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Rationale: Opioid drugs indirectly activate dopamine (DA) neurons in the ventral tegmental area (VTA) through a disinhibition mechanism mediated by mu opioid receptors (MORs) present both on the GABA projection neurons located in the medial tegmental nucleus/tail of the VTA (RMTg/tVTA) and on the VTA GABA interneurons. It is well demonstrated that ethanol, like opioid drugs, provokes VTA DA neuron disinhibition by interacting (through its secondary metabolite, salsolinol) with MORs present in VTA GABA interneurons, but it is not known whether ethanol could disinhibit VTA DA neurons through the MORs present in the RMTg/tVTA., Objectives: The objective of the present study was to determine whether ethanol, directly microinjected into the tVTA/RMTg, is also able to induce VTA DA neurons disinhibition., Methods: Disinhibition of VTA DA neurons was indirectly assessed through the analysis of the motor activity of rats. Cannulae were placed into the tVTA/RMTg to perform microinjections of DAMGO (0.13 nmol), ethanol (150 or 300 nmol) or acetaldehyde (250 nmol) in animals pre-treated with either aCSF or the irreversible antagonist of MORs, beta-funaltrexamine (beta-FNA; 2.5 nmol). After injections, spontaneous activity was monitored for 30 min., Results: Neither ethanol nor acetaldehyde directly administered into the RMTg/tVTA were able to increase the locomotor activity of rats at doses that, in previous studies performed in the posterior VTA, were effective in increasing motor activities. However, microinjections of 0.13 nmol of DAMGO into the tVTA/RMTg significantly increased the locomotor activity of rats. These activating effects were reduced by local pre-treatment of rats with beta-FNA (2.5 nmol)., Conclusions: The tVTA/RMTg does not appear to be a key brain region for the disinhibiting action of ethanol on VTA DA neurons. The absence of dopamine in the tVTA/RMTg extracellular medium, the lack of local ethanol metabolism or both could explain the present results., (© 2023. The Author(s).)

In this work, we report on the in vitro and in vivo pharmacological properties of LP1 analogs to complete the series of structural modifications aimed to generate compounds with improved analgesia. To do that, the phenyl ring in the N -substituent of our lead compound LP1 was replaced by an electron-rich or electron-deficient ring and linked through a propanamide or butyramide spacer at the basic nitrogen of the (-)- cis - N -normetazocine skeleton. In radioligand binding assays, compounds 3 and 7 were found to display nanomolar binding affinity for the μ opioid receptor (MOR) (K i = 5.96 ± 0.08 nM and 1.49 ± 0.24 nM, respectively). In the mouse vas deferens (MVD) assay, compound 3 showed an antagonist effect against DAMGO ([D-Ala 2 , N-MePhe 4 , Gly-ol]-enkephalin), a highly selective MOR prototype agonist, whereas compound 7 produced naloxone reversible effect at MOR. Moreover, compound 7 , as potent as LP1 and DAMGO at MOR, was able to reduce thermal and inflammatory pain assessed by the mouse tail-flick test and rat paw pressure thresholds (PPTs) measured by a Randall-Selitto test.

Consumption of opioids is a growing global health problem. The gold standard for drugs of abuse screening is immunochemical assays. However, this method comes with some disadvantages when screening for a wide variety of opioids. Detection of the binding of a compound at the human μ-opioid receptor (MOR) offers a promising alternative target. Here, we set up a urine assay to allow for detection of compounds that bind at the MOR, thus allowing the assay to be utilized as a screening tool for opioid intake. The assay is based on the incubation of MOR-containing cell membranes with the selective MOR-ligand DAMGO and urine. After filtration, the amount of DAMGO in the eluate is analyzed by liquid chromatography tandem mass spectroscopy (LC-MS/MS). The absence of DAMGO in the eluate corresponds to a competing MOR ligand in the urine sample, thus indicating opiate/opioid intake by the suspect. Sensitivity and specificity were determined by the analysis of 200 consecutive forensic routine casework urine samples. A pronounced displacement of DAMGO was observed in 29 of the 35 opiate/opioid-positive samples. Detection of fentanyl intake proved to be the most challenging aspect. Applying a cut-off value of, e.g., 10% DAMGO binding would lead to a sensitivity of 83% and a specificity of 95%. Consequently, the novel assay proved to be a promising screening tool for opiate/opioid presence in urine samples. The nontargeted approach and possible automation of the assay make it a promising alternative to conventional methods.

The chimeric peptide EN-9 was reported as a κ-opioid/neuropeptide FF receptors bifunctional agonist that modulated chronic pain with no tolerance. Many lines of evidence have shown that the effect of the κ-opioid receptor is mediated by not only its specific activation but also downstream events participation, especially interaction with the μ-opioid receptor pathway in antinociception and tolerance on most occasions. The present study investigated the acute and chronic cross-tolerance of EN-9 with μ-opioid receptor agonist EM-2, DAMGO, and morphine after intracerebroventricularly (i.c.v) injection in the mouse tail-flick test. In the acute tolerance test, EN-9 showed symmetrical acute cross-tolerance to DAMGO but no cross-tolerance to EM2. In the chronic tolerance test, EN-9 had no tolerance after eight days of repeated administration. However, EN-9 illustrated complete cross-tolerance to morphine and symmetrical cross-tolerance to EM2. In addition, inhibition of NPFF receptor could induce the tolerance development of EN-9. These findings indicated that supraspinal EN-9-induced antinociception contains additional components, which are mediated by the downstream μ-opioid receptor pathway both in acute and chronic treatment, whereas the subtypes of μ-opioid receptor or NPFF system pathway involved in antinociceptive effects induced by EN-9 are complex. Identifying the receptor mechanism could help design preferable bifunctional opioid compounds., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Background: Environmental factors contribute to autism spectrum disorder. Fentanyl, one of the most widely used opioid analgesics in anaesthesia, can induce neurotoxicity, but its role in autism remains unknown. We determined whether fentanyl induced autism-like behaviours in young mice and the underlying mechanisms., Methods: Young male and female mice received fentanyl at postnatal days 6, 8, and 10, and performed behavioural tests, including three-chamber social preference, elevated plus maze, grooming behaviour, and open-field test, from postnatal days 30-32. Expression of Grin2b, the gene encoding the GluN2B subunit of the N-methyl-d-aspartate receptor, was assessed in the anterior cingulate cortex of male mice using fluorescence in situ hybridisation histochemistry. We used bisulfite target sequencing to determine Grin2b hypermethylation sites after fentanyl treatment. In the specific activation and rescue experiments, we injected the mu opioid receptor agonist [D-Ala, 2 N-MePhe, 4 Gly-ol]-enkephalin (DAMGO) or Grin2b overexpression lentivirus into the anterior cingulate cortex of male mice., Results: Fentanyl induced autism-like behaviours in both young male and female mice, and downregulated Grin2b expression (0.49-fold [0.08] vs 1.00-fold [0.09]; P<0.01) and GluN2B protein amounts (0.38-fold [0.07] vs 1.00-fold [0.12]; P<0.01) in the anterior cingulate cortex through hypermethylation of Grin2b. The mu-opioid receptor antagonist naloxone and overexpression of Grin2b in anterior cingulate cortex attenuated the fentanyl-induced effects, whereas DAMGO injection into the anterior cingulate cortex induced autism-like behaviours., Conclusions: These data suggest that fentanyl induces autism-like behaviours in young mice via an epigenetic mechanism. Further research is required to determine possible clinical relevance to autism risk., (Copyright © 2022 British Journal of Anaesthesia. Published by Elsevier Ltd. All rights reserved.)

Radioactive ligand binding assays are the most commonly applied method for the determination of binding affinities of compounds at a particular receptor. While they are highly sensitive and high-throughput capable they come with major disadvantages due to the radioactive ligands utilized. Here we present the development of a mass-spectrometry-based binding assay for the determination of binding affinities at the human μ-opioid receptor using non-labelled DAMGO ([D-Ala 2 , N-MePhe 4 , Gly 5 -ol]-enkephalin). The runtime of the LC-MS/MS method was 5.5 min per data point and allowed for the highly sensitive detection of 38.5 fg DAMGO on column. The assay shows low non-specific binding and the equilibrium dissociation constant of DAMGO was 0.57 nM. The assay was applied to determine the K i values of 17 opiates/opioids and the results were in good agreement with the data from radioactive receptor binding assays published in the literature. Additionally, the K i value of six 2-benzylbenzimidazoles, including the widely abused designer opioid isotonitazene, were determined ranging from 0.654 to 72.9 nM. Consequently, the developed assay provides a suitable alternative to radioactive binding assays as it allows for a reliable and rapid determination of receptor binding affinities of e.g. newly emerging designer opioids., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Previous works showed that opioid peptides are produced by olivocochlear efferent neurons, while cochlear hair cells express opioid receptors. It has been proposed that opioids protect the auditory system from damage by intense stimulation, although their use for therapeutic or illicit purposes links to hearing impairment. Therefore, it is relevant to study the effect of opioids in the auditory system to define their functional expression and mechanism of action. This study investigated the modulation of the Ca 2+ currents by opioid peptides in the rat outer hair cells (OHC) using the whole-cell patch-clamp technique. The influence of agonists of the three opioid receptor subtypes (μ, δ, and κ) was studied. The κ opioid receptor agonist U-50488 inhibits the Ca 2+ currents in a partially reversible form. Coincidently, norbinaltorphimine (a κ receptor antagonist) blocked the U-50488 inhibitory effect on the Ca 2+ current. The δ and the μ opioid receptor agonists did not significantly affect the Ca 2+ currents. These results indicate that the κ opioid receptor activation inhibits the Ca 2+ current in OHC, modulating the intracellular Ca 2+ concentration when OHCs depolarize. The modulation of the auditory function by opioids constitutes a relevant mechanism with a potential role in the physiopathology of auditory disturbances., (© 2022 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Subregions within insular cortex and medial prefrontal cortex (mPFC) have been implicated in eating disorders; however, the way these brain regions interact to produce dysfunctional eating is poorly understood. The present study explored how two mPFC subregions, the infralimbic (IL) and prelimbic (PRL) cortices, regulate sucrose hyperphagia elicited specifically by a neurochemical manipulation of the agranular/dysgranular region of gustatory insula (AI/DI). Using intra-AI/DI infusion of the mu-opioid receptor (µ-OR) agonist, DAMGO (1 µg), sucrose hyperphagia was generated in ad-libitum-maintained rats, while in the same rat, either the IL or prelimbic (PRL) subregion of mPFC was inactivated bilaterally with muscimol (30 ng). Intra-IL muscimol markedly potentiated AI/DI DAMGO-induced sucrose hyperphagia by increasing eating bout duration and food consumption per bout. In contrast, PRL attenuated intra-AI/DI DAMGO-driven sucrose intake and feeding duration and eliminated the small DAMGO-induced increase in feeding bout initiation. Intra-IL or -PRL muscimol alone (i.e., without intra-AI/DI DAMGO) did not alter feeding behavior, but slightly reduced exploratory-like rearing in both mPFC subregions. These results reveal anatomical heterogeneity in mPFC regulation of the intense feeding-motivational state engendered by µ-OR signaling in the gustatory insula: IL significantly curtails consummatory activity, while PRL modestly contributes to feeding initiation. Results are discussed with regard to potential circuit-based mechanisms that may underlie the observed results., (© 2022. The Author(s), under exclusive licence to American College of Neuropsychopharmacology.)

The μ-opioid receptors belong to the family of G protein-coupled receptors (GPCRs), and their activation triggers a cascade of intracellular relays with the final effect of analgesia. Classical agonists of this receptor, such as morphine, are the main targets in the treatment of both acute and chronic pain. However, the dangerous side effects, such as respiratory depression or addiction, significantly limit their widespread use. The allosteric centers of the receptors exhibit large structural diversity within particular types and even subtypes. Currently, a considerable interest is aroused by the modulation of μ-opioid receptors. The application of such a technique may result in a reduction in the dose or even discontinuation of classical opiates, thus eliminating the side effects typical of this class of drugs. Our aim is to obtain a series of 1-aryl-5,6(1 H )dioxo-2,3-dihydroimidazo[1,2- a ]imidazole derivatives and provide more information about their activity and selectivity on OP3 (MOP, human mu opioid receptor). The study was based on an observation that some carbonyl derivatives of 1-aryl-2-aminoimidazoline cooperate strongly with morphine or DAMGO in sub-threshold doses, producing similar results to those of normal active doses. To elucidate the possible mechanism of such enhancement, we performed a few in vitro functional tests (involving cAMP and β-arrestin recruitment) and a radioligand binding assay on CHO-K1 cells with the expression of the OP3 receptor. One of the compounds had no orthosteric affinity or intrinsic activity, but inhibited the efficiency of DAMGO. These results allow to conclude that this compound is a negative allosteric modulator (NAM) of the human μ-opioid receptor.

Background and Purpose: Various GPCRs have been described as being modulated in a voltage-dependent manner. Opioid analgesics act via activation of μ receptors in various neurons. As neurons are exposed to large changes in membrane potential, we were interested in studying the effects of depolarization on μ receptor signalling., Experimental Approach: We investigated potential voltage sensitivity of μ receptors in heterologous expression systems (HEK293T cells) using electrophysiology in combination with Förster resonance energy transfer-based assays. Depolarization-induced changes in signalling were also tested in physiological rat tissue containing locus coeruleus neurons. We applied depolarization steps across the physiological range of membrane potentials., Key Results: Studying μ receptor function and signalling in cells, we discovered that morphine-induced signalling was strongly dependent on the membrane potential (V M ). This became apparent at the level of G-protein activation, G-protein coupled inwardly rectifying potassium channel (K ir 3.X) currents and binding of GPCR kinases and arrestin3 to μ receptors by a robust increase in signalling upon membrane depolarization. The pronounced voltage sensitivity of morphine-induced μ receptor activation was also observed at the level of K ir 3.X currents in rat locus coeruleus neurons. The efficacy of peptide ligands to activate μ receptors was not (Met-enkephalin) or only moderately ([D-Ala 2 , N-Me-Phe 4 , Gly 5 -ol]-enkephalin) enhanced upon depolarization. In contrast, depolarization reduced the ability of the analgesic fentanyl to activate μ receptors., Conclusion and Implications: Our results indicate a strong ligand-dependent modulation of μ receptor activity by the membrane potential, suggesting preferential activity of morphine in neurons with high neuronal activity., (© 2020 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Imaging of the mu opioid receptor (MOR) availability with positron emission tomography (PET) is a pertinent challenge in Neuroscience. Both, regulation of receptor expression and occupancy by endogeneous opioids play into cognitive and behavioral phenotypes of healthy function and disease. Receptor expression in the active and inactive states can be measured using high affinity radioagonist and radioantagonist PET tracers, respectively. Occupancy assessment requires radioligands showing competitive and reversible binding with moderate affinity to the MOR, which may lead to physical extinction of the receptor specific signal in vivo. We investigated a moderately potent, selective MOR agonist in rat to test if a radiotracer design paradigm tailored to competition with endogeneous opioids leads to viable imaging results. The benzamide 3,4-dichlorobenzenecarboxylic acid (dimethylamino)cyclohexyl)methyl amide (AH-7921, 1) was synthesized and characterized in rat brain using autoradiography and positron emission tomography. Compound 1 was found to activate with low nanomolar potency the MOR and to a lesser extent KOR as a full agonist. Concentration dependent binding studies with agonist and antagonist radioligands were conducted to assess competition behavior and obtain inhibition constants. Kinetic analysis of 3,4-dichlorobenzene[ 11 C]carboxylic acid (dimethylamino)cyclohexyl)methyl amide binding in rat brain resulted in low but reproducible binding potential in the thalamus (0.8 ± 0.1). A radioactive metabolite was detected in brain (17%, after 15 min). Nonetheless, we conclude that quantitative imaging of MOR availability is possible when using a moderate affinity radiotracer.

We previously developed a model of opioid-induced neuroplasticity in the peripheral terminal of the nociceptor that could contribute to opioid-induced hyperalgesia, type II hyperalgesic priming. Repeated administration of mu-opioid receptor (MOR) agonists, such as DAMGO, at the peripheral terminal of the nociceptor, induces long-lasting plasticity expressed, prototypically as opioid-induced hyperalgesia and prolongation of prostaglandin E2-induced hyperalgesia. In this study, we evaluated the mechanisms involved in the maintenance of type II priming. Opioid receptor antagonist, naloxone, induced hyperalgesia in DAMGO-primed paws. When repeatedly injected, naloxone-induced hyperalgesia, and hyperalgesic priming, supporting the suggestion that maintenance of priming involves changes in MOR signaling. However, the knockdown of MOR with oligodeoxynucleotide antisense did not reverse priming. Mitogen-activated protein kinase and focal adhesion kinase, which are involved in the Src signaling pathway, previously implicated in type II priming, also inhibited the expression, but not maintenance of priming. However, when Src and mitogen-activated protein kinase inhibitors were coadministered, type II priming was reversed, in male rats. A second model of priming, latent sensitization, induced by complete Freund's adjuvant was also reversed, in males. In females, the inhibitor combination was only able to inhibit the expression and maintenance of DAMGO-induced priming when knockdown of G-protein-coupled estrogen receptor 30 (GPR30) in the nociceptor was performed. These findings demonstrate that the maintenance of DAMGO-induced type II priming, and latent sensitization is mediated by an interaction between, Src and MAP kinases, which in females is GPR30 dependent.

Rationale: Using the drinking-in-the-dark (DID) model, we compared the effects of a novel mu-opioid receptor antagonist, GSK1521498, with naltrexone, a licensed treatment of alcohol dependence, on ethanol consumption in mice., Objective: We test the ability of GSK1521498 to reduce alcohol consumption and compare its intrinsic efficacy to that of naltrexone by comparing the two drugs at doses matched for equivalent receptor occupancy., Methods: Thirty-six C57BL/6J mice were tested in a DID procedure. In 2-day cycles, animals experienced one baseline, injection-free session, and one test session when they received two injections, one of test drug and one placebo. All animals received GSK1521498 (0, 0.1, 1 and 3 mg/kg, i.p., 30 min pre-treatment) and naltrexone (0, 0.1, 1 and 3 mg/kg, s.c. 10 min pre-treatment) in a cross-over design. Receptor occupancies following the same doses were determined ex vivo in separate groups by autoradiography, using [3H]DAMGO. Binding in the region of interest was measured integrally by computer-assisted microdensitometry and corrected for non-specific binding., Results: Both GSK1521498 and naltrexone dose-dependently decreased ethanol consumption. When drug doses were matched for 70-75% receptor occupancy, GSK1521498 3 mg/kg, i.p., caused a 2.5-fold greater reduction in alcohol consumption than naltrexone 0.1 mg/kg, s.c. Both GSK1521498 and naltrexone significantly reduced sucrose consumption at a dose of 1 mg/kg but not 0.1 mg/kg. In a test of conditioned taste aversion, GSK1521498 (3 mg/kg) reduced sucrose consumption 24 h following exposure to a conditioning injection., Conclusions: Both opioid receptor antagonists reduced alcohol consumption but GK1521498 has higher intrinsic efficacy than naltrexone.

Methylxanthines like caffeine and theophylline have long been used to treat apnea of prematurity. Despite their success in stimulating neonatal breathing, their mechanism of action remains poorly understood. Methylxanthines can act as both non-specific adenosine receptor antagonists and inhibitors of cAMP-dependent phosphodiesterases, sarcoplasmic/endoplasmic reticulum calcium ATPases or receptor-coupled anion channels, depending on the dose used. Though there is evidence for methylxanthine action at the level of the carotid body, the consensus is that methylxanthines stimulate the respiratory centers of the brainstem. Here we used the in situ neonatal rat working heart-brainstem preparation and the ex vivo neonatal rat carotid body preparation to test the hypothesis that methylxanthines act at the level of the carotid body. We conclude that although the neonatal carotid body has active adenosine receptors, the effects of methylxanthine therapy are likely mediated centrally, predominantly via inhibition of cAMP-dependent phosphodiesterase-4., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The μ-opioid receptor gene, OPRM1, undergoes extensive alternative pre-mRNA splicing, as illustrated by the identification of an array of splice variants generated by both 5' and 3' alternative splicing. The current study reports the identification of another set of splice variants conserved across species that are generated through exon skipping or insertion that encodes proteins containing only a single transmembrane (TM) domain. Using a Tet-Off system, we demonstrated that the truncated single TM variants can dimerize with the full-length 7-TM μ-opioid receptor (MOR-1) in the endoplasmic reticulum, leading to increased expression of MOR-1 at the protein level by a chaperone-like function that minimizes endoplasmic reticulum-associated degradation. In vivo antisense studies suggested that the single TM variants play an important role in morphine analgesia, presumably through modulation of receptor expression levels. Our studies suggest the functional roles of truncated receptors in other G protein-coupled receptor families.

We studied the modulation of morphine-induced mechanical antinociception and side effects by σ₁ receptor inhibition. Both wild-type (WT) and σ₁ receptor knockout (σ₁-KO) mice showed similar responses to paw pressure (100-600 g). The systemic (subcutaneous) or local (intraplantar) administration of σ₁ antagonists (BD-1063, BD-1047, NE-100 and S1RA) was devoid of antinociceptive effects in WT mice. However, σ₁-KO mice exhibited an enhanced mechanical antinociception in response to systemic morphine (1-16 mg/kg). Similarly, systemic treatment of WT mice with σ₁ antagonists markedly potentiated morphine-induced antinociception, and its effects were reversed by the selective σ₁ agonist PRE-084. Although the local administration of morphine (50-200 μg) was devoid of antinociceptive effects in WT mice, it induced dose-dependent antinociception in σ₁-KO mice. This effect was limited to the injected paw. Enhancement of peripheral morphine antinociception was replicated in WT mice locally co-administered with σ₁ antagonists and the opioid. None of the σ₁ antagonists tested enhanced morphine-antinociception in σ₁-KO mice, confirming a σ₁-mediated action. Morphine-induced side-effects (hyperlocomotion and inhibition of gastrointestinal transit) were unaltered in σ₁-KO mice. These results cannot be explained by a direct interaction of σ₁ ligands with μ-opioid receptors or adaptive changes of μ-receptors in σ₁-KO mice, given that [(3)H]DAMGO binding in forebrain, spinal cord, and hind-paw skin membranes was unaltered in mutant mice, and none of the σ₁ drugs tested bound to μ-opioid receptors. These results show that σ₁ receptor inhibition potentiates morphine-induced mechanical analgesia but not its acute side effects, and that this enhanced analgesia can be induced at peripheral level., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Previous studies by our laboratory have demonstrated that the mu opioid receptor antagonist, CTAP, blocks the rewarding effects of cocaine when it is injected directly into the nucleus accumbens or ventral tegmental area (VTA). This finding suggests that cocaine is causing the release of endogenous opioid peptides which activate mu opioid receptors within the nucleus accumbens and VTA. The purpose of the present study was to characterize the dose-response and time-course of mu receptor occupancy following systemic cocaine administration and to determine if release of endogenous opioids by cocaine is mediated by activation of D1 or D2 dopamine receptors. Quantitative in vitro receptor autoradiography was used to measure the regional displacement of (3)H-DAMGO binding following cocaine administration. Adult male Sprague-Dawley rats were given intraperitoneal (i.p.) injections of cocaine and their brains were removed at various times and prepared for mu opioid receptor quantitation. To determine the role of dopamine D1 and D2 receptors in the effect of cocaine on mu receptor occupancy, rats were injected with the selective D1 or D2 receptor antagonists SCH23390 or eticlopride prior to cocaine. For all studies, (3)H-DAMGO binding to mu opioid receptors was measured in the nucleus accumbens, caudate putamen, frontal cortex, olfactory tubercle and VTA. Results demonstrate that cocaine administration caused a time- and dose-dependent reduction in (3)H-DAMGO binding within the nucleus accumbens core and shell. The reduction in mu receptor binding was attenuated by pretreatment with eticlopride. These results suggest that cocaine, acting via D2 dopamine receptors, can cause the release of an endogenous opioid peptide that binds to mu opioid receptors within the nucleus accumbens.

For ectothermic vertebrates, such as reptiles, the effects of opioid receptor subtype activation on breathing are poorly understood. On the basis of previous studies on mammals and lampreys, we hypothesized that mu- and delta-opioid receptor (MOR and DOR, respectively) activation would cause respiratory depression, whereas kappa-opioid receptor (KOR) activation would have no effect. To address this question, we measured respiration in awake, freely swimming adult red-eared slider turtles (Trachemys scripta) before and after injection with agonists for specific opioid receptors. Injection of the MOR agonist [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin acetate salt (DAMGO, 1.5 or 6.5 mg/kg) decreased ventilation (Ve) by 72 +/- 9% and 95 +/- 3%, respectively, 4.0 h after injection as a result of decreased breathing frequency and no change in tidal volume (Vt). DOR agonists, such as [d-Pen(2,5)]-enkephalin hydrate (DPDPE, 5.0 mg/kg) and [d-Ala(2),d-Leu(5)]-enkephalin acetate salt (DADLE, 6.3 mg/kg), decreased Ve by 44 +/- 10% and 89 +/- 4%, respectively, 4.0 h after injection as a result of decreased breathing frequency and no change in Vt. DADLE also increased breath duration by a maximum of 25 +/- 9% at 6.0 h after injection. The KOR agonist U-50488 (6.2 mg/kg) increased Vt by a maximum of 52 +/- 30% at 5.0 h after injection, with variable nonsignificant changes in Ve and breathing frequency. Naloxone injections (0.25-0.5 mg/kg) 1.0 h before opioid agonist injections blocked all DAMGO-dependent effects, DPDPE-dependent frequency depression, and DADLE-dependent breath duration augmentation for 2.0 h after agonist injections. These results show that MOR and DOR activation causes respiratory depression as a result of decreased breathing frequency, whereas Vt is increased after KOR activation.

Chronic and acute activation of mu-opioid receptors (MOR) in hippocampal cornu Ammon 1 (CA1) disrupts rhythmic activity, alters activity-dependent synaptic plasticity and impairs spatial memory formation. In CA1, MORs act by hyperpolarizing inhibitory interneurons and suppressing inhibitory synaptic transmission. MOR modulation of inhibitory synaptic function translates into an increase in excitatory activity in all layers of CA1. However, the exact anatomical sites for MOR actions are not completely known. Therefore, we used voltage-sensitive dye imaging, whole cell patch clamping, photolysis of alpha-carboxy-2-nitrobenzyl ester, trifluoroacetic acid salt (CNB) -caged GABA, and micro-sectioned slices of rat hippocampus to investigate the effect of MOR activation in CA1. First, we investigated the effect of MOR activation using a MOR agonist [d-Ala2, NMe-Phe4, Gly-ol5]-enkephalin (DAMGO) on the direct activation of GABA receptors by photolysis of CNB-caged GABA in all layers of CA1. MOR activation did not affect hyperpolarizations due to direct GABA receptor activation in any layer of CA1, but MOR activation did suppress GABAergic inhibitory postsynaptic potentials suggesting that MOR activation acts by presynaptically inhibiting interneuron function. We next examined whether MOR activation was equivalently effective in all anatomical layers of CA1. To do this, cuts were made between anatomical layers of CA1 and isolated layers were stimulated electrically (five pulses at 20 Hz) to produce excitatory postsynaptic potentials (EPSPs). Under these conditions, MOR activation significantly increased EPSP areas in stratum radiatum (SR), stratum pyramidale (SP) and stratum oriens (SO) relative to stratum lacunosum-moleculare (SLM). When compared with the effect of GABA(A) and GABA(B) receptor antagonists on EPSP areas, the effect of DAMGO was proportionately larger in SR, SP and SO than in SLM. We conclude that MOR activation is more effective at directly modulating activity in SR, SP and SO, and the smaller effect in SLM is likely due to a smaller MOR inhibition of GABA release in SLM.

Preclinical and clinical studies suggest that the administration of the opioid antagonist naltrexone decreases the intake of ethanol. However, the neuroplastic adaptations in the brain associated to reduction of ethanol consumption remains to be elucidated. The aim of the study was to identify gene transcription alterations underlying the attenuation of voluntary ethanol intake by administration of naltrexone in rats. Increasing doses of naltrexone (0.7 mg/kg, 4 days and 1.4 mg/kg/day, 4 days) to rats with acquired high preferring ethanol consumption (>3.5 g of ethanol/kg/day) decreased voluntary ethanol intake (50%). Voluntary ethanol consumption altered mu-opioid receptor function in the cingulate cortex, caudate-putamen (CPu), nucleus accumbens core (Acb C) and shell (Acb S), the expression of tyrosine hydroxylase (TH) in the ventral tegmental area and substantia nigra, proenkephalin (PENK) in the piriform cortex, olfactory tubercle, CPu, Acb C and Acb S, ventromedial nucleus (VMN) and paraventricular nucleus (PVN) of the hypothalamus, corticotropin releasing factor (CRF) in PVN, cannabinoid CB(1) receptor (CB1-R) in the CPu, hippocampus and VMN, and serotonin transporter (5-HTT) in the dorsal and median raphe nuclei. The reduction of ethanol intake induced by naltrexone was associated with a blockade or significant reduction of the changes produced by ethanol in the expression of these genes in key regions related to drug dependence. These results point to a role for the mu-opioid receptor, TH, PENK, CRF, CB1-R, and 5-HTT genes in specific brain regions in the modulation of neuroadaptative mechanisms associated to the decrease of ethanol intake induced by naltrexone.

Aim: Our aim was to judge the importance of candidate pharmacogenetic modulators of the central nervous effects of levomethadone by both magnitude of the modulatory effect and frequency of the mutation to assess the utility of genotyping for clinical levomethadone therapy in a random sample of subjects that, by distribution of genotypes, resembled the clinical setting., Methods: Candidate pharmacogenetic modulators were polymorphisms reported to be of functional consequence and therefore potentially important for metabolism, distribution, or pharmacodynamic action of levomethadone, consisting of genes coding for cytochrome P450 (CYP) 2B6 and 3A, as well as 1A2, 2C8, 2C9, 2C19, and 2D6, for P-glycoprotein (ABCB1), and for mu-opioid receptors (OPRM1). The central nervous effects of levomethadone were investigated by means of measuring pupil size in a random sample of 51 healthy volunteers for 9 hours after oral administration of 0.075 mg/kg levomethadone. Plasma concentrations of levomethadone and its metabolites were assessed concomitantly, and to judge the role of metabolites, the affinities of levomethadone and its metabolites at mu-opioid receptors were estimated by use of displacement of the selective mu-opioid receptor agonist [(3)H]-DAMGO ((3)H-[D-Ala(2),N-MePhe(4),Gly-ol(5)]-enkephalin)., Results: Pupil size decreased to -41.8% +/- 9.6% from baseline at 3.5 +/- 1.1 hours after levomethadone administration. Miosis was still manifest to a lower degree (-25.1% +/- 12.3%) at the end of the observation period. In carriers of the variant 118G allele (118A>G single-nucleotide polymorphism) of the mu-opioid receptor gene (OPRM1), levomethadone had a 1.74 times (95% confidence interval, 1.4-2.2 times) lower miotic potency (P < .001) as compared with noncarriers (concentration at half-maximum effects, 52.3 nmol/L; 95% confidence interval, 36.7-66.2 nmol/L). The maximum percent decrease in pupil diameter from baseline was 44.9% +/- 7.6%, 33% +/- 6.5%, and 24% +/- 6.9% for carriers of the OPRM1 118AA, AG, and GG genotypes, respectively (P < .001 for AG and GG versus wild type, without significant differences between AG and GG). Other candidate polymorphisms in the ABCB1 or in CYP genes had no significant influence on the effects of levomethadone, either because of lack of functional consequences or because of their low allelic frequency. The metabolites of levomethadone did not contribute to the effects as indicated by low metabolite plasma concentrations and their 120- to 1300-fold lower affinities at mu-opioid receptors as compared with levomethadone., Conclusions: Among polymorphisms in OPRM1, ABCB1, and CYP genes previously associated with functional consequences in a different context, the most important pharmacogenetic factor modulating the short-term effects of levomethadone is a polymorphism (OPRM1 118A>G) affecting mu-opioid receptors.

A number of G protein-coupled receptors have been shown to stimulate tuberin phosphorylation, which is critical for the regulation of translation and is apparently involved in neurotrophin-promoted survival of serum-deprived cells. Here, in HEK 293 cells transiently expressing the delta-, kappa-, or mu-opioid receptors, Western blotting analysis using a phosphospecific anti-tuberin antibody revealed a dose- and time-dependent increase in tuberin phosphorylation upon stimulation by specific opioid agonists. In NG108-15, PC12, and SH-SY5Y cells that endogenously express delta-, kappa-, and mu-opioid receptors, respectively, specific opioid agonists also stimulated tuberin phosphorylation in a dose- and time-dependent manner. Pretreatment of cells with pertussis toxin or PI3K inhibitor wortmannin blocked the opioid-stimulated tuberin phosphorylation, implicating the possible involvement of the G(i/o) proteins and the phosphatidylinositol-3 kinase/Akt pathway in opioid-induced tuberin phosphorylation. This is the first study that demonstrates the regulatory role of opioid receptors on tuberin.

We have previously shown that the GABA(B) agonist baclofen (BAC) prevents the expression of morphine (MOR) withdrawal syndrome in male as well as female mice. In addition, we have demonstrated that BAC reestablishes the dopamine levels modified by MOR withdrawal syndrome in male mice. The aim of the present study was to evaluate the micro-opioid receptor binding parameters in striatum and frontal cortex of male and female mice during MOR withdrawal and its prevention with BAC. Prepubertal Swiss-Webster mice of either sex were rendered dependent by intraperitoneal (i.p.) injection of MOR (2 mg/kg) twice daily for 9 days. On the tenth day, dependent animals received naloxone (NAL) (6 mg/kg, i.p.) 60 min after the last dose of MOR and another pool of dependent mice received BAC (2 mg/kg, i.p.) previous to NAL injection. Thirty min after NAL or saline injection mice were sacrificed, brains were collected, and the striatum and frontal cortex were dissected in order to perform binding studies with [(3)H][DAMGO]. The density of micro-opioid receptor increased significantly during MOR withdrawal in male and female striatum as well as in male cortex. In addition, in both brain areas the B(max) was higher in male than in female mice during MOR withdrawal. Finally, BAC pretreatment of MOR withdrawn mice reestablished the levels of micro-opioid receptor by significantly decreasing the B(max) in either sex. In conclusion, although there were sex differences in the micro-opioid receptor density during MOR withdrawal syndrome, BAC was able to reestablish the changes in binding parameters induced by the NAL-precipitated withdrawal in female and male mice.

Background: The inferior colliculus, which receives almost all ascending and descending auditory signals, plays a crucial role in the processing of auditory information. While the majority of the recorded activities in the inferior colliculus are attributed to GABAergic and glutamatergic signalling, other neurotransmitter systems are expressed in this brain area including opiate peptides and their receptors which may play a modulatory role in neuronal communication., Results: Using a perfusion protocol we demonstrate that morphine can inhibit KCl-induced release of [3H]GABA from rat inferior colliculus slices. DAMGO ([D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin) but not DADLE ([D-Ala2, D-Leu5]-enkephalin or U69593 has the same effect as morphine indicating that micro rather than delta or kappa opioid receptors mediate this action. [3H]GABA release was diminished by 16%, and this was not altered by the protein kinase C inhibitor bisindolylmaleimide I. Immunostaining of inferior colliculus cryosections shows extensive staining for glutamic acid decarboxylase, more limited staining for micro opiate receptors and relatively few neurons co-stained for both proteins., Conclusion: The results suggest that micro-opioid receptor ligands can modify neurotransmitter release in a sub population of GABAergic neurons of the inferior colliculus. This could have important physiological implications in the processing of hearing information and/or other functions attributed to the inferior colliculus such as audiogenic seizures and aversive behaviour.

It is well known that the mu opioid agonist, Tyr-D-Ala-Gly-(me) Phe-Gly-ol (DAMGO), increases food intake in rats when injected into a variety of brain sites including the central nucleus of the amygdala (CeA). Immunohistochemical studies measuring c-Fos immunoreactivity (IR) suggest that the CeA contributes to opioid-related feeding. In the current study, we injected 2 nmol of DAMGO and measured food intake, c-Fos IR levels in various brain sites involved in feeding behavior, and mu opioid receptor internalization. We also studied the effect of CeA-injected DAMGO on LiCl-induced increases in c-Fos IR in the amygdala. As was expected, intra-CeA injection of DAMGO increased food intake of rats over a 4-h period. DAMGO injection into the CeA also resulted in mu opioid receptor internalization in the CeA, indicating activation of mu opioid receptor expressing neurons in this site. Administration of DAMGO into the CeA increased c-Fos IR levels in the shell of the nucleus accumbens (NAcc), but not in 17 other brain sites that were studied. We also found that intra-CeA injection of DAMGO, prior to LiCl injection, decreased c-Fos IR levels in the CeA compared to vehicle-injected rats. Thus, intra-CeA administration of DAMGO may increase feeding, in part, by activating neurons in the shell of the nucleus accumbens and by inhibiting activity of selected neurons in the CeA.

In the present work the effect of papaverine, a non specific smooth muscle relaxant, was investigated on the naloxone-precipitated withdrawal contracture of the acute morphine-dependent guinea-pig ileum in vitro. Furthermore, the effect of papaverine was also considered on DAGO (highly selective mu -agonist) and U50-488H (highly selective k-agonist) withdrawal to test whether the possible interaction of papaverine on opioid withdrawal involves mu - and/or k-opioid receptors. Following a 4 min in vitro exposure to opioid agonist, the guinea-pig isolated ileum exhibited a strong contracture after the addition of naloxone. Papaverine treatment (1 x 10(-7) - 5 x 10(-7) - 1 x 10(-6) M) before or after the opioid agonists was able of both preventing and reversing the naloxone-induced contracture after exposure to mu (morphine and DAGO) or k (U50-488H) opiate agonists in a concentration-dependent fascion. Both acetylcholine response and electrical stimulation were not affected by papaverine treatment whereas the final opiate withdrawal was still reduced. The results of the present study indicate that papaverine was able to produce significative influence on the opiate withdrawal in vitro and papaverine was able to exert its effect both at mu and k opioid agonists., (Copyright 2003 John Wiley & Sons, Ltd.)

The effect of dexamethasone on hypotension induced by mu-, kappa- and delta-opioid receptor agonists was investigated in pentobarbital-anaesthetised rats. Morphine (nonselective opioid receptor agonist), DAGO (D-Ala2-N-methyl-[Phe4-Gly5-ol]enkephalin; mu-opioid receptor-selective agonist), U50-488H (trans(+/-)-3,4-dichloro-N-methyl-N-(2[1pyrrolidynyl]cyclohexyl)-benzeneacetamide; kappa-opioid receptor-selective agonist) and deltorphin II (delta-opioid receptor-selective agonist), given intravenously, 5 micromol/kg, induced hypotension in rats. This hypotension was characterised by a fall in mean arterial blood pressure in 1-2 min that recovered in 30 min for morphine and U50-488H and in 5 or 20 min for DAGO and deltorphin II, respectively. Dexamethasone per se at a dose of 7.5 micromol/kg, i.v. did not significantly modify the mean arterial blood pressure of animals. Dexamethasone administration 90 min, but not 30 or 60 min, before the opioid agonists injection, prevented the hypotension induced by morphine or U50-488H, but not that induced by DAGO or deltorphin II. Pretreatment with RU-38486 (mifepristone; 7.5 micromol/kg, i.v.), a glucocorticoid receptor antagonist, 15 min before the steroid, prevented dexamethasone inhibition of hypotension induced by morphine and U50-488H. Furthermore, pretreatment with cycloheximide, a protein synthesis inhibitor (3.5 micromol/kg, i.v.), was also able to abolish the effects of dexamethasone on morphine- and U50-488H-induced hypotension. Results of the present study indicate that dexamethasone inhibited kappa-opioid receptor-mediated hypotension in rats, indicating a further important functional interaction between corticosteroids and the opioid system at kappa receptors. The ability of cycloheximide and RU-38486 to block dexamethasone effects indicates that steroid interference with kappa-opioid receptor-mediated hypotension involves a protein synthesis-dependent mechanism via glucocorticoid receptors.

Reverse-transcribed polymerase chain reaction (RT-PCR) can quantify gene transcripts at low levels and in small samples. Semi-quantitative and quantitative RT-PCR has significant advantages over traditional RNA assays, such as Northern blotting and ribonuclease protection assay. However, owing to the exponential nature of PCR, considerable effort is required to verify linearity of the reaction. Thus, care must be taken to detect small but physiologically relevant changes in gene expression. Using a rapid and highly sensitive RT-PCR method, TaqMan real-time RT-PCR, we determined agonist-induced changes in rat mu opioid receptor (MOR) mRNA levels in cultured cells and compared our results with those obtained by radiolabeled quantitative RT-PCR, which is also highly sensitive but much more time-consuming than TaqMan RT-PCR. Both methods showed up-regulation of agonist-induced MOR. TaqMan RT-PCR showed a similar sensitivity to radiolabeled quantitative RT-PCR and is suitable for the measurement of large numbers of samples. Moreover, no need for radiolabeled compounds is also an advantage of TaqMan PCR. This protocol will probably be useful for quantifying MOR in animal and human tissues.

Rationale: Chronic neuroleptic treatment produces a movement disorder in rats characterized by vacuous chewing movements (VCMs). Neuroleptics also produce a variety of changes in opioid neurotransmission in several regions of the basal ganglia. Rats with the VCM syndrome show elevated mRNA for enkephalin in striatopallidal neurons, suggesting a possible role for enkephalin in the pathophysiology of VCMs., Objective: This study investigated the role of mu-opioid receptor density in the basal ganglia on the expression of VCMs., Methods: Rats were treated with haloperidol for 24 weeks and withdrawn for 9 weeks. Mu (m) receptors were labeled with [3H]-DAMGO., Results: Haloperidol treatment produced a significant reduction in mu-receptor binding in the globus pallidus (P<0.05). There was, however, no relationship between mu-opioid receptor density and VCMs in this or any other region of the basal ganglia., Conclusion: These results replicate prior findings of a neuroleptic-induced reduction in [3H]-DAMGO binding in the globus pallidus. The lack of association between VCMs and [3H]-DAMGO binding in the globus pallidus or any other region suggests that prior reports of enkephalinergic mRNA changes in the striatum are not accompanied by compensatory changes in postsynaptic neurons.

1. Several steroids and related compounds can bind to central opiate receptors in whole-brain mouse homogenates. Among these drugs, the synthetic estrogen, diethylstilbestrol (DES), exhibits one of the highest affinities in binding experiments labeling opiate receptors with the nonselective opiate antagonist, [3H]diprenorphine. 2. In the search for a functional correlate to this biochemical finding, we have studied the effects of DES on the mouse hippocampal slice in vitro preparation. 3. Previously, binding studies were performed in hippocampal homogenates, labeling opiate receptors with [3H]diprenorphine or with the mu-selective opiate agonist, [3H]DAGO. DES inhibited [3H]diprenorphine and [3H]DAGO binding, the IC50 values obtained being (1.03 +/- 0.16) x 10(-5) and (1 +/- 0.8) x 10(-5) M, respectively. 4. In mice hippocampal slices, we measured the extracellular evoked potentials obtained in the CA1 pyramidal cell layer of the hippocampi and the field excitatory postsynaptic potentials (EPSP) obtained in the stratum radiatum. The presence of DES (10(-5) M) induced an increase in the amplitude of the population spikes measured in the pyramidal layer without modifying the field EPSP. This effect is similar to that obtained in the presence of DAGO in this preparation. The effect produced by DES was not modified by the presence of the opiate competitive antagonist, naloxone (10(-5) M), or by the opiate alkylating agent, beta-chlornaltrexamine (10(-5) M). Conversely, in the presence of the transcription inhibitor, actinomycin D (5 micrograms/ml), the effect produced by DES was inhibited. 5. Our results with DES support the general idea that estrogens increase central excitability. Although diethylstilbestrol can bind to opiate receptors in the hippocampus, the effect induced by this estrogen on hippocampal excitability seems unrelated to a direct action on opiate receptors, and an intracellular effect is suggested.

Opioid binding properties of Tyr-D-Ser-Gly-Phe-Leu-Thr-NH-NH-Gly-Mal (DSLET-Mal), a novel enkephalin-framed affinity label, was determined in rat brain membranes. In competition studies the ligand showed high affinity for the delta opioid sites, labelled by [(3)H][Ile(5,6)]deltorphin II (K(i) = 8 nM), whereas its binding to the mu ([(3)H]DAMGO) and kappa ([(3)H]EKC) sites was weaker. Preincubation of the rat brain membranes with DSLET-Mal at micromolar concentrations resulted in a wash-resistant and dose-dependent inhibition of the [(3)H][Ile(5,6)]deltorphin II binding sites (96% blocking at 10 microM concentration). Intracerebroventricular (ICV) administration of DSLET-Mal reduced the density of delta opioid receptors and had no effect on mu and kappa receptors, as determined by saturation binding studies. [Ile(5, 6)]deltorphin II-stimulated [(35)S]GTPgammaS binding was determined in membrane preparations of different brain areas of the ICV-treated animals. In both frontal cortex and hippocampus DSLET-Mal significantly decreased G protein activation by the delta agonist, having no effect on DAMGO stimulated [(35)S]GTPgammaS binding. DSLET-Mal had qualitatively similar effects on both receptor binding and G protein activation. These characteristics of the compound studied suggest that DSLET-Mal can serve as an affinity label for further studies of the delta-opioid receptors., (Copyright 1999 Academic Press.)

The aim of this paper is to study the influence of salmon calcitonin (SCT) on opioid analgesia when opioid transduction pathways are functionally uncoupled from Gi/o proteins by treatment with pertussis toxin (PTX). The antinociceptive effect of morphine and three selective opioid agonists, [D-Ala2,N-Me-Phe2,Gly5-ol]enkephalin (DAMGO) (OP(3-mu receptor agonist), [D-Pen2.5]-enkephalin (OP-1-delta receptor agonist) and trans-( +/- )-3,4-dichloro-N-methyl-N-[2-1(-pyrrolidinyl)-cyclohexyl]-benzene-acetam ide methane sulfonate (U-50, 488H) (OP1-kappareceptor agonist) was evaluated, using the tail flick test, in mice treated with PTX or with PTX and SCT. PTX blocked the antinociceptive effect of the opioids, being the antinociception similar in control animals and in mice treated with PTX and SCT. Thus, SCT prevents the effect of the blockade of Gi/o-proteins. From this it could be suggested that calcitonin activates alternative antinociceptive mechanisms that are not dependent on Gi/o-proteins.

A series of pyrido- and pyrimidomorphinans (6a-h and 7a-g) were synthesized from naltrexone and evaluated for binding and biological activity at the opioid receptors. The unsubstituted pyridine 6a displayed high affinities at opioid delta, mu, and kappa receptors with K(i) values of 0.78, 1.5, and 8.8 nM, respectively. Compound 6a was devoid of agonist activity in the mouse vas deferens (MVD) and guinea pig ileum (GPI) preparations but was found to display moderate to weak antagonist activity in the MVD and GPI with K(e) values of 37 and 164 nM, respectively. The pyrimidomorphinans in general displayed lower binding potencies and delta receptor binding selectivities than their pyridine counterparts. Incorporation of aryl groups as putative delta address mimics on the pyrido- and pyrimidomorphinan framework gave ligands with significant differences in binding affinity and intrinsic activity. Attachment of a phenyl group at the 4'-position of 6a or the equivalent 6'-position of 7a led to dramatic reduction in binding potencies at all the three opioid receptors, indicating the existence of a somewhat similar steric constraint at the ligand binding sites of delta, mu, and kappa receptors. In contrast, the introduction of a phenyl group at the 5'-position of 6a did not cause any reduction in the binding affinity at the delta receptor. In comparison to the unsubstituted pyridine 6a, the 5'-phenylpyridine 6c showed improvements in mu/delta and kappa/delta binding selectivity ratios as well as in the delta antagonist potency in the MVD. Interestingly, introduction of a chlorine atom at the para position of the pendant 5'-phenyl group of 6c not only provided further improvements in delta antagonist potency in the MVD but also shifted the intrinsic activity profile of 6c from an antagonist to that of a mu agonist in the GPI. Compound 6d thus possesses the characteristics of a nonpeptide mu agonist/delta antagonist ligand with high affinity at the delta receptor (K(i) = 2.2 nM), high antagonist potency in the MVD (K(e) = 0.66 nM), and moderate agonist potency in the GPI (IC(50) = 163 nM). Antinociceptive evaluations in mice showed that intracerebroventricular (icv) injections of 6d produced a partial agonist effect in the 55 degrees C tail-flick assay and a full agonist effect in the acetic acid writhing assay (A(50) = 7.5 nmol). No signs of overt toxicity were observed with this compound in the dose ranges tested. Moreover, repeated icv injections of an A(90) dose did not induce any significant development of antinociceptive tolerance in the acetic acid writhing assay. The potent delta antagonist component of this mixed mu agonist/delta antagonist may be responsible for the diminished propensity to produce tolerance that this compound displays.

Morphine and other micro opioids regulate a number of intracellular signaling pathways, including the one mediated by phospholipase C (PLC). By studying PLC beta3-deficient mice, we have established a strong link between PLC and mu opioid-mediated responses at both the behavioral and cellular levels. Mice lacking PLC beta3, when compared with the wild type, exhibited up to a 10-fold decrease in the ED(50) value for morphine in producing antinociception. The reduced ED(50) value was unlikely a result of changes in opioid receptor number or affinity because no differences were found in whole-brain B(max) and K(d) values for mu, kappa, and delta opioid receptors between wild-type and PLC beta3-null mice. We also found that opioid regulation of voltage-sensitive Ca(2+) channels in primary sensory neurons (dorsal root ganglion) was different between the two genotypes. Consistent with the behavioral findings, the specific mu agonist [D-Ala(2),(Me)Phe(4),Gly(ol)(5)]enkephalin (DAMGO) induced a greater whole-cell current reduction in a greater proportion of neurons isolated from the PLC beta3-null mice than from the wild type. In addition, reconstitution of recombinant PLC protein back into PLC beta3-deficient dorsal root ganglion neurons reduced DAMGO responses to those of wild-type neurons. In neurons of both genotypes, activation of protein kinase C with phorbol esters markedly reduced DAMGO-mediated Ca(2+) current reduction. These data demonstrate that PLC beta3 constitutes a significant pathway involved in negative modulation of mu opioid responses, perhaps via protein kinase C, and suggests the possibility that differences in opioid sensitivity among individuals could be, in part, because of genetic factors.

Dynamin plays a critical role in the membrane fission mechanism that mediates regulated endocytosis of many G protein-coupled receptors. In addition, dynamin is required for ligand-induced activation of mitogen-activated protein kinase by certain receptors, raising a general question about the role of dynamin in mitogenic signal transduction. Here we report that endocytosis of mu and delta opioid receptors is not required for efficient ligand-induced activation of mitogen-activated protein kinase. Nevertheless, mitogenic signaling mediated by these receptors is specifically dynamin-dependent. Thus a functional role of dynamin in mitogenic signaling can be dissociated from its role in receptor-mediated endocytosis, suggesting a previously unidentified and distinct role of dynamin in signal transduction by certain G protein-coupled receptors.

Cells immunoreactive for the mu-opioid receptor (MOR) in laminae I-II of the spinal cord were identified as small neurons with rostro-caudal dendrites. In spinal cord slices, [D-Ala2,MePhe4-Gly-ol5]enkephalin (DAMGO) or etorphine (1 microM) caused naloxone-sensitive MOR endocytosis in 100% of these neurons, whereas the selective delta- and kappa-opioid agonists [D-Pen2,5]enkephalin (DPDPE) and spiradoline mesylate (U-62,066), respectively, produced negligible internalization at 1 microM. The EC50 for DAMGO was 30 nM, similar to its potency to inhibit cAMP accumulation and to increase [gamma-35S]GTP binding. MOR internalization followed an exponential timecourse with a half-life of 1.7 min. MOR internalization in spinal cord slices was faster and occurred at lower agonist concentrations than in MOR-transfected cells, suggesting that spinal cord neurons have a more effective coupling of MORs to intracellular components mediating endocytosis.

1. The actions of opioid receptor agonists on synaptic transmission in substantia gelatinosa (SG) neurones in adult (6- to 10-week-old) rat spinal cord slices were examined by use of the blind whole-cell patch-clamp technique. 2. Both the mu-receptor agonist DAMGO (1 microM) and the delta-receptor agonist DPDPE (1 microM) reduced the amplitude of glutamatergic excitatory postsynaptic currents (EPSCs) which were monosynaptically evoked by stimulating Adelta afferent fibres. Both also decreased the frequency of miniature EPSCs without affecting their amplitude. 3. In contrast, the kappa-receptor agonist U-69593 (1 microM) had little effect on the evoked and miniature EPSCs. 4. The effects of DAMGO and DPDPE were not seen in the presence of the mu-receptor antagonist CTAP (1 microM) and the delta-receptor antagonist naltrindole (1 microM), respectively. 5. Neither DAMGO nor DPDPE at 1 microM affected the responses of SG neurones to bath-applied AMPA (10 microM). 6. Evoked and miniature inhibitory postsynaptic currents (IPSCs), mediated by either the GABAA or the glycine receptor, were unaffected by the mu-, delta- and kappa-receptor agonists. Similar results were also obtained in SG neurones in young adult (3- to 4-week-old) rat spinal cord slices. 7. These results indicate that opioids suppress excitatory but not inhibitory synaptic transmission, possibly through the activation of mu- and delta- but not kappa-receptors in adult rat spinal cord SG neurones; these actions are presynaptic in origin. Such an action of opioids may be a possible mechanism for the antinociception produced by their intrathecal administration.

The effects of opioid peptides on the osmotic release of neurohypophyseal hormones, arginine vasotocin (AVT) and mesotocin (MT), were determined in 2-day-old chicks. Experiment 1 examined the effect of a variety of doses of naloxone, an opioid antagonist, on chicks administered isotonic or hypertonic solution. Plasma osmolality in chicks administered hypertonic solution was significantly higher than that in groups administered isotonic solution. None of the doses of naloxone affected plasma osmolality in response to isotonic and hypertonic solution. Plasma levels of AVT increased in hypertonic solution and this response was further enhanced by naloxone injection as the doses increased. The hypertonic solution alone did not affect plasma levels of MT, but additional treatment with naloxone slightly increased plasma levels of MT. Experiment 2 examined the effect of DAMGO ([d-Ala(2), N-Me-Phe(4),Gly-ol]-enkepha lin), a specific mu receptor agonist. Relatively high plasma osmolality caused by hypertonic solution was not affected by additional treatment with DAMGO. Plasma levels of AVT in response to hypertonic solution and to additional treatment with naloxone were reduced by higher doses of DAMGO. Experiment 3 examined the effect of naloxone on chicks administered different concentrations of NaCl. Administration of hypertonic solution resulted in an increase in plasma osmolality and plasma levels of AVT. Naloxone administration enhanced the increase in plasma AVT levels in response to hypertonic solution. Experiment 4 examined the effect of naloxone on different kinds of hypertonic solution, 0.15 M NaCl, 1.5 M NaCl, 2.55 M urea, and 1.95 M sucrose. The increases in plasma osmolality resulting from the administration of the urea and sucrose solutions were the same as those in the chicks injected with 1.5 M NaCl. In sucrose-treated chicks, plasma levels of AVT increased in chicks administered naloxone but not in chicks injected with normal saline. In contrast, no significant changes in plasma levels of AVT were observed in urea treatment with or without naloxone. In Experiments 3 and 4, plasma levels of MT after administration of hypertonic solutions did not change. However, naloxone administration enhanced plasma levels of MT in osmotically stimulated chicks. The results of the present study suggest that opioid peptides attenuate the increase in plasma AVT and MT in hypertonic states., (Copyright 1999 Academic Press.)

We used the whole cell open-patch or perforated-patch technique to characterize mu-opioid modulation of Ca(2+) current (I(Ca)) in nodose sensory neurons and in a specific subpopulation of nodose cells, aortic baroreceptor neurons. The mu-opiate receptor agonist Tyr-D-Ala-Gly-MePhe-Gly-ol enkephalin (DAGO) inhibited I(Ca) in 95% of neonatal [postnatal day (P)1-P3] nodose neurons. To the contrary, only 64% of juvenile cells (P20-P35) and 61% of adult cells (P60-P110) responded to DAGO. DAGO-mediated inhibition of I(Ca) was naloxone sensitive, irreversible in the presence of guanosine 5'-O-(3-thiotriphosphate), absent with guanosine 5'-O-(2-thiodiphosphate), and eliminated with pertussis toxin; DAGO's inhibition of I(Ca) was G protein mediated. Incubation of neurons with omega-conotoxin GVIA eliminated the effect of DAGO in neonatal but not in juvenile cells. In the latter, DAGO reduced 37% of the current remaining in the presence of omega-conotoxin. In the subset of nodose neurons, aortic baroafferents, the effect of DAGO was concentration dependent, with an IC(50) of 1.82 x 10(-8) M. DAGO slowed activation of I(Ca), but activation curves constructed from tail currents were the same with and without DAGO (100 nM). In summary, mu-opiate modulation of I(Ca) in nodose neurons was demonstrated in three age groups, including specifically labeled baroafferents. The demonstration of a mechanism of action of mu-opioids on baroreceptor afferents provides a basis for the attenuation of the baroreflex that occurs at the level of the nucleus tractus solitarii.

NAC-1 is an mRNA that is increased selectively in the nucleus accumbens after acute and repeated cocaine administration. Antisense or control oligonucleotides were microinjected into the nucleus accumbens of rats to define the role of NAC-1 in the behavioral responses to acute systemic cocaine. Antisense oligonucleotides decreased NAC-1 mRNA levels by 26% and markedly enhanced the motor stimulant response to an acute cocaine injection compared to sense oligonucleotide microinjections. The augmentation in cocaine motor behavior produced by NAC-1 antisense pretreatment in the nucleus accumbens was not associated with increased dopamine release as estimated by microdialysis. In contrast, the behavioral response to dopamine microinjection into the nucleus accumbens was increased after antisense oligonucleotide treatment, while the motor response to mu-opioid receptor stimulation was unaltered. These data suggest that the induction of NAC-1 by cocaine may be a compensatory mechanism that minimizes the behavioral impact of cocaine administration by regulating postsynaptic dopamine transmission within the nucleus accumbens., (Copyright 1999 Wiley-Liss, Inc.)

Rationale: The pedunculopontine tegmental nucleus (PPTg) has been implicated in drug reward, particularly in the development of dependence. However, little is known of the receptor systems within this nucleus which might be involved. Furthermore, some research suggests that the PPTg may also be part of the neuronal circuitry involved in established drug-taking behavior., Objective: The objective of these experiments was to examine the role of mu-opioid and nicotinic cholinergic mechanisms in the PPTg in cocaine self-administration., Methods: Microinfusions of mu-opioid and nicotinic receptor selective compounds were made into the PPTg of rats trained to self-administer cocaine intravenously, in the vicinity of cholinergic cells which are known to project to the midbrain dopamine neurons of the ventral tegmental area (VTA)., Results: The mu-opioid selective agonist DAMGO, tested at doses of 0, 0.05 and 0.5 microg, produced a dose-related reduction in the number of cocaine infusions obtained during the 1-h self-administration sessions. The mu-selective antagonist CTOP (0-2 microg) and nicotine (0-10 microg) did not produce significant changes in cocaine self-administration. Microinfusions of the nicotinic antagonist dihydro-beta-erythroidine (0-30 microg) produced a small but significant increase in cocaine-maintained responding., Conclusions: These data show that mu-opioid mechanisms in the PPTg can influence cocaine self-administration markedly. Moreover, the data demonstrate that PPTg circuitry can influence drug reward in already-established drug-reinforced behavior, as well as during the development of dependence (as shown by previous research).

Opiate analgesia, tolerance, and addiction are mediated by drug-induced activation of the mu opioid receptor. A fundamental question in addiction biology is why exogenous opiate drugs have a high liability for inducing tolerance and addiction while native ligands do not. Studies indicate that highly addictive opiate drugs such as morphine are deficient in their ability to induce the desensitization and endocytosis of receptors. Here, we demonstrate that this regulatory mechanism reveals an independent functional property of opiate drugs that can be distinguished from previously established agonist properties. Moreover, this property correlates with agonist propensity to promote physiological tolerance, suggesting a fundamental revision of our understanding of the role of receptor endocytosis in the biology of opiate drug action and addiction.

Opioid effects on cell division in the embryonic cerebral cortex were examined using two experimental approaches: (i) the presence of opioid receptors in the embryonic day 16 mouse neocortex was tested using immunohistochemical techniques; (ii) the values of the indices of [3H]thymidine pulse labelled cells and mitotic indices were estimated in the ventricular zone of the embryonic day 16 mouse neocortex 2.5, 4.5 and 8.5 h after administration to pregnant females of selected opioid receptor agonists or the opioid antagonist naloxone. The immunohistochemical study demonstrated that distinct subpopulations of the ventricular zone cells express mu, delta or kappa opioid receptors. Acute exposure of mouse embryos to mu, delta and kappa opioid receptor agonists or naloxone differentially affects the indices of [3H] thymidine pulse labelled cells and mitotic indices indicating changes in the cell cycle composition. Treatment with the mu opioid receptor agonist D-Ala2-MePhe4, Gly-ol5-enkephalin (DAGO), or the partially selective kappa opioid receptor agonist bremazocine, increased the [3H]thymidine labelling and mitotic indices. In contrast, the delta receptor agonist (D-Ser8)-leucine enkephalin-Thr (DSLET) produced a decrease in the labelled cell indices and mitotic indices. Naloxone provided a biphasic effect: a decrease in the values of labelled cell indices 2.5 h after naloxone administration, followed by an increase in the values of the indices at 4.5 and 8.5 h. These results suggest that the endogenous embryonic/maternal opioid systems are involved in the regulation of cell division in the ventricular zone of the late embryonic cortex.

Monoclonal antibodies generated against multiple antigenic peptides of the N-terminal sequence (3LVPSARAELQSSPLV17) of the cloned delta opioid receptor immunoprecipitated a 58 kDa protein from CHAPS-solubilized NG108-15 membranes. The immunoprecipitates bound [3H]DPDPE--but not [3H]DAMGO--with a Kd of 6.4 nM and a Bmax of 75 pM. Western blot analysis revealed a distinct band of 58 kDa. The antibodies inhibited basal and PGE1-stimulated cAMP levels, and mimicked the effect of agonists manifest in a compensatory increase in cAMP formation. The antibody will be potentially useful in the analysis of functional epitopes on the delta opioid receptor.

1. The overall effect of the mu-opioid receptor agonist DAMGO (Tyr-D-Ala-Gly-MePhe-Gly-ol) on ventrolateral periaqueductal grey (PAG) neurons in brain slices was studied using the whole-cell patch-clamp recording technique. 2. Under current-clamp conditions, DAMGO (1 microM) increased cell firing in many PAG neurons even though the opioid induced hyperpolarization and inhibited excitatory postsynaptic potentials (EPSPs) in these cells. 3. The increase in cell activity by DAMGO was observed in both transverse and horizontal slices. The increase persisted when the membrane potential was re-depolarized to the control level. Thus, different planes of sections or the removal of Na+ channel inactivation could not account for the observation. 4. The GABA antagonist bicuculline caused cell firing, mimicking the excitatory effect of DAMGO. Unlike DAMGO, however, bicuculline depolarized PAG cells. 5. Under voltage-clamp conditions, with the same driving force, the evoked inhibitory postsynaptic currents (IPSCs) in these neurons were 2.3 times larger than the evoked excitatory postsynaptic currents (EPSCs). Furthermore, DAMGO inhibited IPSCs by 60.7% while it inhibited EPSCs by 35.3%. 6. We propose that the overall effect of an opioid depends on the dynamic balance of its excitatory and inhibitory actions. In the PAG, the blockade of the inhibitory drive of GABAergic inputs by DAMGO is large. It overcomes the DAMGO-induced hyperpolarization and inhibition of EPSCs and thus results in the excitation of these neurons.

Differential effects of neuropeptide Y (NPY) and mu-opioid DAMGO on 'palatability' vs. 'energy'. A variety of studies suggest that NPY is an important manager of energy metabolism. In contrast, the opioid peptides appear to influence the 'rewarding' aspects of feeding. In the current study, we stimulated feeding by injecting NPY (110 pmol) or the mu-opioid agonist DAMGO (2 nmol) into the paraventricular nucleus of rats. Following injection, rats were given free access to laboratory chow and a 10% sucrose solution. Animals injected with saline derived 10% of their kilocalories from the chow and 90% from the sucrose solution (total kcal/4 h=12.2+/-1. 0). Those rats injected with NPY derived 48% of their energy from chow and 52% from the sucrose solution (total kcal/4 h=24.8+/-1.7). The DAMGO-injected rats derived only 15% of their kilocalories from chow and the remainder from the sucrose solution (total kcal/4 h=23. 0+/-2.3). Thus, while NPY and DAMGO both stimulated energy intake compared to saline controls (P<0.0001), the effect on intake of a palatable dilute energy solution (0.4 kcal/g) vs. a 'bland' laboratory chow (3.95 kcal/g) was different. The results of this study reinforce the notion that NPY has a major effect on energy needs, whereas opioids influence the 'rewarding' characteristics of foods., (Copyright 1999 Elsevier Science B.V.)

In addition to its central actions, morphine has important peripheral effects. To examine peripheral analgesic mechanisms, we developed a topical opioid paradigm in which the tail was immersed in a dimethyl sulfoxide (DMSO) solution containing various drugs. Alone, DMSO was inactive in the tail-flick assay in mice. DMSO solutions containing morphine and peptides such as [D-Ala2,MePhe4, Gly(ol)5]enkephalin (DAMGO) produced a potent, dose-dependent analgesia with the radiant heat tail-flick assay. The actions of the drugs were local. Analgesia was observed only in regions of the tail exposed to the solution and not in more proximal unexposed portions of the tail. Immersion of the tail in a solution containing either 125I-labeled morphine or 125I-labeled DAMGO revealed no detectable uptake of radioactivity into the brain, spinal cord, or blood. In the tail, radioactivity was limited only to the regions actually immersed in the solutions. The topical drugs potentiated systemic agents, similar to the previously established synergy between peripheral and central sites of action. Local tolerance was rapidly produced by repeated daily exposure of the tail to morphine. Topical morphine tolerance was effectively blocked by the N-methyl-D-aspartate (NMDA) antagonist MK801 given either systemically or topically but not intrathecally. The ability of a topical NMDA antagonist to block local morphine tolerance suggests that peripheral NMDA receptors mediate topical morphine tolerance. Morphine was cross-tolerant to DAMGO, but not to morphine-6beta-glucuronide, implying different mechanisms of action. These observations are significant in the design and use of opioids clinically.

The influence of membrane microviscosity on mu-opioid agonist and antagonist binding, as well as agonist efficacy, was examined in membranes prepared from SH-SY5Y cells and from a C6 glioma cell line stably expressing the rat mu-opioid receptor (C6mu). Addition of cholesteryl hemisuccinate (CHS) to cell membranes increased membrane microviscosity and reduced the inhibitory effect of sodium and guanine nucleotides on the affinity of the full agonists sufentanil and [D-Ala2,N-MePhe4,Gly-ol5]enkephalin (DAMGO) for the mu-opioid receptor. Binding of the antagonists [3H]naltrexone and [3H]diprenorphine and the partial agonist nalbuphine was unaffected by CHS. The effect of CHS on agonist binding was reversed by subsequent addition of cis-vaccenic acid, suggesting that the effect of CHS is the result of increased membrane microviscosity and not a specific sterol-receptor interaction. CHS addition increased the potency of DAMGO to stimulate guanosine-5'-O-(3-[35S]thio)triphosphate binding by fourfold, whereas the potency of nalbuphine was unaffected. However, nalbuphine efficacy relative to that of the full agonist DAMGO was strongly increased in CHS-treated membranes compared with that in control membranes. Membrane rigidification also resulted in an increased efficacy for the partial agonists meperidine, profadol, and butorphanol relative to that of DAMGO as measured by agonist-stimulated GTPase activity in control and CHS-modified membranes. These findings support a regulatory role for membrane microviscosity in receptor-mediated G protein activation.

Morphine significantly stimulated invertebrate immunocyte intracellular calcium level increases in a concentration-dependent manner in cells preloaded with Fura 2/AM. Morphine's action was blocked by prior exposure of the cells to the opiate receptor antagonist naloxone. Various opioid peptides did not exhibit this ability, indicating a morphine-mu 3 mediated process. In comparing the sequence of events concerning morphine's action in stimulating both [Ca2+]i and NO production in these cells, we found that the first event precedes the second by 42 +/- 7 s. The opiate stimulation of [Ca2+]i- was attenuated in cells leached of calcium. strongly suggesting that intracellular calcium levels regulate cNOS activity in invertebrate immunocytes.