The Foundational Science of Endogenous Opioids and Their Receptors.

The function of endogenous opioids spans from initiating behaviors that are critical for survival, to responding to rapidly changing environmental conditions. A network of interconnected systems throughout the body characterizes the endogenous opioid system (EOS). EOS receptors for beta-endorphin, enkephalin, dynorphin, and endomorphin underpin the diverse functions of the EOS across biological systems. This chapter presents a succinct yet comprehensive summary of the structure of the EOS, EOS receptors, and their relationship to other biological systems.

Interactions Between Endogenous Opioids and the Immune System.

The endogenous opioid system, which consists of opioid receptors and their ligands, is widely expressed in the nervous system and also found in the immune system. As a part of the body's defense machinery, the immune system is heavily regulated by endogenous opioid peptides. Many types of immune cells, including macrophages, dendritic cells, neutrophils, and lymphocytes are influenced by endogenous opioids, which affect cell activation, differentiation, proliferation, apoptosis, phagocytosis, and cytokine production. Additionally, immune cells also synthesize and secrete endogenous opioid peptides and participate peripheral analgesia. This chapter is structured into two sections. Part one focuses on immunoregulatory functions of central endogenous opioids; and part two describes how opioid peptide-containing immune cells participate in local analgesia.

Role of Endorphins in Breast Cancer Pathogenesis and Recovery.

Understanding the relationship between stress and breast cancer development is essential to preventing and alleviating the cancer. Recent research has shed light on the cognitive, physiological, cellular, and molecular underpinnings of how the endorphin pathway and stress pathway affect breast cancer. This chapter consists of two parts. Part 1 will discuss the role of endorphins in breast cancer development. This includes a discussion of three topics: (1) the neurophysiological effect of endorphins on breast tumor growth in vivo, along with further experiments that will deepen our knowledge of how ß-endorphin affects breast cancer; (2) how both the opioid receptor and somatostatin receptor classes alter intracellular signaling in breast cancer cells; and (3) genetic alleles in the opioid signaling pathway that are correlated with increased breast cancer risk. Part 2 will discuss the role of endorphins in recovery from breast cancer. This includes a discussion of three topics: (1) the relationship between breast cancer diagnosis and depression; (2) the effectiveness of cognitive behavioral therapy in reducing stress in breast cancer patients; and (3) the effect of psychotherapy and exercise on preserving telomere length in breast cancer patients.

The Opioid Growth Factor in Growth Regulation and Immune Responses in Cancer.

It has become apparent that endogenous opioids act not only as neurotransmitters and neuromodulators, but have multiple functions in the body. Activation of the opioid system by opiate drugs is associated with a risk of cancer development through direct stimulation of tumor cell proliferation and through immunosuppression. In contrast, the endogenous peptide opioid [Met5]-enkephalin, now commonly referred to as Opioid Growth Factor (OGF), negatively regulates cell proliferation in a wide number of cells during development, homeostasis, and neoplasia. This action is mediated through the opioid growth factor receptor, originally designated the zeta (ζ) opioid receptor. Further, contrary to the traditional notion of opiates as immunosuppressive, endogenous OGF has been shown to possess a number of positive immunomodulatory properties and may provide a beneficial effect in cancer by augmenting the activity of cells involved in both innate and acquired immunity. Taken together, the evidence supports consideration of opioid peptides such as OGF as new strategies for cancer therapy.

The Endogenous Opioid System as a Pathway of Positive Emotions.

Pleasant emotions take a variety of forms and are a key part of the human experience. Although negative emotions have often been a focus of research, positive emotions, e.g., joy, pleasure, and love, have recently gained more attention. Each of these emotions is rich and complex in its own right. However, positive emotions appear to serve key evolutionary functions, which are mediated by complex biological substrates. This chapter summarizes key research and explores the biological underpinnings of positive emotions, with an emphasis on the roles that endogenous opioids play in the experience, expression, and development of positive emotions. The necessity of emphasizing positive emotions in research is also discussed.

The Role of Endogenous Opioids in Cardioprotection.

The opioid system involves opioid receptors (OPRs) and endogenous opioid peptides.This chapter will focus on the distribution of OPRs in the cardiovascular system, the expression pattern in the heart, the activation by opioid peptides, and the effects of OPRs activation with potential relevance in cardiovascular performance. In the heart, OPRs are co-expressed with beta adrenergic receptors (ß-ARs) in the G-protein-coupled receptor (GPCR) superfamily, functionally cross-talk with ß-Ars and modify catecholamine-induced effects. They are involved in cardiac contractility, energy metabolism, myocyte survival or death, vascular resistance. The effects of the opioid system in the regulation of systemic circulation at both the central and peripheral level are presented. The pathways are discussed under physiological (i.e., aging) and pathological conditions (atherosclerosis, heart failure, essential hypertension, ischemic stress). Stimulation of OPRs not only inhibits cardiac excitation-contraction coupling, but also protects the heart against hypoxic and ischemic injury. An enhanced sensitivity to opioids of endocrine organs and neuronal systems is operative in hypertensive patients. The opioid system can be pharmacologically engaged to selectively mimic these responses via cardiac and nervous signaling. The clinical opportunities for the use of cardioprotective effects of opioids require future investigations to provide more specific details of the impact on cardiac performance and electrophysiological properties.

Enantioselective de novo synthesis of 14-hydroxy-6-oxomorphinans.

The enantioselective de novo synthesis of pharmacologically important 14-hydroxy-6-oxomorphinans is described. 4,5-Desoxynaltrexone and 4,5-desoxynaloxone were prepared using this route and their biological activities against the opioid receptors were measured.

Morphine timing-dependent modulation of TRPV1 phosphorylation correlates with differential morphine effects on myocardial ischemia/reperfusion injury.

Opioids are used for pain relief in patients suffering from acute myocardial ischemia or infarction. Clinical and laboratory studies demonstrate that morphine treated patients or the experimental animal model suffering acute myocardial ischemia and reperfusion, may worsen myocardial viability. As transient receptor potential vanilloid 1 (TRPV1) plays important roles in pain sensation and cardio-protection, we query whether opioids may exacerbate myocardial viability via interaction with TRPV1 activity in the pain relief. We found the co-expressions of TRPV1 and opioid µ, δ and κ receptors in adult rat cardiomyocytes. Intravenous injection of morphine (0.3 mg/kg) at 20 min after induction of myocardial ischemia, in the rat model of acute myocardial ischemia and reperfusion, induced significant reduction of phosphorylated TRPV1 (p-TRPV1) in the ventricular myocardium and increase in serum cardiac troponin I (cTnI), compared with the ischemia/reperfusion controls (all P < 0.05). The effects of morphine were completely reversed by selective opioid µ, δ and κ receptor antagonists. While significant upregulation of p-TRPV1 (P < 0.05) and improvement of ±dP/dt max (all P < 0.05) were detected in the animals giving the same dose of morphine before induction of myocardial ischemia. The changes in p-TRPV1 correlate with the alterations of cTnI (r = -0.5840, P = 0.0283) and ±dP/dt max (r = 0.8084, P = 0.0005 and r = -0.8133, P = 0.0004, respectively). The findings of this study may indicate that potentiation and attenuation of TRPV1 sensitivity correlate with the improvement of the cardiac performance and the aggravation of myocardial viability, respectively, by giving morphine before and during myocardial ischemia and reperfusion.

Low doses of acetaminophen produce antidepressive-like effects through the opioid system in mice.

Acetaminophen (paracetamol) is one of the most popular analgesics for the management of fever and pain but few reports have investigated its antidepressant-like effect. Moreover, the role of the opioidergic pathway has been indicated in depression pathophysiology. This study aimed to examine the involvement of the opioid receptors in the antidepressant-like effect of acetaminophen after acute and sub-chronic administration using mice forced swimming test (FST). Our finding showed that administration of acetaminophen (50 and 100 mg/kg, i.p.) 30 min before the FST produced an antidepressant effect which was reduced by naloxone (1 mg/kg, i.p., a nonselective opioid receptor antagonist). Moreover, we observed that acetaminophen in higher doses (200 and 400 mg/kg) was ineffective. Also, the response of the non-effective dose of acetaminophen (25 mg/kg) was potentiated by the non-effective dose of morphine (0.1 mg/kg) in the FST that was antagonized by naloxone. Also, in contrast to morphine (10 mg/kg), acetaminophen (100 mg/kg, i.p.) induced neither tolerance to the anti-immobility behavior nor withdrawal syndrome after repeated administration. In addition, RT-PCR showed that hippocampal mu- and kappa-opioid receptor mRNA expression increased in mice after repeated administration of acetaminophen; however, morphine therapy for 6 days did not affect kappa-opioid receptor expression. Our findings demonstrated that acetaminophen in lower doses but not high doses revealed an antidepressant-like activity without inducing tolerance and withdrawal syndromes. Moreover, the observed effect of acetaminophen may be via altering the opioid system, particularly hippocampal mu- and kappa-receptors.

In Vitro and In Vivo Pharmacological Profiles of LENART01, a Dermorphin-Ranatensin Hybrid Peptide.

Diverse chemical and pharmacological strategies are currently being explored to minimize the unwanted side effects of currently used opioid analgesics while achieving effective pain relief. The use of multitarget ligands with activity at more than one receptor represents a promising therapeutic approach. We recently reported a bifunctional peptide-based hybrid LENART01 combining dermorphin and ranatensin pharmacophores, which displays activity to the mu-opioid receptor (MOR) and dopamine D2 receptor (D2R) in rat brains and spinal cords. In this study, we investigated the in vitro binding and functional activities to the human MOR and the in vivo pharmacology of LENART01 in mice after subcutaneous administration. In vitro binding assays showed LENART01 to bind and be selective to the human MOR over the other opioid receptor subtypes and delta, kappa and nociceptin receptors. In the [35S]GTPγS binding assay, LENART01 acted as a potent and full agonist to the human MOR. In mice, LENART01 produced dose-dependent antinociceptive effects in formalin-induced inflammatory pain, with increased potency than morphine. Antinociceptive effects were reversed by naloxone, indicating MOR activation in vivo. Behavioral studies also demonstrated LENART01's properties to induce less adverse effects without locomotor dysfunction and withdrawal syndrome compared to conventional opioid analgesics, such as morphine. LENART01 is the first peptide-based MOR-D2R ligand known to date and the first dual MOR-dopamine D2R ligand for which in vivo pharmacology is reported with antinociceptive efficacy and reduced opioid-related side effects. Our current findings may pave the way to new pain therapeutics with limited side effects in acute and chronic use.

Antinociceptive effects of Raphanus sativus sprouts involve the opioid and 5-HT1A serotonin receptors, cAMP/cGMP pathways, and the central activity of sulforaphane.

Raphanus sativus L. cv. Sango, commonly known as red radish, is widely consumed around the world as a vegetable, but its benefit in pain relief is not sufficiently investigated. This study aimed to evaluate the antinociceptive effects of R. sativus and a possible mechanism of action. An aqueous extract of R. sativus sprouts (AERSS) was investigated by parenteral (10, 30, and 100 mg kg-1, i.p.) and enteral (500 mg kg-1, p.o.) administration in the neurogenic and inflammatory phases of the formalin test, where gastric damage was also evaluated as a possible adverse effect. Ketorolac (5 mg kg-1, i.p.) was used as the reference drug. Endogenous opioid and 5-HT1A serotonin receptors, as well as the cAMP/NO-cGMP pathways, were explored in the study of a possible mechanism of action by using their corresponding antagonists: naloxone, 1 mg kg-1, i.p., WAY100635, 1 mg kg-1, i.p., and enzymatic activators or inhibitors, respectively. Sulforaphane (SFN), a known bioactive metabolite, was analyzed using electroencephalography (EEG) to evidence its central involvement. A significant and dose-dependent antinociceptive activity was observed with the AERSS resembling the antinociceptive effect of the reference drug, with an equivalent significant response with a dose of 500 mg kg-1, p.o. without causing gastric damage. The participation of the endogenous opioid and 5-HT1A serotonin receptors at central and peripheral levels was also observed, with a differential participation of cAMP/NO-cGMP. SFN as one metabolite produced significant changes in the EEG analysis, reinforcing its effects on the CNS. Our preclinical evidence supports the benefits of consuming Raphanus sativus cv. Sango sprouts for pain relief.

Opioid receptor antagonists reduce motivated wheel-running behavior in mice.

We hypothesized that opioid receptor antagonists would inhibit motivated behavior produced by a natural reward. To evaluate motivated responses to a natural reward, mice were given access to running wheels for 71.5 h in a multi-configuration testing apparatus. In addition to a running wheel activity, locomotor activity (outside of the wheel), food and water intake, and access to a food container were measured in the apparatus. Mice were also tested separately for novel-object exploration to investigate whether naloxone affects behavior unrelated to natural reward. In untreated mice wheel running increased from day 1 to day 3. The selective µ-opioid receptor antagonist ß-funaltrexamine (ß-FNA) (5 mg/kg) slightly decreased wheel running, but did not affect the increase in wheel running from day 1 to day 3. The non-selective opioid receptor antagonist naloxone produced a greater reduction in wheel running than ß-FNA and eliminated the increase in wheel running that occurred over time in the other groups. Analysis of food access, locomotor behavior, and behavior in the novel-object test suggested that the reduction in wheel running was selective for this highly reinforcing behavior. These results indicate that opioid receptor antagonism reduces responses to the natural rewarding effects of wheel running and that these effects involve multiple opioid receptors since the non-selective opioid receptor antagonist had greater effects than the selective µ-opioid receptor antagonist. It is possible that at the doses employed, other receptor systems than opioid receptors might be involved, at least in part, in the effect of naloxone and ß-FNA.

Apneic response to fentanyl in adult rats: Role of laryngeal afferents.

Intravenous (systemic) bolus injection of fentanyl (FNT) reportedly induces an immediate vagal-mediated apnea; however, the precise origin of vagal afferents responsible for this apnea remains unknown. We tested whether intralaryngeal (local) application of FNT would also trigger an apnea and whether the apneic response to both local and systemic administration of FNT was laryngeal afferent-mediated. Cardiorespiratory responses to FNT were recorded in anesthetized male adult rats with and without bilateral sectioning of the superior laryngeal nerve (SLNx) or peri-SLN capsaicin treatment (SLNcap) to block local C-fiber signal conduction. Opioid mu-receptor (MOR)-immunoreactivity was detected in laryngeal C- and myelinated neurons. We found that local and systemic administration of FNT elicited an immediate apnea. SLNx, rather than SLNcap, abolished the apneic response to local FNT application though MORs were abundantly expressed in both laryngeal C- and myelinated neurons. Importantly, SLNx failed to affect the apneic response to systemic FNT administration. These results lead to the conclusion that laryngeal afferents' MORs are responsible for the apneic response to local, but not systemic, administration of FNT.

Effects of dopamine and opioid receptor antagonism on the neural processing of social and nonsocial rewards.

Rewards are a broad category of stimuli inducing approach behavior to aid survival. Extensive evidence from animal research has shown that wanting (the motivation to pursue a reward) and liking (the pleasure associated with its consumption) are mostly regulated by dopaminergic and opioidergic activity in dedicated brain areas. However, less is known about the neuroanatomy of dopaminergic and opioidergic regulation of reward processing in humans, especially when considering different types of rewards (i.e., social and nonsocial). To fill this gap of knowledge, we combined dopaminergic and opioidergic antagonism (via amisulpride and naltrexone administration) with functional neuroimaging to investigate the neurochemical and neuroanatomical bases of wanting and liking of matched nonsocial (food) and social (interpersonal touch) rewards, using a randomized, between-subject, placebo-controlled, double-blind design. While no drug effect was observed at the behavioral level, brain activity was modulated by the administered compounds. In particular, opioid antagonism, compared to placebo, reduced activity in the medial orbitofrontal cortex during consumption of the most valued social and nonsocial rewards. Dopamine antagonism, however, had no clear effects on brain activity in response to reward anticipation. These findings provide insights into the neurobiology of human reward processing and suggest a similar opioidergic regulation of the neural responses to social and nonsocial reward consumption.

The Role of δ2-Opioid Receptors in the Regulation of Tolerance of Isolated Cardiomyocytes to Hypoxia and Reoxygenation.

Hypoxia (20 min) and reoxygenation (30 min) were simulated on isolated rat cardiomyocytes to evaluate the cytoprotective effect of selective δ2-opioid receptor agonist deltorphin II, opioid receptor antagonist naloxone methiodide, µ-opioid receptor antagonist CTAP, κ-opioid receptor antagonist nor-binaltorphimine, ε1-opioid receptor antagonist BNTX, and δ2-opioid receptors naltriben. Deltorphin II was administered 5 min before reoxygenation, antagonists were administered 10 min before reoxygenation. The cytoprotective effect of deltorphin II was assessed by the number of cardiomyocytes survived after hypoxia/reoxygenation, as well as by the lactate dehydrogenase content in the incubation medium. It has been established that the cytoprotective effect of deltorphin II occurs at a concentration of 64 nmol/liter and is associated with activation of δ2-opioid receptors.

Genetic mouse models in opioid research: current status and future directions.

Synthetic and semi-synthetic opioids are prescribed for the management of severe pain conditions, but their long-term use is often leading to physical dependence and addiction disorders. Understanding the complex neurobiology of the opioid system in preclinical models will be essential for the development of safe and efficacious analgesics. With rising numbers of synthetic opioid users and overdose cases, a better understanding of the neuroanatomical and cellular pathways associated with physical dependence and addiction is expected to guide treatment approaches for opioid use disorders. In this commentary, we highlight the importance of advanced genetic mouse models for studying the regional effects of opioid receptors, and we discuss the need of genetic mouse models for the investigation of the regional, circuit and cell compartment-specific role of intracellular mediators of opioid actions.

Traumatic Brain Injury Induces Nociceptin/Orphanin FQ and Nociceptin Opioid Peptide Receptor Expression within 24 Hours.

Traumatic brain injury (TBI) is a major cause of mortality and disability around the world, for which no treatment has been found. Nociceptin/Orphanin FQ (N/OFQ) and the nociceptin opioid peptide (NOP) receptor are rapidly increased in response to fluid percussion, stab injury, and controlled cortical impact (CCI) TBI. TBI-induced upregulation of N/OFQ contributes to cerebrovascular impairment, increased excitotoxicity, and neurobehavioral deficits. Our objective was to identify changes in N/OFQ and NOP receptor peptide, protein, and mRNA relative to the expression of injury markers and extracellular regulated kinase (ERK) 24 h following mild (mTBI) and moderate TBI (ModTBI) in wildtype (WT) and NOP receptor-knockout (KO) rats. N/OFQ was quantified by radioimmunoassay, mRNA expression was assessed using real-time PCR and protein levels were determined by immunoblot analysis. This study revealed increased N/OFQ mRNA and peptide levels in the CSF and ipsilateral tissue of WT, but not KO, rats 24 h post-TBI; NOP receptor mRNA increased after ModTBI. Cofilin-1 activation increased in the brain tissue of WT but not KO rats, ERK activation increased in all rats following ModTBI; no changes in injury marker levels were noted in brain tissue at this time. In conclusion, this study elucidates transcriptional and translational changes in the N/OFQ-NOP receptor system relative to TBI-induced neurological deficits and initiation of signaling cascades that support the investigation of the NOP receptor as a therapeutic target for TBI.

An examination of the effects of nucleus accumbens core nociceptin on appetitive and consummatory motivation for food.

The nucleus accumbens (NAc) is a critical region for regulating the appetitive and consummatory aspects of motivated behavior. Previous work has shown differential effects of NAc µ-, δ-, and κ- receptor stimulation on food intake and for shifting motivation within an effort-based choice (EBC) task. However, the motivational role of the nociceptin opioid peptide (NOP) receptor, a fourth member of the opioid receptor family, is less well understood. These experiments therefore characterized the effect of NAc injections of nociceptin, the endogenous ligand for the NOP receptor, on consummatory and appetitive motivation. Three groups of male Sprague-Dawley rats received nociceptin injections into the NAc core prior to testing in a progressive ratio lever pressing task, an EBC task, or a palatable feeding assay. In the feeding experiment, 10 nmol of nociceptin increased consumption in the first 30 min, but this increase was not sustained through the end of the 2-hr session. Additionally, nociceptin injections did not alter breakpoint in the progressive ratio task. However, in the EBC task, nociceptin significantly decreased breakpoint for sugar pellets without affecting consumption of rat chow. These data suggest that NAc NOP receptor stimulation transiently increases consummatory motivation toward palatable diets and inhibits appetitive motivation when alternate food options are freely available. This pattern of effects contrasts with those obtained following NAc stimulation of other opioid receptors, suggesting that the four opioid receptor classes each serve unique roles in modulating food-directed motivation within the NAc core.

The Opioid Receptor Influences Circadian Rhythms in Human Keratinocytes through the ß-Arrestin Pathway.

The recent emphasis on circadian rhythmicity in critical skin cell functions related to homeostasis, regeneration and aging has shed light on the importance of the PER2 circadian clock gene as a vital antitumor gene. Furthermore, delta-opioid receptors (DOPrs) have been identified as playing a crucial role in skin differentiation, proliferation and migration, which are not only essential for wound healing but also contribute to cancer development. In this study, we propose a significant association between cutaneous opioid receptor (OPr) activity and circadian rhythmicity. To investigate this link, we conducted a 48 h circadian rhythm experiment, during which RNA samples were collected every 5 h. We discovered that the activation of DOPr by its endogenous agonist Met-Enkephalin in N/TERT-1 keratinocytes, synchronized by dexamethasone, resulted in a statistically significant 5.6 h delay in the expression of the core clock gene PER2. Confocal microscopy further confirmed the simultaneous nuclear localization of the DOPr-ß-arrestin-1 complex. Additionally, DOPr activation not only enhanced but also induced a phase shift in the rhythmic binding of ß-arrestin-1 to the PER2 promoter. Furthermore, we observed that ß-arrestin-1 regulates the transcription of its target genes, including PER2, by facilitating histone-4 acetylation. Through the ChIP assay, we determined that Met-Enkephalin enhances ß-arrestin-1 binding to acetylated H4 in the PER2 promoter. In summary, our findings suggest that DOPr activation leads to a phase shift in PER2 expression via ß-arrestin-1-facilitated chromatin remodeling. Consequently, these results indicate that DOPr, much like its role in wound healing, may also play a part in cancer development by influencing PER2.

Novel 1-(1-Arylimiazolin-2-Yl)-3-Arylalkilurea Derivatives with Modulatory Activity on Opioid MOP Receptors.

µ-opioid receptor ligands such as morphine and fentanyl are the most known and potent painkillers. However, the severe side effects seen with their use significantly limit their widespread use. The continuous broadening of knowledge about the properties of the interactions of the MOP receptor (human mu opioid receptor, OP3) with ligands and specific intracellular signaling pathways allows for the designation of new directions of research with respect to compounds with analgesic effects in a mechanism different from classical ligands. Allosteric modulation is an extremely promising line of research. Compounds with modulator properties may provide a safer alternative to the currently used opioids. The aim of our research was to obtain a series of urea derivatives of 1-aryl-2-aminoimidazoline and to determine their activity, mechanism of biological action and selectivity toward the MOP receptor. The obtained compounds were subjected to functional tests (cAMP accumulation and ß-arrestin recruitment) in vitro. One of the obtained compounds, when administered alone, did not show any biological activity, while when co-administered with DAMGO, it inhibited ß-arrestin recruitment. These results indicate that this compound is a negative allosteric modulator (NAM) of the human MOP receptor.