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Disturbances in the function of the mesostriatal dopamine system may contribute to the development and maintenance of chronic pain, including its sensory and emotional/cognitive aspects. In the present study, we assessed the influence of chronic constriction injury (CCI) of the sciatic nerve on the expression of genes coding for dopamine and opioid receptors as well as opioid propeptides in the mouse mesostriatal system, particularly in the nucleus accumbens. We demonstrated bilateral increases in mRNA levels of the dopamine D1 and D2 receptors (the latter accompanied by elevated protein level), opioid propeptides proenkephalin and prodynorphin, as well as delta and kappa (but not mu) opioid receptors in the nucleus accumbens at 7 to 14 days after CCI. These results show that CCI-induced neuropathic pain is accompanied by a major transcriptional dysregulation of molecules involved in dopaminergic and opioidergic signaling in the striatum/nucleus accumbens. Possible functional consequences of these changes include opposite effects of upregulated enkephalin/delta opioid receptor signaling vs. dynorphin/kappa opioid receptor signaling, with the former most likely having an analgesic effect and the latter exacerbating pain and contributing to pain-related negative emotional states.

Social isolation stress (SIS) as a type of chronic stress could induce depressive- and anxiety-like behaviors. Our study evaluates the role of opioid system on negative behavioral impacts of SIS in male NMRI mice. We investigated effects of morphine, a nonselective opioid receptor (OR) agonist, naltrexone (NLX), an OR antagonist, naltrindole (NLT), a delta opioid receptor (DOR) antagonist, SNC80, a DOR agonist, U-69593, a kappa opioid receptor (KOR) agonist, nor-Binaltorphimine, a selective KOR antagonist and cyprodime hydrochloride a selective mu opioid receptor (MOR) antagonist on depressive- and anxiety-like behaviors. Using RT-PCR we evaluated ORs gene expression in mice brain. Our findings showed that SIS induced anxiety- and depressive-like behavior in the forced swimming test, open field test, splash test and hole-board test. Moreover, administration of SNC-80 significantly mitigated anxiety- and depressive-like behaviors. NLT decreased grooming-activity in the splash test. Excitingly, administration of agents affecting KOR failed to alter the negative effects of SIS. RT-PCR demonstrated that MOR and KOR gene expression decreased in socially isolated mice; however, SIS did not affect DORs expression. Our findings suggest that SIS at least in part, probably via altering endogenous opioids particularly MORs and KORs but not DORs mediated negative impacts on behavior; also, it could be concluded that DORs might be considered as a novel target for studying depression and anxiety., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Regulation of μ-, δ- and κ-opioid receptor protein level in spleen lymphocytes when stimulated by mitogen is not known. To answer the question whether these cells do express opioid receptor (OR) proteins, primary, fresh rat spleen lymphocytes were prepared and stimulated for 48 h with mitogenic dose of Con A. The unstimulated lymphocytes did not express μ- and δ-OR proteins in detectable amounts, however, stimulation with Con A resulted in appearance of clearly detectable immunoblot signals of both μ-OR and δ-OR. κ-OR were detected already in primary cells and increased 2.4-fold in Con A-stimulated cells. These results were supported by data obtained by flow cytometry analysis indicating a dramatic increase in number of μ-, δ- and κ-OR expressing cells after mitogen stimulation. The newly synthesized μ-, δ- and κ-OR in Con A-stimulated spleen lymphocytes were present in the cells interior and not functionally mature, at least in terms of their ability to enhance activity of trimeric G proteins determined by three different protocols of agonist-stimulated, high-affinity [ 35 S]GTPγS binding assay. The up-regulation of μ-, δ- and κ-OR was associated with specific decrease of their cognate trimeric G proteins, G i 1α/G i 2α; the other Gα and Gβ subunits were unchanged. The level of β-arrestin-1/2 was also decreased in Con A-stimulated splenocytes. We conclude that up-regulation of OR expression level in spleen lymphocytes by Con A proceeds in conjunction with down-regulation of their intracellular signaling partners, G i 1α/G i 2α proteins and β-arrestin-1/2. These regulatory proteins are expressed in high amounts already in unstimulated cells and decreased by mitogen stimulation., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background and Purpose: To better understand opioid signalling in visceral nociceptors, we examined the expression and selective activation of μ and δ opioid receptors by dorsal root ganglia (DRG) neurons innervating the mouse colon., Experimental Approach: DRG neurons projecting to the colon were identified by retrograde tracing. δ receptor-GFP reporter mice, in situ hybridization, single-cell RT-PCR and μ receptor-specific antibodies were used to characterize expression of μ and δ receptors. Voltage-gated Ca 2+ currents and neuronal excitability were recorded in small diameter nociceptive neurons (capacitance <30 pF) by patch clamp and ex vivo single-unit afferent recordings were obtained from the colon., Key Results: In situ hybridization of oprm1 expression in Fast Blue-labelled DRG neurons was observed in 61% of neurons. μ and δ receptors were expressed by 36-46% of colon DRG neurons, and co-expressed by ~25% of neurons. μ and δ receptor agonists inhibited Ca 2+ currents in DRG, effects blocked by opioid antagonists. One or both agonists inhibited action potential firing by colonic afferent endings. Incubation of neurons with supernatants from inflamed colon segments inhibited Ca 2+ currents and neuronal excitability. Antagonists of μ, but not δ receptors, inhibited the effects of these supernatant on Ca 2+ currents, whereas both antagonists inhibited their actions on neuronal excitability., Conclusions and Implications: A significant number of small diameter colonic nociceptors co-express μ and δ receptors and are inhibited by agonists and endogenous opioids in inflamed tissues. Thus, opioids that act at μ or δ receptors, or their heterodimers may be effective in treating visceral pain., (© 2018 The British Pharmacological Society.)

Cellular interactions between delta and mu opioid receptors (DORs and MORs), including heteromerization, are thought to regulate opioid analgesia. However, the identity of the nociceptive neurons in which such interactions could occur in vivo remains elusive. Here we show that DOR-MOR co-expression is limited to small populations of excitatory interneurons and projection neurons in the spinal cord dorsal horn and unexpectedly predominates in ventral horn motor circuits. Similarly, DOR-MOR co-expression is rare in parabrachial, amygdalar, and cortical brain regions processing nociceptive information. We further demonstrate that in the discrete DOR-MOR co-expressing nociceptive neurons, the two receptors internalize and function independently. Finally, conditional knockout experiments revealed that DORs selectively regulate mechanical pain by controlling the excitability of somatostatin-positive dorsal horn interneurons. Collectively, our results illuminate the functional organization of DORs and MORs in CNS pain circuits and reappraise the importance of DOR-MOR cellular interactions for developing novel opioid analgesics., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

The functional diversity of primary afferent neurons of the dorsal root ganglia (DRG) generates a variety of qualitatively and quantitatively distinct somatosensory experiences, from shooting pain to pleasant touch. In recent years, the identification of dozens of genetic markers specifically expressed by subpopulations of DRG neurons has dramatically improved our understanding of this diversity and provided the tools to manipulate their activity and uncover their molecular identity and function. Opioid receptors have long been known to be expressed by discrete populations of DRG neurons, in which they regulate cell excitability and neurotransmitter release. We review recent insights into the identity of the DRG neurons that express the delta opioid receptor (DOR) and the ion channel mechanisms that DOR engages in these cells to regulate sensory input. We highlight recent findings derived from DORGFP reporter mice and from in situ hybridization and RNA sequencing studies in wild-type mice that revealed DOR presence in cutaneous mechanosensory afferents eliciting touch and implicated in tactile allodynia. Mechanistically, we describe how DOR modulates opening of voltage-gated calcium channels (VGCCs) to control glutamatergic neurotransmission between somatosensory neurons and postsynaptic neurons in the spinal cord dorsal horn. We additionally discuss other potential signaling mechanisms, including those involving potassium channels, which DOR may engage to fine tune somatosensation. We conclude by discussing how this knowledge may explain the analgesic properties of DOR agonists against mechanical pain and uncovers an unanticipated specialized function for DOR in cutaneous mechanosensation.

Delta opioid receptor (DOR) displays a unique, highly conserved, structure and an original pattern of distribution in the central nervous system, pointing to a distinct and specific functional role among opioid peptide receptors. Over the last 15 years, in vivo pharmacology and genetic models have allowed significant advances in the understanding of this role. In this review, we will focus on the involvement of DOR in modulating different types of hippocampal- and striatal-dependent learning processes as well as motor function, motivation, and reward. Remarkably, DOR seems to play a key role in balancing hippocampal and striatal functions, with major implications for the control of cognitive performance and motor function under healthy and pathological conditions.

Brain injuries, such as cerebral hypoxia-ischemia (H-I), induce a regenerative response from the neural stem/progenitors (NSPs) of the subventricular zone (SVZ); however, the mechanisms that regulate this expansion have not yet been fully elucidated. The Notch- Delta-Serrate-Lag2 (DSL) signaling pathway is considered essential for the maintenance of neural stem cells, but it is not known if it is necessary for the expansion of the NSPs subsequent to perinatal H-I injury. Therefore, the aim of this study was to investigate whether this pathway contributes to NSP expansion in the SVZ after H-I and, if so, to establish whether this pathway is directly induced by H-I or regulated by paracrine factors. Here we report that Notch1 receptor induction and one of its ligands, Delta-like 1, precedes NSP expansion after perinatal H-I in P6 rat pups and that this increase occurs specifically in the most medial cell layers of the SVZ where the stem cells reside. Pharmacologically inhibiting Notch signaling in vivo diminished NSP expansion. With an in vitro model of H-I, Notch1 was not induced directly by hypoxia, but was stimulated by soluble factors, specifically leukemia inhibitory factor, produced by astrocytes within the SVZ. These data confirm the importance both of the Notch-DSL signaling pathway in the expansion of NSPs after H-I and in the role of the support cells in their niche. They further support the body of evidence that indicates that leukemia inhibitory factor is a key injury-induced cytokine that is stimulating the regenerative response of the NSPs. © 2016 Wiley Periodicals, Inc., Competing Interests: The authors have no conflicts of interest to report., (© 2016 Wiley Periodicals, Inc.)

δ‑opioid receptor (DOR) belongs to the family of G protein‑coupled receptors (GPCRs). Numerous studies have shown that DOR is widely distributed in human peripheral tissues and is closely related to the development and progression of certain malignant tumours. However, there is controversy in the literature regarding whether DOR has an impact on the development and progression of human breast cancer. The present study comprehensively elaborates on the biological functions of DOR by determining the distribution of DOR expression in breast cancer tissues and cells and by further verifying the effects of DOR on breast cancer progression. DOR was found to be highly expressed in human breast cancer tissues and cells. In addition, the high expression level of DOR positively correlated with tumour grade and clinical stage and negatively correlated with breast cancer metastasis and prognosis. Upregulating and activating DOR promoted the proliferation of human breast cancer cells in a concentration‑dependent manner within a specific concentration range, whereas downregulating or inhibiting DOR activation significantly suppressed cell proliferation. The majority of tumour cells were arrested in G1 phase, and some cells exhibited apoptosis. DOR upregulation and activation induced protein kinase C (PKC) activation, resulting in increased phosphorylation levels of extracellular signal‑regulated kinases (ERKs). After inhibition of the PKC/ERK signalling pathway, the effects of DOR on breast cancer were significantly attenuated in vivo and in vitro. In summary, DOR is highly expressed in breast cancer and is closely related to its progression. These results suggest that DOR may serve as a potential biomarker for the early diagnosis of breast cancer and may be a viable molecular target for therapeutic intervention.

The aim of this study was to investigate the immunological mechanisms by which synthetic methionine enkephalin (MENK) exerts therapeutic effects on tumor growth. Our findings in vivo or in vitro show that MENK treatment either in vivo or in vitro could up-regulate the percentages of CD8+T cells, induce markers of activated T cells, increased cytotoxic activity against mouse S180 tumor cells and increase secretion of IFNγ. In addition, the adoptively transferred CD8+T cells, after either in vitro or in vivo treatment with MENK, result in significantly increased survival of S180 tumor-bearing mice and significant shrinkage in tumor growth. Opioid receptors are detected on normal CD8+T cells and exposure to MENK leads to increased expression of opioid receptors. Interaction between MENK and the opioid receptors on CD8+T cells appears to be essential for the activation of CTL, since the addition of naltrexone (NTX), an opioid receptor antagonist, significantly inhibits all of the effects of MENK. The evidence obtained indicates that the MENK-induced T cell signaling is associated with a significant up-regulation of Ca2+ influx into the cytoplasm and the translocation of NFAT2 into nucleus, and these signaling effects are also inhibited by naltrexone., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Although biochemical and physiological evidence suggests a strong interaction between striatal CB1 cannabinoid (CB1 R) and D2 dopamine (D2 R) receptors, the mechanisms are poorly understood. We targeted medium spiny neurons of the indirect pathway using shRNA to knockdown either CB1 R or D2 R. Chronic reduction in either receptor resulted in deficits in gene and protein expression for the alternative receptor and concomitantly increased expression of the cannabinoid receptor interacting protein 1a (CRIP1a), suggesting a novel role for CRIP1a in dopaminergic systems. Both CB1 R and D2 R knockdown reduced striatal dopaminergic-stimulated [(35) S]GTPγS binding, and D2 R knockdown reduced pallidal WIN55212-2-stimulated [(35) S]GTPγS binding. Decreased D2 R and CB1 R activity was associated with decreased striatal phosphoERK. A decrease in mRNA for opioid peptide precursors pDYN and pENK accompanied knockdown of CB1 Rs or D2 Rs, and over-expression of CRIP1a. Down-regulation in opioid peptide mRNAs was followed in time by increased DOR1 but not MOR1 expression, leading to increased [D-Pen2, D-Pen5]-enkephalin-stimulated [(35) S]GTPγS binding in the striatum. We conclude that mechanisms intrinsic to striatal medium spiny neurons or extrinsic via the indirect pathway adjust for changes in CB1 R or D2 R levels by modifying the expression and signaling capabilities of the alternative receptor as well as CRIP1a and the DELTA opioid system., (© 2013 International Society for Neurochemistry.)

Calcium signaling plays a major role in the function of cells. Measurement of intracellular calcium mobilization is a robust assay that can be performed in a high-throughput manner to study the effect of compounds on potential drug targets. Pharmaceutical companies frequently use calcium signaling assays to screen compound libraries on G-protein-coupled receptors (GPCRs). In this chapter we describe the application of FLIPR technology to the evaluation of GPCR-induced calcium mobilization. We also include the implications of GPCR hetero-oligomerization and the identification of heteromeric receptors as novel drug targets on high-throughput calcium screening.

Methionine enkephalin (MENK), the endogenous neuropeptide, is known to exert direct effects on the neuroendocrine and the immune systems and participates in regulation of various functions of cells related to both the innate and adaptive immune systems. Dendritic cells (DCs) play important role in initiating and regulating T cell responses. The aim of this work is to investigate the effects of MENK on differentiation, maturation, and function of DCs derived from murine bone marrow progenitors (BM-derived DCs). Our result showed that MENK could induce BM-derived DCs to polarize predominantly to mDC subtype, rather than pDC both in vivo and in vitro, and this was in favor of Th1 response. BM-derived DCs, after treatment with MENK, up-regulated the expressions of MHC class II and key costimulatory molecules. Result by RT-PCR showed MENK could increase expressions of delta and kappa receptors on BM-derived DCs. Also MENK promoted BM-derived DCs to secret higher levels of proinflammatory cytokines of IL-12p70, TNF-α. Furthermore, differentiated BM-derived DCs treated with MENK displayed higher activity to induce allogeneic T cell proliferation and MENK also inhibited tumor growth in vivo and induced apoptosis of tumor cells in vitro. Thus, it is concluded that MENK could be an effective inducer of BM-derived DCs and might be a new therapeutic agent for cancer, as well as other immune handicapped disease. Also we may consider MENK as a potential adjuvant in vaccine preparation.

Certain cognitive deficits in schizophrenia have been linked to dysfunction of prefrontal cortical (PFC) γ-aminobutyric acid (GABA) neurons and appear neurodevelopmental in nature. Since opioids suppress GABA neuron activity, we conducted the first study to determine 1) whether the μ opioid receptor (MOR), δ opioid receptor (DOR), and opioid ligand proenkephalin are altered in the PFC of a large cohort of schizophrenia subjects and 2) the postnatal developmental trajectory in monkey PFC of opioid markers that are altered in schizophrenia. We used quantitative polymerase chain reaction to measure mRNA levels from 42 schizophrenia and 42 matched healthy comparison subjects; 18 monkeys chronically exposed to haloperidol, olanzapine, or placebo; and 49 monkeys aged 1 week-11.5 years. We found higher levels for MOR mRNA (+27%) in schizophrenia but no differences in DOR or proenkephalin mRNAs. Elevated MOR mRNA levels in schizophrenia did not appear to be explained by substance abuse, psychotropic medications, or illness chronicity. Finally, MOR mRNA levels declined through early postnatal development, stabilized shortly before adolescence and increased across adulthood in monkey PFC. In schizophrenia, higher MOR mRNA levels may contribute to suppressed PFC GABA neuron activity and might be attributable to alterations in the postnatal developmental trajectory of MOR signaling.

The hippocampal formation (HF) is an important site at which stress circuits and endogenous opioid systems intersect, likely playing a critical role in the interaction between stress and drug addiction. Prior study findings suggest that the stress-related neuropeptide corticotropin releasing factor (CRF) and the delta opioid receptor (DOR) may localize to similar neuronal populations within HF lamina. Here, hippocampal sections of male and cycling female adult Sprague-Dawley rats were processed for immunolabeling using antisera directed against the DOR and CRF peptide, as well as interneuron subtype markers somatostatin or parvalbumin, and analyzed by fluorescence and electron microscopy. Both DOR- and CRF-labeling was observed in interneurons in the CA1, CA3, and dentate hilus. Males and normal cycling females displayed a similar number of CRF immunoreactive neurons co-labeled with DOR and a similar average number of CRF-labeled neurons in the dentate hilus and stratum oriens of CA1 and CA3. In addition, 70% of DOR/CRF dual-labeled neurons in the hilar region co-labeled with somatostatin, suggesting a role for these interneurons in regulating perforant path input to dentate granule cells. Ultrastructural analysis of CRF-labeled axon terminals within the hilar region revealed that proestrus females have a similar number of CRF-labeled axon terminals that contain DORs compared to males but an increased number of CRF-labeled axon terminals without DORs. Taken together, these findings suggest that while DORs are anatomically positioned to modulate CRF immunoreactive interneuron activity and CRF peptide release, their ability to exert such regulatory activity may be compromised in females when estrogen levels are high., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

Both delta-opioid receptor (DOPr) and cannabinoid-2 receptor (CB2R) agonists attenuate neuropathic pain, but the precise mechanism implicated in these effects is not completely elucidated. We investigated whether nitric oxide synthesized by neuronal (NOS1) or inducible (NOS2) nitric-oxide synthases could modulate DOPr and/or CB2R antiallodynic and antihyperalgesic effects through the peripheral nitric oxide-cGMP-protein kinase G (PKG) pathway activation and affect their expression during neuropathic pain. In wild-type (WT) mice at 21 days after chronic constriction of sciatic nerve, we evaluated the effects of [d-Pen(2),d-Pen(5)]-enkephalin (DPDPE); (2-methyl-1-propyl-1H-indol-3-yl)-1-naphthalenylmethanone (JWH-015); and a NOS1 [N-[(4S)-4-amino-5-[(2-aminoethyl)amino]pentyl]-N'-nitroguanidine tris(trifluoroacetate) salt; NANT], NOS2 [l-N(6)-(1-iminoethyl)-lysine; l-NIL], l-guanylate cyclase [1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; ODQ], or PKG [(Rp)-8-(para-chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate; Rp-8-pCPT-cGMPs] inhibitor administered alone or combined. Expression of DOPr and CB2R mRNA in the spinal cord and dorsal root ganglia of naive and nerve-injured WT, NOS1-knockout (KO), and NOS2-KO mice, also was assessed. The subplantar administration of NANT, l-NIL, ODQ, or Rp-8-pCPT-cGMPs dose-dependently inhibited neuropathic pain and enhanced the local effects of DPDPE or JWH-015. Moreover, although the basal levels of DOPr and CB2R mRNA were similar between WT and NOS-KO animals, nerve injury only decreased (DOPr) or increased (CB2R) their expression in the dorsal root ganglia of WT and NOS2-KO mice, and not in NOS1-KO mice. Results suggest that inactivation of the nitric oxide-cGMP-PKG peripheral pathway triggered by NOS1 and NOS2 enhanced the peripheral actions of DOPr and CB2R agonists and that nitric oxide synthesized by NOS1 is implicated in the peripheral regulation of DOPr and CB2R gene transcription during neuropathic pain.

Sarco(endo)plasmic reticulum calcium ATPase (SERCA)2b maintains the cellular Ca(2+) homeostasis by transferring Ca(2+) from the cytosol to the lumen of the endoplasmic reticulum (ER). Recently, SERCA2b has also been shown to be involved in the biosynthesis of secreted and membrane proteins via direct protein-protein interactions, involving components of the ER folding and quality-control machinery, as well as newly synthesized G protein-coupled receptors. Here we demonstrate that the human delta opioid receptor (hdeltaOR) exists in a ternary complex with SERCA2b and the ER molecular chaperone calnexin. The interaction between SERCA2b and hdeltaOR in vivo did not require calnexin as it was independent of the C-terminal calnexin-interacting domain of SERCA2b. However, the receptor was able to mediate co-immunoprecipitation of calnexin with the C-terminally truncated SERCA2b. The association of SERCA2b with hdeltaOR was regulated in vitro by Ca(2+) and ATP in a manner that was opposite to the calnexin-hdeltaOR interaction. Importantly, co-expression of the catalytically inactive SERCA2b(D351A) or calnexin binding-compromised SERCA2bDeltaC mutants with the receptor decreased the expression of mature receptors in a manner that did not directly relate to changes in the ER Ca(2+) concentration. We conclude that dynamic interactions among SERCA2b, calnexin and the hdeltaOR precursor orchestrate receptor biogenesis and are regulated by Ca(2+) and ATP. We further hypothesize that the primary role of SERCA2b in this process is to act as a Ca(2+) sensor in the vicinity of active translocons, integrating protein folding with local fluctuations of ER Ca(2+) levels.

The delta opioid receptor (DOR) agonist [D-Ala2, D-Leu5] enkephalin (DADLE) has been implicated as a novel neuroprotective agent in the CNS. The current study was designed to evaluate the effects of intracerebroventricular (ICV) application of DADLE on neurological outcomes following asphyxial cardiac arrest (CA) in rats. Male Sprague-Dawley rats were randomly assigned to four groups: Sham group, CA group, DADLE group (DADLE+CA), and Naltrindole group (Naltrindole and DADLE+CA). All drugs were administered into the left cerebroventricle 30 min before CA. CA was induced by 8-min asphyxiation and the animals were resuscitated with a standardized method. DOR protein expression in the hippocampus was significantly increased in the CA group at 1 h after restoration of spontaneous circulation (ROSC) compared with the Sham group. As time progressed, expression of DOR proteins decreased gradually in the CA group. Treatment with DADLE alone or co-administration with Naltrindole reversed the down-regulation of DOR proteins in the hippocampus induced by CA at 24 h after ROSC. Compared with the CA group, the DADLE group had persistently better neurological functional recovery, as assessed by neurological deficit score (NDS) and Morris water maze trials. The number of surviving hippocampal CA1 neurons in the DADLE group was significantly higher than those in the CA group. However, administration of Naltrindole abolished most of the neuroprotective effects of DADLE. We conclude that ICV administration of DADLE 30 min before asphyxial CA has significant protective effects in attenuating hippocampal CA1 neuronal damage and neurological impairments, and that DADLE executes its effects mainly through DOR., (2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

By sustained activation of mu-opioid receptors (MORs), chronic opioids cause analgesic tolerance, physical dependence, and opioid addiction, common clinical problems for which an effective treatment is still lacking. Chronic opioids recruit delta-opioid receptors (DORs) to plasma membrane through exocytotic trafficking, but the role of this new DOR and its interaction with existing MOR in brain functions and in these clinical problems remain largely unknown. In this study, we investigated the mechanisms underlying synaptic and behavioral actions of chronic morphine-induced DORs and their interaction with MORs in nucleus raphe magnus (NRM) neurons important for opioid analgesia. We found that the emerged DOR inhibited GABAergic IPSCs through both the phospholipase A(2) (PLA(2)) and cAMP/protein kinase A (PKA) signaling pathways. MOR inhibition of IPSCs, normally mediated predominantly by the PLA(2) pathway, was additionally mediated by the cAMP/PKA pathway, with MOR potency significantly increased after chronic morphine treatment. Isobologram analysis revealed a synergistic DOR-MOR interaction in their IPSC inhibition, which was dependent on upregulated activities of both the PLA(2) and cAMP/PKA pathways. Furthermore, DOR and MOR agonists microinjected into the NRM in vivo also produced a PLA(2)-dependent synergism in their antinociceptive effects. These findings suggest that the cAMP/PKA pathway, upregulated by chronic opioids, becomes more important in the mechanisms of both MOR and DOR inhibition of GABA synaptic transmission after chronic opioid exposure, and DORs and MORs are synergic both synaptically and behaviorally in producing analgesic effects in a PLA(2)-dependent fashion, supporting the potential therapeutic use of DOR agonists in pain management under chronic opioid conditions.

Tricyclic antidepressants (TCAs) have been reported to interact with the opioid system, but their pharmacological activity at opioid receptors has not yet been elucidated. In the present study, we investigated the actions of amoxapine, amitriptyline, nortriptyline, desipramine, and imipramine at distinct cloned and native opioid receptors. In Chinese hamster ovary (CHO) cells expressing delta-opioid receptors (CHO/DOR), TCAs displaced [3H]naltrindole binding and stimulated guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding at micromolar concentrations with amoxapine displaying the highest potency and efficacy. Amoxapine and amitriptyline inhibited cyclic AMP formation and induced the phosphorylation of signaling molecules along the extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphatidylinositol-3 kinase pathways. Amoxapine also activated delta-opioid receptors in rat dorsal striatum and nucleus accumbens and human frontal cortex. In CHO cells expressing kappa-opioid receptors (CHO/KOR), TCAs, but not amoxapine, exhibited higher receptor affinity and more potent stimulation of [(35)S]GTPgammaS binding than in CHO/DOR and effectively inhibited cyclic AMP accumulation. Amitriptyline regulated ERK1/2 phosphorylation and activity in CHO/KOR and C6 glioma cells endogenously expressing kappa-opioid receptors, and this effect was attenuated by the kappa-opioid antagonist nor-binaltorphimine. In rat nucleus accumbens, amitriptyline slightly inhibited adenylyl cyclase activity and counteracted the inhibitory effect of the full kappa agonist trans-(-)-3,4dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide (U50,488). At the cloned mu-opioid receptor, TCAs showed low affinity and no significant agonist activity. These results show that TCAs differentially regulate opioid receptors with a preferential agonist activity on either delta or kappa subtypes and suggest that this property may contribute to their therapeutic and/or side effects.

The opioid system is known to play a role in various aspects of learning and memory in diverse species of mammals and birds. Earlier studies have localized the endogenous opioids, met- and leu-enkephalin, in the song control regions of male zebra finches (Taenopygia guttata), a sexually dimorphic species of songbirds wherein only males sing. Recent research has shown that levels of enkephalin increase in some of the song control regions during singing and that blocking opioid receptors with the antagonist naloxone decreases the frequency of singing in songbirds. However, the distribution of receptors specific to the opioid system has not been studied in zebra finches. In the present study, we used reverse transcriptase PCR (RT-PCR) and quantitative real-time PCR (qRT-PCR) to demonstrate that the song control regions lateral magnocellular nucleus of the anterior nidopallium (LMAN), Area X, MSt (medial striatum), HVC and RA (robust nucleus of the arcopallium) expressed higher levels of mu-OR mRNA compared to delta-OR mRNA. In situ hybridization was used to demonstrate that neither LMAN nor Area X could be delineated from the surrounding brain regions [anterior nidopallium (ANP) and MSt, respectively], based on OR mRNA expression. However, HVC and RA neurons expressed marginally higher levels of mu-OR mRNA compared to the posterior nidopallium, which was confirmed by immunohistochemical localization. We also found that the dorsolateral subdivision of DLM (dorsolateral nucleus of the medial thalamus) demonstrated high levels of mu-OR immunoreactivity. Our results suggest that the ORs may be involved in modulating different aspects of vocalization and/or gating auditory input, as well as motor control.

Activation of micro-opioid receptors makes animals hyperphagic and increases their preference for a high-fat diet. Previous studies have suggested that this receptor population plays a role in mediating the hyperphagia that is associated with food deprivation. In this paper, we tested the hypothesis that food deprivation will increase the expression of micro-opioid receptors in the ventral medial hypothalamus and arcuate nucleus (VMH/ARC). Food deprivation resulted in a significant increase in the mRNA expression of micro-opioid receptors in the VMH/ARC and the lateral hypothalamus (LH) after 48 h of fasting but not after 24 or 12 h of fasting in either the light or dark. We did not observe a change in the mRNA expression of kappa- or delta-opioid receptors after food deprivation. When food-deprived animals were given a choice between a low-fat diet and a high-fat diet, they were hyperphagic and consumed significantly more of the high-fat diet. When the micro-opioid receptors were blocked with beta-funaltrexamine (selective mu-opioid receptor antagonist), prior to giving food-deprived animals access to both a low-fat and high-fat diet, it significantly decreased the percentage of high-fat diet consumed. These data demonstrate that hypothalamic micro-opioid receptors may contribute to the hyperphagia and increased preference for a high-fat diet that is associated with food deprivation.

Pharmacological and behavioral studies suggest that spinal delta- and kappa-opioid antinociceptive systems are functionally associated with ovarian sex steroids. These interactions can be demonstrated specifically during pregnancy or hormone-simulated pregnancy (HSP). The analgesia associated with both conditions can be abolished by blockade of either spinal kappa-opioid receptors or delta-opioid receptors (DOR). Furthermore, both dynorphin (DYN) release (J Pharmacol Exp Ther 298:1213-1220, 2001) and the processing of the DYN precursor (J Neurochem 65:1374-1380, 1995) are significantly increased in the spinal cord during HSP. We undertook the current study to determine whether DYN, DOR, and estrogen receptor alpha (ERalpha) share anatomical relationships that permit their direct interaction. Coexpression of DOR or ERalpha by DYN neurons was assessed using fluorescence immunohistochemistry and a synaptosomal release assay. Findings show that ERalpha and DYN are coexpressed. Moreover, in the spinal cord of HSP animals, there were significant increases in the number of DYN-immunoreactive (DYN-ir) cells, ERalpha-ir cells, cells double-labeled for DYN-ir and ERalpha-ir and the proportion of DYN-ir cells coexpressing ERalpha. Some varicose fibers in the spinal cord dorsal horn and intermediate gray matter that expressed DYN-ir also expressed DOR-ir. Activation of DORs located on DYN terminals was sufficient to inhibit K(+)-evoked DYN release. These data define, at least in part, the anatomical substrates that may be relevant to the antinociception of gestation and its hormonal simulation. Furthermore, they provide a framework for understanding sex-based nociception and antinociception and suggest novel strategies for treating pain.

Endogenous opioid peptides are locally produced at the inflammatory site where antigens are captured and processed by dendritic cells (DCs). Subsequently, maturing DCs migrate towards draining lymph nodes to initiate T cell response. Given the primordial role of DCs in adaptive immune response, we examined whether opioids may affect the migratory response of DCs. We found that the delta opioid receptor (DOR) mRNA was expressed at low level in bone marrow-derived immature DCs and up-regulated upon DC maturation. Moreover, DOR agonists triggered DC chemotaxis in vitro. In vivo, enkephalins prevented the egress of mature DCs injected into the peritoneal cavity of normal mice. This effect was inhibited by blocking opioid receptors on mature DCs. The cross-talk between CCR7 and DOR receptors that are both up-regulated during DC maturation was then examined. Whereas opioids did not alter the migratory responsiveness to CCR7 ligands, DOR-mediated mobilization of mature DCs was inhibited by CCL19 and CCL21 suggesting that the opioid chemotactic activity decreases as the concentration of the chemokines increases.

Dimerization has emerged as a common mechanism for regulating the function of G protein-coupled receptors (GPCR). Among these are chemokine receptors, which detect various chemokines and regulate a range of physiological process, including immune cell trafficking, cancer cell migration, and neuronal patterning. Homo- and heterodimerization in response to chemokine binding has been shown to be required for the initiation or alteration of signaling by a number of chemokine receptors. In this issue of the European Journal of Immunology, a new study indicates that the formation of heterodimers of chemokine receptor CXCR4 and the delta-opioid receptor (DOR) prevents each of them from actively signaling, suggesting a novel mechanism for silencing GPCR function.

Zebrafish is a novel experimental model that has been used in developmental studies as well as in the study of pathological processes involved in human diseases. It has been demonstrated that the endogenous opioid system is involved in developmental mechanisms. We have studied the relationship between the different embryonic stages and opioid receptor expression for the four known opioid receptors in zebrafish (mu, delta 1, delta 2 and kappa). The mu opioid receptor is detected at higher levels than the other opioid receptors before the midblastula transition and during the segmentation period. The delta duplicate 2 exhibits only one peak of expression at 21 h postfertilization (hpf), when the motor nervous system is forming. The kappa receptor is expressed at very low levels. In situ hybridization studies at 24 hpf show that the opioid receptors are widely distributed in zebrafish CNS and at 48 hpf their localization is detected in more defined structures. Our results support specific implications of the opioid receptors in developmental processes such as morphogenesis of the CNS, neurogenesis, neuroprotection and development of neuromuscular and digestive system. Pain-related alterations can be a consequence of changes in the endogenous opioid system during development, hence we provide important information that might help to solve pain-related pathological situations.

This study was designed to test the hypothesis that inhibition of agonist-induced delta-receptor down-regulation would block the development of opioid tolerance in a cell-based model. A human embryonic kidney 293 cell line was established that expressed an epitope-tagged delta-opioid receptor (DOR). Treatment of DOR cells with Tyr-d-Ala-Gly-Phe-d-Leu-enkephalin (DADL) resulted in a time-dependent decrease in the B(max) of delta-opioid receptor binding sites and immunoreactive receptor protein. When cells were coincubated with the proteasome inhibitor N-benzyloxycarbonyl-l-leucyl-l-leucyl-l-leucinal (ZLLL) and DADL, the magnitude of the agonist-induced decrease in B(max) and immunoreactive receptor protein was reduced compared with DADL treatment alone. Acute treatment of DOR cells with DADL caused a 3-fold increase in the level of phosphorylated mitogen-activated protein (MAP) kinase. Prior exposure of DOR cells to DADL completely abrogated the agonist-induced activation of MAP kinase. When DOR cells were coincubated with DADL and ZLLL, the proteasome inhibitor prevented the loss of agonist activation of MAP kinase. Acute treatment of DOR cell membranes with DADL stimulated [(35)S]guanosine 5'-3-O-(thio-)triphosphate (GTPgammaS) binding. When DOR cells were preincubated with DADL, the agonist-induced increase in [(35)S]GTPgammaS binding was attenuated. Coincubation of ZLLL and agonist partially prevented the decreased responsiveness to agonist stimulation. The results of this study demonstrated that inhibition of agonist-induced down regulation with a proteasome inhibitor attenuated opioid tolerance in a cellular model, and suggest that coadministration of a proteasome inhibitor with chronic opioid agonist treatment may be useful for limiting opioid tolerance in vivo.

Knockout and pharmacological studies demonstrated that the activation of delta opioid peptide (DOP) receptors produces antidepressant-like effects in rodents. Here we report the results obtained with the novel DOP ligand H-Dmt-Tic-NH-CH(2)-Bid (UFP-502). UFP-502 bound with high affinity (pK(i) 9.43) to recombinant DOP receptors displaying moderate selectivity over MOP and KOP. In CHO(hDOP) [(35)S]GTPgammaS binding and mouse vas deferens experiments, UFP-502 behaved as a potent (pEC(50) 10.09 and 10.70, respectively) full agonist. In these preparations, naloxone, naltrindole and N,N(CH(3))(2)Dmt-Tic-OH showed similar pA(2) values against UFP-502 and DPDPE and the same rank order of potency. In vivo in mice, UFP-502 mimicked DPDPE actions, producing a significant reduction of immobility time after intracerebroventricular administration in the forced swimming test and a clear antinociceptive effect after intrathecal injection in the tail withdrawal assay. However, while the effects of DPDPE were fully prevented by naltrindole those evoked by UFP-502 were unaffected (tail withdrawal assay) or only partially reversed (forced swimming test). In conclusion, UFP-502 represents a novel and useful chemical template for the design of selective agonists for the DOP receptor.

We report the computer-aided design, chemical synthesis, and biological evaluation of a novel family of delta opioid receptor (DOR) antagonists containing a 1,2,4-triazole core structure that are structurally distinct from other known opioid receptor active ligands. Among those delta antagonists sharing this core structure, 8 exhibited strong binding affinity (K(i) = 50 nM) for the DOR and appreciable selectivity for delta over mu and kappa opioid receptors (delta/mu = 80; delta/kappa > 200).

Glutamate transporters play an essential role in regulating temporal and spatial glutamate concentration in the synaptic cleft. The excitatory amino acid carrier 1 (EAAC1) is a neuronal-specific transporter. Opioid receptors are found to modulate the glutamatergic system in brain, but the regulatory mechanism remains to be investigated. Here we report that the delta-opioid receptor (DOR) co-expressed with EAAC1 in Xenopus oocytes, but not the micro-opioid receptor, down-regulates EAAC1 function, and that [d-Pen(2,5)]-enkephalin stimulation of DOR can counteract the down-regulation of the EAAC1-mediated uptake. Results from co-immunoprecipitation and immunofluorescence microscopy in both oocytes and rat hippocampal neurons indicate co-localization and suggest direct interaction between DOR and EAAC1. The results suggest that DOR can reduce EAAC1 function by direct protein-protein interaction and that activation of DOR releases the inhibitory interaction. We suggest that this mechanism may be important for modulation of the glutamatergic system following opioid stimulation.

The present study demonstrates that perikaryaldelta-opioid receptors (deltaORs) in rat dorsal root ganglion (DRG) neurons bind and internalize opioid ligands circulating in the CSF. Using confocal and electron microscopy, we found that prolonged morphine treatment increased the cell surface density of these perikaryal deltaORs and, by way of consequence, receptor-mediated internalization of the fluorescent deltorphin (DLT) analog omega-Bodipy 576/589 deltorphin-I 5-aminopentylamide (Fluo-DLT) in all three types of DRG neurons (small, medium, and large). In contrast, chronic inflammatory pain induced by the injection of complete Freund's adjuvant (CFA) into one hindpaw selectively increased Fluo-DLT internalization in small and medium-sized DRG neurons ipsilateral to the inflammation. Based on our previous studies in the spinal cord of mu-opioid receptor (muOR) knock-out mice, it may be assumed that the enhanced membrane recruitment of deltaORs observed after sustained morphine is attributable to stimulation of muORs. However, the selectivity of the effect induced by inflammatory pain suggests that it involves a different mechanism, namely a modality-specific and pain-related activation of C and Adelta fibers. Indeed, stimulation by capsaicin of transient receptor potential vanilloid 1 receptors, which are selectively expressed by small diameter (< 600 microm2) DRG neurons, increased Fluo-DLT internalization exclusively in this cell population. The present results, therefore, demonstrate that DRG neurons express perikaryal deltaORs accessible to CSF-circulating ligands and that the density and, hence, presumably also the responsiveness, of these receptors may be modulated by both pain-related stimuli and sustained exposure to muOR agonists.

The study evaluates the effects of kappa- (KOR), delta- (DOR), and mu-opioid receptor (MOR) agonists on the inhibition of plasma extravasation during acute and chronic intestinal inflammation in mice. The antiexudative effects of KOR and DOR agonists in animals treated with nitric oxide synthase (NOS) inhibitors and their protein levels in the gut (whole jejunum and mucosa) and spinal cord of mice with chronic intestinal inflammation were also measured. Inflammation was induced by the intragastric administration of one (acute) or two (chronic) doses of croton oil. Plasma extravasation was measured using Evans blue and protein levels by Western blot and immunoprecipitation. Plasma extravasation was significantly increased 2.7 times during chronic inflammation. The potency of the KOR agonist trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolydinyl)cyclohexyl]-benzeneazetamine (U50,488H) inhibiting plasma extravasation was enhanced 26.3 times during chronic compared with acute inflammation. [d-Pen(2),d-Pen(5)]-Enkephalin (DPDPE) (a DOR agonist) was also 11.8 times more potent during chronic inflammation, whereas the antiexudative effects of fentanyl (a MOR agonist) were not significantly altered. Receptor-specific antagonists reversed the effects. Protein levels of KOR and DOR in the whole jejunum and mucosa were significantly increased after chronic inflammation. Treatment with NOS inhibitors N(omega)-nitro-l-arginine methyl ester or l-N(6)-(1-iminoethyl)-lysine hydrochloride diminished plasma extravasation and inhibited the increased antiexudative effects of U50,488H and DPDPE during chronic intestinal inflammation. The data show that the enhanced antiexudative effects of KOR and DOR agonists could be related to an increased expression of KOR and DOR in the gut and that the release of nitric oxide may play a role augmenting the effects of opioids during chronic inflammation.

The development of neuropathic pain is associated with multiple changes in gene expression occurring in the dorsal root ganglia (DRG) and spinal cord. The goal of this study was to evaluate whether the disruption of CB1 cannabinoid receptor gene modulates the changes induced by neuropathic pain in the expression of mu- (MOR), delta- (DOR) and kappa-opioid receptors (KOR) mRNA levels in the DRG and spinal cord. The induction of c-fos expression in the lumbar and sacral regions of the spinal cord was also evaluated in these animals. Opioid receptors mRNA levels were determined by using real-time PCR and Fos protein levels by immunohistochemistry. Nerve injury significantly reduced the expression of MOR in the DRG and the lumbar section of the spinal cord from CB1 cannabinoid knockout (KO) mice and wild-type littermates (WT). In contrast, mRNA levels of DOR and KOR were not significantly changed in any of the different sections analysed. Furthermore, sciatic nerve injury evoked a similar increase of c-fos expression in lumbar and sacral regions of the spinal cord of both KO and WT. In all instances, no significant differences were observed between WT and KO mice. These data revealed specific changes induced by neuropathic pain in MOR expression and c-fos levels in the DRG and/or spinal cord that were not modified by the genetic disruption of CB1 cannabinoid receptors.

Mice lacking Na+/H+ exchanger 1 (NHE1) show a unique epilepsy phenotype although the underlying mechanisms remain unclear. Since expression of delta-opioid receptor (DOR) may be involved in control of epileptic activity, we conducted immunohistochemistry and autoradiography to investigate whether DOR expression is dys-regulated in the brain of NHE1 null mouse. Immunohistochemistry showed a decline in DOR expression in hippocampus and cortex. Autoradiographic results confirmed that the density of DOR was decreased in most cortical and hippocampal regions such as striate and temporal cortex, hippocampal CA1 and CA3 regions (reduced by 27.7 +/- 6.4%, 29.4 +/- 5.1%, 40.7 +/- 4.4% and 20.6 +/- 5.7%, respectively, P < 0.05). These data demonstrate that NHE1 null mutation leads to a reduction of DOR expression in the cortical and hippocampal regions, which provides a new clue for the genesis of epilepsy.

Objective: To investigate the expression and regulation of the kappa-opioid receptor (KOR) and the delta-opioid receptor (DOR) in fibroblast-like synoviocytes (FLS) from patients with osteoarthritis (OA) and rheumatoid arthritis (RA), and to explore the potential antiarthritic mechanisms of peripheral KORs., Methods: FLS isolated from synovial tissues of 6 OA patients, 8 RA patients, and 2 healthy individuals were exposed to the selective KOR agonist U69593, the selective DOR agonist SNC 80, and kappa-opioid dynorphin A in the presence or absence of the KOR antagonist nor-binaltorphimine, the DOR antagonist naltrindole, and the proinflammatory cytokines tumor necrosis factor alpha (TNFalpha) and interleukin-1beta (IL-1beta). The expression of KOR and DOR in OA and RA FLS was evaluated on the messenger RNA (mRNA) and protein levels with TaqMan real-time reverse transcriptase-polymerase chain reaction and immunofluorescence staining, respectively. KOR/DOR-mediated activation of ERK-1 and ERK-2 was investigated by Western blotting., Results: We detected functional KOR and DOR in normal FLS and observed a reduction of both receptors in OA and RA FLS, which was more distinct in RA FLS. U69593 enhanced KOR mRNA expression in both OA and RA FLS in a KOR antagonist-reversible manner. However, the dose required for maximal enhancement in RA FLS was 10 times higher than that required in OA FLS. TNFalpha and IL-1beta both suppressed the expression of DOR and KOR mRNA in both OA and RA FLS., Conclusion: DOR and KOR are constitutively present in normal FLS and are suppressed under inflammatory conditions, such as RA and OA. Most interestingly, the KOR agonist U69593 may exert an antiarthritic effect via up-regulation of KOR in OA and RA FLS.

It has previously been suggested that exogenous growth hormone (GH) affect quality of life and higher brain functions through the endogenous opioid system. Recently, we showed that GH down-regulate 72 and 48 kDa delta opioid receptor (DOR) proteins in the adult rat cerebral cortex and cerebellum. In the present study, we found that an antiserum raised against the N-terminus of the DOR also recognizes a 36 kDa protein, not recognized by a C-terminus-directed antiserum. We aimed to investigate the identity of the 72, 48 and 36 kDa proteins and to further study the effects of GH on their expression in different brain regions. The expression was studied in hypophysectomized (Hx) and untreated normal female rats. One subgroup of Hx rats received GH as a daily subcutaneous injection for 19 days. Our data show that treatment with GH in Hx rats normalized the expression of the 72 kDa protein in the cerebral cortex, whereas no significant effect were observed for the 48 or 36 kDa proteins. However, GH significantly reduced the ratio between the 72 and 36 kDa proteins in different brain regions of Hx rats. Our data suggest that GH reduces the levels of a 72 kDa DOR that likely represents a dimeric form of a 36 kDa DOR post-translationally truncated at the C-terminus, and that altered receptor dimerization may be involved in GH induced effects in the central nervous system.

Background: Neuropeptide FF (NPFF) plays a role in physiological pain sensation and opioid analgesia. For example, NPFF potentiates opiate-induced analgesia and the delta opioid receptor antagonist naltrindole inhibits NPFF-induced antinociception. The nature of the interactions between NPFF and opioid receptors seems to be complex and the molecular mechanisms behind the observed physiological effects are not known., Results: We used a stable Chinese hamster ovary cell line expressing c-MYC-tagged human delta opioid receptor to study the interactions at the molecular level. Our results imply that NPFF can directly modulate the activation of delta opioid receptor in the absence of NPFF receptors. The modulatory effect, though only moderate, was consistently detected with several methods. The agonist-induced receptor trafficking was changed in the presence of (1DMe)NPYF, a stable NPFF-analogue. (1DMe)NPYF enhanced the receptor activation and recovery; opioid antagonists inhibited the effects, indicating that they were delta opioid receptor-mediated. The binding experiments with a novel ligand, Terbium-labeled deltorphin I, showed that (1DMe)NPYF modulated the binding of delta opioid receptor ligands. The levels of phosphorylated mitogen-activated protein kinase and intracellular cAMP were studied to clarify the effects of NPFF on the opioid signaling mechanisms. Application of (1DMe)NPYF together with a delta opioid receptor agonist enhanced the signaling via both pathways studied. Concomitantly to the receptor trafficking, the time-course of the activation of the signaling was altered., Conclusion: In addition to working via indirect mechanisms on the opioid systems, NPFF may exert a direct modulatory effect on the delta opioid receptor. NPFF may be a multi-functional neuropeptide that regulates several neuronal systems depending on the site of action.

An in vivo fluorescent deltorphin (Fluo-DLT) internalization assay was used to assess the distribution and regulation of pharmacologically available delta opioid receptors (deltaORs) in the rat lumbar (L4-5) spinal cord. Under basal conditions, intrathecal injection of Fluo-DLT resulted in the labeling of numerous deltaOR-internalizing neurons throughout dorsal and ventral horns. The distribution and number of Fluo-DLT-labeled perikaryal profiles were consistent with that of deltaOR-expressing neurons, as revealed by in situ hybridization and immunohistochemistry, suggesting that a large proportion of these cells was responsive to intrathecally administered deltaOR agonists. Pretreatment of rats with morphine for 48 hr resulted in a selective increase in Fluo-DLT-labeled perikaryal profiles within the dorsal horn. These changes were not accompanied by corresponding augmentations in either deltaOR mRNA or (125)I-deltorphin-II binding levels, suggesting that they were attributable to higher densities of cell surface deltaOR available for internalization rather than to enhanced production of the receptor. Unilateral dorsal rhizotomy also resulted in increased Fluo-DLT internalization in the ipsilateral dorsal horn when compared with the side contralateral to the deafferentation or to non-deafferented controls, suggesting that deltaOR trafficking in dorsal horn neurons may be regulated by afferent inputs. Furthermore, morphine treatment no longer increased Fluo-DLT internalization on either side of the spinal cord after unilateral dorsal rhizotomy, indicating that microOR-induced changes in the cell surface availability of deltaOR depend on the integrity of primary afferent inputs. Together, these results suggest that regulation of deltaOR responsiveness through microOR activation in this region is linked to somatosensory information processing.

It has been widely accepted that repeated administration of kappa-opioid receptor agonists leads to the development of antinociceptive tolerance. The present study was designed to investigate the effect of repeated administration of a selective kappa-opioid receptor agonist (1S-trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide hydrochloride ((-)U-50,488H) on the mu- and delta-opioid receptor agonist-induced antinociception and G-protein activation in mice. The mice were injected either subcutaneously (s.c.) or intracerebroventricularly (i.c.v.) pretreated with saline or (-)U-50,488H once a day for seven consecutive days. Two hours after the last injection, the mice were challenged by either mu- or delta-opioid receptor agonist for the antinociceptive assay. Repeated treatment with (-)U-50,488H (s.c. or i.c.v.) significantly enhanced antinociceptive effect of both mu-opioid receptor agonist (morphine) and delta-opioid receptor agonists ([d-Ala2]deltorphin (DELT) and (+)-4-[(alphaR)-alpha-((2S,5R)-4-allyl-2,5-dime thyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC-80) compared to saline-treated groups. Under these conditions, repeated s.c. injection of (-)U-50,488H significantly enhanced both mu- and delta-opioid receptor agonist-stimulated [35S]GTPgammaS binding in the membrane of the thalamus. On the contrary, either repeated administration of morphine (s.c. or i.c.v.) or SNC-80 failed to affect the kappa-opioid receptor agonist-induced antinociception and G-protein activation. Taken together, these results suggest that repeated stimulation of kappa-opioid receptor markedly increases the functional mu- and delta-opioid receptors, whereas repeated stimulation of either mu- or delta-opioid receptor had no direct effect on kappa-opioidergic function in mice.

Opioids are thought to control the excitability of hippocampal principal neurons indirectly by inhibiting GABAergic interneurons. However, direct inhibition of hippocampal principal neurons by opioids has also been reported. To understand better the neuromodulatory role of opioids in rat hippocampal circuits, we analyzed types of micro- and delta-opioid receptor (MOR, DOR)-expressing hippocampal neurons. Most MOR-immunoreactive neurons in the granular and pyramidal cell layers exhibited multipolar morphologies characteristic of GABAergic neurons. Virtually all neurons in the hippocampal formation expressing high MOR mRNA levels cocontained the mRNA for glutamic acid decarboxylase (GAD). Most parvalbumin-, several calretinin-, and several pre-proenkephalin-containing neurons expressed the MOR gene in the hippocampal formation. Expression of high DOR mRNA levels was restricted to GAD-positive neurons in the principal cell layers, oriens layer and hilus. More than 90% of the parvalbumin-positive neurons in the hippocampal formation strongly expressed the DOR gene. Granule cells expressing vesicular glutamate transporter 1 (VGLUT1) mRNA contained very low MOR and DOR transcript levels. In VGLUT1-positive pyramidal cells, weak DOR but no MOR gene expression was detected. Whereas most somatostatinergic hilar neurons were negative for MOR and DOR mRNA, somatostatinergic oriens layer neurons frequently expressed these receptors. Taken together, weak expression of MOR and DOR genes in hippocampal principal cells is in concordance with direct opioid-mediated inhibition of principal cells. However, strong expression of the MOR and DOR genes in the hippocampus is restricted to gamma-aminobutyric acid (GABA)ergic neurons, with DORs being selectively expressed in the parvalbumin- and somatostatin-containing subpopulations. Activation of MOR and/or DOR in parvalbumin- and somatostatin-containing neurons, which provide GABAergic inhibition to the perisomatic and distal dendritic regions of principal cells, respectively, is likely to facilitate principal cell excitation., (Copyright 2003 Wiley-Liss, Inc.)

To assess the validity of rodent models for investigating the role of delta opioid receptors (DOR) in analgesia, the distribution of DOR binding and mRNA were compared between rodent and primate spinal cord and dorsal root ganglia (DRG), using receptor autoradiography and in situ hybridization, respectively. In mouse and rat spinal cord, [(125)I]-deltorphin-labeled DOR binding sites were detected throughout the gray matter. In contrast, in primate and particularly in human spinal cord, DOR binding was mainly present in laminae I-II, with little to no binding in deeper layers. Accordingly, in rodent spinal cord, DOR mRNA was expressed by a large number of neurons distributed throughout the ventral and dorsal horns, whereas in the primate, DOR expression was significantly lower, as evidenced by a moderate number of labeled cells throughout the gray matter in monkey and by only few labeled cells in human, mainly in Clarke's column and lamina IX. Major species differences in DOR expression were also observed in primary afferent cells bodies. In rat DRG, intense DOR mRNA hybridization was primarily observed over large ganglion cells immunopositive for neurofilament 200. In contrast, in monkey and human DRG, DOR mRNA was primarily detected over small and medium-sized ganglion cells. These results demonstrate major differences in the expression and distribution of DOR in the spinal cord and DRG between mammalian species. Specifically, they point to a progressive specialization of DOR toward the regulation of primary somatosensory, namely nociceptive, inputs during phylogeny and suggest that the effects of DOR agonists in rodents may not be entirely predictive of their action in humans., (Copyright 2003 Wiley-Liss, Inc.)

Opioid receptors are expressed in a cell type-specific manner. Here we show that the mouse delta-opioid receptor (mDOR) gene is regulated by promoter region CpG methylation. The mDOR promoter containing a putative CpG island is highly methylated in Neuro2A cells, correlating with the repression of this gene in these cells. This is in contrast with the unmethylated state of the mDOR promoter in NS20Y cells, which express a high level of mDOR. Repression of mDOR transcription in Neuro2A cells could be partially relieved by chemically induced demethylation with 5-aza-2'-deoxycytidine. In addition, in vitro methylation of the luciferase reporter gene driven by the mDOR promoter resulted in an inhibition of transcription in NS20Y cells. Methyl-CpG-binding protein complex 1 (MeCP1) has been implicated in methylation-mediated transcriptional repression of several genes. Electrophoretic mobility shift assays showed that fully methylated, but not unmethylated, mDOR promoter fragment formed a MeCP1-like protein complex that contained methyl-CpG-binding domain protein 2 (MBD2) and Sp3. Furthermore, the expression level of Sp3 was decreased when Neuro2A cells were demethylated with 5-aza-2'-deoxycytidine, and increasing Sp3 levels in Schneider's Drosophila line 2 cells led to the repression of mDOR promoter activity when the promoter was methylated. These results demonstrate that Sp3 and MBD2 are involved in the transcriptional repression of mDOR in Neuro2A cells through binding to the methylated CpG sites in the promoter region and may play a role in the cell type-specific expression of mDOR.

Linuron is an herbicide with weak androgen receptor (AR) antagonist activity. Exposure to linuron from gestation days (GD) 12 to 21 perturbs androgen-dependent male reproductive development. In utero exposure to 50-mg/kg/day linuron induces malformations of the epididymis and the vas deferens. The objective of this study was to identify alterations in gene expression within the testis and epididymis associated with abnormal Wolffian duct development and to correlate changes in gene expression with the gross morphology of the affected epididymides. Pregnant Sprague-Dawley rats were administered either corn oil vehicle or linuron (50 mg/kg/day) by gavage from GD 12 to 21 (n = 3-6 controls, n = 5-10 linuron-treated dams per time point). Changes in gene expression were evaluated in testes on GD 21 and in epididymides on GD 21 and postnatal day (PND) 7, using cDNA microarrays and confirmed by real-time reverse transcriptase polymerase chain reaction (RT-PCR) analyses. RNA was isolated from intact epididymides with reduced or no ductal coiling from the linuron groups, and epididymides with noncontiguous ducts were excluded. In the fetal testis, exposure to linuron did not result in reduced mRNA expression of the AR or that of several steroidogenic enzymes, supporting the hypothesis that linuron does not reduce fetal testosterone production. Linuron induced a significant decrease in AR mRNA expression in GD 21 epididymides. Significant changes in mRNA expression in GD 21 and PND 7 epididymides were also identified in the epidermal growth factor (EGF), insulin-like growth factor 1 (IGF-1), bone morphogenetic protein (BMP), fibroblast growth factor (FGF), and Notch signaling pathways. These pathways are involved in tissue morphogenesis. Changes in the expression of AR and IGF-1 receptors were detected by immunostaining in malformed epididymides from linuron-exposed rats. Linuron induced changes in epididymal gene expression suggestive of altered paracrine interactions between the mesenchyme and epithelial cells during development. The EGF, Notch, IGF-1, BMP4, and FGF signaling pathways may be involved in normal testosterone-mediated development of the Wolffian duct.

Repeated amphetamine administration results in behavioral sensitization, an enduring behavioral transformation expressed after short and long periods of withdrawal. To investigate the participation of the opioid system in amphetamine-induced behavioral sensitization, we studied the effect of naloxone, an opioid receptor antagonist, on the expression of behavioral sensitization tested after short- (2 days) and long-term (14 days) withdrawal periods. In addition, using quantitative competitive RT-PCR, we examined the levels of mu-opioid receptor (MOR) and delta-opioid receptor (DOR) mRNA in the nucleus accumbens shell (NAcSh) and ventral tegmental area (VTA) of behaviorally sensitized rats, at these two withdrawal times. This study showed that whereas naloxone did not modify the expression of behavioral sensitization tested after 2 days of withdrawal, it completely blocked the expression when tested after 14 days of withdrawal. DOR and MOR mRNA levels were not modified in the NAcSh of rats expressing behavioral sensitization after 2 or 14 days of withdrawal. Conversely, DOR and MOR mRNA levels were elevated in the VTA of animals expressing behavioral sensitization after 2 days of withdrawal. However, whereas DOR mRNA returned to control levels, MOR mRNA levels remained elevated in animals expressing behavioral sensitization after 14 days of withdrawal. These results indicate a striking difference between the role played by opioid receptors in the expression of amphetamine-induced behavioral sensitization, when tested after short- or long-term withdrawal periods. In addition, our results support the notion that repeated amphetamine-induced changes in opioid receptor expression may contribute to the perpetuation of psychostimulant abuse and/or relapse.

Hormones released from the pituitary have been shown to regulate the expression of different proteins in the central nervous system. We wanted to examine whether peripheral administration of bovine growth hormone (bGH) regulates the expression of delta-opioid receptor (DOR) in the cerebral cortex and cerebellum. Expression of the DOR protein was quantified using Western blot densitometry. DOR mRNA was quantified with a solution hybridization RNase protection assay. Hypophysectomized (Hx) and untreated normal female rats were included in the study. All Hx rats were hormonally treated with cortisol (400 microg/kg/day) and L-thyroxine (10 microg/kg/day) for 19 days. Hypophysectomy resulted in a threefold increase in cerebral cortex and a twofold increase in cerebellum of the DOR protein compared with normal rats. One subgroup of Hx rats received bGH (1 mg/kg body weight) as a daily subcutaneous injection for 19 days. This treatment normalized the levels of DOR protein in the cerebral cortex and cerebellum. Immunohistochemical experiments showed that GH decreased DOR expression especially in layers II-VI in cerebral cortex and in stratum moleculare in cerebellum. Quantification of DOR mRNA by solution hybridization RNase protection assay corresponded to the DOR protein measurements. We conclude that the expression of DORs in cerebral cortex and cerebellum is regulated by GH., (Copyright 2002 Wiley-Liss, Inc.)

The role of endogenous opioid peptides and receptors has recently been investigated using knockout mice. Although the affinities of opioid peptides for opioid receptors has been known for many years there is still some uncertainty over which receptor is the endogenous target for each peptide. To address this issue we have studied using quantitative autoradiography the levels of all four opioid receptor subtypes (micro, delta, kappa and opioid receptor-like 1 [ORL1]) in brains sectioned from enkephalin and dynorphin knockouts, as well as from double knockouts. Because receptor up-regulation has been observed when its cognate ligand-peptide is genetically ablated, regional changes in receptor binding in knockout mice may reflect areas where the peptide ligand is tonically active at its receptor or played a role in receptor regulation. In addition, the study aimed to correlate previously observed behaviour in these animals with receptor modulation. Marked region-specific up-regulation of the micro, delta, and kappa opioid receptors but not ORL1 receptors was observed in proenkephalin and prodynorphin knockouts. In proenkephalin knockouts this was most pronounced for the micro- and delta-receptor and in prodynorphin knockouts for the kappa-receptor. Combinatorial double knockouts did not show any changes in addition to those observed in single knockouts. The largest changes were observed in limbic regions and our results suggest that proenkephalin peptides are tonically active at micro and delta-receptors predominantly in these areas. Prodynorphin peptides appear to regulate mostly the kappa-receptor but they are also modulators of micro- and delta-receptors.

Previous studies have demonstrated that naive splenic mouse T cells express no or only very low levels of the delta-type opioid receptor (delta OR), but stimulation of mouse splenocytes with Con A results in induction of delta OR mRNA and protein. In this report we have shown that stimulation of highly purified populations of naive mouse T cells with anti-CD3 mAb alone results in T cell activation, as evidenced by sustained IL-2 secretion and cell proliferation, but fails to elicit delta OR expression. However, delta OR expression is induced by costimulation of these very pure T cells with anti-CD3 and anti-CD28 mAbs. The delta OR induction by anti-CD3 and anti-CD28 costimulation was completely blocked by inhibition of phosphatidylinositol 3-kinase with wortmannin. Because phosphatidylinositol 3-kinase activation in T cells is linked to costimulation, these results suggest that induction of delta OR expression during T cell activation is strictly dependent on costimulation. It also appears that costimulatory receptors other than CD28 can provide the signaling required for delta OR expression because delta OR mRNA was induced by Con A stimulation of splenocytes from CD28-deficient mice.

Intestinal inflammation enhances the inhibitory effects of mu- and delta-opioids in the gut, possibly related to an increased receptor expression. We evaluated the effects of opioids after intraperitoneal administration of antisense oligodeoxynucleotides to mu- and/or delta-opioid receptor mRNA. Inflammation was induced in mice by intragastric administration of croton oil; gastrointestinal transit was assessed with charcoal and permeability with [51Cr]etylenediaminetetraacetate ([51Cr]EDTA). Baseline values were unaltered after antisense oligodeoxynucleotides. In controls, antisense oligodeoxynucleotides to mu-opioid receptor mRNA decreased the antitransit effects of morphine (27%) and [N-MePhe3D-Pro4]morphiceptin (PL017) (26%), and the reduction was significantly greater during inflammation (50% and 47%). A similar effect was observed on permeability (control: 41-21% decrease; inflamed: 66-45%). In both assays, antisense oligodeoxynucleotides to delta-opioid receptor mRNA also reduced the effects of [D-Pen2,5]enkephalin (DPDPE) in a higher percentage during inflammation (43-32% controls, 60-49% inflamed). We show that antisense oligodeoxynucleotides to mu- and/or delta-opioid receptor mRNA are efficiently blocking the intestinal effects of opioids during inflammation, suggesting that an increased transcription of these receptors in the gut mediates the enhanced effects of opioids during inflammation.

The delta opioid receptors (DORs) modulate T cell proliferation, IL-2 production, chemotaxis, and intracellular signaling. Moreover, in DOR-transfected Jurkat cells, delta opioids have been shown to suppress HIV-1 p24 Ag expression. These observations led us to characterize the expression of DORs by human peripheral blood T cells and to determine whether a specific DOR agonist, benzamide,4-([2,5-dimethyl-4-(2-propenyl)-1-piperazinyl](3-methoxyphenyl)methyl]-N,-,(2S[1(S*),2alpha,5beta])-(9Cl) (SNC-80), can suppress p24 Ag expression by HIV-1-infected CD4+ T cells obtained from normal donors. By immunofluorescence flow cytometry, PHA stimulated the expression of DOR from 1.94 +/- 0.70 (mean +/- SEM) to 20.70 +/- 1.88% of the PBMC population by 48 h (p < 0.0001). DOR expression was approximately 40% of both the PHA-stimulated CD4+ and CD8+ T cell subsets, and virtually all DORs were found on these subsets. To determine whether activated DORs suppress HIV-1 expression, PBMC were prestimulated with PHA, and then CD4+ T cells were purified, pretreated with SNC-80, and infected with HIV-1. In a concentration-dependent manner, SNC-80 inhibited production of p24 Ag. SNC-80 10(-10) M maximally suppressed (approximately 50%) both lymphocytotropic (HIV-1 MN) and monocytotropic (SF162) strains; higher concentrations were less effective. Naltrindole, a selective DOR antagonist, abolished the inhibitory effects of SNC-80. Kinetic studies indicated that 24-h pre- or postincubation with SNC-80, relative to infection with HIV-1, eliminated its suppressive effects. Thus, stimulating the DORs expressed by activated CD4+ T cells significantly suppressed the expression of HIV-1. These findings suggest that opioid immunomodulation directed at host T cells may be adjunctive to standard antiviral approaches to HIV-1 infection.

Background: The kappa and delta opioid receptors were characterized in longitudinal muscle strip of guinea-pig ileum (GPI, mu, kappa), mouse (MVD, delta, mu, kappa) and rabbit (LVD, kappa) vas deferens and rabbit ear artery (ART, delta, kappa) with particular attention to the presence of receptor subtypes., Material and Methods: For this purpose, type/subtype selective agonist and antagonist were used such as [D-Ala2, D-Leu5]-enkephalin (delta agonist) [D-Pen2,5]-enkephalin (delta 1 agonist), deltorphin II (delta 2 agonist) naltriben (NTB, delta 2 antagonist), BOC-YPGFLT(OtBu) (delta antagonist) on the one hand and ethylketocyclazocine (EKC, kappa/(agonist), PD-117 302 (kappa 1 agonist), [Met5]-enkephalin-Arg6, Phe7 (ME-RF, delta/kappa 2/(agonist) and its amide ME-RF-NH2, kappa 2/(agonist), naltrexone (NTX, mu > delta = kappa antagonist) and norbinaltorphimine (nBNI, kappa antagonist) on the other hand., Results and Conclusion: In MVD the Ke of NTB against different (agonists revealed no receptor type heterogeneity. In LVD the Ke of nBNI but not of NTX against EKC versus the ones against ME-RF and ME-RF-NH2 indicated inhomogenous (receptor population. The (receptor antagonist BOC-YPGFLT(OtBu) antagonized the action of ME-RF-NH2 in ART but not in MVD or LVD indicating a special receptor subtype in ART (kappa, possibly delta).