Release Of Opioid Peptides Research Articles

Cytotoxic T cells modulate inflammation and endogenous opioid analgesia in chronic arthritis

BackgroundThis study examined the development of chronic pain, a cardinal symptom of rheumatoid arthritis (RA), in mice with antigen- and collagen-induced arthritis (ACIA). Since the role of CD8+ T cells in arthritis is controversial, we investigated the consequences of CD8-depletion on arthritis development and opioid modulation of pain in this novel model of chronic autoimmune arthritis.MethodsDisease severity in control and CD8-depleted animals was determined by histological assessment of knee-joint sections and measurement of autoantibody formation. Pain was evaluated by measuring mechanical allodynia and thermal hyperalgesia in von Frey and Hargreaves tests, respectively. The production and release of endogenous opioids and inflammatory cytokines was assessed in immunoassays.ResultsIn ACIA, mice display persistent mechanical allodynia and thermal hyperalgesia for more than 2 months after induction of arthritis. The blockade of peripheral opioid receptors with naloxone-methiodide (NLXM) transiently increased thermal hyperalgesia, indicating that endogenous opioid peptides were released in the arthritic joint to inhibit pain. CD8+ T cell depletion did not affect autoantibody formation or severity of joint inflammation, but serum levels of the pro-inflammatory cytokines TNFα and IL-17 were increased. The release of opioid peptides from explanted arthritic knee cells and the NLXM effect were significantly reduced in the absence of CD8+ T cells.ConclusionsWe have successfully modeled the development of chronic pain, a hallmark of RA, in ACIA. Furthermore, we detected a yet unknown protective role of CD8+ T cells in chronic ACIA since pro-inflammatory cytokines rose and opioid peptide release decreased in the absence of these cells.

Propionic acid metabolism, ASD, and vitamin B12: Is there a role for environmental nitrous oxide?

Foley et al. (2014) published their findings in this journal on the role of prenatal exposure to propionic acid (PPA) and behavioral outcomes in treated rat pups. The authors show that PPA treated pups displayed subtle differences in behavior including nest seeking, novel object recognition, and locomotor activity. Others have previously proposed that PPA infusion in rat could represent a valid animal model of ASD since many of the diagnostic criteria for the disorder spectrum manifest under such conditions. A pathogenic makeover of gut microbiome to facilitate the growth of microbes capable of producing PPA, like Clostridia species, has been proposed as an infectious contributing etiology to the PPA model of ASD, however the reason for this pathogenic microbial overgrowth is not clear. This discussion highlights a previously identified novel environmental factor (i.e., nitrous oxide, N2O) in the etiopathogenesis of ASD and related neuropathology and posits that altered PPA metabolism in ASD may represent a key manifestation of this particular exposure. Trace environmental exposure to N2O may induce release of endogenous opioid peptides that have been shown to confer a virulence advantage to certain microbes, like Pseudomonas aeruginosa. Pathogenic overproduction of PPA in ASD may be a compensatory mechanism to curb this enhanced virulence potential. Therefore, future research on the PPA model of ASD should consider its role as a consequence of environmental exposure to N2O.

Commentary: 'Unhealthy diet,' nutrient status, and ADHD symptoms: a confounding role for environmental nitrous oxide exposure - reflections on Rijlaarsdam etal. (2016).

Rijlaarsdam etal. (2016) recently published their findings utilizing a longitudinal design showing that prenatal 'unhealthy diet' was positively associated with IGF2 DNA methylation at birth across both youth cohorts. However, only in the EOP youth was prenatal 'unhealthy diet' positively associated with ADHD symptoms presumably through IGF2 DNA hypermethylation. Rijlaarsdam etal.'s () choice to assess high fat and sugar diet with the Food Frequency Questionnaire (FFQ) may offer some indication as to prenatal nutrient status, as the foods identified by the FFQ in their study are relatively low in free choline. It has been shown that gestational choline deficiency in rats leads to hypermethylation of IGF2. Consistent with the literature describing an association between air pollution and cognitive neurodevelopmental impairment, the author of this commentary has previously proposed through empirical investigation that chronic environmental exposure to the trace levels of the pervasive air pollutant, nitrous oxide (N2 O), may facilitate core features of neurodevelopmental disorders, like ADHD. Impaired acetylcholine synthesis in rats exposed to N2 O has been shown, with a 53% reduction in [1-2H2,2-2H2] choline. Low-dose N2 O exposure is also thought to stimulate central release of opioid peptides, like dynorphin, which play a role in significantly increasing food intake behavior and/or modulating sucrose intake. Taken altogether, these studies present a strong confounder to the interpretation made by Rijlaarsdam etal. () that prenatal 'unhealthy diet' may play a role in the onset of ADHD symptoms in youth with EOP conduct problems through induction of IGF2 DNA hypermethylation. While the 'unhealthy diet' may represent possible maternal nutrient deficiencies during gestation, it is also possible that exposure to air pollutants, particularly N2 O, may not only directly reduce fetal cholinergic status thereby enhancing IGF2 DNA hypermethylation but may also significantly modulate maternal food intake behaviors (i.e. sucrose).

Mechanisms of Expression and Release of Endogenous Opioids in Peripheral Tissues

The topic of this review is the current understanding of the mechanisms of neuro-immune interactions responsible for suppression of pain by endogenous opioids. Special attention is paid to the events occurring in peripheral tissues after injury, excitation of high-threshold afferent inputs, and generation of nociceptive impulsation. The release of opioid peptides from immune cells migrating to the focus of inflammation and “inhabiting” the latter provides (at least partially) significant reduction of the excitability of nociceptive sensory neurons. Exogenous opioid ligands that do not cross the bloodbrain barrier also selectively modulate the excitability of primary afferents. Thus, sensory neurons in the peripheral tissues are an essential target for the action of endogenous opioids. There are reasons to believe that the clinical use of peripherally acting opioids can help one to substantially avoid the negative side effects caused by the action of conventional analgesics (opioids and anticonvulsants). Modern concepts on the mechanisms of secretion and release of peripherally acting opioids and their effects on inflammation and pain, the role of the immune response in antinociception, and prospects for the use of the above opioids in the treatment of pain-related phenomena are discussed.

Leukocyte opioid receptors mediate analgesia via Ca2+-regulated release of opioid peptides

Opioids are the most powerful analgesics. As pain is driven by sensory transmission and opioid receptors couple to inhibitory G proteins, according to the classical concept, opioids alleviate pain by activating receptors on neurons and blocking the release of excitatory mediators (e.g., substance P). Here we show that analgesia can be mediated by opioid receptors in immune cells. We propose that activation of leukocyte opioid receptors leads to the secretion of opioid peptides Met-enkephalin, β-endorphin and dynorphin A (1–17), which subsequently act at local neuronal receptors, to relieve pain. In a mouse model of neuropathic pain induced by a chronic constriction injury of the sciatic nerve, exogenous agonists of δ-, μ- and κ-opioid receptors injected at the damaged nerve infiltrated by opioid peptide- and receptor-expressing leukocytes, produced analgesia, as assessed with von Frey filaments. The analgesia was attenuated by pharmacological or genetic inactivation of opioid peptides, and by leukocyte depletion. This decrease in analgesia was restored by the transfer of wild-type, but not opioid receptor-lacking leukocytes. Ex vivo, exogenous opioids triggered secretion of opioid peptides from wild-type immune cells isolated from damaged nerves, which was diminished by blockade of Gαi/o or Gβγ (but not Gαs) proteins, by chelator of intracellular (but not extracellular) Ca2+, by blockers of phospholipase C (PLC) and inositol 1,4,5-trisphosphate (IP3) receptors, and was partially attenuated by protein kinase C inhibitor. Similarly, the leukocyte depletion-induced decrease in exogenous opioid analgesia was re-established by transfer of immune cells ex vivo pretreated with extracellular Ca2+ chelator, but was unaltered by leukocytes pretreated with intracellular Ca2+ chelator or blockers of Gαi/o and Gβγ proteins. Thus, both ex vivo opioid peptide release and in vivo analgesia were mediated by leukocyte opioid receptors coupled to the Gαi/o–Gβγ protein–PLC–IP3 receptors–intracellular Ca2+ pathway. Our findings suggest that opioid receptors in immune cells are important targets for the control of pathological pain.

Imaging endogenous opioid peptide release with [11C]carfentanil and [3H]diprenorphine: influence of agonist-induced internalization.

Understanding the cellular processes underpinning the changes in binding observed during positron emission tomography neurotransmitter release studies may aid translation of these methodologies to other neurotransmitter systems. We compared the sensitivities of opioid receptor radioligands, carfentanil, and diprenorphine, to amphetamine-induced endogenous opioid peptide (EOP) release and methadone administration in the rat. We also investigated whether agonist-induced internalization was involved in reductions in observed binding using subcellular fractionation and confocal microscopy. After radioligand administration, significant reductions in [(11)C]carfentanil, but not [(3)H]diprenorphine, uptake were observed after methadone and amphetamine pretreatment. Subcellular fractionation and in vitro radioligand binding studies showed that amphetamine pretreatment only decreased total [(11)C]carfentanil binding. In vitro saturation binding studies conducted in buffers representative of the internalization pathway suggested that μ-receptors are significantly less able to bind the radioligands in endosomal compared with extracellular compartments. Finally, a significant increase in μ-receptor-early endosome co-localization in the hypothalamus was observed after amphetamine and methadone treatment using double-labeling confocal microscopy, with no changes in δ- or κ-receptor co-localization. These data indicate carfentanil may be superior to diprenorphine when imaging EOP release in vivo, and that alterations in the ability to bind internalized receptors may be a predictor of ligand sensitivity to endogenous neurotransmitter release.

CXCL10 controls inflammatory pain via opioid peptide-containing macrophages in electroacupuncture.

Acupuncture is widely used for pain treatment in patients with osteoarthritis or low back pain, but molecular mechanisms remain largely enigmatic. In the early phase of inflammation neutrophilic chemokines direct opioid-containing neutrophils in the inflamed tissue and stimulate opioid peptide release and antinociception. In this study the molecular pathway and neuroimmune connections in complete Freund's adjuvant (CFA)-induced hind paw inflammation and electroacupuncture for peripheral pain control were analyzed. Free moving Wistar rats with hind paw inflammation were treated twice with electroacupuncture at GB30 (Huan Tiao - gall bladder meridian) (day 0 and 1) and analyzed for mechanical and thermal nociceptive thresholds. The cytokine profiles as well as the expression of opioid peptides were quantified in the inflamed paw. Electroacupuncture elicited long-term antinociception blocked by local injection of anti-opioid peptide antibodies (beta-endorphin, met-enkephalin, dynorphin A). The treatment altered the cytokine profile towards an anti-inflammatory pattern but augmented interferon (IFN)-gamma and the chemokine CXCL10 (IP-10: interferon gamma-inducible protein) protein and mRNA expression with concomitant increased numbers of opioid peptide-containing CXCR3+ macrophages. In rats with CFA hind paw inflammation without acupuncture repeated injection of CXCL10 triggered opioid-mediated antinociception and increase opioid-containing macrophages. Conversely, neutralization of CXCL10 time-dependently decreased electroacupuncture-induced antinociception and the number of infiltrating opioid peptide-expressing CXCR3+ macrophages. In summary, we describe a novel function of the chemokine CXCL10 - as a regulator for an increase of opioid-containing macrophages and antinociceptive mediator in inflammatory pain and as a key chemokine regulated by electroacupuncture.

TGF-β and Opioid Receptor Signaling Crosstalk Results in Improvement of Endogenous and Exogenous Opioid Analgesia under Pathological Pain Conditions

Transforming growth factor-β1 (TGF-β1) protects against neuroinflammatory events underlying neuropathic pain. TGF-β signaling enhancement is a phenotypic characteristic of mice lacking the TGF-β pseudoreceptor BAMBI (BMP and activin membrane-bound inhibitor), which leads to an increased synaptic release of opioid peptides and to a naloxone-reversible hypoalgesic/antiallodynic phenotype. Herein, we investigated the following: (1) the effects of BAMBI deficiency on opioid receptor expression, functional efficacy, and analgesic responses to endogenous and exogenous opioids; and (2) the involvement of the opioid system in the antiallodynic effect of TGF-β1. BAMBI-KO mice were subjected to neuropathic pain by sciatic nerve crash injury (SNI). Gene (PCR) and protein (Western blot) expressions of μ- and δ-opioid receptors were determined in the spinal cord. The inhibitory effects of agonists on the adenylyl cyclase pathway were investigated. Two weeks after SNI, wild-type mice developed mechanical allodynia and the functionality of μ-opioid receptors was reduced. By this time, BAMBI-KO mice were protected against allodynia and exhibited increased expression and function of opioid receptors. Four weeks after SNI, when mice of both genotypes had developed neuropathic pain, the analgesic responses induced by morphine and RB101 (an inhibitor of enkephalin-degrading enzymes, which increases the synaptic levels of enkephalins) were enhanced in BAMBI-KO mice. Similar results were obtained in the formalin-induced chemical-inflammatory pain model. Subcutaneous TGF-β1 infusion prevented pain development after SNI. The antiallodynic effect of TGF-β1 was naloxone-sensitive. In conclusion, modulation of the endogenous opioid system by TGF-β signaling improves the analgesic effectiveness of exogenous and endogenous opioids under pathological pain conditions.

Antinociceptive Effect of Docosahexaenoic Acid (DHA) through Long Fatty Acid Receptor G Protein-coupled Receptor 40 (GPR40)

Fatty acids, one class of essential nutrients for humans, are an important source of energy and an essential component of cell membranes. They also function as signal transduction molecules in a variety of biological phenomena. The important functional role of fatty acids in both onset and suppression of pain has become increasingly apparent in recent years. Recently, we have also demonstrated that the release of an endogenous opioid peptide, β-endorphin, plays an important role in the induction of docosahexaenoic acid (DHA)-induced antinociception. It is well known that fatty acids affect intracellular and intercellular signaling as well as the membrane fluidity of neurons. In addition to intracellular actions, unbound free fatty acids (FFAs) can also carry out extracellular signaling by stimulating the G protein-coupled receptor (GPCR). Among these receptors, G protein-coupled receptor 40 (GPR40) has been reported to be activated by long-chain fatty acids such as DHA, eicosapentaenoic acid (EPA) and arachidonic acid. In the peripheral area, GPR40 is preferentially expressed in pancreatic β-cells and is known to relate to the secretion of hormone and peptides. On the other hand, even though this receptor is widely distributed in the central nervous system, reports studying the role and functions of GPR40 in the brain have not been found. In this review, we summarize the findings of our recent study about the long-chain fatty acid receptor GPR40 as a novel pain regulatory system.

Peripheral interactions between cannabinoid and opioid systems contribute to the antinociceptive effect of crotalphine.

Crotalphine is an antinociceptive peptide that, despite its opioid-like activity, does not induce some of the characteristic side effects of opioids, and its amino acid sequence has no homology to any known opioid peptide. Here, we evaluated the involvement of the peripheral cannabinoid system in the crotalphine effect and its interaction with the opioid system. Hyperalgesia was evaluated using the rat paw pressure test. Involvement of the cannabinoid system was determined using a selective cannabinoid receptor antagonist. Cannabinoid and opioid receptor activation were evaluated in paw slices by immunofluorescence assays using conformation state-sensitive antibodies. The release of endogenous opioid peptides from skin tissue was measured using a commercial enzyme immunoassay (EIA). Both p.o. (0.008-1.0 μg·kg(-1) ) and intraplantar (0.0006 μg per paw) administration of crotalphine induced antinociception in PGE2 -induced hyperalgesia. Antinociception by p.o. crotalphine (1 μg·kg(-1) ) was blocked by AM630 (50 μg per paw), a CB2 receptor antagonist, and by antiserum anti-dynorphin A (1 μg per paw). Immunoassay studies confirmed that crotalphine increased the activation of both κ-opioid (51.7%) and CB2 (28.5%) receptors in paw tissue. The local release of dynorphin A from paw skin was confirmed by in vitro EIA and blocked by AM630. Crotalphine-induced antinociception involves peripheral CB2 cannabinoid receptors and local release of dynorphin A, which is dependent on CB2 receptor activation. These results enhance our understanding of the mechanisms involved in the peripheral effect of crotalphine, as well as the interaction between the opioid and cannabinoid systems.

Toll like Receptor (TLR)-4 as a Regulator of Peripheral Endogenous Opioid-Mediated Analgesia in Inflammation

BackgroundLeukocytes containing opioid peptides locally control inflammatory pain. In the early phase of complete Freund’s adjuvant (CFA)-induced hind paw inflammation, formyl peptides (derived e.g. from Mycobacterium butyricum) trigger the release of opioid peptides from neutrophils contributing to tonic basal antinociception. In the later phase we hypothesized that toll-like-receptor-(TLR)-4 activation of monocytes/macrophages triggers opioid peptide release and thereby stimulates peripheral opioid-dependent antinociception.ResultsIn Wistar rats with CFA hind paw inflammation in the later inflammatory phase (48–96 h) systemic leukocyte depletion by cyclophosphamide (CTX) or locally injected naloxone (NLX) further decreased mechanical and thermal nociceptive thresholds. In vitro β-endorphin (β-END) content increased during human monocyte differentiation as well as in anti-inflammatory CD14+CD16- or non-classical M2 macrophages. Monocytes expressing TLR4 dose-dependently released β-END after stimulation with lipopolysaccharide (LPS) dependent on intracellular calcium. Despite TLR4 expression proinflammatory M1 and anti-inflammatory M2 macrophages only secreted opioid peptides in response to ionomycin, a calcium ionophore. Intraplantar injection of LPS as a TLR4 agonist into the inflamed paw elicited an immediate opioid- and dose-dependent antinociception, which was blocked by TAK-242, a small-molecule inhibitor of TLR4, or by peripheral applied NLX. In the later phase LPS lowered mechanical and thermal nociceptive thresholds. Furthermore, local peripheral TLR4 blockade worsened thermal and mechanical nociceptive pain thresholds in CFA inflammation.ConclusionEndogenous opioids from monocytes/macrophages mediate endogenous antinociception in the late phase of inflammation. Peripheral TLR4 stimulation acts as a transient counter-regulatory mechanism for inflammatory pain in vivo, and increases the release of opioid peptides from monocytes in vitro. TLR4 antagonists as new treatments for sepsis and neuropathic pain might unexpectedly transiently enhance pain by impairing peripheral opioid analgesia.

Neuroimmune interactions after nerve injury and opioid-mediated pain control

Here, we investigated pain relief resulting from neuroimmune interactions associated with peripheral nerve damage. Our hypothesis is based on a new concept that activation of opioid receptors on immune cells leads to the release of opioid peptides, which activate neuronal receptors, to improve neuropathic pain. As a model of neuropathy we applied chronic constriction injury of the sciatic nerve in mice. Mechanical sensitivity was evaluated with von Frey filaments. Immune cells infiltrating damaged nerves were isolated 2 days following the injury, and the secretion of opioid peptide Met-enkephalin was examined using radioimmunoassay. In vivo we found that selective agonists of mu-, delta- and kappa-opioid receptors injected at the nerve injury site produced analgesia. This effect was attenuated when immune cells infiltrating damaged nerves were depleted. In vitro, the opioid receptor agonists dose-dependently secreted Met-enkephalin from leukocytes. This release was abrogated by the respective opioid receptor antagonists, by blocking Gαi and Gβγ subunits, phospholipase C, and inositol 1,4,5-trisphosphate receptors, and by removing Ca 2+ from intracellular, but not extracellular, stores. Our results suggest that activation of leukocytic Gαi-coupled opioid receptors induces Ca 2+ -regulated secretion of endogenous opioid peptides, which might contribute to the amelioration of neuropathic pain. These findings provide mechanistic evidence supporting the idea that damaged peripheral nerves infiltrated by opioid-containing immune cells might be a promising target for the control painful inflammatory neuropathies.

Sweetened-Fat Intake Sensitizes Gamma-Aminobutyric Acid—Mediated Feeding Responses Elicited from the Nucleus Accumbens Shell

There is much interest in exploring whether reward-driven feeding can produce druglike plasticity in the brain. The gamma-aminobutyric acid (GABA) system in the nucleus accumbens (Acb) shell, which modulates hypothalamic feeding systems, is well placed to "usurp" homeostatic control of feeding. Nevertheless, it is unknown whether feeding-induced neuroadaptations occur in this system. Separate groups of ad libitum-maintained rats were exposed to daily bouts of sweetened-fat intake, predator stress, or intra-Acb shell infusions of either d-amphetamine (2 or 10 μg) or the μ-opioid agonist D-[Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO, 2.5 μg), then challenged with intra-Acb shell infusion of the GABAA agonist, muscimol (10 ng). Exposure to sweetened fat robustly sensitized muscimol-induced feeding. Sensitization was present 1 week after cessation of the palatable feeding regimen but had abated by 2 weeks. Rats exposed to sweetened fat did not show an altered feeding response to food deprivation. Repeated intra-Acb shell infusions of DAMGO (2.5 μg) also sensitized intra-Acb shell muscimol-driven feeding. However, neither repeated intra-Acb shell d-amphetamine infusions (2 or 10 μg) nor intermittent exposure to an aversive stimulus (predator stress) altered sensitivity to muscimol. Palatable feeding engenders hypersensitivity of Acb shell GABA responses; this effect may involve feeding-induced release of opioid peptides. Heightened arousal, aversive experiences, or increased catecholamine transmission alone are insufficient to produce the effect, and a hunger-induced feeding drive is insufficient to reveal the effect. These findings reveal a novel type of food-induced neuroadaptation within the Acb; possible implications for understanding crossover effects between food reward and drug reward are discussed.

Sucrose-induced analgesia in mice: Role of nitric oxide and opioid receptor-mediated system

Background:The mechanism of action of sweet substance-induced analgesia is thought to involve activation of the endogenous opioid system. The nitric oxide (NO) pathway has a pivotal role in pain modulation of analgesic compounds such as opioids.Objectives:We investigated the role of NO and the opioid receptor-mediated system in the analgesic effect of sucrose ingestion in mice.Materials and Methods:We evaluated the effect of intraperitoneal administration of 10 mg/kg of NO synthase inhibitor, N-nitro-L-arginine methyl ester (L-NAME) and 20 mg/kg of opioid receptor antagonist, naltrexone on the tail flick response in sucrose ingesting mice.Results:Sucrose ingestion for 12 days induced a statistically significant increase in the latency of tail flick response which was unmodified by L-NAME, but partially inhibited by naltrexone administration.Conclusions:Sucrose-induced nociception may be explained by facilitating the release of endogenous opioid peptides. Contrary to some previously studied pain models, the NO/cyclic guanosine monophosphate (cGMP) pathway had no role in thermal hyperalgesia in our study. We recommend further studies on the involvement of NO in other animals and pain models.

Involvement of the endogenous opioid system in the psychopharmacological actions of ethanol: the role of acetaldehyde

Significant evidence implicates the endogenous opioid system (EOS) (opioid peptides and receptors) in the mechanisms underlying the psychopharmacological effects of ethanol. Ethanol modulates opioidergic signaling and function at different levels, including biosynthesis, release, and degradation of opioid peptides, as well as binding of endogenous ligands to opioid receptors. The role of β-endorphin and µ-opioid receptors (OR) have been suggested to be of particular importance in mediating some of the behavioral effects of ethanol, including psychomotor stimulation and sensitization, consumption and conditioned place preference (CPP). Ethanol increases the release of β-endorphin from the hypothalamic arcuate nucleus (NArc), which can modulate activity of other neurotransmitter systems such as mesolimbic dopamine (DA). The precise mechanism by which ethanol induces a release of β-endorphin, thereby inducing behavioral responses, remains to be elucidated. The present review summarizes accumulative data suggesting that the first metabolite of ethanol, the psychoactive compound acetaldehyde, could participate in such mechanism. Two lines of research involving acetaldehyde are reviewed: (1) implications of the formation of acetaldehyde in brain areas such as the NArc, with high expression of ethanol metabolizing enzymes and presence of cell bodies of endorphinic neurons and (2) the formation of condensation products between DA and acetaldehyde such as salsolinol, which exerts its actions via OR.

OA08.02. Differential acute effects of real and sham acupuncture on mu-opioid receptor availability in treatment naive and conditioned chronic pain patients

Purpose Previous studies suggest acupuncture analgesic effects involve the release of endogenous opioid peptides. We have previously shown that while both acupuncture and sham reduced clinical pain in fibromyalgia (FM), only acupuncture increased mu-opioid receptor (MOR) baseline receptor availability in vivo (binding potential, BP). Here we investigated whether real and sham acupuncture were associated with differential changes in MOR BP in FM patients that were naive to acupuncture as compared to conditioned patients.

In VivoVisualization of Delta Opioid Receptors upon Physiological Activation Uncovers a Distinct Internalization Profile

G-protein-coupled receptors (GPCRs) mediate numerous physiological functions and represent prime therapeutic targets. Receptor trafficking upon agonist stimulation is critical for GPCR function, but examining this process in vivo remains a true challenge. Using knock-in mice expressing functional fluorescent delta opioid receptors under the control of the endogenous promoter, we visualized in vivo internalization of this native GPCR upon physiological stimulation. We developed a paradigm in which animals were made dependent on morphine in a drug-paired context. When re-exposed to this context in a drug-free state, mice showed context-dependent withdrawal signs and activation of the hippocampus. Receptor internalization was transiently detected in a subset of CA1 neurons, uncovering regionally restricted opioid peptide release. Importantly, a pool of surface receptors always remained, which contrasts with the in vivo profile previously established for exogenous drug-induced internalization. Therefore, a distinct response is observed at the receptor level upon a physiological or pharmacological stimulation. Altogether, direct in vivo GPCR visualization enables mapping receptor stimulation promoted by a behavioral challenge and represents a powerful approach to study endogenous GPCR physiology.

Nitrous oxide-induced NO-dependent neuronal release of β-endorphin from the rat arcuate nucleus and periaqueductal gray

Nitrous oxide (N 2O)-induced antinociception is thought to result from nitric oxide (NO)-dependent neuronal release of endogenous opioid peptides in the central nervous system. The present study employed microdialysis to determine whether exposure to N 2O stimulates proopiomelanocortin (POMC) neurons to release β-endorphin in the arcuate nucleus (ARC) of the hypothalamus and the periaqueductal gray (PAG) of the midbrain. Male Sprague–Dawley rats were stereotaxically implanted with microdialysis probes in the ARC or PAG. Exposure to 70% N 2O significantly increased dialysate levels of oxidation products of NO as well as β-endorphin, compared to levels in fractions collected under room air. These increases in the ARC and PAG were abolished by systemic pretreatment with l-N G-nitro arginine methyl ester ( l-NAME). These findings suggest an association between increased NO activity and the stimulated release of β-endorphin during exposure of rats to N 2O.

Anesthesia Literature Review

Anesthesia Literature Review

Recruitment of opioid peptide-containing neutrophils is independent of formyl peptide receptors

In complete Freund's adjuvans (CFA) inflammation opioid containing neutrophils release opioid peptides upon stimulation and mediate peripheral analgesia. Neutrophil migration is regulated partially by chemokines, but other mediators e.g. formyl peptides could also contribute. In vitro, formyl peptides but not Mycobacterium butyricum (CFA component) induced migration of neutrophils. In contrast, local formyl peptide injection did not induce leukocyte recruitment in vivo due to insufficient up-regulation of adhesion molecule expression. Furthermore, leukocyte recruitment and peripheral opioid-mediated analgesia were unaffected by systemic formyl peptide receptor blockade in CFA inflammation. Thus, while formyl peptides do not regulate migration they directly stimulate opioid peptide release.