Kappa opioid receptor antagonism and chronic antidepressant treatment have beneficial activities on social interactions and grooming deficits during heroin abstinence.

Addiction is a chronic brain disorder that progressively invades all aspects of personal life. Accordingly, addiction to opiates severely impairs interpersonal relationships, and the resulting social isolation strongly contributes to the severity and chronicity of the disease. Uncovering new therapeutic strategies that address this aspect of addiction is therefore of great clinical relevance. We recently established a mouse model of heroin addiction in which, following chronic heroin exposure, 'abstinent' mice progressively develop a strong and long-lasting social avoidance phenotype. Here, we explored and compared the efficacy of two pharmacological interventions in this mouse model. Because clinical studies indicate some efficacy of antidepressants on emotional dysfunction associated with addiction, we first used a chronic 4-week treatment with the serotonergic antidepressant fluoxetine, as a reference. In addition, considering prodepressant effects recently associated with kappa opioid receptor signaling, we also investigated the kappa opioid receptor antagonist norbinaltorphimine (norBNI). Finally, we assessed whether fluoxetine and norBNI could reverse abstinence-induced social avoidance after it has established. Altogether, our results show that two interspaced norBNI administrations are sufficient both to prevent and to reverse social impairment in heroin abstinent animals. Therefore, kappa opioid receptor antagonism may represent a useful approach to alleviate social dysfunction in addicted individuals.

Mu and delta opioid receptor knockout mice show increased colonic sensitivity.

BACKGROUND: Opiates act through opioid receptors to diminish pain. Here, we investigated whether mu (MOR) and delta (DOR) receptor endogenous activity assessed in the whole mouse body or in particular at peripheral receptors on primary nociceptive neurons, control colonic pain. METHODS: We compared global MOR and DOR receptor knockout (KO) mice, mice with a conditional deletion of MOR and DOR in Nav1.8-positive nociceptive primary afferent neurons (cKO), and control floxed mice of both genders for visceral sensitivity. Visceromotor responses to colorectal distension (CRD) and macroscopic colon scores were recorded on naïve mice and mice with acute colitis induced by 3% dextran sodium sulphate (DSS) for 5 days. Transcript expression for opioid genes and cytokines was measured by quantitative RT-PCR. RESULTS: Naïve MOR and DOR global KO mice show increased visceral sensitivity that was not observed in cKO mice. MOR and preproenkephalin (Penk) were the most expressed opioid genes in colon. MOR KO mice had augmented kappa opioid receptor and Tumour-Necrosis-Factor-α and diminished Penk transcript levels while DOR, preprodynorphin and Interleukin-1ß were unchanged. Global MOR KO females had a thicker colon than floxed females. No alteration was detected in DOR mutant animals. A 5-day DSS treatment led to comparable hypersensitivity in the different mouse lines. CONCLUSION: Our results suggest that mu and delta opioid receptor global endogenous activity but not activity at the peripheral Nav1.8 neurons contribute to visceral sensitivity in naïve mice, and that endogenous MOR and DOR tones were insufficient to elicit analgesia after 5-day DSS-induced colitis. SIGNIFICANCE: Knockout mice for mu and delta opioid receptor have augmented colon sensitivity in the CRD assay. It shows endogenous mu and delta opioid analgesia that may be explored as potential targets for alleviating chronic intestinal pain.

Orphanin FQ/nociceptin binds to functionally coupled ORL1 receptors on human immune cell lines and alters peripheral blood mononuclear cell proliferation.

Orphanin FQ/nociceptin (OFQ/N) has been shown to modulate nociception, responses to stress and anxiety. We investigated OFQ/N function in human immune cells. We find that monocytic U937, T lymphocytic CEM, and MOLT-4 cell lines express OFQ/N binding sites at levels comparable to that of human SH-SY5Y neuroblastoma cells. We show that OFQ/N receptors are functionally coupled to G proteins in these cells. Finally OFQ/N decreases proliferation of phytohemagglutinin-stimulated peripheral blood mononuclear cells in vitro at doses ranging from 10(-13) to 10(-8) M. Thus, our data suggest that OFQ/N and OFQ/N receptor may act as an immunomodulatory system.

A single nucleotide polymorphic mutation in the human mu-opioid receptor severely impairs receptor signaling.

Large scale sequencing of the human mu-opioid receptor (hMOR) gene has revealed polymorphic mutations that occur within the coding region. We have investigated whether the mutations N40D in the extracellular N-terminal region, N152D in the third transmembrane domain, and R265H and S268P in the third intracellular loop alter functional properties of the receptor expressed in mammalian cells. The N152D receptor was produced at low densities. Binding affinities of structurally diverse opioids (morphine, diprenorphine, DAMGO and CTOP) and the main endogenous opioid peptides (beta-endorphin, [Met]enkephalin, and dynorphin A) were not markedly changed in mutant receptors (<3-fold). Receptor signaling was strongly impaired in the S268P mutant, with a reduction of efficacy and potency of several agonists (DAMGO, beta-endorphin, and morphine) in two distinct functional assays. Signaling at N40D and R265H mutants was highly similar to wild type, and none of the mutations induced detectable constitutive activity. DAMGO-induced down-regulation of receptor-binding sites, following 20 h of treatment, was identical in wild-type and mutant receptors. Our data show that natural sequence variations in hMOR gene have little influence on ligand binding or receptor down-regulation but could otherwise modify receptor density and signaling. Importantly, the S268P mutation represents a loss-of-function mutation for the human mu-opioid receptor, which may have an incidence on opioid-regulated behaviors or drug addiction in vivo.

Regulation of kappa-opioid receptor mRNA level by cyclic AMP and growth factors in cultured rat glial cells.

The mRNA of the kappa-opioid receptor (KOR) has been found recently in cultured astrocytes and in microglia. By using RT-PCR and Southern hybridization, we confirmed these observations and, in addition, we observed that KOR mRNA was expressed in oligodendrocytes and in the precursors of astrocytes and oligodendrocytes. KOR mRNA level was the highest in the immature astrocytes and decreased with their maturation. Very few data are available on the regulation of KOR level by extracellular signals. Therefore, we examined the effect of three growth factors known to be present in the adult brain, basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF-BB) and leukemia inhibitory factor (LIF) and of two cyclic AMP (cAMP) generating systems, the cAMP analog, 8-(4-chlorophenylthio)-cAMP and forskolin, on this level. It was found that in astrocytes, KOR mRNA level decreased dramatically under the effect of cAMP and less under the effect of bFGF while it did not change significantly after LIF treatment. In oligodendrocytes, it also decreased with cAMP, but increased under the effect of bFGF and PDGF-BB. In microglia, a decrease was observed with cAMP and lipopolysaccharides (LPS), the most used activators of macrophages. These results shed new evidence on the expression of opioid receptor mRNA in the glial cells of the rat CNS. The regulation of KOR mRNA level under the effect of extracellular signals suggests that opioids take part in dynamic processes in glial cells, possibly related to glial-neuron communication.

The human delta-opioid receptor: genomic organization, cDNA cloning, functional expression, and distribution in human brain.

We have used the mouse delta-opioid receptor (mDOR) cDNA to isolate the mDOR gene and its human homologue. In both species the coding region is interrupted by two introns with conserved exon-intron boundaries located after transmembrane domains 1 and 4. Using the polymerase chain reaction and primers based on the sequence of the cloned human delta-opioid receptor (hDOR) gene, we have obtained a full length cDNA encoding the hDOR from SH-SY5Y neuroblastoma cells. The cDNA sequence is 100% identical to the cloned human genomic sequence and 94% identical to the mouse sequence at the protein level. When expressed in COS cells, hDOR displays nanomolar affinities for delta-selective ligands, whereas the affinities for mu- and kappa-selective ligands are in the micromolar range. The delta agonists [D-Ala2, D-Leu5]enkephalin, cyclic [D-penicillamine2,D-penicillamine5]enkephalin, and BW373U86 efficiently decrease forskolin-induced cAMP levels in hDOR-expressing COS cells, indicating functional coupling of the receptor. The distribution of hDOR mRNA in human brain was investigated using delta-selective reverse transcription-polymerase chain reaction amplification, followed by Southern hybridization with a delta-specific probe. The transcript is found in cortical areas, including olfactory bulb, hippocampus, and amygdala, as well as in basal ganglia and hypothalamus. No expression is detected in internal globus pallidus, thalamus, any investigated brainstem structure, or pituitary gland. Taken together, our results indicate similar structural, pharmacological, functional, and anatomical properties for the hDOR and the mDOR and therefore support the use of rodent models for the study of these receptors in opioid function.