Preoperative brain µ-opioid receptor availability predicts weight development following bariatric surgery in women.

Bariatric surgery is the most effective method for weight loss in morbid obesity. There is significant individual variability in the weight loss outcomes, yet factors leading to postoperative weight loss or weight regain remain elusive. Alterations in the µ-opioid receptor (MOR) and dopamine D2 receptor (D2R) systems are associated with obesity and appetite control, and the magnitude of initial brain receptor system perturbation may predict long-term surgical weight loss outcomes. We tested this hypothesis by studying 19 morbidly obese women (mean BMI 40) scheduled to undergo bariatric surgery. We measured their preoperative MOR and D2R availabilities using positron emission tomography with [11C]carfentanil and [11C]raclopride, respectively, and then assessed their weight development association with regional MOR and D2R availabilities at 24-month follow-up. MOR availability in the amygdala consistently predicted weight development throughout the follow-up period, but no associations were found for D2R. This is the first study to our knowledge to demonstrate that neuroreceptor markers prior to bariatric surgery are associated with postoperative weight development. Postoperative weight regain may derive from dysfunction in the opioid system, and weight loss outcomes after bariatric surgery may be partially predicted based on preoperative brain receptor availability, opening up new potential for treatment possibilities.

Association of OPRM1 Functional Coding Variant With Opioid Use Disorder: A Genome-Wide Association Study.

Importance: With the current opioid crisis, it is important to improve understanding of the biological mechanisms of opioid use disorder (OUD). Objectives: To detect genetic risk variants for OUD and determine genetic correlations and causal association with OUD and other traits. Design, Setting, and Participants: A genome-wide association study of electronic health record-defined OUD in the Million Veteran Program sample was conducted, comprising 8529 affected European American individuals and 71 200 opioid-exposed European American controls (defined by electronic health record trajectory analysis) and 4032 affected African American individuals and 26 029 opioid-exposed African American controls. Participants were enrolled from January 10, 2011, to May 21, 2018, with electronic health record data for OUD diagnosis from October 1, 1999, to February 7, 2018. Million Veteran Program results and additional OUD case-control genome-wide association study results from the Yale-Penn and Study of Addiction: Genetics and Environment samples were meta-analyzed (total numbers: European American individuals, 10 544 OUD cases and 72 163 opioid-exposed controls; African American individuals, 5212 cases and 26 876 controls). Data on Yale-Penn participants were collected from February 14, 1999, to April 1, 2017, and data on Study of Addiction: Genetics and Environment participants were collected from 1990 to 2007. The key result was replicated in 2 independent cohorts: proxy-phenotype buprenorphine treatment in the UK Biobank and newly genotyped Yale-Penn participants. Genetic correlations between OUD and other traits were tested, and mendelian randomization analysis was conducted to identify potential causal associations. Main Outcomes and Measures: Main outcomes were International Classification of Diseases, Ninth Revision-diagnosed OUD or International Statistical Classification of Diseases and Related Health Problems, Tenth Revision-diagnosed OUD (Million Veteran Program), and DSM-IV-defined opioid dependence (Yale-Penn and Study of Addiction: Genetics and Environment). Results: A total of 114 759 individuals (101 016 men [88%]; mean [SD] age, 60.1 [12.8] years) were included. In 82 707 European American individuals, a functional coding variant (rs1799971, encoding Asn40Asp) in OPRM1 (µ-opioid receptor gene, the main biological target for opioid drugs; OMIM 600018) reached genome-wide significance (G allele: ß = -0.066 [SE = 0.012]; P = 1.51 × 10-8). The finding was replicated in 2 independent samples. Single-nucleotide polymorphism-based heritability of OUD was 11.3% (SE = 1.8%). Opioid use disorder was genetically correlated with 83 traits, including multiple substance use traits, psychiatric illnesses, cognitive performance, and others. Mendelian randomization analysis revealed the following associations with OUD: risk of tobacco smoking, depression, neuroticism, worry neuroticism subcluster, and cognitive performance. No genome-wide significant association was detected for African American individuals or in transpopulation meta-analysis. Conclusions and Relevance: This genome-wide meta-analysis identified a significant association of OUD with an OPRM1 variant, which was replicated in 2 independent samples. Post-genome-wide association study analysis revealed associated pleiotropic characteristics. Recruitment of additional individuals with OUD for future studies-especially those of non-European ancestry-is a crucial next step in identifying additional significant risk loci.

Overexpression of µ-Opioid Receptors in Peripheral Afferents, but Not in Combination with Enkephalin, Decreases Neuropathic Pain Behavior and Enhances Opioid Analgesia in Mouse.

BACKGROUND: The current study used recombinant herpes simplex virus type I to increase expression of µ-opiate receptors and the opioid ligand preproenkephalin in peripheral nerve fibers in a mouse model of neuropathic pain. It was predicted that viral vector delivery of a combination of genes encoding the µ-opioid receptor and preproenkephalin would attenuate neuropathic pain and enhance opioid analgesia. The behavioral effects would be paralleled by changes in response properties of primary afferent neurons. METHODS: Recombinant herpes simplex virus type 1 containing cDNA sequences of the µ-opioid receptor, human preproenkephalin, a combination, or Escherichia coli lacZ gene marker (as a control) was used to investigate the role of peripheral opioids in neuropathic pain behaviors. RESULTS: Inoculation with the µ-opioid receptor viral vector (n = 13) reversed mechanical allodynia and thermal hyperalgesia and produced leftward shifts in loperamide (ED50 = 0.6 ± 0.2 mg/kg vs. ED50 = 0.9 ± 0.2 mg/kg for control group, n = 8, means ± SD) and morphine dose-response curves (ED50 = 0.3 ± 0.5 mg/kg vs. ED50 = 1.1 ± 0.1 mg/kg for control group). In µ-opioid receptor viral vector inoculated C-fibers, heat-evoked responses (n = 12) and ongoing spontaneous activity (n = 18) were decreased after morphine application. Inoculation with both µ-opioid receptor and preproenkephalin viral vectors did not alter mechanical and thermal responses. CONCLUSIONS: Increasing primary afferent expression of opioid receptors can decrease neuropathic pain-associated behaviors and increase systemic opioid analgesia through inhibition of peripheral afferent fiber activity.

Sleep quality and OPRM1 polymorphisms: a cross-sectional study among opioid-naive individuals

ABSTRACT O sistema opioidérgico envolve a regulação do sono e da vigília. É possível, portanto, que os polimorfismos genéticos no OPRM1 influenciem na qualidade do sono. Este estudo investigou a associação de polimorfismos do OPRM1 com a qualidade subjetiva do sono entre indivíduos sem tratamento prévio com opióides. Este estudo observacional de corte transversal envolveu 161 homens que nunca haviam se tornado opióides (média de idade = 27,74 anos; variação: 18 a 63 anos). A qualidade subjetiva do sono foi avaliada com a versão traduzida e validada em malaio do Índice de Qualidade do Sono de Pittsburgh (PSQI). O DNA foi extraído do sangue total e submetido à reação em cadeia da polimerase (PCR) para dois polimorfismos OPRM1 (118A> G e IVS2 + 691G> C). Sujeitos combinados com 118A e IVS2 + 691Galelos (haplótipo AC) apresentaram escores significativamente mais baixos do PSQI [média (DP) = 4,29 (1,76)] em comparação àqueles sem o haplótipo [4,99 (2,50)] (p = 0,004). Por outro lado, os indivíduos com genótipo heterozigótico combinado (GC / AG diplotipo) apresentaram escores significativamente mais altos do PSQI em comparação àqueles sem o diplótipo [6,04 (2,48) vs 4,54 (2,22), p = 0,004]. Em indivíduos sem tratamento prévio com opiáceos, o haplótipo AC e o diplótipo GC / AG para os polimorfismos 118A> G e IVS2 + 691G> C do OPRM1 estão associados a uma melhor e pior qualidade do sono, respectivamente.

Opioid Receptors in Psoriatic Skin: Relationship with Itch.

Psoriasis is an inflammatory immunogenetic skin disease, often accompanied by itch. Opioid receptors are known regulators of itch sensation in the central nervous system. In the brain, µ-opioid receptors may potentiate itch, while activation of κ-opioid receptors may reduce or even alleviate itch; however, the role of opioid receptors in itch perception in the skin is poorly understood. To further elucidate the role of opioid receptors in the neurobiology of psoriatic itch, punch biopsies of non-lesional and lesional skin of patients with psoriasis and healthy controls were studied. Real-time polymerase chain reaction and immunofluorescence microscopy were used to detect opioid receptor genes and protein expression, respectively. The OPRK1/κ-opioid receptor pathway was found to be downregulated in lesional skin of psoriasis, correlating positively with itch sensation. In contrast, the OPRM1/µ-opioid receptor system was uniformly expressed by epidermal keratinocytes in all analysed groups. These findings suggest that imbalance of epidermal opioid receptors may result in disordered neuroepidermal homeostasis in psoriasis, which could potentiate transmission of itch.

Opiate receptor blockade on human granulosa cells inhibits VEGF release.

The objectives of this study were to determine whether the main opioid receptor (OPRM1) is present on human granulosa cells and if exogenous opiates and their antagonists can influence granulosa cell vascular endothelial growth factor (VEGF) production via OPRM1. Granulosa cells were isolated from women undergoing oocyte retrieval for IVF. Complementary to the primary cells, experiments were conducted using COV434, a well-characterized human granulosa cell line. Identification and localization of opiate receptor subtypes was carried out using Western blot and flow cytometry. The effect of opiate antagonist on granulosa cell VEGF secretion was assessed by enzyme-linked immunosorbent assay. For the first time, the presence of OPRM1 on human granulosa cells is reported. Blocking of opiate signalling using naloxone, a specific OPRM1 antagonist, significantly reduced granulosa cell-derived VEGF levels in both COV434 and granulosa-luteal cells (P < 0.01). The presence of opiate receptors and opiate signalling in granulosa cells suggest a possible role in VEGF production. Targeting this signalling pathway could prove promising as a new clinical option in the prevention and treatment of ovarian hyperstimulation syndrome.

OPRM1 receptor as new biomarker to help the prediction of post mastectomy pain and recurrence in breast cancer.

Breast cancer is the most common type of cancer among women worldwide. Short-term postsurgical recovery is complicated by many factors, including imbalanced inflammatory and immune response, acute pain associated with functional impairment, and chronic postmastectomy pain (CPMP), developed by about 25-60% of patients. Opioids, most common drugs used for treatment of cancer pain, are immunosuppressive, and therefore, they might directly and/or indirectly influence long-term cancer recurrence. Moreover, they also produce endocrinopathy, which consists primarily of hypothalamic-pituitary-gonadal axis or hypothalamic-pituitary-adrenal axis dysfunction. The interindividual variability in both CPMP and opioid response is believed to be largely underlined by genetic variability in the gene locus for µ-opioid receptor (OPRM1) that modulates opioid pharmacodynamics. For this reason, OPRM1 genotype may play a key role both in short-term postmastectomy outcome and in long-term follow-up, becoming a new biomarker for breast cancer recurrence in patients suffering from chronic postmastectomy pain managed by opioid therapy. Hence OPRM1 might be used in near future to customize the opioid therapy, avoiding not only opioid side effects but also the disease progression. In this review we evaluate the literature state of the art on this topic and possible steps towards obtaining the safest individualized postmastectomy analgesic therapy. Therefore, a personalized pain treatment strategy might be useful to both manage pain and control cancer disease progression.

Opioid requirement, opioid receptor expression, and clinical outcomes in patients with advanced prostate cancer.

BACKGROUND: Preclinical studies show that opioids stimulate angiogenesis and tumor progression through the mu opioid receptor (MOR). Although MOR is overexpressed in several human malignancies, the effect of chronic opioid requirement on cancer progression or survival has not been examined in humans. METHODS: We performed a retrospective analysis on 113 patients identified in the Minneapolis VA Tumor Registry (test cohort) and 480 patients from the national VA Central Cancer Registry (validation cohort) who had been diagnosed with stage IV prostate cancer between 1995 and 2010 to examine whether MOR expression or opioid requirement is associated with disease progression and survival. All opioids were converted to oral morphine equivalents for comparison. Laser scanning confocal microscopy was used to analyze MOR immunoreactivity in prostate cancer biopsies. The effects of variables on outcomes were analyzed in univariable and multivariable models. RESULTS: In patients with metastatic prostate cancer, MOR expression and opioid requirement were independently associated with inferior progression-free survival (hazard ratio [HR] 1.65, 95% confidence interval [CI] 1.33-2.07, P<.001 and HR 1.08, 95% CI 1.03-1.13, P<.001, respectively) and overall survival (HR 1.55, 95% CI 1.20-1.99, P<.001 and HR 1.05, 95% CI 1.00-1.10, P = .031, respectively). The validation cohort confirmed that increasing opioid requirement was associated with worse overall survival (HR 1.005, 95% CI 1.002-1.008, P = .001). CONCLUSION: Higher MOR expression and greater opioid requirement are associated with shorter progression-free survival and overall survival in patients with metastatic prostate cancer. Nevertheless, clinical practice should not be changed until prospective randomized trials show that opioid use is associated with inferior clinical outcomes, and that abrogation of the peripheral activities of opioids ameliorates this effect.

Cellular events during arthritis-induced hyperalgesia are mediated by interleukin-6 and p38 MAPK and their effects on the expression of spinal mu-opioid receptors.

Activation of mitogen-activated protein kinase (MAPK) enzymes in nociceptive plasticity has been extensively studied. P38 MAPK enzyme, which can be activated by cytokines, acts as a crucial intracellular regulator of environmental changes. The aim of this study was to elucidate the cellular events during arthritis-induced hyperalgesia that are mediated by interleukin-6 and p38 MAPK, and their effects on the expression of spinal mu-opioid receptors (MORs), in different stages of arthritis in male Wistar rats. Complete Freund's adjuvant (CFA)-induced arthritis (AA) was caused by subcutaneous injection of CFA into the rats' hindpaw. Anti-IL-6 antibody and p38 MAPK phosphorylation inhibitor were administered during 21 days of study. Spinal MOR, p38, and phosphorylated-p38 (pp38) proteins expressions were detected by Western blotting. Daily treatment with anti-IL-6 antibody and p38 MAPK phosphorylation inhibitor, SB203580, significantly decreased paw edema in AA group. Daily anti-IL-6 and SB203580 administration caused a significant reduction in hyperalgesia in the first week of the study, but increased hyperalgesia in the next 2 weeks in experimental groups compared to the AA control group. Expression of pp38 MAPK protein significantly decreased on the 3, 7, 14, and 21 days in AA+SB203580 and AA+anti-IL6 groups compared to AA group. Additionally, daily treatment with anti-IL6 antibody and SB203580 in AA group caused significantly decrease in spinal MOR expression compared to AA control group. The results of our study can confirm that activated spinal p38 MAPK enzyme may play an important role in cellular IL-6 signaling pathways in hyperalgesia variation during different stages of AA inflammation. Also, it can be suggested that at least a part of p38 MAPK effects on hyperalgesia is mediated by spinal MOR expression variation.

Development of a cyclic adenosine monophosphate assay for Gi-coupled G protein-coupled receptors by utilizing the endogenous calcitonin activity in Chinese hamster ovary cells.

Activation of G(i)-coupled G protein-coupled receptor (GPCRs) by their ligands leads to inhibition of adenylyl cyclase (AC) and reduction of cyclic adenosine monophosphate (cAMP) levels in cells. The traditional cAMP assay for G(i)-coupled GPCRs commonly uses forskolin, a nonspecific AC activator, to increase the basal cAMP level in cells to create an assay window for ligand detection. However, there is still a need to develop a nonforskolin-based cAMP assay because of the challenges inherent in titrating the concentration of forskolin to achieve a reliable assay window, along with issues related to the cAMP-independent effects of forskolin. Herein, we describe such an assay by utilizing the endogenous activity of the calcitonin receptor in Chinese hamster ovary (CHO) cells. The calcitonin receptor is a G(s)-coupled GPCR that, when activated by calcitonin, leads to the stimulation of AC and increases cAMP in cells. Thus, we use calcitonin, instead of forskolin, to increase the basal cAMP level in CHO cells to achieve an assay window. We demonstrated that calcitonin peptides robustly increased cAMP accumulation in several CHO cell lines stably expressing well-known G(i)-coupled GPCRs, such as the Dopamine D2 receptor, the Opioid µ receptor, or the Cannabinoid receptor-1. Agonists of these G(i)-coupled GPCRs attenuated calcitonin-induced cAMP production in their receptor stable cell lines. On the other hand, antagonists and/or inverse agonists blocked the effects of their agonists on calcitonin-induced cAMP production. This calcitonin-based cAMP assay has been demonstrated to be sensitive and robust and exhibited acceptable assay windows (signal/noise ratio) and, thus, can be applied to screen for agonists and antagonists/inverse agonists of G(i)-coupled GPCRs in high-throughput screening formats.

Nuclear expression of mu-opioid receptors in a human mesothelial cell line.

1 Possibly acting via mu-opioid receptors (MORs), morphine inhibits the formation of experimentally induced postoperative abdominal adhesions in rats. Mesothelial cells may participate in adhesion formation by secreting mediators that interfere negatively with fibrinolysis. Morphine may prevent adhesions by inhibiting the release of pro-adhesion mediators from mesothelial cells. This study aimed to investigate whether human mesothelial cells express MOR-1; if so, such could constitute a site of action for morphine in adhesion prevention. 2 Cells from Met-5A, a human mesothelial cell line were seeded and prepared for immunocytochemistry and Western blotting. 3 Immunocytochemistry showed MOR-1 expression in mesothelial cells, predominantly in the nuclei. Western blotting showed two bands (c. 35 and 50 kDa) which correspond to those obtained with a control lysate from cells known to express MORs. In addition, we found MOR-1 expression with nuclear and cytoplasmatic localization in biopsies from human abdominal adhesions. 4 The current findings may suggest that morphine could interact directly with mesothelial cells via MOR-1 receptors, and thereby modulate adhesion formation, possibly by interfering with the release of pro-adhesion factors from these cells.

Rapid modulation of micro-opioid receptor signaling in primary sensory neurons.

Management of pain by opioid analgesics is confounded by central adverse effects that limit clinical dosages. Consequently, there is considerable interest to understand peripheral analgesic effects of opioids. The actions of opioids on peripheral sensory neurons have been difficult to study because of a general lack of effect of opioid agonists on nociceptor function in culture despite documented presence of opioid receptors. In this study, the micro-opioid receptor agonist, [D-Ala(2),N-MePhe(4),Gly-ol(5)]-enkephalin (DAMGO), did not alter guanosine 5'-O-(3-[(35)S]thio)-triphosphate (GTPgamma[(35)S]) binding, adenylyl cyclase activity, or neuropeptide release in primary cultures of rat trigeminal ganglion (TG). However, after brief exposure to bradykinin (BK), DAMGO stimulated GTPgamma[(35)S] binding and inhibited both prostaglandin E(2) (PGE(2))-stimulated adenylyl cyclase activity and BK/PGE(2)-stimulated neuropeptide release. The effect of BK was blocked by the B(2) antagonist HOE 140 [D-Arg[Hyp(3),Thi(5),D-Tic(7),Oic(8)]-bradykinin], but not by the B(1) antagonist, Lys-[Leu8]des-Arg9-BK, and was mimicked by the protease-activated receptor-2 agonist, Ser-Leu-Ile-Gly-Arg-Leu-NH(2), and by activation of protein kinase C (PKC) or by administration of arachidonic acid (AA). The enhanced responsiveness of micro-opioid receptor signaling by BK priming was blocked by both cyclooxygenase and PKC inhibitors; however, the effect of AA was blocked only by a cyclooxygenase inhibitor. The results indicate that micro-opioid receptor signaling in primary sensory TG neurons is enhanced by activation of phospholipase C-coupled receptors via a cyclooxygenase-dependent AA metabolite that is downstream of PKC.

Beta-endorphin, Met-enkephalin and corresponding opioid receptors within synovium of patients with joint trauma, osteoarthritis and rheumatoid arthritis.

OBJECTIVE: Intra-articularly applied opioid agonists or antagonists modulate pain after knee surgery and in chronic arthritis. Therefore, the expression of beta-endorphin (END), Met-enkephalin (ENK), and mu and delta opioid receptors (ORs) within synovium of patients with joint trauma (JT), osteoarthritis (OA) and rheumatoid arthritis (RA) were examined. METHODS: Synovial samples were subjected to double immunohistochemical analysis of opioid peptides with immune cell markers, and of ORs with the neuronal markers calcitonin gene-related peptide (CGRP) and tyrosine hydroxylase (TH). RESULTS: END and ENK were expressed by macrophage-like (CD68(+)) and fibroblast-like (CD68(-)) cells within synovial lining layers of all disorders. In the sublining layers, END and ENK were mostly expressed by granulocytes in patients with JT, and by macrophages/monocytes, lymphocytes and plasma cells in those with OA and RA. Overall, END- and ENK-immunoreactive (IR) cells were more abundant in patients with RA than in those with OA and JT. ORs were found on nerve fibres and immune cells in all patients. OR-IR nerve fibres were significantly more abundant in patients with RA than in those with OA and JT. muORs and deltaORs were coexpressed with CGRP but not with TH. CONCLUSIONS: Parallel to the severity of inflammation, END and ENK in immune cells and their receptors on sensory nerve terminals are more abundant in patients with RA than in those with JT and OA. These findings are consistent with the notion that, with prolonged and enhanced inflammation, the immune and peripheral nervous systems upregulate sensory nerves expressing ORs and their ligands to counterbalance pain and inflammation.

Regulation of psychostimulant-induced preprodynorphin, c-fos and zif/268 messenger RNA expression in the rat dorsal striatum by mu opioid receptor blockade.

Several studies have shown that psychostimulants can induce differential immediate early gene and neuropeptide expression in the patch versus matrix compartments of dorsal striatum. The patch compartment contains a high density of mu opioid receptors and activation of these receptors may contribute to psychostimulant-induced gene expression in the patch versus matrix compartments of dorsal striatum. However, the contribution of mu opioid receptor activation to psychostimulant-induced changes in gene expression in the patch compartment of dorsal striatum has not been examined. The current study examined the role of mu opioid receptors in psychostimulant induction of preprodynorphin, c-fos and zif/268 messenger RNA expression in the patch versus matrix compartments of dorsal striatum. Male Sprague-Dawley rats were treated with the mu opioid receptor antagonist, clocinnamox (1 mg/kg, s.c.), 24 h prior to treatment with cocaine (30 mg/kg, i.p.) or methamphetamine (15 mg/kg, s.c.) and sacrificed 45 min or 3 h later. Mu opioid receptor antagonism blocked psychostimulant-induced preprodynorphin messenger RNA expression only in the rostral patch compartment, whereas psychostimulant-induced zif/268 messenger RNA expression in the patch and matrix compartments was attenuated throughout the dorsal striatum. Clocinnamox pretreatment had no effect on stimulant-induced increases in c-fos expression. These data suggest that mu opioid receptor activation plays a specific role in psychostimulant-induced preprodynorphin messenger RNA expression in the rostral patch compartment and zif/268 messenger RNA expression throughout dorsal striatum.

Proinflammatory chemokines, such as C-C chemokine ligand 3, desensitize mu-opioid receptors on dorsal root ganglia neurons.

Pain is one of the hallmarks of inflammation. Opioid receptors mediate antipain responses in both the peripheral nervous system and CNS. In the present study, pretreatment of CCR1: mu-opioid receptor/HEK293 cells with CCL3 (MIP-1alpha) induced internalization of mu-opioid receptors and severely impaired the mu-opioid receptor-mediated inhibition of cAMP accumulation. Immunohistochemical staining showed that CCR1 and mu-opioid receptors were coexpressed on small to medium diameter neurons in rat dorsal root ganglion. Analysis of ligand-induced calcium flux showed that both types of receptors were functional. Pretreatment of neurons with CCL3 exhibited an impaired [D-Ala(2),N-MePhe(4),Gly-o15]enkephalin-elicited calcium response, indicative of the heterologous desensitization of mu-opioid receptors. Other chemokines, such as CCL2, CCL5, and CXCL8, exhibited similar inhibitory effects. Our data indicate that proinflammatory chemokines are capable of desensitizing mu-opioid receptors on peripheral sensory neurons, providing a novel potential mechanism for peripheral inflammation-induced hyperalgesia.

Abdominal surgery induces mu opioid receptor endocytosis in enteric neurons of the guinea-pig ileum.

Immunohistochemistry and confocal microscopy were used to investigate mu opioid receptor (muOR) internalization in enteric neurons of the guinea-pig ileum following abdominal surgery. The following surgical procedures were performed under halothane or isofluorane anesthesia: a) midline abdominal skin incision, b) laparotomy or c) laparotomy with intestinal manipulation. Gastrointestinal transit was evaluated by using a non-absorbable marker and measuring fecal pellet output. In neurons from normal and control (anesthesia alone) animals, muOR was predominantly at the cell surface. muOR endocytosis following skin incision was not significantly different from controls (21.2+/-3.5% vs. 13.7+/-2.1%, mean+/-S.E.M.), whereas it was significantly increased by laparotomy (46.5+/-6.1%; P<0.01 vs. controls) or laparotomy plus intestinal manipulation (40.5+/-6.1%; P<0.01 vs. controls) 30 min following surgery compared with controls. muOR endocytosis remained elevated at 4 h (38.6+/-1.2%; P<0.01 vs. controls), whereas it was similar to controls at 6 and 12 h (17.5+/-5.8% and 11.2+/-3.0%). muOR endocytosis occurred in cholinergic and nitrergic neurons. Gastrointestinal transit was significantly delayed by laparotomy or laparotomy plus intestinal manipulation (12.8+/-1.2 and 13.8+/-0.6 h vs. 7.0+/-0.5 in controls; P<0.01), but was not significantly changed by skin incision (8.2+/-0.6 h). The findings of the present study support the concept that the noxious stimulation caused by abdominal surgery induces release of endogenous opioids thus resulting in muOR endocytosis in neurochemically distinct enteric neurons. muOR internalization can serve as indirect evidence of opioid release and as a means to visualize neuronal pathways activated by opioids.

Quantitative autoradiography of adenosine receptors in brains of chronic naltrexone-treated mice.

1. Manipulation of micro opioid receptor expression either by chronic morphine treatment or by deletion of the gene encoding micro opioid receptors leads to changes in adenosine receptor expression. Chronic administration of the opioid receptor antagonist naltrexone leads to upregulation of micro receptor binding in the brain. 2. To investigate if there are any compensatory alterations in adenosine systems in the brains of chronic naltrexone-treated mice, we carried out quantitative autoradiographic mapping of A(1) and A(2A) adenosine receptors in the brains of mice treated for 1 week with naltrexone (8 mg(-1) kg(-1) day(-1)), administered subcutaneously via osmotic minipump. 3. Adjacent coronal brain sections were cut from chronic saline- and naltrexone-treated mice for the determination of binding of [(3)H] D-Ala(2)-MePhe(4)-Gly-ol(5) enkephalin ([(3)H] DAMGO), [(3)H]1,3-dipropyl-8-cyclopentylxanthine ([(3)H] DPCPX) or [(3)H] 2-[p-(2-carbonylethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosine ([(3)H] CGS21680) to micro, A(1) and A(2A) receptors, respectively. 4. A significant increase in micro and A(1) receptor binding was detected in chronic naltrexone-treated brains. The changes in micro receptors were significant in several regions, but changes in A(1) were relatively smaller but showed significant upregulation collectively. No significant change in A(2A) receptor binding was detected in chronic naltrexone-treated brains. 5. The results show that blockade of opioid receptors causes upregulation of A(1) receptors, but not A(2A) receptors, by as yet undefined mechanisms.

Mu opioid receptors are in discrete hippocampal interneuron subpopulations.

In the rat hippocampal formation, application of mu opioid receptor (MOR) agonists disinhibits principal cells, promoting excitation-dependent processes such as epileptogenesis and long-term potentiation. However, the precise location of MORs in particular inhibitory circuits, has not been determined, and the roles of MORs in endogenous functioning are unclear. To address these issues, the distribution of MOR-like immunoreactivity (-li) was examined in several populations of inhibitory hippocampal neurons in the CA1 region using light and electron microscopy. We found that MOR-li was present in many parvalbumin-containing basket cells, but absent from cholecystokinin-labeled basket cells. MOR-li was also commonly in interneurons containing somatostatin-li or neuropeptide Y-li that resembled the "oriens-lacunosum-moleculare" (O-LM) interneurons innervating pyramidal cell distal dendrites. Finally, MOR-li was in some vasoactive intestinal peptide- or calretinin-containing profiles resembling interneurons that primarily innervate other interneurons. These findings indicate that MOR-containing neurons form a neurochemically and functionally heterogeneous subset of hippocampal GABAergic neurons. MORs are most frequently on interneurons that are specialized to inhibit pyramidal cells, and are on a limited number of interneurons that target other interneurons. Moreover, the distribution of MORs to different neuronal types in several laminae, some relatively far from endogenous opioids, suggests normal functional roles that are different from the actions seen with exogenous agonists such as morphine.

Identification of delta- and mu-type opioid receptors on human and murine dendritic cells.

The purpose of this study was to evaluate mu- and delta-opioid receptors (OR) on human and murine dendritic cells (DC). Expression of mu- and delta-OR mRNA on DC was demonstrated by RT-PCR. The immunocytochemical and Western blot analyses revealed the expression of OR protein in DC. Radioreceptor assay demonstrated the specific saturated temperature-dependent binding of [3H]-labeled opioid ligand on DC and B(max)=2.8+/-0.3 fmol/10(6) cells and K(D)=4.8+/-1.0 nM were calculated by a Scatchard analysis. Finally, OR ligands DADLE and DAGO dose-dependently modulated the capacity of DC to induce T cell proliferation in an MLR assay. Importantly, expression of functional OR on DC was significantly increased upon TNF-alpha-induced DC maturation. Thus, these data suggest a new mechanism of opioid-dependent neuroendocrine immunomodulation.

Co-distribution of Fos- and mu opioid receptor immunoreactivity within the rat septopreoptic area and hypothalamus during acute glucose deprivation: effects of the mu receptor antagonist CTOP.

Mu opioid receptors occur throughout the brain, but central sites where ligand neuromodulatory effects occur during glucopenia have not been identified. The present studies investigated whether septal, preoptic, and hypothalamic neurons that express immunoreactivity for this receptor are transcriptionally activated in response to the glucose antimetabolite, 2-deoxy-D-glucose (2DG), and if intracerebroventricular (icv) administration of the selective mu receptor antagonist, CTOP, modifies this functional response to glucose substrate imbalance. Neurons labeled for mu receptor-immunoreactivity (-ir) were observed in the lateral septal nucleus (LS), medial septum (MS), anterior division of the stria terminalis (BSTa), median preoptic nucleus (MEPO), medial preoptic nucleus (MPN), parastrial nucleus (PS), anterior hypothalamic periventricular nucleus (PVa), and lateral hypothalamic area (LPO). 2DG injection (400 mg/kg i.p.) resulted in co-labeling of mu receptor-positive neurons in the LS, MS, BSTa, MEPO, PVa, and LPO for nuclear Fos-ir. Icv delivery of CTOP decreased mean numbers of co-labeled neurons in the LS, MS, BSTa, and MEPO. These results provide evidence for transactivational effects of glucopenia on mu opioid receptor-expressing neurons within the septum, preoptic area, and hypothalamus, and suggest that the functional status of these receptors within discrete septopreoptic sites may be critical for maximal glucoprivic induction of the Fos stimulus-transcription cascade within local cells. These results thus support the view that the neural loci described above may serve as substrates for regulatory effects of mu opioid receptor ligands on central compensatory activities during acute glucose deprivation.