Region Specificity in Endogenous Opioid Peptides and Mu-opioid Receptor Gene Expression in Rat Brain Areas Involved in Addiction After Frequent Morphine Treatment

Backgrounds: Molecular mechanisms involved in adverse effects of morphine, including tolerance and dependence, have remained elusive. We examined possible alterations in the gene expression of proenkephalin (Penk), prodynorphin (Pdyn), and mu-opioid receptor (Oprm1) in reward brain areas following frequent morphine treatment. Methods: Two groups of male Wistar rats were used. The groups received either saline (1 mL/kg) or morphine (10 mg/kg) twice daily for eight days. On day 8, rats were decapitated, brain areas involved in addiction were dissected, including the midbrain, striatum, prefrontal cortex (PFC), hippocampus, and hypothalamus, and gene expression was evaluated with real-time PCR. Results: Prolonged morphine treatment decreased Penk, Pdyn, and Oprm1 gene expressions in the midbrain but upregulated them in the striatum compared to the control group treated with saline. Significant increases in Pdyn and Oprm1 gene expressions were detected in the PFC, but there was no significant difference in Penk gene expression between the two groups. Besides, Pdyn gene expression was decreased in the hippocampus and hypothalamus; however, no significant differences in Penk and Oprm1 gene expressions were detected between the groups in these areas. Conclusions: The expression of endogenous opioid peptides and receptors after frequent use of morphine follows a region specificity in brain areas involved in addiction. These alterations may result in new physiological setpoints outside the normal range, which need to be considered when using morphine in medicine.

In Vitro Analyses of Spinach-Derived Opioid Peptides, Rubiscolins: Receptor Selectivity and Intracellular Activities through G Protein- and β-Arrestin-Mediated Pathways

Activated opioid receptors transmit internal signals through two major pathways: the G-protein-mediated pathway, which exerts analgesia, and the β-arrestin-mediated pathway, which leads to unfavorable side effects. Hence, G-protein-biased opioid agonists are preferable as opioid analgesics. Rubiscolins, the spinach-derived naturally occurring opioid peptides, are selective δ opioid receptor agonists, and their p.o. administration exhibits antinociceptive effects. Although the potency and effect of rubiscolins as G-protein-biased molecules are partially confirmed, their in vitro profiles remain unclear. We, therefore, evaluated the properties of rubiscolins, in detail, through several analyses, including the CellKeyTM assay, cADDis® cAMP assay, and PathHunter® β-arrestin recruitment assay, using cells stably expressing µ, δ, κ, or µ/δ heteromer opioid receptors. In the CellKeyTM assay, rubiscolins showed selective agonistic effects for δ opioid receptor and little agonistic or antagonistic effects for µ and κ opioid receptors. Furthermore, rubiscolins were found to be G-protein-biased δ opioid receptor agonists based on the results obtained in cADDis® cAMP and PathHunter® β-arrestin recruitment assays. Finally, we found, for the first time, that they are also partially agonistic for the µ/δ dimers. In conclusion, rubiscolins could serve as attractive seeds, as δ opioid receptor-specific agonists, for the development of novel opioid analgesics with reduced side effects.

Special Issue of International Journal of Molecular Sciences “Opioid Receptors and Endorphinergic Systems 2.0”

Opioid peptides exhibit a wide-ranging tissue distribution and control multiple tissue functions not only through reflex mechanisms involving the central nervous system or the modulation of neurotransmitter release, but also by acting directly at the cellular level by targeting selected receptor subtypes (μ, δ, and κ are among the most frequently expressed) [...]

Casomorphins and Gliadorphins Have Diverse Systemic Effects Spanning Gut, Brain and Internal Organs

Food-derived opioid peptides include digestive products derived from cereal and dairy diets. If these opioid peptides breach the intestinal barrier, typically linked to permeability and constrained biosynthesis of dipeptidyl peptidase-4 (DPP4), they can attach to opioid receptors. The widespread presence of opioid receptors spanning gut, brain, and internal organs is fundamental to the diverse and systemic effects of food-derived opioids, with effects being evidential across many health conditions. However, manifestation delays following low-intensity long-term exposure create major challenges for clinical trials. Accordingly, it has been easiest to demonstrate causal relationships in digestion-based research where some impacts occur rapidly. Within this environment, the role of the microbiome is evidential but challenging to further elucidate, with microbiome effects ranging across gut-condition indicators and modulators, and potentially as systemic causal factors. Elucidation requires a systemic framework that acknowledges that public-health effects of food-derived opioids are complex with varying genetic susceptibility and confounding factors, together with system-wide interactions and feedbacks. The specific role of the microbiome within this puzzle remains a medical frontier. The easiest albeit challenging nutritional strategy to modify risk is reduced intake of foods containing embedded opioids. In future, constituent modification within specific foods to reduce embedded opioids may become feasible.

The immune-opioid axis in prediabetes: predicting prediabetes with insulin resistance by plasma interleukin-10 and endomorphin-2 to kappa-opioid receptors ratio

Background Prediabetes is characterized by a hemoglobin A1c of 5.7–6.4% and fasting blood glucose of 100–125 mg/dl. A high percentage of prediabetes subjects develop type 2 diabetes mellitus in the next years. The effects of opioid peptides and their receptors, in addition to immunological cytokines, on prediabetes are not well understood. Therefore, molecular, physiological, and clinical studies are required to link the opioid system, immune system, and insulin resistance (IR) in prediabetes. We hypothesize that opioid peptides (endomorphin-2 (EM2), and β-endorphin (βEP)), and their receptors (µ-opioid receptors (MOR) and κ-opioid receptors (KOR)), in addition to the inflammatory cytokines (IL-6) and anti-inflammatory cytokine (IL-10), affect IR parameters in patients with prediabetes. Methods Sixty prediabetes patients with IR (prediabetes+IR) and sixty prediabetes patients without IR (prediabetes-IR), in addition to 58 controls, have participated in the study. IL-6, IL-10, EM2, βEP, MOR, and KOR were measured by the ELISA technique. Results In general, most prediabetes subjects have dyslipidemia. The IL-6, IL-10, β-endorphin, MOR, and endomorphin-2 were higher in the prediabetes subgroups than the control group. The immune system was activated in the prediabetes in an IR-dependent manner. Prediabetes+IR can be predicted by the increased levels of IL-10, βEP, and EM2 and by the combination of IL-10 and EM2/KOR with good sensitivity and specificity. Conclusion Opioid peptides and their receptors were upregulated in patients with prediabetes, depending on the significance of IR and the immune cytokines. The intercorrelation between the immune system, EOS, and insulin in prediabetes was confirmed.

An Hypothesis for CXCL1/CXCR2 Signaling Regulating Neutrophil-Derived Opioid Peptides Involved in Acupuncture for Inflammatory Pain

Increasing evidences demonstrate that acupuncture is effective in treating inflammatory pain. Recent studies have found that peripheral endogenous opioid peptides in the area of inflammation are involved in acupuncture-treating inflammatory pain. However, the source of endogenous opioid peptides in local area of inflammation and the mechanism of acupuncture regulating these opioid peptides remain unclear. Studies have demonstrated that neutrophils infiltrated in the inflamed tissue contain and release opioid peptides. Chemokine (C-X-C motif) ligand 1 (CXCL1) is one of the key neutrophil chemokines and can promote the blood neutrophil recruitment to the area of inflammation. In our previous experiments, we found that acupuncture could alleviate inflammatory pain and significantly increase the concentration of chemokine CXCL1 in the blood of rats with inflammatory pain. So we suppose that increased concentration of CXCL1 by acupuncture could activate the blood opioid-containing neutrophils via its main receptor chemokine receptor type 2 (CXCR2) and promote them recruit to the inflamed tissue to release opioid peptides, participating in the analgesic effect of acupuncture.

Positive allosteric modulation of the mu-opioid receptor produces analgesia with reduced side effects

Positive allosteric modulators (PAMs) of the mu-opioid receptor (MOR) have been hypothesized as potentially safer analgesics than traditional opioid drugs. This is based on the idea that PAMs will promote the action of endogenous opioid peptides while preserving their temporal and spatial release patterns and so have an improved therapeutic index. However, this hypothesis has never been tested. Here, we show that a mu-PAM, BMS-986122, enhances the ability of the endogenous opioid Methionine-enkephalin (Met-Enk) to stimulate G protein activity in mouse brain homogenates without activity on its own and to enhance G protein activation to a greater extent than β-arrestin recruitment in Chinese hamster ovary (CHO) cells expressing human mu-opioid receptors. Moreover, BMS-986122 increases the potency of Met-Enk to inhibit GABA release in the periaqueductal gray, an important site for antinociception. We describe in vivo experiments demonstrating that the mu-PAM produces antinociception in mouse models of acute noxious heat pain as well as inflammatory pain. These effects are blocked by MOR antagonists and are consistent with the hypothesis that in vivo mu-PAMs enhance the activity of endogenous opioid peptides. Because BMS-986122 does not bind to the orthosteric site and has no inherent agonist action at endogenously expressed levels of MOR, it produces a reduced level of morphine-like side effects of constipation, reward as measured by conditioned place preference, and respiratory depression. These data provide a rationale for the further exploration of the action and safety of mu-PAMs as an innovative approach to pain management.

Endogenous Opioid Peptides and Alternatively Spliced Mu Opioid Receptor Seven Transmembrane Carboxyl-Terminal Variants

There exist three main types of endogenous opioid peptides, enkephalins, dynorphins and β-endorphin, all of which are derived from their precursors. These endogenous opioid peptides act through opioid receptors, including mu opioid receptor (MOR), delta opioid receptor (DOR) and kappa opioid receptor (KOR), and play important roles not only in analgesia, but also many other biological processes such as reward, stress response, feeding and emotion. The MOR gene, OPRM1, undergoes extensive alternative pre-mRNA splicing, generating multiple splice variants or isoforms. One type of these splice variants, the full-length 7 transmembrane (TM) Carboxyl (C)-terminal variants, has the same receptor structures but contains different intracellular C-terminal tails. The pharmacological functions of several endogenous opioid peptides through the mouse, rat and human OPRM1 7TM C-terminal variants have been considerably investigated together with various mu opioid ligands. The current review focuses on the studies of these endogenous opioid peptides and summarizes the results from early pharmacological studies, including receptor binding affinity and G protein activation, and recent studies of β-arrestin2 recruitment and biased signaling, aiming to provide new insights into the mechanisms and functions of endogenous opioid peptides, which are mediated through the OPRM1 7TM C-terminal splice variants.