Agonist-induced phosphorylation bar code and differential post-activation signaling of the delta opioid receptor revealed by phosphosite-specific antibodies.

The δ-opioid receptor (DOP) is an attractive pharmacological target due to its potent analgesic, anxiolytic and anti-depressant activity in chronic pain models. However, some but not all selective DOP agonists also produce severe adverse effects such as seizures. Thus, the development of novel agonists requires a profound understanding of their effects on DOP phosphorylation, post-activation signaling and dephosphorylation. Here we show that agonist-induced DOP phosphorylation at threonine 361 (T361) and serine 363 (S363) proceeds with a temporal hierarchy, with S363 as primary site of phosphorylation. This phosphorylation is mediated by G protein-coupled receptor kinases 2 and 3 (GRK2/3) followed by DOP endocytosis and desensitization. DOP dephosphorylation occurs within minutes and is predominantly mediated by protein phosphatases (PP) 1α and 1ß. A comparison of structurally diverse DOP agonists and clinically used opioids demonstrated high correlation between G protein-dependent signaling efficacies and receptor internalization. In vivo, DOP agonists induce receptor phosphorylation in a dose-dependent and agonist-selective manner that could be blocked by naltrexone in DOP-eGFP mice. Together, our studies provide novel tools and insights for ligand-activated DOP signaling in vitro and in vivo and suggest that DOP agonist efficacies may determine receptor post-activation signaling.

δ-Opioid Receptors, microRNAs, and Neuroinflammation in Cerebral Ischemia/Hypoxia.

Hypoxia and ischemia are the main underlying pathogenesis of stroke and other neurological disorders. Cerebral hypoxia and/or ischemia (e.g., stroke) can lead to neuronal injury/death and eventually cause serious neurological disorders or even death in the patients. Despite knowing these serious consequences, there are limited neuroprotective strategies against hypoxic and ischemic insults in clinical settings. Recent studies indicate that microRNAs (miRNAs) are of great importance in regulating cerebral responses to hypoxic/ischemic stress in addition to the neuroprotective effect of the δ-opioid receptor (DOR). Moreover, new discovery shows that DOR can regulate miRNA expression and inhibit inflammatory responses to hypoxia/ischemia. We, therefore, summarize available data in current literature regarding the role of DOR and miRNAs in regulating the neuroinflammatory responses in this article. In particular, we focus on microglia activation, cytokine production, and the relevant signaling pathways triggered by cerebral hypoxia/ischemia. The intent of this review article is to provide a novel clue for developing new strategies against neuroinflammatory injury resulting from cerebral hypoxia/ischemia.

DADLE enhances viability and anti-inflammatory effect of human MSCs subjected to 'serum free' apoptotic condition in part via the DOR/PI3K/AKT pathway.

AIM: Nutritional deprivation and inflammation-rich zones are the major causative reasons for poor survivability of transplanted mesenchymal stem cells (MSCs). Therefore in the present study, we demonstrated the cytoprotective and anti-inflammatory effects of activated delta (δ)-opioid receptor (DOR) with synthetic peptide [D-Ala2, D-Leu5]-enkephalin (DADLE) treatment on human MSCs cultured in serum-starved condition. MAIN METHODS: Cell viability was measured using MTT and Annexin V/PI assays. Expressions of pro-apoptotic (Bcl2) and anti-apoptotic genes (Bax/Bad), levels of activated p44/42 MAPK, Akt, PI3-kinase-p110γ and cleaved caspase-3 were determined by qPCR and western blot. Levels of secreted cytokines were measured by ELISA. KEY FINDINGS: In comparison to the control, DADLE significantly increased cell survivability under serum deprived condition as confirmed by MTT (71% vs 45%) and Annexin V/PI assays (25.9% vs 3.7%). Significant up-regulation of pro-apoptotic Bcl2 (~2.1 folds), down-regulations of anti-apoptotic Bax/Bad (~2.6/2.7 folds) as well as of cleaved caspase-3, increased expression of PI3kinase subunit p110γ and activation of Akt (Ser473) were observed following DADLE treatment in cells under 'serum deprivation' stress. In addition, DADLE treated hMSCs secreted increased levels of anti-inflammatory cytokines (IL10/IL4/TGF-ß) under serum deprived condition. LPS stimulated macrophages showed abated release of pro-inflammatory cytokines (IL1/TNFα/IL6) when grown in hMSC conditioned 'serum deprived' media treated with DADLE. Both the cytoprotective and anti-inflammatory effects of DADLE were inhibited by the DOR specific antagonist naltrindole. SIGNIFICANCE: The DOR signaling pathway improved cell viability and enhanced anti-inflammatory effect of hMSCs subjected to 'serum deprivation' stress that could have potential therapeutic benefits in reparative medicine.

Clathrin light chains' role in selective endocytosis influences antibody isotype switching.

Clathrin, a cytosolic protein composed of heavy and light chain subunits, assembles into a vesicle coat, controlling receptor-mediated endocytosis. To establish clathrin light chain (CLC) function in vivo, we engineered mice lacking CLCa, the major CLC isoform in B lymphocytes, generating animals with CLC-deficient B cells. In CLCa-null mice, the germinal centers have fewer B cells, and they are enriched for IgA-producing cells. This enhanced switch to IgA production in the absence of CLCa was attributable to increased transforming growth factor ß receptor 2 (TGFßR2) signaling resulting from defective endocytosis. Internalization of C-X-C chemokine receptor 4 (CXCR4), but not CXCR5, was affected in CLCa-null B cells, and CLC depletion from cell lines affected endocytosis of the δ-opioid receptor, but not the ß2-adrenergic receptor, defining a role for CLCs in the uptake of a subset of signaling receptors. This instance of clathrin subunit deletion in vertebrates demonstrates that CLCs contribute to clathrin's role in vivo by influencing cargo selectivity, a function previously assigned exclusively to adaptor molecules.

The immunomodulation mediated by a delta-opioid receptor for [Met(5)]-enkephalin in oyster Crassostrea gigas.

Opioid receptors (OR) are a group of G protein-coupled receptors with opioids as ligands, which play an important role in triggering the second messengers to modulate immune response in vertebrate immunocytes. In the present study, the full length cDNA of a homologue of δ-opioid receptor (DOR) for [Met(5)]-enkaphalin was cloned from oyster Crassostrea gigas (designated as CgDOR), which was 1104 bp encoding a peptide of 367 amino acids containing a conserved 7tm_1 domain. After the stimulation of [Met(5)]-enkephalin, the concentration of second messengers Ca(2+) and cAMP in the HEK293T cells decreased significantly (p <0.05) with the expression of CgDOR. However, this trend was reverted with the addition of DOR antagonist BNTX. The CgDOR transcripts were ubiquitously detected in the tested tissues including haemocytes, gonad, mantle, kidney, gill, adductor muscle and hepatopancreas, with the highest expression level in the hepatopancreas. After LPS stimulation, the expression level of CgDOR mRNA began to increase (4.05-fold, p <0.05) at 6 h, and reached the highest level (5.00-fold, p <0.05) at 12 h. Haemocyte phagocytic and antibacterial activities increased significantly after [Met(5)]-enkephalin stimulation, whereas the increase was repressed with the addition of DOR antagonist BNTX. These results collectively suggested that CgDOR for [Met(5)]-enkephalin could modulate the haemocyte phagocytic and antibacterial functions through the second messengers Ca(2+) and cAMP, which might be requisite for pathogen elimination and homeostasis maintenance in oyster.

Immunotherapy of cancer via mediation of cytotoxic T lymphocytes by methionine enkephalin (MENK).

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.

[Role of dopamine D1- and D2-receptors in the delta1-opioidergic immunostimulation].

The study has shown that activation of delta1-opioid receptors by a highly selective peptide agonist DPDPE (100 microg/kg) results in a significant increase of the immune response to antigen (SRBC, 5 x 10(8)) in CBA mice. SCH-23390 (1 mg/kg), a selective antagonist of the postsynaptic dopamine D1-receptors, and selective D2-blocker haloperidol (1 mg/kg) prevented immunostimulating effect of DPDPE. Comparison of effects of the antagonists suggests that delta1-opioidergic immunostimulation has more significant impact due to involvement of dopamine D1-receptors.

Dopaminergic amacrine cells express opioid receptors in the mouse retina.

The presence of opioid receptors has been confirmed by a variety of techniques in vertebrate retinas including those of mammals; however, in most reports, the location of these receptors has been limited to retinal regions rather than specific cell types. Concurrently, our knowledge of the physiological functions of opioid signaling in the retina is based on only a handful of studies. To date, the best-documented opioid effect is the modulation of retinal dopamine release, which has been shown in a variety of vertebrate species. Nonetheless, it is not known if opioids can affect dopaminergic amacrine cells (DACs) directly, via opioid receptors expressed by DACs. This study, using immunohistochemical methods, sought to determine whether (1) µ- and δ-opioid receptors (MORs and DORs, respectively) are present in the mouse retina, and if present, (2) are they expressed by DACs. We found that MOR and DOR immunolabeling were associated with multiple cell types in the inner retina, suggesting that opioids might influence visual information processing at multiple sites within the mammalian retinal circuitry. Specifically, colabeling studies with the DAC molecular marker anti-tyrosine hydroxylase antibody showed that both MOR and DOR immunolabeling localize to DACs. These findings predict that opioids can affect DACs in the mouse retina directly, via MOR and DOR signaling, and might modulate dopamine release as reported in other mammalian and nonmammalian retinas.

Opiate antagonist prevents µ- and δ-opiate receptor dimerization to facilitate ability of agonist to control ethanol-altered natural killer cell functions and mammary tumor growth.

In the natural killer (NK) cells, δ-opiate receptor (DOR) and µ-opioid receptor (MOR) interact in a feedback manner to regulate cytolytic function with an unknown mechanism. Using RNK16 cells, a rat NK cell line, we show that MOR and DOR monomer and dimer proteins existed in these cells and that chronic treatment with a receptor antagonist reduced protein levels of the targeted receptor but increased levels of opposing receptor monomer and homodimer. The opposing receptor-enhancing effects of MOR and DOR antagonists were abolished following receptor gene knockdown by siRNA. Ethanol treatment increased MOR and DOR heterodimers while it decreased the cellular levels of MOR and DOR monomers and homodimers. The opioid receptor homodimerization was associated with an increased receptor binding, and heterodimerization was associated with a decreased receptor binding and the production of cytotoxic factors. Similarly, in vivo, opioid receptor dimerization, ligand binding of receptors, and cell function in immune cells were promoted by chronic treatment with an opiate antagonist but suppressed by chronic ethanol feeding. Additionally, a combined treatment of an MOR antagonist and a DOR agonist was able to reverse the immune suppressive effect of ethanol and reduce the growth and progression of mammary tumors in rats. These data identify a role of receptor dimerization in the mechanism of DOR and MOR feedback interaction in NK cells, and they further elucidate the potential for the use of a combined opioid antagonist and agonist therapy for the treatment of immune incompetence and cancer and alcohol-related diseases.

Influence of endogenous opioid systems on T lymphocytes as assessed by the knockout of mu, delta and kappa opioid receptors.

Here, we evaluated the influence of endogenous opioid activation on immune responses by examining consequences of all three opioid receptor gene (mu, delta and kappa) inactivation. In triple-opioid receptor knockout mice, splenocytes and thymocytes numbers, lymphocyte subsets as well as proliferation and cytokines induced by in vitro stimulation of T lymphocytes were measured. Compared with wild-type mice, similar lymphocyte distribution in thymus and spleen as well as comparable T lymphocyte proliferation were observed, while lower levels of IL-2 and IFNγ as well as higher levels of IL-4 and IL-10 were found in triple-opioid receptor knockout mice. Together, our results indicate a shift from TH1 to TH2 cytokines in triple-opioid receptor knockout animals, suggesting that global endogenous opioid tone drives T lymphocytes toward a TH1 profile under non-pathological conditions.

Autoantibodies to the delta-opioid receptor function as opioid agonists and display immunomodulatory activity.

In this report, we show that affinity purified human anti-delta opioid receptor (DOR) autoantibodies from IVIG are specific to DOR and possess agonistic properties displayed by their ability to dramatically decrease forskolin stimulated cAMP accumulation. Anti-DOR autoantibody also caused phosphorylation of the opioid receptor. Anti-DOR autoantibody treatment showed a significant reduction in CXCR4 gene expression as well as surface protein expression. In contrast, anti-DOR autoantibody treatment significantly upregulated CCR5 gene and protein expression. The presence of anti-DOR autoantibodies in IVIG and their potent immunomodulatory activity is further evidence to support the cross-talk between the neuroendocrine and immune systems.

Delta opioid receptor-mediated immunoregulatory role of methionine-enkephalin in freshwater teleost Channa punctatus (Bloch.).

The immunoregulatory role of methionine-enkephalin (Met-enk) is well studied in mammals, but has not been explored in ectotherms despite the fact that this peptide is highly conserved in vertebrates. The present study demonstrates the diverse effects of Met-enk depending on its concentration and specific function of splenic phagocytes in the freshwater fish Channa punctatus. Although Met-enk increased both phagocytic as well as respiratory burst activity, the concentration-related response was opposite to each other. It had the maximum stimulatory effect on phagocytosis at 10(-9)M, while the same concentration was least effective in increasing superoxide production. Similarly, Met-enk at concentrations lower or higher than 10(-9)M was either ineffective or less effective in case of phagocytosis, while highly effective in stimulating superoxide production. On the other hand, concentration-independent inhibitory effect of Met-enk was observed in case of nitrite production. Nonetheless, Met-enk regulated all the functions of phagocyte through opioid receptors since non-specific opioid receptor antagonist naltrexone completely blocked the effect of Met-enk on phagocytosis, superoxide and nitrite production by splenic phagocytes of C. punctatus. Among selective opioid receptor antagonists, delta-opioid receptor antagonist naltrindole completely antagonized the effect of Met-enk on phagocytosis, superoxide and nitrite production, while mu- and kappa-opioid receptor antagonist, CTAP and norbinaltorphimine, respectively, were ineffective in influencing any of the functions. This suggests that Met-enk modulates splenic phagocyte functions in the fish C. punctatus via delta-opioid receptor. This is further substantiated by using highly selective delta-opioid receptor agonist, SNC80.

Autoantibodies against opioid or glutamate receptors are associated with changes in morphine reward and physical dependence in mice.

BACKGROUND: Possible interactions between nervous and immune systems during opioid addiction remain elusive. Recombinant mu-delta opioid receptors (MDOR) and the glutamate receptor 1 (GluR1) subunit of amino-3-hydroxy-5-methyl-4-isoxazole propionic acid glutamate receptors are involved in acute and chronic effects of morphine. Elevated levels of autoantibodies (aAbs) to these receptors were demonstrated in heroin human addicts and in animal models. This study characterized the role of aAbs to these receptors in behavioral modulations recruited during opioid tolerance and sensitization. METHODS AND FINDINGS: Male CD-1 mice, immunized with either MDOR or GluR1 peptide fragments (80 microg intraperitoneal (i.p.)), were examined for spontaneous behavior and response to morphine (5 mg/kg i.p.). Spontaneous home-cage activity, novelty-induced self-grooming and morphine-induced hyperactivity were higher in GluR1 mice compared to Vehicle subjects, whereas MDOR immunization was associated with an increased morphine-induced conditioned place preference. In response to escalating doses of morphine (from 10 to 60 mg/kg i.p., twice daily) and naloxone-precipitated withdrawal (1 mg/kg subcutaneous), GluR1 mice exhibited a more marked stereotyped sniffing behavior and less body tremors compared to Vehicle subjects, whereas less sniffing and teeth chattering were found in MDOR mice. The expected downregulation of mu receptor binding sites, induced by chronic morphine in vehicle subjects, was completely absent following MDOR immunization. CONCLUSIONS: These findings indicate an altered response to morphine-related reinforcing and aversive effects in MDOR mice and altered coping with the environment in GluR1 mice. Circulating aAbs to specific neuroreceptors may alter the response to opiates and play a role as determinants of vulnerability to opiate addiction.

Thymus exclusivity: all the right conditions for T cells.

A fraction of primitive "lymphoid" precursors retain plasticity for myeloid differentiation. In this issue of Immunity, Laiosa et al. describe that Notch-Delta signals can protect thymic precursors from reprogramming into the myeloid lineage, antagonizing the enforced myeloid transcription factors such as PU.1 and C/EBPalpha.

Specific changes in levels of autoantibodies to glutamate and opiate receptors induced by morphine administration in rats.

Several groups of brain receptors are involved in the mechanisms underlying the development of opiate addiction, but the interactions occurring between these neuroreceptors and the immune system, including potential autoimmune responses, remain poorly understood. We studied in rats the effects of repeated administration of different psychotropic drugs on serum levels of autoantibodies (aAbs) to the mu delta-opiate receptor (MDOR), as well as to the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) GluR1 and to the N-methyl-D-aspartate (NMDA) NR2 subunits of the glutamate receptor, as analyzed by ELISA. We found that repeated administration of morphine significantly elevated aAbs levels to MDOR and to the AMPA GluR1 subunit, but not to the NMDA NR2 subunit. In contrast, a similar regimen of a psychostimulant drug, such as D-amphetamine, or a commonly abused substance, such as nicotine, had no effect on these aAbs levels. A nonspecific elevating effect on aAbs to the brain structural protein S100B was observed for all drugs tested versus controls. These observations support the hypothesis that, following opiate administration, specific interactions between nervous and immune systems occur. Therefore, together with further investigations on their potential functional consequences, we propose a thorough exploration of aAbs to MDOR and to AMPA GluR1 subunit as early biomarkers signaling opiate addiction.

Stress-induced modulation of NK activity during influenza viral infection: role of glucocorticoids and opioids.

Activation of the hypothalamic-pituitary-adrenal axis (HPA) and sympathetic nervous system by stress has been shown to modulate both innate and adaptive immunity during an experimental influenza A/PR8 viral infection. HPA activation alters levels of glucocorticoids (GC) and opioids which are associated with suppression of lymphoid cellularity and NK activity. These experiments were designed to investigate the role that stress-induced GC and opioids have in modulating NK activity during an influenza viral infection. C57BL/6 mice were treated daily with mifepristone (RU486), a GC receptor antagonist or naltrexone (NTX), a non-specific opioid receptor antagonist. Mice were infected intranasally with A/PR8 virus and underwent daily restraint stress (RST). Phenotypic analysis of splenic cell populations and NK cytotoxicity were assessed at 3 days post-infection. RST of infected mice significantly suppressed splenic CD3(-)DX5+ cellularity and NK cytolytic activity. RU486 administration fully restored splenic NK cellularity but not cytolytic activity. NTX administration restored NK cytolytic activity but not splenic NK cell number. A similar restoration in NK cytolytic activity was observed after administration of beta-funaltrexamine (FNA), a mu-specific opioid receptor antagonist, but not the delta- or kappa-specific opioid receptor antagonists naltrindole or nor-binaltorphimine, respectively. Co-administration of RU486 and NTX restored both NK cellularity and cytolytic activity. The restoration of RST-induced suppression of NK activity by RU486 and NTX or FNA suggests that glucocorticoids modulate NK cellularity and opioids that bind to the mu-opioid receptor modulate NK cytotoxicity during periods of stress and viral infection.

Methionine-enkephalin stimulates hydrogen peroxide and nitric oxide production in rat peritoneal macrophages: interaction of mu, delta and kappa opioid receptors.

OBJECTIVE: Methionine-enkephalin (MET) modulates various functions of macrophages related to both immune and inflammatory reactions in a naloxone reversible manner, suggesting that opioid receptors are involved in the regulation of macrophage activity. Since an endogenous opioid ligand might interact with more than one type of opioid receptor, the receptor interaction determines its effect on a particular function. METHODS: In the present study we have investigated the involvement of different opioid receptor types/subtypes in MET-induced modulation of H(2)O(2) and NO production in macrophages. Thioglycollate-elicited or resident rat peritoneal macrophages were treated in vitro with MET and/or specific antagonists of delta(1,2), delta(1), delta(2), mu and kappa opioid receptors. RESULTS: MET increased H(2)O(2)production in phorbol myristate acetate-stimulated rat peritoneal macrophages mainly through delta(1) opioid receptor. MET also enhanced NO production in rat peritoneal macrophages stimulated with lipopolysaccharide through delta(1) and mu opioid receptors. The blockade of mu and kappa receptor facilitated a potentiating effect of MET on H(2)O(2) release, and blockade of kappa receptor further raised the MET-induced increase of NO production in macrophages. CONCLUSION: It is concluded that both negative and positive functional interaction between delta, mu and kappa opioid receptors regulate the influence of MET on H(2)O(2) and NO production in rat peritoneal macrophages.

Pharmacological characterization of AR-M1000390 at human delta opioid receptors.

We investigated the pharmacological properties of a newly synthesised delta agonist AR-M1000390, derived from SNC-80 ((+)-4-[(alpha R)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethyl-benzamide), in the neuroblastoma cell line SK-N-BE expressing only human delta-opioid receptors. Binding and functional experiments showed a weak affinity (K(i) = 106 +/- 34 nM) correlated with a weak potency (EC(50) = 111 +/- 31 nM) to inhibit the forskolin-stimulated cAMP accumulation. Sustained activation of opioid receptors in the presence of the maximal inhibitory concentration of AR-M1000390 produced a rapid and strong desensitization. In order to examine the contribution of internalization and down-regulation in the desensitization processes, binding and functional experiments were conducted in the presence or in the absence of hypertonic sucrose solution to block clathrin-dependent opioid receptor endocytosis. We observed both the inability of AR-M1000390 to down-regulate opioid receptors and the absence of any effect of sucrose on desensitization. The lack of delta-opioid receptor internalization by AR-M1000390 was further corroborated by confocal microscopy using antibodies directed either against the endogenous delta-opioid receptors or the FLAG-tagged delta-opioid receptors stably expressed in the SK-N-BE cells. These data suggest that uncoupling rather than internalization is responsible for delta-opioid receptors desensitization by AR-M1000390.

Phosphorylation of activating transcription factor in murine splenocytes through delta opioid receptors.

Delta opioid receptors (DORs) modulate TCR signaling through the mitogen-activated protein kinases (MAPKs), ERKs 1 and 2. These studies determined whether a DOR agonist alone ([D-Ala(2)-D-Leu(5)]enkephalin; DADLE) affects phosphorylation of the activating transcription factor (ATF-2) and its interaction with the MAPK, c-Jun NH(2)-terminal kinase (JNK). DOR expression was induced on murine splenocytes by anti-CD3 and then quiescent cells were treated with DADLE. DADLE, itself, dose-dependently induced maximal phosphorylation of ATF-2 within 5-10min; naltrindole, a specific antagonist, abolished this. Anti-ATF-2 immunoprecipitates from control and DADLE-treated splenocytes showed a dominant 59kDa phosphorylated band and a 71kDa band. DADLE stimulated phosphorylation of both bands, although the 71kDa band was selectively immunoprecipitated by anti-JNK. Thus, DADLE stimulated phosphorylation of 71kDa ATF-2 and its association with JNK, suggesting that JNK is activated through DORs. Along with previous observations, these studies suggest that lymphocyte DORs can affect the activation of MAPKs by TCR-independent stimulation (e.g., JNK) or indirectly by modulating TCR-dependent stimulation (e.g., ERK).