, 48, and 72 hours postoperatively was statistically lower in the epidural analgesia group compared to the intravenous analgesia group (p < 0.001). However, there was no significant difference in pain scores (p = 0.422). Additionally, perioperative hypotension requiring treatment, as well as nausea and vomiting, were significantly higher in the epidural analgesia group compared to the intravenous analgesia group (p < 0.001). CONCLUSIONS: Epidural analgesia is superior to intravenous morphine in terms of reducing postoperative opioid consumption within the initial 72 h after open liver resection. Nevertheless, perioperative hypotension, which necessitates management, should be approached with consideration and vigilance. TRIAL REGISTRATION: The study was registered in the Clinical Trials Registry at www. CLINICALTRIALS: gov/ , NCT number: NCT06301932.

Morphine promotes non-small cell lung cancer progression by downregulating E-cadherin via the PI3K/AKT/mTOR pathway.

Morphine has been suggested to affect cancer cell dynamics and decrease survival rates in lung cancer patients at specific doses, but the precise mechanisms poorly understood. In this study, we aimed to investigate the molecular mechanisms by which morphine modulates the malignant characteristics of non-small cell lung cancer. Cell proliferation was assessed via the Cell Counting Kit-8 assay, and cell migration and invasion were examined via wound healing and Transwell assays. We employed immunofluorescence staining to evaluate E-cadherin expression in A549 and Lewis lung cancer (LLC) cell lines and immunohistochemistry to evaluate E-cadherin expression in nude mice tumours. Additionally, the in vivo effects of morphine on lung cancer progression were explored in a xenograft tumour experiments, in which naloxone was used as a morphine antagonist. Western blot analysis was performed to detect E-cadherin, phosphorylated mTOR (p-mTOR), mTOR, phosphorylated AKT (p-AKT), AKT, phosphorylated PI3K (p-PI3K), and PI3K protein levels in A549 and LLC cells as well as in tumour samples. Morphine (10 µM) significantly increased the proliferation of A549 and LLC cells in vitro (p < 0.05). It also enhanced the migratory and invasive capacities of these cell lines (p < 0.01). Mechanistically, morphine treatment (10 µM) led to a reduction in the expression of E-cadherin, and an increase in the phosphorylation of PI3K, AKT, and mTOR in A549 and LLC cells (p < 0.01). Morphine treatment (1.5 mg/kg) also reduced E-cadherin expression in xenograft tumours and promoted tumour growth in vivo (p < 0.05). This effect was reversed by naloxone (0.1 mg/kg). The results demonstrated that morphine stimulates the malignant proliferation of A549 and LLC cell lines and promotes xenograft tumour growth. Perhaps by specifically targeting MOR, morphine triggers a signalling cascade that activates the PI3K/AKT/mTOR pathway while inhibiting the EMT marker E-cadherin, which may consequently promote the progression of lung cancer.

Inhibition of Dorsal Root Ganglia Transient Receptor Potential Ankyrin 1 Upregulation Contributes to the Protective Effect of Morphine Against Gastric Mucosal Damage Induced by Water-Immersion Restraint Stress.

The pathogenesis mechanism of acute gastric mucosal lesions (AGML) is still unclear; further exploration is urgently needed to find a new therapeutic target. This study aimed to investigate whether morphine might regulate the expression and function of transient receptor potential ankyrin 1 (TRPA1) through a cyclic adenosine monophosphate/protein kinase A (cAMP/PKA)-dependent pathway, thereby alleviating gastric mucosal lesions caused by water-immersion restraint stress (WIRS). Rats were administered with intrathecal morphine, TRPA1 antagonist (HC-030031), µ-opioid receptor antagonist, or protein kinase A inhibitor (H-89), respectively, before WIRS. After 6 hours of WIRS, microscopic lesions, hematoxylin and eosin staining, and transmission electron microscopy were applied to assess the damage of the gastric mucosa. Real-time polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay were conducted to detect the levels of TRPA1 and substance P (SP) in the dorsal root ganglia (DRG) and gastric tissues. In addition, immunofluorescence was used to explore the possible co-expression of TRPA1 and µ-opioid receptors in the DRG. The results indicated that WIRS upregulated TRPA1 and SP in gastric mucosa, and HC-030031 or H-89 could alleviate gastric mucosal lesions caused by WIRS (P < .0001). Morphine was found to suppress both WIRS-induced gastric mucosal lesions (P < .0001) and the upregulation of TRPA1 (P = .0086) and SP (P = .0013). Both TRPA1 and SP play important roles in the pathogenesis of WIRS-induced AGML. Exogenous gastroprotective strategies reduce elevated levels of TRPA1 via the cAMP/PKA-dependent pathway. Inhibition of TRPA1 upregulation in the DRG is critical for intrathecal morphine preconditioning-induced gastric protection.

Morphine-responsive neurons that regulate mechanical antinociception.

Opioids are widely used, effective analgesics to manage severe acute and chronic pain, although they have recently come under scrutiny because of epidemic levels of abuse. While these compounds act on numerous central and peripheral pain pathways, the neuroanatomical substrate for opioid analgesia is not fully understood. By means of single-cell transcriptomics and manipulation of morphine-responsive neurons, we have identified an ensemble of neurons in the rostral ventromedial medulla (RVM) that regulates mechanical nociception in mice. Among these, forced activation or silencing of excitatory RVMBDNF projection neurons mimicked or completely reversed morphine-induced mechanical antinociception, respectively, via a brain-derived neurotrophic factor (BDNF)/tropomyosin receptor kinase B (TrkB)-dependent mechanism and activation of inhibitory spinal galanin-positive neurons. Our results reveal a specific RVM-spinal circuit that scales mechanical nociception whose function confers the antinociceptive properties of morphine.

Non-pulmonary complications of intrathecal morphine administration: a systematic review and meta-analysis with meta-regression.

BACKGROUND: Intrathecal morphine provides effective analgesia for a range of operations. However, widespread implementation into clinical practice is hampered by concerns for potential side-effects. We undertook a systematic review, meta-analysis, and meta-regression with the primary objective of determining whether a threshold dose for non-pulmonary complications could be defined and whether an association could be established between dose and complication rates when intrathecal morphine is administered for perioperative or obstetric analgesia. METHODS: We systematically searched the literature for randomised controlled trials comparing intrathecal morphine vs control in patients undergoing any type of surgery under general or spinal anaesthesia, or women in labour. Primary outcomes were rates of postoperative nausea and vomiting, pruritus, and urinary retention within the first 24 postoperative hours, analysed according to doses (1-100 µg; 101-200 µg; 201-500 µg; >500 µg), type of surgery, and anaesthetic strategy. Trials were excluded if doses were not specified. RESULTS: Our analysis included 168 trials with 9917 patients. The rates of postoperative nausea and vomiting, pruritus, and urinary retention were significantly increased in the intrathecal morphine group, with an odds ratio (95% confidence interval) of 1.52 (1.29-1.79), P<0.0001; 6.11 (5.25-7.10), P<0.0001; and 1.73 (1.17-2.56), P=0.005, respectively. Meta-regression could not establish an association between dose and rates of non-pulmonary complications. There was no subgroup difference according to surgery for any outcome. The quality of evidence was low (Grading of Recommendations Assessment, Development, and Evaluation [GRADE] system). CONCLUSIONS: Intrathecal morphine significantly increased postoperative nausea and vomiting, pruritus, and urinary retention after surgery or labour in a dose-independent manner. SYSTEMATIC REVIEW PROTOCOL: PROSPERO (CRD42023387838).

The autoimmune nature of morphine addiction.

The onset of the disease as a morphine addiction is associated with the appearance in the patient's body of antibodies directed against opiate receptors (ORs). Once anti-opiate receptor antibodies (anti-OR antibodies) appear in the blood they will tend to bind to ORs. Such binding will cause blocking of physiological functions of OR. The blockage is felt by a morphine addict as withdrawal syndrome. To get rid of this harmful condition, the addict increases the dose of morphine taken. This is where tolerance manifests itself. The drug addict is forced to increase the dose of morphine from time to time because of the body responds by producing the more and more anti-OR antibodies. The immunological nature of morphine addiction is the reason for lifelong changes in the body's reactivity to the drug. An addict can be cured if he gets rid of B- and T-memory cells, which specifically react to ORs.

A novel intervention of molecular hydrogen on the unbalance of the gut microbiome in opioid addiction: Experimental and human studies.

The gut-brain axis mediates the interaction pathway between microbiota and opioid addiction. In recent years, many studies have shown that molecular hydrogen has therapeutic and preventive effects on various diseases. This study aimed to investigate whether molecular hydrogen could serve as pharmacological intervention agent to reduce risks of reinstatement of opioid seeking and explore the mechanism of gut microbiota base on animal experiments and human studies. Morphine-induced conditioned place preference (CPP) was constructed to establish acquisition, extinction, and reinstatement stage, and the potential impact of H2 on the behaviors related to morphine-induced drug extinction was determined using both free accessible and confined CPP extinction paradigms. The effects of morphine on microbial diversity and composition of microbiota, as well as the subsequent changes after H2 intervention, were assessed using 16 S rRNA gene sequencing. Short-Chain Fatty Acids (SCFAs) in mice serum were detected by gas chromatography-mass spectrometry (GC-MS). Meanwhile, we also conducted molecular hydrogen intervention and gut microbiota testing in opioid-addicted individuals. Our results revealed that molecular hydrogen could enhance the extinction of morphine-related behavior, reducing morphine reinstatement. Gut microbes may be a potential mechanism behind the therapeutic effects of molecular hydrogen on morphine addiction. Additionally, molecular hydrogen improved symptoms of depression and anxiety, as well as gut microbial features, in individuals with opioid addiction. This study supports molecular hydrogen as a novel and effective intervention for morphine-induced addiction and reveals the mechanism of gut microbiota.

Functional differences in the mu opioid receptor SNP 118A>G are dependent on receptor splice-variant and agonist-specific recruitment of ß-arrestin.

The OPRM1 gene codes for the mu opioid receptor (MOR) and polymorphisms are associated with complex patient clinical responses. The most studied single nucleotide polymorphism (SNP) in OPRM1 is adenine (A) substituted by guanine (G) at position 118 (118A>G, rs1799971) leading to a substitution of asparagine (Asn) for aspartic acid (Asp) at position 40 in the N terminus of the resulting protein. To date, no structural explanation for the associated clinical responses resulting from the 118A>G polymorphism has been proposed. We utilized computational modeling paired with functional cellular assays to predict unstructured N- and C-terminal regions of MOR-1. Using molecular docking and post-docking energy minimizations with morphine, we show that the extracellular substitution of Asn at position 40 alters the cytoplasmic C-terminal conformation, while leaving the G-protein binding interface unaffected. A real-time BRET assay measuring G-protein and ß-arrestin association with MOR r generated data that tested this prediction. Consistent with this in silico prediction, we show changes in morphine-mediated ß-arrestin association with receptor variants with little change in morphine-mediated G-protein association comparing MOR-1 wild type (WT) to MOR-1118A>G. We tested the system with different opioid agonists, the OPRM1 118A>G SNP, and different MOR splice variants (MOR-1 and MOR-1O). These results are consistent with the observation that patients with the 118A>G OPRM1 allele respond more readily to fentanyl than to morphine. In conclusion, the 118A>G substitution alters receptor responses to opioids through variable C-terminal domain movements that are agonist and splice variant dependent.