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Opioid receptors (ORs) precisely modulate behavior when activated by native peptide ligands but distort behaviors to produce pathology when activated by non-peptide drugs. A fundamental question is how drugs differ from peptides in their actions on target neurons. Here, we show that drugs differ in the subcellular location at which they activate ORs. We develop a genetically encoded biosensor that directly detects ligand-induced activation of ORs and uncover a real-time map of the spatiotemporal organization of OR activation in living neurons. Peptide agonists produce a characteristic activation pattern initiated in the plasma membrane and propagating to endosomes after receptor internalization. Drugs produce a different activation pattern by additionally driving OR activation in the somatic Golgi apparatus and Golgi elements extending throughout the dendritic arbor. These results establish an approach to probe the cellular basis of neuromodulation and reveal that drugs distort the spatiotemporal landscape of neuronal OR activation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background/aims: To develop a suitable hepatocyte-like cell model that could be a substitute for primary hepatocytes with essential transporter expression and functions. Induced hepatocyte-like (iHep) cells directly reprogrammed from mice fibroblast cells were fully characterized., Methods: Naïve iHep cells were transfected with nuclear hepatocyte factor 4 alpha (Hnf4α) and treated with selected small molecules. Sandwich cultured configuration was applied. The mRNA and protein expression of transporters were determined by Real Time PCR and confocal. The functional transporters were estimated by drug biliary excretion measurement. The inhibition of bile acid efflux transporters by cholestatic drugs were assessed., Results: The expression and function of p-glycoprotein (P-gp), bile salt efflux pump (Bsep), multidrug resistance-associated protein 2 (Mrp2), Na+-dependent taurocholate cotransporting polypeptide (Ntcp), and organic anion transporter polypedtides (Oatps) in iHep cells were significantly improved after transfection of hepatocyte nuclear factor 4 alpha (Hnf4α) and treatment with selected inducers. In vitro intrinsic biliary clearances (CLb,int) of optimized iHep cells for rosuvastatin, methotrexate, d8-TCA (deuterium-labeled sodium taurocholate acid) and DPDPE ([D-Pen2,5] enkephalin hydrate) correlated well with that of sandwich-cultured primary mouse hepatocytes (SCMHs) (r2 = 0.984). Cholestatic drugs were evaluated and the results were compared well with primary mice hepatocytes., Conclusion: The optimized iHep cells expressed functional drug transporters and were comparable to primary mice hepatocytes. This study suggested direct reprogramming could provide a potential alternative to primary hepatocytes for drug candidate hepatobiliary disposition and hepatotoxicity screening., (© 2016 S. Karger AG, Basel.)

Physical exercise stimulates the release of endogenous opioid peptides supposed to be responsible for changes in mood, anxiety, and performance. Exercise alters sensitivity to these effects that modify the efficacy at the opioid receptor. Although there is evidence that relates exercise to neuropeptide expression in the brain, the effects of exercise on opioid receptor binding and signal transduction mechanisms downstream of these receptors have not been explored. Here, we characterized the binding and G protein activation of mu opioid receptor, kappa opioid receptor or delta opioid receptor in several brain regions following acute (7 days) and chronic (30 days) exercise. As regards short- (acute) or long-term effects (chronic) of exercise, overall, higher opioid receptor binding was observed in acute-exercise animals and the opposite was found in the chronic-exercise animals. The binding of [(35) S]GTPγS under basal conditions (absence of agonists) was elevated in sensorimotor cortex and hippocampus, an effect more evident after chronic exercise. Divergence of findings was observed for mu opioid receptor, kappa opioid receptor, and delta opioid receptor receptor activation in our study. Our results support existing evidence of opioid receptor binding and G protein activation occurring differentially in brain regions in response to diverse exercise stimuli. We characterized the binding and G protein activation of mu, kappa, and delta opioid receptors in several brain regions following acute (7 days) and chronic (30 days) exercise. Higher opioid receptor binding was observed in the acute exercise animal group and opposite findings in the chronic exercise group. Higher G protein activation under basal conditions was noted in rats submitted to chronic exercise, as visible in the depicted pseudo-color autoradiograms., (© 2014 International Society for Neurochemistry.)

Kappa-opioid receptor (κ) antagonists are potential therapeutic agents for a range of psychiatric disorders. The feasibility of developing κ-antagonists has been limited by the pharmacodynamic properties of prototypic κ-selective antagonists; that is, they inhibit receptor signaling for weeks after a single administration. To address this issue, novel trans-(3R,4R)-dimethyl-4-(3-hydroxyphenyl) piperidine derivatives, based on JDTic, were designed using soft-drug principles. The aim was to determine if the phenylpiperidine-based series of κ-antagonists was amenable to incorporation of a potentially metabolically labile group, while retaining good affinity and selectivity for the κ-receptor. Opioid receptor binding affinity and selectivity of three novel compounds (BU09057, BU09058, and BU09059) were tested. BU09059, which most closely resembles JDTic, had nanomolar affinity for the κ-receptor, with 15-fold and 616-fold selectivity over μ- and δ-receptors, respectively. In isolated tissues, BU09059 was a potent and selective κ-antagonist (pA2 8.62) compared with BU09057 (pA2 6.87) and BU09058 (pA2 6.76) which were not κ-selective. In vivo, BU09059 (3 and 10 mg/kg) significantly blocked U50,488-induced antinociception and was as potent as, but shorter acting than, the prototypic selective κ-antagonist norBNI. These data show that a new JDTic analogue, BU09059, retains high affinity and selectivity for the κ-receptor and has a shorter duration of κ-antagonist action in vivo.

Chronic L-3,4-dihydroxyphenylalanine (L-DOPA) treatment of Parkinson's disease induces in time numerous side effects, such as abnormal involuntary movements called L-DOPA-induced dyskinesias (LIDs). An involvement of glutamate transmission, dopamine transmission and opioid transmission in striatal output pathways has been hypothesized for the induction of LIDs. Interestingly, our previous experiments indicated that some striatal δ-opioid receptors are located on terminals of glutamatergic corticostriatal neurons and that stimulation of these receptors modulates the release of glutamate and dopamine. The present study was performed to test the involvement of δ-opioid receptors, and more precisely of those located on corticostriatal neurons, in abnormal involuntary movements induced by L-DOPA in hemiparkinsonian rats. The effects of a selective agonist, [D-Pen(2), D-Pen(5)]-enkephalin (DPDPE) and a selective antagonist (naltrindole) of δ-opioid receptors on LIDs were investigated in animals submitted or not to a corticostriatal deafferentation. Our results indicate that DPDPE and naltrindole respectively enhanced and reduced LIDs in animals in which the ipsilateral cortex was preserved intact. However, the lesion of the ipsilateral cortex prevented the stimulant effect of DPDPE on LIDs. The [(3)H]-DPDPE binding to striatal membranes prepared from the whole striatum was also studied. A significant increase in density of δ-opioid receptors was found in the striatum of dyskinetic animals as compared to non-dyskinetic animals but this difference was abolished by the corticostriatal deafferentation. These results indicate that δ-opioid transmission modulates the expression of LIDs in rodents and suggest that the δ-opioid receptors involved in this effect are located on terminals of corticostriatal neurons., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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.

To evaluate potential mechanisms of clinical hepatotoxicity, 4 endothelin receptor antagonists (ERAs) were examined for substrate activity and inhibition of hepatic uptake and efflux transporters in sandwich-cultured human hepatocytes. The 4 transporters studied were sodium-dependent taurocholate cotransporter (NTCP), organic anion transporter (OATP), bile salt export pump (BSEP), and multidrug resistance-associated protein 2 (MRP2). ERA transporter inhibition was examined using the substrates taurocholate (for NTCP and BSEP), [(3)H]estradiol-17beta-D-glucuronide (for OATP), and [2-D-penicillamine, 5-D-penicillamine]enkephalin (for MRP2). ERA substrate activity was evaluated using probe inhibitors ritonavir (OATP and BSEP), bromosulfalein (OATP), erythromycin (P-glycoprotein), probenecid (MRP2 and OATP), and cyclosporin (NTCP). ERAs were tested at 2, 20, and 100 micromol*L-1 for inhibition and at 2 micromol*L-1 as substrates. OATP, NTCP, or BSEP transport activity was not reduced by ambrisentan or darusentan. Bosentan and sitaxsentan attenuated NTCP transport at higher concentrations. Only sitaxsentan decreased OATP transport (52%), and only bosentan reduced BSEP transport (78%). MRP2 transport activity was unaltered. OATP inhibitors decreased influx of all ERAs. Darusentan influx was least affected (84%-100% of control), whereas bosentan was most affected (32%-58% of control). NTCP did not contribute to influx of ERAs. Only bosentan and darusentan were shown as substrates for both BSEP and P-glycoprotein efflux. All ERAs tested were substrates for at least one hepatic transporter. Bosentan and sitaxsentan, but not ambrisentan and darusentan, inhibited human hepatic transporters, which provides a potential mechanism for the increased hepatotoxicity observed for these agents in the clinical setting.

The human liver-specific organic anion transporting polypeptides (OATPs) 1B1 and 1B3 are involved in the elimination of numerous xenobiotics and drugs. Although dogs are frequently used for toxicologic and pharmacokinetic characterization of novel drugs, nothing is known about their OATP1B1/1B3 ortholog. Therefore, we cloned and characterized the first canine organic anion transporting polypeptide from dog liver, termed Oatp1b4. The isolated Oatp1b4 cDNA comprises 3661 base pairs (bp) with an open reading frame of 2076bp, encoding a 692-amino acid protein with a molecular mass of approximately 85kDa. The Oatp1b4 gene is approximately 61kb long and has a similar organization as the human OATP1B1 and OATP1B3 with 13 exons identical in length. Northern blot analysis shows that Oatp1b4 is predominantly expressed in the liver. Oatp1b4 mediates sodium-independent transport of typical organic anions including bromosulfophthalein (BSP), [D-penicillamine(2,5)]enkephalin (DPDPE), estradiol-17beta-glucuronide (E17betaG), estrone-3-sulfate and taurocholate. In addition, Oatp1b4 transports the OATP1B3-specific substrate cholecystokinin octapeptide (CCK-8). Kinetic studies showed that Oatp1b4-mediated E17betaG and estrone-3-sulfate transports were monophasic with K(m) values of 5+/-1microM and 33+/-4microM, respectively. In conclusion, the cloned canine Oatp1b4 will provide additional molecular basis to further characterize the species difference of the OATP1B family members.

This study investigated the role of a multispecific organic anion transporter, Oatp1a4/Slco1a4, in drug transport across the blood-brain barrier. In vitro transport studies using human embryonic kidney 293 cells expressing mouse Oatp1a4 identified the following compounds as Oatp1a4 substrates: pitavastatin (K(m) = 8.3 microM), rosuvastatin (K(m) = 12 microM), pravastatin, taurocholate (K(m) = 40 microM), digoxin, ochratoxin A, and [d-penicillamine(2,5)]-enkephalin. Double immunohistochemical staining of Oatp1a4 with P-glycoprotein (P-gp) or glial fibrillary acidic protein demonstrated that Oatp1a4 signals colocalized with P-gp signals partly but not with glial fibrillary acidic protein, suggesting that Oatp1a4 is expressed in both the luminal and the abluminal membranes of mouse brain capillary endothelial cells. The brain-to-blood transport of pitavastatin, rosuvastatin, pravastatin, and taurocholate after microinjection into the cerebral cortex was significantly decreased in Oatp1a4(-/-) mice compared with that in wild-type mice. The blood-to-brain transport of pitavastatin, rosuvastatin, taurocholate, and ochratoxin A, determined by in situ brain perfusion, was significantly lower in Oatp1a4(-/-) mice than in wild-type mice, whereas transport of pravastatin and [D-penicillamine(2,5)]-enkephalin was unchanged. The blood-to-brain transport of digoxin was significantly lower in Oatp1a4(-/-) mice than in wild-type mice only when P-gp was inhibited by N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918). Taken together, these results show that Oatp1a4 can mediate the brain-to-blood and blood-to-brain transport of its substrate drugs across the blood-brain barrier. The brain-to-plasma ratio of taurocholate, pitavastatin, and rosuvastatin was close to the capillary volume in wild-type mice, and it was not affected by Oatp1a4 dysfunction. Whether Oatp1a4 can deliver drugs from the blood to the brain remains controversial.

Abstract An important limitation in the clinical use of opiates is progressive loss of analgesic efficacy over time. Development of analgesic tolerance is tightly linked to receptor desensitization. In the case of delta opioid receptors (DOR), desensitization is especially swift because receptors are rapidly internalized and are poorly recycled to the membrane. In the present study, we investigated whether Src activity contributed to this sorting pattern and to functional desensitization of DORs. A first series of experiments demonstrated that agonist binding activates Src and destabilizes a constitutive complex formed by the spontaneous association of DORs with the kinase. Src contribution to DOR desensitization was then established by showing that pre-treatment with Src inhibitor PP2 (20 microM; 1 hr) or transfection of a dominant negative Src mutant preserved DOR signalling following sustained exposure to an agonist. This protection was afforded without interfering with endocytosis, but suboptimal internalization interfered with PP2 ability to preserve DOR signalling, suggesting a post-endocytic site of action for the kinase. This assumption was confirmed by demonstrating that Src inhibition by PP2 or its silencing by siRNA increased membrane recovery of internalized DORs and was further corroborated by showing that inhibition of recycling by monensin or dominant negative Rab11 (Rab11S25N) abolished the ability of Src blockers to prevent desensitization. Finally, Src inhibitors accelerated recovery of DOR-Galphal3 coupling after desensitization. Taken together, these results indicate that Src dynamically regulates DOR recycling and by doing so contributes to desensitization of these receptors.

The CXCR4 chemokine receptor and the delta opioid receptor (DOR) are pertussis toxin-sensitive G protein-coupled receptors (GPCR). Both are widely distributed in brain tissues and immune cells, and have key roles in inflammation processes and in pain sensation on proximal nerve endings. We show that in immune cells expressing CXCR4 and DOR, simultaneous addition of their ligands CXCL12 and [D-Pen2, D-Pen5]enkephalin does not trigger receptor function. This treatment does not affect ligand binding or receptor expression, nor does it promote heterologous desensitization. Our data indicate that CXCR4 and DOR form heterodimeric complexes that are dynamically regulated by the ligands. This is compatible with a model in which GPCR oligomerization leads to suppression of signaling, promoting a dominant negative effect. Knockdown of CXCR4 and DOR signaling by heterodimerization might have repercussions on physiological and pathological processes such as inflammation, pain sensation and HIV-1 infection.

The antinociceptive effect of i.t. administered N(alpha)-amidino-Tyr-d-Arg-Phe-beta-Ala (amidino-TAPA), an N-terminal tetrapeptide analog of dermorphin, was characterized in ddY mice. In the opioid receptor ligand-binding assays using mouse brain membranes, amidino-TAPA showed a very high affinity for mu-opioid receptors, a low affinity to delta-opioid receptors and no affinity for kappa-opioid receptors. In the mouse tail-flick test, i.t. treatment with amidino-TAPA produced a potent antinociception. The antinociception induced by amidino-TAPA was significantly attenuated by i.t. pretreatment with the mu-opioid receptor antagonist beta-funaltrexamine, the kappa-opioid receptor antagonist nor-binaltorphimine and the delta-opioid receptor antagonist naltrindole. Moreover, the antinociception induced by amidino-TAPA was significantly attenuated by i.t. pretreatment with antisera against the endogenous kappa-opioid peptides dynorphin A, dynorphin B and alpha-neo-endorphin; and the endogenous delta-opioid peptide [Leu(5)]enkephalin. In mice lacking prodynorphin, the precursor of the endogenous kappa-opioid peptides, the antinociceptive effect of amidino-TAPA was significantly attenuated compared to that in wild-type C57BL/6J mice. However, there was no difference in G-protein activation by amidino-TAPA in the spinal cord membranes from prodynorphin knockout mice and C57BL/6J mice. The present results suggest that the spinal antinociception induced by the mu-opioid receptor selective peptide amidino-TAPA is mediated in part by the release of endogenous opioid peptides in the spinal cord, which is caused by the direct stimulation of mu-opioid receptors.

Two full-length cDNAs, encoding delta (delta) and mu (mu) opioid receptors, were cloned from the brain of the rough-skinned newt Taricha granulosa, complementing previous work from our laboratory describing the cloning of newt brain kappa (kappa) and ORL1 opioid receptors. The newt delta receptor shares 82% amino acid sequence identity with a frog delta receptor and lower (68-70%) identity with orthologous receptors cloned from mammals and zebrafish. The newt mu receptor shares 79% sequence identity with a frog mu receptor, 72% identity with mammalian mu receptors, and 66-69% identity with mu receptors cloned from teleost fishes. Membranes isolated from COS-7 cells transiently expressing the newt delta receptor possessed a single, high-affinity (Kd = 2.4 nM) binding site for the nonselective opioid antagonist [3H]naloxone. In competition binding assays, the newt delta receptor displayed highest affinity for Met-enkephalin, relatively low affinity for Leu-enkephalin, beta-endorphin, and [D-penicillamine, D-penicillamine] enkephalin (DPDPE) (a delta-selective agonist in mammals), and very low affinity for mu-, kappa-, or ORL1-selective agonists. COS-7 cells expressing the newt mu receptor also possessed a high-affinity (Kd = 0.44 nM) naloxone-binding site that showed highest affinity for beta-endorphin, moderate-to-low affinity for Met-enkephalin and Leu-enkephalin and DAMGO (a mu-selective agonist in mammals), and very low affinity for DPDPE and kappa- or ORL1-selective agonists. COS-7 cells expressing either receptor type (delta or mu) showed very high affinity (Kd = 0.1-0.3 nM) for the nonselective opioid antagonist diprenorphine. Taricha granulosa expresses the same four subtypes (delta, mu, kappa, and ORL1) of opioid receptors found in other vertebrate classes, but ligand selectivity appears less stringent in the newt than has been documented in mammals.

Rapid and extensive biliary excretion of [D-penicillamine2,5]enkephalin (DPDPE) in rats as the unchanged peptide suggests that multiple transport proteins may be involved in the hepatobiliary disposition of this zwitterionic peptide. Although DPDPE is a P-glycoprotein substrate, the role of other transport proteins in the hepatic clearance of DPDPE has not been established. Furthermore, the ability of various experimental approaches to quantitate the contribution of a specific hepatic uptake or excretion process when multiple transport systems are involved has not been addressed. 3H-DPDPE uptake in suspended Wistar rat hepatocytes was primarily (>95%) due to temperature-dependent transport mechanisms; similar results were obtained in suspended hepatocytes from Mrp2-deficient (TR-) rats. Pharmacokinetic modeling revealed that saturable and linear processes were involved in 3H-DPDPE uptake in hepatocytes. The use of transport modulators suggested that hepatic uptake of 3H-DPDPE was mediated by Oatp1a1, Oatp1a4, and likely Oatp1b2. Accumulation of 3H-DPDPE in sandwich-cultured (SC) hepatocytes was rapid; uptake of 3H-DPDPE in SC rat hepatocytes from control and TR- rats was similar. However, the biliary excretion index and biliary clearance decreased by 83 and 85%, respectively, in TR- SC rat hepatocytes, indicating that DPDPE is an Mrp2 substrate. Rate constants for uptake and excretion of 3H-DPDPE in SC rat hepatocytes were determined by pharmacokinetic modeling; data were consistent with basolateral excretion of 3H-DPDPE from the hepatocyte. These results demonstrate the complexities of hepatobiliary disposition when multiple transport mechanisms are involved for a given substrate and emphasize the necessity of multi-experimental approaches for the comprehensive resolution of these processes.

N3-(2',5 '-Dimethoxyphenacyl)arabinofuranosyluracil (N3-(2',5 '-DiMeOPhAc)AraU) is a pyrimidine nucleoside derivative which possesses antinociceptive effect by intracerebroventricular (i.c.v.) administration to mice. The compound (100 microM) significantly decreased the specific binding of [3H]D-Ala2, N-Me-Phe4, Gly5-ol-enkephalin (DAMGO) and [3H]D-Pen2, D-Pen5-enkephalin (DPDPE) at the mu- and delta-opioid receptor, respectively, but has no effect on the binding of [3H]U-69,593 at the kappa-opioid receptor of rat brain slices. The autoradiographic studies also demonstrated that [3H]DAMGO and [3H]DPDPE bindings on the rat brain slice were displaced by N3-(2',5'-DiMeOPhAc)AraU. These results indicate that N3-(2',5'-DiMeOPhAc)AraU interacts with mu- and delta-opioid receptors.

Pyrene possesses unique spectroscopic properties such as a high quantum yield, a long half-life in the excited state, and the ability to form excimers when in proximity to each other in the excited state. These properties allow pyrenylalanine, which is a pyrene moiety incorporated into an amino acid, to be used as a fluorescent probe in peptides and proteins. The common route for the synthesis of pyrenylalanine involves 5 steps, with subsequent separation of the two isomers by recrystallization. This paper reports a novel 3-step asymmetric synthesis of pyrenylalanine with high enantioselectivity, good yields, and facile isomer purification. After synthesis, pyrenylalanine was incorporated into a series of opioid, CCK, and melanotropin peptide ligands in order to study the effects of aromaticity, lipophilicity, and steric properties on their potency and efficacy at their corresponding biological receptors. The change in binding and efficacy of the labeled ligands as compared to the unlabeled ligands demonstrates the possible role of lipophilicity/aromaticity in the binding and signal transduction of the ligand-receptor interaction.

We studied the effect of a novel delta-opioid receptor antagonist N,N(CH(3))(2)Dmt-Tic-OH (Me(2)-Dmt-Tic-OH) on voluntary ethanol intake in an alcohol-preferring AA (Alko, Alcohol) rat line using a 4-hour limited access paradigm. Acute injections of Me(2)-Dmt-Tic-OH (10 and 30 mg/kg, i.p.) did not reduce 1-hour or 4-hour ethanol intake. Subtype non-selective opioid receptor antagonist naltrexone [0.1 and 0.3 mg/kg, subcutaneously (s.c.)] significantly reduced 1-hour ethanol drinking but had no effect on 4-hour ethanol consumption. Locomotor stimulation induced by the delta-opioid receptor agonist Tyr-D-Pen-Gly-Phe-D-Pen (DPDPE; 15 microg, intracerebroventricularly) was significantly attenuated by Me(2)-Dmt-Tic-OH (10 and 30 mg/kg, i.p.), which confirmed its efficacy as a delta-opioid receptor antagonist in rat brain. Our results support the idea that delta-opioid receptors do not mediate alcohol reward in AA rats.

Compound trans-4-(p-bromophenyl)-4-(dimethylamino)-1-(2-thiophen-2-yl-ethyl)-cyclohexanol (C8813), structurally unrelated to morphine, is a novel analgesic. The present study examined the antinociception, opioid receptor selectivity and in vitro activity of C8813. The antinociceptive activity was evaluated using mouse hot plate and acetic acid writhing tests. In mouse hot plate test, the antinociceptive ED(50) of C8813 was 11.5 microg/kg, being 591 times and 3.4 times more potent than morphine and fentanyl respectively. In mouse writhing test, the antinociceptive ED(50) of C8813 was 16.9 microg/kg, being 55 times and 2.3 times more active than morphine and fentanyl respectively. In the opioid receptor binding assay, C8813 showed high affinity for mu-opioid receptor (K(i) = 1.37 nM) and delta-opioid receptor (K(i) = 3.24 nM) but almost no affinity for kappa-opioid receptor (at 1 microM). In the bioassay, the inhibitory effect of C8813 in the guinea-pig ileum (GPI) was 16.5 times more potent than in the mouse vas deferens (MVD). The inhibitory effects of C8813 in the GPI and MVD could be antagonized by mu-opioid receptor antagonist naloxone and delta-opioid receptor antagonist ICI174,864 respectively. However, the inhibitory effect of C8813 in the rabbit vas deferens was very weak. These results indicated that C8813 was a potent analgesic and a high affinity agonist for the mu- and delta-opioid receptors.

Imipramine (CAS 113-52-0) is being utilized widely for the treatment of major depression. In recent years, there has been evidence of the involvement of the endogenous opioid system in major depression and its treatment. There is some evidence indicating that opioid receptors could be involved in the antidepressant mechanism of action. Regarding this topic, mood-related behavior of endogenous enkephalins seems to be mediated by delta-opioid receptors. In this work, the effects of subacute (5 day) and chronic (15 day) treatments of imipramine on the density and the affinity of the delta-receptors in the striatum and in the parietal and frontal cortices of the rat brain are described. Studied parameters (Bmax and Kd) were calculated by a saturation binding assay with the delta-opioid agonists [3H]-DPDPE (tyrosyl-2,6-3H(N)-(2-D-penicillamine-5-D-penicillamine)-enkephalin) as specific ligand and DSLET ([D-serine2]-D-leucine-enkephalin-threonine) as non-radioactive competing ligand. It was found that 15 days treatment significantly decreased the delta-opioid receptor density,without changing the affinity, in the frontal cortex of the rat brain. That decrease was confirmed by delta-opioid receptor immunostaining. These results suggest that delta-opioid receptors could play a role in the chronic action mechanism of imipramine.

The properties of two prospective positron emission tomography (PET) ligands for the delta-opioid receptor, N1'-fluoroethyl-naltrindole (BU97001) and N1'-fluoroethyl-(14-formylamino)-naltrindole (BU97018) were investigated. Both were antagonists in the mouse vas deferens, and showed high affinity and selectivity, 1.81 nM and 3.09 nM respectively. [3H]BU97001 binding to rat whole brain was also of high affinity, K(D) of 0.42 nM of and B(MAX) of 59.95 fmol mg of protein(-1). In autoradiographic studies, it was found to bind to brain areas previously shown to be associated with the delta-opioid receptor and good correlations were found to exist with naltrindole and DPDPE. BU97018 and especially BU97001 appear to show good potential as delta-opioid receptor PET ligands with the incorporation of 18F.

1. There is evidence for interactions between mu and delta opioid systems both in vitro and in vivo. This work examines the hypothesis that interaction between these two receptors can occur intracellularly at the level of G protein in human neuroblastoma SH-SY5Y cells. 2. The [(35)S]GTP gamma S binding assay was used to measure G protein activation following agonist occupation of opioid receptors. The agonists DAMGO (EC(50), 45 nM) and SNC80 (EC(50), 32 nM) were found to be completely selective for stimulation of [(35)S]-GTP gamma S binding through mu and delta opioid receptors respectively. Maximal stimulation of [(35)S]-GTP gamma S binding produced by SNC80 was 57% of that seen with DAMGO. When combined with a maximally effective concentration of DAMGO, SNC80 caused no additional [(35)S]-GTP gamma S binding. This effect was also seen when measured at the level of adenylyl cyclase. 3. Receptor activation increased the dissociation of pre-bound [(35)S]-GTP gamma S. In addition, the delta agonist SNC80 promoted the dissociation of [(35)S]-GTP gamma S from G proteins initially labelled using the mu agonist DAMGO. Conversely, DAMGO promoted the dissociation of [(35)S]-GTP gamma S from G proteins initially labelled using SNC80. 4. Tolerance to DAMGO and SNC80 in membranes from cells exposed to agonist for 18 h was homologous and there was no evidence for alteration in G protein activity. 5. The findings support the hypothesis that mu- and delta-opioid receptors share a common G protein pool, possibly through a close organization of the two receptors and G protein at the plasma membrane.

Specific binding sites for [125I]beta-endorphin and the delta1-opioid [3H][D-pen(2), D-pen(5)]enkephalin (DPDPE) were quantified using autoradiography in soleus and extensor digitorum longus (EDL) muscles of lean and obese-diabetic (ob/ob) mice. The density of binding was significantly higher in obese-diabetic than lean mice. The uptake of 2-deoxy-D-[1-3H]deoxyglucose, a nonmetabolized glucose analogue, into isolated soleus and EDL muscles was stimulated by beta-endorphin, beta-endorphin 1-27, and DPDPE, but not by the delta2-opioid deltorphin II. Both beta-endorphin and DPDPE stimulated deoxyglucose uptake in obese-diabetic mice. Thus, glucose transport in skeletal muscle may be partly mediated via delta1-opioid receptors. The increased receptor density in obese-diabetic mice may be an adaptive response., (Copyright 2001 by W.B. Saunders Company)

mu- and delta-Opioid agonists interact in a synergistic manner to produce analgesia in several animal models. Additionally, receptor binding studies using membranes derived from brain tissue indicate that interactions between mu- and delta-opioid receptors might be responsible for the observation of multiple opioid receptor subtypes. To examine potential interactions between mu- and delta-opioid receptors, we examined receptor binding and functional characteristics of mu-, delta-, or both mu- and delta-opioid receptors stably transfected in rat pituitary GH(3) cells (GH(3)MOR, GH(3)DOR, and GH(3)MORDOR, respectively). Saturation and competition binding experiments revealed that coexpression of mu- and delta-opioid receptors resulted in the appearance of multiple affinity states for mu- but not delta-opioid receptors. Additionally, coadministration of selective mu- and delta-opioid agonists in GH(3)MORDOR cells resulted in a synergistic competition with [(3)H][D-Pen(2,5)]enkephalin (DPDPE) for delta-opioid receptors. Finally, when equally effective concentrations of [D-Ala(2),N-MePhe(4),Gly-ol(5)]enkephalin (DAMGO) and two different delta-opioid agonists (DPDPE or 2-methyl-4a alpha-(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12a alpha-octahydroquinolino-[2,3,3-g]-isoquinoline; TAN67) were coadministered in GH(3)MORDOR cells, a synergistic inhibition of adenylyl cyclase activity was observed. These results strongly suggest that cotransfection of mu- and delta-opioid receptors alters the binding and functional characteristics of the receptors. Therefore, we propose that the simultaneous exposure of GH(3)MORDOR cells to selective mu- and delta-opioid agonists produces an interaction between receptors resulting in enhanced receptor binding. This effect is translated into an augmented ability of these agonists to inhibit adenylyl cyclase activity. Similar interactions occurring in neurons that express both mu- and delta-opioid receptors could explain observations of multiple opioid receptor subtypes in receptor binding studies and the synergistic interaction of mu- and delta-opioids in analgesic assays.

In this study, receptor binding profiles of opioid ligands for subtypes of opioid delta-receptors were examined employing [3H]D-Pen2,D-Pen5-enkephalin ([3H]DPDPE) and [3H]Ile(5,6)-deltorphin II ([3H]Ile-Delt II) in human cerebral cortex membranes. [3H]DPDPE, a representative ligand for delta1 sites, labeled a single population of binding sites with apparent affinity constant (Kd) of 2.72 +/- 0.21 nM and maximal binding capacity (Bmax) value of 20.78 +/- 3.13 fmol/mg protein. Homologous competition curve of [3H]Ile-Delt II, a representative ligand for delta2 sites, was best fit by the one-site model (Kd = 0.82 +/- 0.07 nM). Bmax value (43.65 +/- 2.41 fmol/mg) for [3H]Ile-Delt II was significantly greater than that for [3H]DPDPE. DPDPE, [D-Ala2,D-Leu5]enkephalin (DADLE) and 7-benzylidenaltrexone (BNTX) were more potent in competing for the binding sites of [3H]DPDPE than for those of [3H]Ile-Delt II. On the other hand, deltorphin II (Delt II), [D-Ser2,Leu5,Thr6]enkephalin (DSLET), naltriben (NTB) and naltrindole (NTI) were found to be equipotent in competing for [3H]DPDPE and [3H]Ile-Delt II binding sites. These results indicate that both subtypes of opioid delta-receptors, delta1 and delta2, exist in human cerebral cortex with different ligand binding profiles.

We evaluated smooth muscle from human internal mammary artery and rat aorta for the presence of delta opioid receptors. Radioligand receptor competition studies using the delta-receptor selective agonist, [3H]-[D-Ala2, Met5] enkephalinamide (DAMA) suggested the expression of a high affinity binding site in rat and human blood vessels that was consistent with the delta-2 opioid receptor subtype. Using RT-PCR with primers to the cloned delta opioid receptor (DOR), a cDNA fragment identical to the known DOR sequence was obtained from the smooth muscle cell line, A-10. Stimulation of A-10 cells with DAMA resulted in a significant mobilization of intracellular calcium and membrane depolarization. Exposure of aortic rings denuded of endothelium to DAMA induced a naltrindole-senstive increase in contractile tone. These data demonstrate the presence of a functional DOR in vascular smooth muscle and a direct impact of opioids on vascular contractile tone.

Structural changes accompanying the binding of ligands to the cloned human delta-opioid receptor immobilized in a solid-supported lipid bilayer have been investigated using coupled plasmon-waveguide resonance spectroscopy. This highly sensitive technique directly monitors mass density, conformation, and molecular orientation changes occurring in anisotropic thin films and allows direct determination of binding constants. Although both agonist binding and antagonist binding to the receptor cause increases in molecular ordering within the proteolipid membrane, only agonist binding induces an increase in thickness and molecular packing density of the membrane. This is a consequence of mass movements perpendicular to the plane of the bilayer occurring within the lipid and receptor components. These results are consistent with models of receptor function that involve changes in the orientation of transmembrane helices.

We hypothesized that the selectivity profile of the rat mu-opioid receptor for opioid receptor-selective ligands is determined by the nature of the amino acid residues at highly divergent sites in the ligand-binding pocket. To determine which characteristics of these residues contribute to opioid receptor ligand selectivity, we made various mutant receptors that replaced the Lys(303) and Trp(318) residues near the extracellular interface of transmembrane domains VI and VII, respectively. Ligand binding determinations using transiently transfected monkey kidney epithelial (COS-1) cells show that Lys(303) mutations cause little change in the receptor binding profile, whereas the Trp(318) mutant receptors have considerably lower affinity for micro-opioid receptor-selective ligands and greatly increased affinity for delta-opioid receptor-selective ligands. The nature of these mutations show that this effect is not due to sterics or charge alone. [35S]guanosine-5'-O-(3-thio)-triphosphate ([35S]GTPgammaS) activity assays show that these residues may influence functional, as well as binding selection. We conclude that a primary role for Trp(318) is to form a basis for ligand selectivity.

The existence of dimers and oligomers for many G protein-coupled receptors has been described by us and others. Since many G protein-coupled receptor subtypes are highly homologous to each other, we examined whether closely related receptors may interact with each other directly and thus have the potential to create novel signaling units. Using mu- and delta-opioid receptors, we show that each receptor expressed individually was pharmacologically distinct and could be visualized following electrophoresis as monomers, homodimers, homotetramers, and higher molecular mass oligomers. When mu- and delta-opioid receptors were coexpressed, the highly selective synthetic agonists for each had reduced potency and altered rank order, whereas endomorphin-1 and Leu-enkephalin had enhanced affinity, suggesting the formation of a novel binding pocket. No heterodimers were visualized in the membranes coexpressing mu- and delta-receptors by the methods available. However, hetero-oligomers were identified by the ability to co-immunoprecipitate mu-receptors with delta-receptors and vice versa using differentially epitope-tagged receptors. In contrast to the individually expressed mu- and delta-receptors, the coexpressed receptors showed insensitivity to pertussis toxin and continued signal transduction, likely due to interaction with a different subtype of G protein. In this study, we provide, for the first time, evidence for the direct interaction of mu- and delta-opioid receptors to form oligomers, with the generation of novel pharmacology and G protein coupling properties.

Background: The in vitro adaptive responses of delta opiate receptors (DOR) to chronic ethanol treatment have been well documented. The acute effects of ethanol on these receptors are not well characterized beyond its effect on ligand binding. The aim of this study was to evaluate the acute effects of clinically relevant concentrations of ethanol (50-200 mm) on the saturation binding kinetics, receptor/ligand internalization, and agonist stimulation of G-protein coupling in N18TG2 cells expressing the Flag epitope-tagged mouse DOR., Methods: Confocal microscopy was used to localize Flag epitope-tagged DOR in N18TG2 cells. Saturation binding assays at 4 degrees C and 37 degrees C were conducted in the absence or presence of ethanol on cells not pretreated or pretreated with ethanol for 30 min at 37 degrees C. Highly specific delta agonist, DPDPE ([D-Pen2,D-Pen5]enkephalin), was used in these studies. The effect of ethanol on agonist stimulation of G-protein coupling was examined using [35S]GTPgammaS (guanosine-5'-O-(3-thio)triphosphate) binding to membranes. Agonist-mediated receptor internalization was examined using flow cytometry of cells labeled with the antiserum directed against the Flag epitope, and the ligand internalization was examined using [3H]DPDPE., Results: Ethanol decreased the binding of the agonist [3H]DPDPE, and not the antagonist [3H]diprenorphine, in a dose-dependent manner. These effects were temperature-dependent. Ethanol reversibly inhibited agonist stimulation of [35S]GTPgammaS binding. In non-pretreated cells, ethanol decreased the rate of receptor/ligand internalization, but this effect was not seen in ethanol pretreated cells. Taken together, these results suggest that pretreatment of N18TG2 cells with ethanol induces compensatory mechanisms that allow the receptor to function efficiently in its presence., Conclusion: Acute ethanol decreased the binding, agonist-mediated functional coupling and receptor/ligand internalization in N18TG2 cells expressing epitope-tagged DOR. In these cells, 30-min pretreatment with ethanol was sufficient to reverse these effects.

A series of 4-diarylaminotropanes has been prepared. Both endo and exo diastereomeric forms bound to the delta opioid receptor but the endo isomers were more potent and selective versus the mu opioid receptor than the exo isomers. The most potent delta opioid agonist (14) exhibited a delta opioid Ki of 0.2 nM and was 860-fold selective over mu.

Opioid receptors are known to couple to G-proteins and to inhibit adenylyl cyclase. Receptor activation of G-proteins can be measured by agonist-stimulated [35S]guanylyl-5'-O-(gamma-thio)-triphosphate (GTP gamma S-) binding in brain sections to localize neuroanatomically functional coupling of receptors to intracellular signal transduction mechanisms. In the present study the selective mu-, delta- and kappa 1-opioid agonists DAMGO ([D-Ala2,N-Me-Phe4, Gly-ol5]-enkephalin), DPDPE ([D-Pen2,5]-enkephalin) and enadoline (CI-977) were used to stimulate [35S]GTP gamma S-binding in human brain sections of frontal cortex and cerebellum. In human frontal cortex mu- and delta- opioid stimulated [35S]GTP gamma S-binding was evenly distributed throughout the gray matter, while kappa(1)-opioid stimulated [35S]GTP gamma S-binding was detected predominantly in lamina V and VI. In the cerebellar cortex stimulated [35S]GTP gamma S-binding revealed functional coupling of mu- and kappa 1-opioid receptors in the molecular layer.

The previously described cyclic mu opioid receptor-selective tetrapeptide Tyr-c[D-Cys-Phe-D-Pen]NH2 (Et) (JOM-6) was modified at residues 1 and 3 by substitution with various natural and synthetic amino acids, and/or by alteration of the cyclic system. Effects on mu and delta opioid receptor binding affinities, and on potencies and efficacies as measured by the [35S]-GTPgammaS assay, were evaluated. Affinities at mu and delta receptors were not influenced dramatically by substitution of Tyr1 with conformationally restricted phenolic amino acids. In the [35S]-GTPgammaS assay, all of the peptides tested exhibited a maximal response comparable with that of fentanyl at the mu opioid receptor, and all showed high potency, in the range 0.4-9nM. However, potency changes did not always correlate with affinity, suggesting that the conformation required for binding and the conformation required for activation of the opioid receptors are different. At the delta opioid receptor, none of the peptides were able to produce a response equivalent to that of the full delta agonist BW 373,U86 and only one had an EC50 value of less than 100nM. Lastly, we have identified a peptide, D-Hat-c[D-Cys-Phe-D-Pen]NH2 (Et), with high potency and > 1,000-fold functional selectivity for the mu over delta opioid receptor as measured by the [35S]-GTPgammaS assay.

Repeated prenatal exposure to cocaine attenuates arousal and cardiorespiratory functions in neonates. This study explored the possible role of brainstem mu- and delta-opioid systems in these effects. Medullary sections were obtained from 6 to 7 (young) and 20 to 21-day-old (older) piglets either unexposed or exposed prenatally to a 2-mg/kg intravenous cocaine hydrochloride dose, injected to the pregnant sow four times a day during the last third of gestation. Mu- and delta-opioid receptor binding was assessed by quantitative autoradiography using, respectively, 125I-DAMGO (Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol) and 125I-DPDPE (Tyr-D-Pen-Gly-pCl-Phe-D-Pen). At control, delta-, but not mu-opioid, receptor density increased with postnatal age. In contrast, cocaine-induced mu-, but not delta-opioid, receptor density increased 1) in the dorsal motor vagal (dmnX) and facial (nF) nuclei, and, at borderline significance level, in the cardiorespiratory-related gigantocellular reticular nucleus (nRG) of the young, and 2) in the spinal trigeminal nucleus and tract (nSp5), and in the cardiorespiratory-related medial solitary tract (nTSm) and lateral reticular (nRL) nuclei of both age groups. These findings support a possible participation of the mu-opioid system in the attenuation of arousal and cardiorespiration after repeated prenatal exposure to cocaine.

Twenty N- and/or C-modified Dmt-Tic analogues yielded similar K(i) values with either [(3)H]DPDPE (delta(1) agonist) or [(3)H]N, N(Me)(2)-Dmt-Tic-OH (delta antagonist). N-Methylation enhanced delta antagonism while N-piperidine-1-yl, N-pyrrolidine-1-yl, and N-pyrrole-1-yl were detrimental. Dmt-Tic-X (X = -NHNH(2), -NHCH(3), -NH-1-adamantyl, -NH-tBu, -NH-5-tetrazolyl) had high delta affinities (K(i) = 0.16 to 1 nM) with variable mu affinities to yield nonselective or weakly mu-selective analogues. N, N-(Me)(2)Dmt-Tic-NH-1-adamantane exhibited dual delta and mu receptor affinities (K(i)delta = 0.16 nM and K(i)mu = 1.12 nM) and potent delta antagonism (pA(2) = 9.06) with mu agonism (IC(50) = 16 nM). H-Dmt-betaHTic-OH (methylene bridge between C(alpha) of Tic and carboxylate function) yielded a biostable peptide with high delta affinity (K(i) = 0.85 nM) and delta antagonism (pA(2) = 8.85) without mu bioactivity. Dmt-Tic-Ala-X (X = -NHCH(3), -OCH(3), -NH-1-adamantyl, -NHtBu) exhibited high delta affinities (K(i) = 0.06 to 0.2 nM) and elevated mu affinities (K(i) = 2.5 to 11 nM), but only H-Dmt-Tic-Ala-NH-1-adamantane and H-Dmt-Tic-Ala-NHtBu yielded delta receptor antagonism (pA(2) = 9.29 and 9.16, respectively). Thus, Dmt-Tic with hydrophobic C-terminal substituents enhanced mu affinity to provide delta antagonists with dual receptor affinities and bifunctional activity.

The cDNA coding for the bovine mu-opioid receptor has been cloned and sequenced. Conserved sequences from murine delta-receptor cDNA were used as primers in polymerase chain reaction (PCR) to amplify cDNA, prepared by reverse transcription of bovine brain mRNA. This cDNA was used to probe a bovine brain library. The partial sequence obtained was extended to provide the full length clone by PCR. The cDNA has an open reading frame of 1203 base pairs (bp) with a 3'-untranslated region of 1900 bp and a 5'-untranslated region of 265 bp. The protein contains 401 amino acids and has 94% amino acid identity with the human and 91% with the rat mu-opioid receptor. It has the putative seven transmembrane domains, characteristic of G protein-coupled receptors and contains 5 potential N-linked glycosylation sites near the N-terminus. Several potential phosphorylation sites and a putative palmitoylation site are also present. The receptor was stably expressed in HEK293 cells. The binding profile was found to be that of a typical mu receptor, i. e., mu agonists and antagonists, but not delta and kappa ligands, bound with high affinity. Functional assays, namely, opioid stimulation of [35S]GTPgammaS binding and inhibition of forskolin-activated adenylyl cyclase, were also found to be highly specific for mu-opioid agonists. The receptor was downregulated by chronic exposure to mu agonists but not delta or kappa agonists. Evidence is presented indicating that the cloned receptor is the same as the bovine mu receptor previously purified to homogeneity in our laboratory. No evidence was found for genes for multiple mu-type opioid receptors.

Gene targeting was used to delete exon 2 of mouse DOR-1, which encodes the delta opioid receptor. Essentially all 3H-[D-Pen2,D-Pen5]enkephalin (3H-DPDPE) and 3H-[D-Ala2,D-Glu4]deltorphin (3H-deltorphin-2) binding is absent from mutant mice, demonstrating that DOR-1 encodes both delta1 and delta2 receptor subtypes. Homozygous mutant mice display markedly reduced spinal delta analgesia, but peptide delta agonists retain supraspinal analgesic potency that is only partially antagonized by naltrindole. Retained DPDPE analgesia is also demonstrated upon formalin testing, while the nonpeptide delta agonist BW373U69 exhibits enhanced activity in DOR-1 mutant mice. Together, these findings suggest the existence of a second delta-like analgesic system. Finally, DOR-1 mutant mice do not develop analgesic tolerance to morphine, genetically demonstrating a central role for DOR-1 in this process.