A one-pot multicomponent approach to a new series of morphine derivatives and their biological evaluation.

A novel and facile domino reaction has been developed to synthesize a variety of new derivatives from hydromorphone, amines and paraformaldehyde in good yields in a catalyst-free fashion with high atom efficiency. The products show a mixed MOR/DOR biological characteristic which makes them valuable for further study as opioid analgesics.

Kappa Opioid Receptor-Induced Aversion Requires p38 MAPK Activation in VTA Dopamine Neurons.

The endogenous dynorphin-κ opioid receptor (KOR) system encodes the dysphoric component of the stress response and controls the risk of depression-like and addiction behaviors; however, the molecular and neural circuit mechanisms are not understood. In this study, we report that KOR activation of p38α MAPK in ventral tegmental (VTA) dopaminergic neurons was required for conditioned place aversion (CPA) in mice. Conditional genetic deletion of floxed KOR or floxed p38α MAPK by Cre recombinase expression in dopaminergic neurons blocked place aversion to the KOR agonist U50,488. Selective viral rescue by wild-type KOR expression in dopaminergic neurons of KOR(-/-) mice restored U50,488-CPA, whereas expression of a mutated form of KOR that could not initiate p38α MAPK activation did not. Surprisingly, while p38α MAPK inactivation blocked U50,488-CPA, p38α MAPK was not required for KOR inhibition of evoked dopamine release measured by fast scan cyclic voltammetry in the nucleus accumbens. In contrast, KOR activation acutely inhibited VTA dopaminergic neuron firing, and repeated exposure attenuated the opioid response. This adaptation to repeated exposure was blocked by conditional deletion of p38α MAPK, which also blocked KOR-induced tyrosine phosphorylation of the inwardly rectifying potassium channel (GIRK) subunit Kir3.1 in VTA dopaminergic neurons. Consistent with the reduced response, GIRK phosphorylation at this amino terminal tyrosine residue (Y12) enhances channel deactivation. Thus, contrary to prevailing expectations, these results suggest that κ opioid-induced aversion requires regulation of VTA dopaminergic neuron somatic excitability through a p38α MAPK effect on GIRK deactivation kinetics rather than by presynaptically inhibiting dopamine release. SIGNIFICANCE STATEMENT: Kappa opioid receptor (KOR) agonists have the potential to be effective, nonaddictive analgesics, but their therapeutic utility is greatly limited by adverse effects on mood. Understanding how KOR activation produces dysphoria is key to the development of better analgesics and to defining how the endogenous dynorphin opioids produce their depression-like effects. Results in this study show that the aversive effects of κ receptor activation required arrestin-dependent p38α MAPK activation in dopamine neurons but did not require inhibition of dopamine release in the nucleus accumbens. Thus, contrary to the prevailing view, inhibition of mesolimbic dopamine release does not mediate the aversive effects of KOR activation and functionally selective κ opioids that do not activate arrestin signaling may be effective analgesics lacking dysphoric effects.

κ-Opioid receptor mediates the antinociceptive effect of nitrous oxide in mice.

BACKGROUND: Our previous reports demonstrated that genetic deletion of µ-opioid receptor has no influence on the anaesthetic and antinociceptive effects of nitrous oxide (N2O) in mice, and that an antagonist selective for κ-opioid receptor (KOP), but not that selective for δ-opioid receptor, suppresses the antinociceptive effect of N2O. However, it is not known whether genetic deletion of KOP affects the N2O actions. METHODS: We measured the minimum alveolar concentration (MAC) of volatile anaesthetics in the absence and presence of N2O. The antinociceptive action of N2O was tested by an acetic acid-writhing test and a hot-plate test. The number of c-Fos-immunopositive cells in sections from the lumbar spinal cord was counted to test whether the descending inhibitory system participates in the pharmacological action of N2O. The hypnotic action of N2O was assessed by measuring the N2O-induced decrease in the EC50 for loss of the righting reflex (EC50-LORR) of sevoflurane. RESULTS: Sevoflurane MAC was not significantly reduced by N2O and its antinociceptive action was almost completely abolished in KOP-knockout (KO) mice. The N2O-induced increase in c-Fos-immunopositive cells in laminae III-IV of the lumbar spinal cord was significant in wild-type (WT), but not in KOP-KO mice. In contrast, sevoflurane EC50-LORR was similarly reduced by N2O in WT and KOP-KO mice. CONCLUSIONS: Our study suggests that N2O demonstrates its antinociceptive action and reduces sevoflurane MAC in mice through KOP activation, whereas its hypnotic potency is not dependent on KOP activation.

Mice deficient for delta- and mu-opioid receptors exhibit opposing alterations of emotional responses.

The role of the opioid system in controlling pain, reward and addiction is well established, but its role in regulating other emotional responses is poorly documented in pharmacology. The mu-, delta- and kappa- opioid receptors (encoded by Oprm, Oprd1 and Oprk1, respectively) mediate the biological activity of opioids. We have generated Oprd1-deficient mice and compared the behavioural responses of mice lacking Oprd1, Oprm (ref. 6) and Oprk1 (ref. 7) in several models of anxiety and depression. Our data show no detectable phenotype in Oprk1-/- mutants, suggesting that kappa-receptors do not have a role in this aspect of opioid function; opposing phenotypes in Oprm-/- and Oprd1-/- mutants which contrasts with the classical notion of similar activities of mu- and delta-receptors; and consistent anxiogenic- and depressive-like responses in Oprd1-/- mice, indicating that delta-receptor activity contributes to improvement of mood states. We conclude that the Oprd1-encoded receptor, which has been proposed to be a promising target for the clinical management of pain, should also be considered in the treatment of drug addiction and other mood-related disorders.

Lyophilized Kratom Tea as a Therapeutic Option for Opioid Dependence.

BACKGROUND: Made as a tea, the Thai traditional drug "kratom" reportedly possesses pharmacological actions that include both a coca-like stimulant effect and opium-like depressant effect. Kratom has been used as a substitute for opium in physically-dependent subjects. The objective of this study was to evaluate the antinociception, somatic and physical dependence produced by kratom tea, and then assess if the tea ameliorated withdrawal in opioid physically-dependent subjects. METHODS: Lyophilized kratom tea (LKT) was evaluated in C57BL/6J and opioid receptor knockout mice after oral administration. Antinociceptive activity was measured in the 55 °C warm-water tail-withdrawal assay. Potential locomotor impairment, respiratory depression and locomotor hyperlocomotion, and place preference induced by oral LKT were assessed in the rotarod, Comprehensive Lab Animal Monitoring System, and conditioned place preference assays, respectively. Naloxone-precipitated withdrawal was used to determine potential physical dependence in mice repeatedly treated with saline or escalating doses of morphine or LKT, and LKT amelioration of morphine withdrawal. Data were analyzed using one- and two-way ANOVA. RESULTS: Oral administration of LKT resulted in dose-dependent antinociception (≥1 g/kg, p.o.) absent in mice lacking the mu-opioid receptor (MOR) and reduced in mice lacking the kappa-opioid receptor. These doses of LKT did not alter coordinated locomotion or induce conditioned place preference, and only briefly reduced respiration. Repeated administration of LKT did not produce physical dependence, but significantly decreased naloxone-precipitated withdrawal in morphine dependent mice. CONCLUSIONS: The present study confirms the MOR agonist activity and therapeutic effect of LKT for the treatment of pain and opioid physical dependence.

Stress-induced p38 mitogen-activated protein kinase activation mediates kappa-opioid-dependent dysphoria.

The molecular mechanisms mediating stress-induced dysphoria in humans and conditioned place aversion in rodents are unknown. Here, we show that repeated swim stress caused activation of both kappa-opioid receptor (KOR) and p38 mitogen-activated protein kinase (MAPK) coexpressed in GABAergic neurons in the nucleus accumbens, cortex, and hippocampus. Sites of activation were visualized using phosphoselective antibodies against activated kappa receptors (KOR-P) and against phospho-p38 MAPK. Surprisingly, the increase in P-p38-IR caused by swim-stress exposure was completely KOR dependent; P-p38-IR did not increase in KOR(-/-) knock-out mice subjected to the same swim-paradigm or in wild-type mice pretreated with the KOR antagonist norbinaltorphimine. To understand the relationship between p38 activation and the behavioral effects after KOR activation, we administered the p38 inhibitor SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)-1H-imidazole (i.c.v.)] and found that it selectively blocked the conditioned place aversion caused by the kappa agonist trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide (U50488) and the KOR-dependent swim stress-induced immobility while not affecting kappa-opioid analgesia or nonselectively affecting associative learning. We found that the mechanism linking KOR and p38 activation in vivo was consistent with our previous in vitro data suggesting that beta-arrestin recruitment is required; mice lacking G-protein-coupled receptor kinase 3 also failed to increase p-p38-IR after KOR activation in vivo, failed to show swim stress-induced immobility, or develop conditioned place aversion to U50488. Our results indicate that activation of p38 MAPK signaling by the endogenous dynorphin-kappa-opioid system likely constitutes a key component of the molecular mechanisms mediating the aversive properties of stress.

Sciatic nerve ligation-induced proliferation of spinal cord astrocytes is mediated by kappa opioid activation of p38 mitogen-activated protein kinase.

Partial sciatic nerve ligation (pSNL) markedly increased glial fibrillary acidic protein immunoreactivity (GFAP-IR) 1 week after lesion in the L4-L5 spinal dorsal horn of wild-type, but not in dynorphin knock-out, mice lacking kappa opioid receptors (KOR-/-) or in wild-type mice pretreated with the KOR antagonist nor-binaltorphimine (norBNI). A direct effect of KOR on glial cell proliferation was suggested by the findings that primary cultures of type II GFAP-immunoreactive astrocytes isolated from mouse spinal cord express KOR. Sustained treatment with the kappa agonist U50,488 (trans-3,4-dichloro-N-methyl-N-[2-(1-pyrolytinil)-cyclohexyl]-benzeneacetamide methane sulfonate) significantly increased the proliferation rate of GFAP-immunoreactive astrocytes isolated from wild-type mice, and this effect was blocked by norBNI pretreatment. Proliferation of cultured type II astrocytes may have been stimulated by mitogen-activated protein kinase (MAPK) activation by KOR because (1) U50,488 treatment increased phospho-p38 MAPK-immunoreactivity 247 +/- 44% over untreated cells, (2) the increase in phospho-p38 induced by U50,488 was blocked by norBNI and not evident in KOR-/- cultures, and (3) GFAP-immunoreactive astrocyte proliferation induced by U50,488 was blocked by the p38 MAPK inhibitor SB 203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole]. Similar mechanisms of astrocyte activation may also be responsible in vivo because intrathecal injection of SB 203580 blocked the increased GFAP-IR in lumbar spinal cord induced by pSNL. Although the relationship between kappa-stimulated astrocyte proliferation and neuropathic pain mechanisms was not directly established in these studies, the results support the hypothesis that KOR activation induces spinal astrocyte proliferation, which may contribute to cellular reorganization after sciatic nerve damage.

Non-canonical Opioid Signaling Inhibits Itch Transmission in the Spinal Cord of Mice.

Chronic itch or pruritus is a debilitating disorder that is refractory to conventional anti-histamine treatment. Kappa opioid receptor (KOR) agonists have been used to treat chronic itch, but the underlying mechanism remains elusive. Here, we find that KOR and gastrin-releasing peptide receptor (GRPR) overlap in the spinal cord, and KOR activation attenuated GRPR-mediated histamine-independent acute and chronic itch in mice. Notably, canonical KOR-mediated Gαi signaling is not required for desensitizing GRPR function. In vivo and in vitro studies suggest that KOR activation results in the translocation of Ca2+-independent protein kinase C (PKC)δ from the cytosol to the plasma membrane, which in turn phosphorylates and inhibits GRPR activity. A blockade of phospholipase C (PLC) in HEK293 cells prevented KOR-agonist-induced PKCδ translocation and GRPR phosphorylation, suggesting a role of PLC signaling in KOR-mediated GRPR desensitization. These data suggest that a KOR-PLC-PKCδ-GRPR signaling pathway in the spinal cord may underlie KOR-agonists-induced anti-pruritus therapies.

Essential role of mu opioid receptor in the regulation of delta opioid receptor-mediated antihyperalgesia.

Analgesic effects of delta opioid receptor (DOR) -selective agonists are enhanced during persistent inflammation and arthritis. Although the underlying mechanisms are still unknown, membrane density of DOR was shown to be increased 72 h after induction of inflammation, an effect abolished in mu opioid receptor (MOR) -knockout (KO) mice [Morinville A, Cahill CM, Kieffer B, Collier B, Beaudet A (2004b) Mu-opioid receptor knockout prevents changes in delta-opioid receptor trafficking induced by chronic inflammatory pain. Pain 109:266-273]. In this study, we demonstrated a crucial role of MOR in DOR-mediated antihyperalgesia. Intrathecal administration of the DOR selective agonist deltorphin II failed to induce antihyperalgesic effects in MOR-KO mice, whereas it dose-dependently reversed thermal hyperalgesia in wild-type mice. The antihyperalgesic effects of deltorphin II were blocked by naltrindole but not d-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH(2) (CTOP) suggesting that this agonist was mainly acting through DOR. SNC80-induced antihyperalgesic effects in MOR-KO mice were also attenuated as compared with littermate controls. In contrast, kappa opioid receptor knockout did not affect deltorphin II-induced antihyperalgesia. As evaluated using mice lacking endogenous opioid peptides, the regulation of DOR's effects was also independent of beta-endorphin, enkephalins, or dynorphin opioids known to be released during persistent inflammation. We therefore conclude that DOR-mediated antihyperalgesia is dependent on MOR expression but that activation of MOR by endogenous opioids is probably not required.

A role for the mu opioid receptor in the antidepressant effects of buprenorphine.

Buprenorphine (BPN), a mixed opioid drug with high affinity for mu (MOR) and kappa (KOR) opioid receptors, has been shown to produce behavioral responses in rodents that are similar to those of antidepressant and anxiolytic drugs. Although recent studies have identified KORs as a primary mediator of BPN's effects in rodent models of depressive-like behavior, the role of MORs in BPN's behavioral effects has not been as well explored. The current studies investigated the role of MORs in mediating conditioned approach behavior in the novelty-induced hypophagia (NIH) test, a behavioral measure previously shown to be sensitive to chronic treatment with antidepressant drugs. The effects of BPN were evaluated in the NIH test 24h post-administration in mice with genetic deletion of the MOR (Oprm1-/-) or KOR (Oprk1-/-), or after pharmacological blockade with the non-selective opioid receptor antagonist naltrexone and selective MOR antagonist cyprodime. We found that behavioral responses to BPN in the NIH test were blocked in Oprm1-/- mice, but not in Oprk1-/- mice. Both cyprodime and naltrexone significantly reduced approach latency at doses experimentally proven to antagonize the MOR. In contrast the selective MOR agonist morphine and the selective KOR antagonist nor-BNI were both ineffective. Moreover, antinociceptive studies revealed persistence of the MOR antagonist properties of BPN at 24h post-administration, the period of behavioral reactivity. These data support modulation of MOR activity as a key component of BPN's antidepressant-like effects in the NIH paradigm.

Endogenous kappa-opioid receptor systems regulate mesoaccumbal dopamine dynamics and vulnerability to cocaine.

Genetic and pharmacological approaches were used to examine kappa-opioid receptor (KOR-1) regulation of dopamine (DA) dynamics in the nucleus accumbens and vulnerability to cocaine. Microdialysis revealed that basal DA release and DA extraction fraction (Ed), an indirect measure of DA uptake, are enhanced in KOR-1 knock-out mice. Analysis of DA uptake revealed a decreased Km but unchanged Vmax in knock-outs. Knock-out mice exhibited an augmented locomotor response to cocaine, which did not differ from that of wild-types administered a behavioral sensitizing cocaine treatment. The ability of cocaine to increase DA was enhanced in knock-outs, whereas c-fos induction was decreased. Although repeated cocaine administration to wild types produced behavioral sensitization, knock-outs exhibited no additional enhancement of behavior. Administration of the long-acting KOR antagonist nor-binaltorphimine to wild-type mice increased DA dynamics. However, the effects varied with the duration of KOR-1 blockade. Basal DA release was increased whereas Ed was unaltered after 1 h blockade. After 24 h, release and Ed were increased. The behavioral and neurochemical effects of cocaine were enhanced at both time points. These data demonstrate the existence of an endogenous KOR-1 system that tonically inhibits mesoaccumbal DA neurotransmission. Its loss induces neuroadaptations characteristic of "cocaine-sensitized" animals, indicating a critical role of KOR-1 in attenuating responsiveness to cocaine. The increased DA uptake after pharmacological inactivation or gene deletion highlights the plasticity of mesoaccumbal DA neurons and suggests that loss of KOR-1 and the resultant disinhibition of DA neurons trigger short- and long-term DA transporter adaptations that maintain normal DA levels, despite enhanced release.

Prior activation of kappa opioid receptors by U50,488 mimics repeated forced swim stress to potentiate cocaine place preference conditioning.

Repeated forced-swim stress (FSS) produced analgesia, immobility and potentiation of cocaine-conditioned place preference (CPP) in wild-type C57Bl/6 mice, but not in littermates lacking the kappa opioid receptor (KOR) gene. These results were surprising because kappa agonists are known to produce conditioned place aversion and to suppress cocaine-CPP when coadministered with cocaine. The possibility that disruption of the kappa system blocked the stress response by adversely affecting the hypothalamic-pituitary axis was examined by measuring plasma corticosterone levels. However, disruption of the dynorphin/kappa system by gene deletion or receptor antagonism did not reduce the FSS-induced elevation of plasma corticosterone levels. A second explanation for the difference is that kappa receptor activation caused by FSS occurred prior to cocaine conditioning rather than contemporaneously. To test this hypothesis, we measured the effects of the kappa agonist (trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide (U50,488) administered to mice at various intervals preceding cocaine conditioning. The results showed that the interaction between the kappa system and cocaine reinforcement depended on the timing of the drug pairing. Mice given U50,488 60 min prior to cocaine showed a robust, nor-BNI-sensitive potentiation of cocaine-CPP, whereas administration 15 min before cocaine significantly suppressed cocaine-CPP. In the absence of cocaine, U50,488 given 60 min prior to saline conditioning produced no place preference, whereas administration 15 min before saline conditioning produced significant place aversion. The results of this study suggest that kappa receptor activation induced by FSS prior to the cocaine-conditioning session may be both necessary and sufficient for potentiation of the reinforcing actions of cocaine.

Absence of delta -9-tetrahydrocannabinol dysphoric effects in dynorphin-deficient mice.

The involvement of dynorphin on Delta-9-tetrahydrocannabinol (THC) and morphine responses has been investigated by using mice with a targeted inactivation of the prodynorphin (Pdyn) gene. Dynorphin-deficient mice show specific changes in the behavioral effects of THC, including a reduction of spinal THC analgesia and the absence of THC-induced conditioned place aversion. In contrast, acute and chronic opioid effects were normal. The lack of negative motivational effects of THC in the absence of dynorphin demonstrates that this endogenous opioid peptide mediates the dysphoric effects of marijuana.

Motivational effects of cannabinoids are mediated by mu-opioid and kappa-opioid receptors.

Repeated THC administration produces motivational and somatic adaptive changes leading to dependence in rodents. To investigate the molecular basis for cannabinoid dependence and its possible relationship with the endogenous opioid system, we explored delta9-tetrahydrocannabinol (THC) activity in mice lacking mu-, delta- or kappa-opioid receptor genes. Acute THC-induced hypothermia, antinociception, and hypolocomotion remained unaffected in these mice, whereas THC tolerance and withdrawal were minimally modified in mutant animals. In contrast, profound phenotypic changes are observed in several place conditioning protocols that reveal both THC rewarding and aversive properties. Absence of microreceptors abolishes THC place preference. Deletion of kappa receptors ablates THC place aversion and furthermore unmasks THC place preference. Thus, an opposing activity of mu- and kappa-opioid receptors in modulating reward pathways forms the basis for the dual euphoric-dysphoric activity of THC.

Autoradiography in opioid triple knockout mice reveals opioid and opioid receptor like binding of naloxone benzoylhydrazone.

Naloxone benzoylhydrazone (NalBzoH) is a ligand used to study opioid receptors. It has been suggested to act at a novel kappa3 receptor but also appears to bind to classical opioid receptors, and possibly the ORL1 receptor. We have used opioid receptor triple knockout mice, deficient in genes coding for the mu, delta and kappa-receptor, to characterise the relative contributions of opioid and ORL1 activity to the binding of this ligand, by carrying out receptor autoradiography with [3H]NalBzoH. As competing ligands we have used diprenorphine and nociceptin at 1 microM, alone or in combination, to determine the contribution of opioid and ORL1 receptor binding. At 4 nM [3H]NalBzoH showed labelling in wild-type brains indicative of broad spectrum classical opioid receptor binding. In the triple knockout brains all labelling was completely absent, suggesting that at this concentration there is no binding to ORL1 sites. However at 50 nM [3H]NalBzoH showed labelling in triple knockout brains with a distribution pattern indicative of ORL1 labelling. Quantitative analysis showed that nociceptin displaced typically 30% of the residual labelling in knockout brains whilst diprenorphine had relatively little effect. The data show that at 50 nM NalBzoH no binding was detected other than to classical opioid receptors or to ORL1 in an approximate ratio of 2:1.

Neurofilament proteins and cAMP pathway in brains of mu-, delta- or kappa-opioid receptor gene knock-out mice: effects of chronic morphine administration.

Opiate addiction is associated with abnormalities of neurofilament (NF) proteins and upregulation of cAMP signaling in the brain, which may modulate neuronal plasticity. This study investigated, using gene-targeted mice lacking mu-, delta- or kappa-opioid receptors, the role of these receptors in modulating the basal activity and the chronic effects of morphine on both intracellular targets. In WT mice, chronic treatment (5 days) with morphine (20-100 mg/kg) resulted in decreases in the immunodensity of neurofilament (NF)-L in the cerebral cortex (14-23%). In contrast, chronic morphine did not decrease NF-L in cortices of mu-, delta-, and kappa-KO mice, suggesting the involvement of the three types of opioid receptors in this effect of morphine. Also, the marked increase in phosphorylated NF-H induced by chronic morphine in WT mice (two-fold) was abolished in mu -KO mice. In cortex and/or striatum of mu-, delta- and kappa-KO mice, the basal immunodensities of Galphai1/2 proteins, the catalytic isoform (Calpha) of protein kinase A (PKA) and the total content of cAMP response element-binding protein (CREB, the nuclear target of PKA) were not different from those of WT mice. In contrast, phosphorylated CREB (the active form of this transcription factor) was reduced in cortex and/or striatum (23-26%) of mu- and delta-KO mice, but not in kappa-KO animals. These results suggest that the endogenous opioid tone acting on mu-/delta-receptors tonically stimulate CREB activation in the brain. In cortex and/or striatum of WT mice, chronic morphine did not induce upregulation of the main components of the cAMP signaling pathway. In contrast, chronic morphine treatment in mu-KO mice, but not in delta- or kappa-KO, resulted in a paradoxical upregulation of Galphai1/2 (12-19%), PKA (19-21%,) and phosphorylated CREB (21-73%), but not total CREB, in cortex and/or striatum. The induction of heterologous receptor adaptations in mu-KO mice may explain this paradoxical effect of morphine.

Enhanced humoral response in kappa-opioid receptor knockout mice.

Opiates are major analgesics and addictive drugs described also as immunomodulators. Here, we investigated the contribution of kappa-opioid receptor (KOR) activity in immunity in vivo by studying immune responses in KOR knockout mice. These animals displayed a modest reduction in thymus cellularity and CD4(+) cell ratio, parallel to a slight increase in immature CD4(+)CD8(+) lymphocytes. In spleen, KOR null animals showed augmented cell number with no change in cell distribution. T and B lymphocyte proliferative capabilities in vitro, Natural Killer activity and steady-state Ig levels were unchanged in KOR-/- mice. We immunized the mice with the antigen keyhole limpet hemocyanin (KLH). Compared to wild-type (WT) mice, KOR-/- animals produced significant higher levels of antigen-specific total Ig, IgM, IgG1 and IgG2a antibodies. This enhancement of humoral activity was not observed in mu-opioid receptor and delta-opioid receptor knockout animals. These results show that endogenous activation of kappa-opioid receptors may exert a tonic inhibition of antibody (Ab) response.

Autoradiographic mapping of the opioid receptor-like 1 (ORL1) receptor in the brains of mu-, delta- or kappa-opioid receptor knockout mice.

The opioid receptor-like 1 (ORL1) receptor shares a high degree of sequence homology with the classical mu-, delta- and kappa-opioid receptors and a functional mutual opposition between these receptors has been suggested. To further address this possible interaction we have used mu-, delta- and kappa-opioid receptor knockout mice to determine autoradiographically if there are any changes in the number or distribution of the ORL1 receptor, labelled with [(3)H]nociceptin, in the brains of mice deficient in each of the opioid receptors. An up-regulation of ORL1 expression was observed across all brain regions in delta-knockouts with cortical regions typically showing a 15-30% increase in binding that was most marked in heterozygous mice. In contrast, ORL1 receptor expression was down-regulated in virtually all brain structures in heterozygous kappa-knockouts although the magnitude of this change was not as great as for the delta-knockouts. No significant alterations in ORL1 receptor expression were observed across brain regions in mu-receptor knockout mice and there were no qualitative differences in ORL1 receptor expression in any groups. These data suggest there are interactions between the ORL1 system and the classical opioid receptors and that the interactions are receptor-specific. The greater differences observed in heterozygous mice suggest that these interactions might be most relevant when there is only partial loss of receptor function.

Opposite alterations of NPFF1 and NPFF2 neuropeptide FF receptor density in the triple MOR/DOR/KOR-opioid receptor knockout mouse brains.

Mice lacking the mu-delta-kappa-opioid receptor (MOR/DOR/KOR) genes and their corresponding wild-type littermates have been used to quantify NPFF(1) and NPFF(2) (neuropeptide FF) receptors by in vitro autoradiography in the central nervous tissues. Adjacent coronal sections were labelled with [125I]YVP ([125I]YVPNLPQRF-NH(2)) and [125I]EYF ([125I]EYWSLAAPQRF-NH(2)) as specific radioligands for NPFF(1) and NPFF(2) receptors, respectively. NPFF(2) receptors are predominantly expressed in both genotypes, but their density increases significantly in non cortical regions of mutant mice: 64% in the amygdaloid area, 89, 308, 1214 and 49% in the nucleus of the vertical limb of the diagonal band, substantia nigra, the vestibular nucleus and the spinal cord, respectively. In contrast, the density of the NPFF(1) subtype is lower than NPFF(2) in both genotypes and significantly decreased in some brain areas of mutant mice: -99, -90 and -90% in the nucleus of the vertical limb of the diagonal band, substantia nigra and the spinal cord, respectively. This study shows that mice lacking opioid receptors have brain region-dependent increases (NPFF(2)) and decreases (NPFF(1)) in NPFF receptors densities and suggests a different functional participation of each NPFF receptor subtype in the actions of opioids.

Improgan, a cimetidine analog, induces morphine-like antinociception in opioid receptor-knockout mice.

Improgan is an analog of the H(2) antagonist cimetidine that does not act on known histamine receptors, but induces highly effective analgesia in rodents following intracerebroventricular (icv) administration. Since the mechanism of action of this compound remains unknown, improgan analgesia was characterized presently with the tail immersion nociceptive test in mutant mice lacking either the mu (exon 1 of MOR-1), delta (exon 2 of DOR-1) or kappa (exon 3 of KOR-1) opioid receptor. Improgan (30 microg, icv) induced reversible, maximal analgesia in both sexes of all three genotypes (+/+, +/- and -/-) of MOR-1 mutant mice 10 and 20 min after administration, whereas morphine analgesia was reduced (+/-) or abolished (-/-) in these subjects. In DOR-1 mutant mice, improgan was equally effective in all three genotypes, despite the reduction (+/-) or complete loss (-/-) of delta opioid receptor (3H-[D-Pen(2), D-Pen(5)]enkephalin, DPDPE) binding. Similarly, improgan analgesia was equivalent in all three genotypes of KOR-1 mutant mice, whereas kappa-mediated analgesia (U50,488) and kappa opioid (3H-U69,593) binding were abolished in the homozygous (-/-) mice. These studies demonstrate that improgan analgesia does not require intact MOR-1, DOR-1, or KOR-1 genes, and support the hypothesis that improgan-like analgesics act in the CNS by non-opioid mechanisms.