enkephalin--ala(2)-mephe(4)-gly(5)- and Morphine-Dependence

A series of 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-(isoquinoline-3'-carboxamido)morphinan (NAQ) analogues were synthesized and pharmacologically characterized to study their structure-activity relationship at the mu opioid receptor (MOR). The competition binding assay showed two-atom spacer and aromatic side chain were optimal for MOR selectivity. Meanwhile, substitutions at the 1'- and/or 4'-position of the isoquinoline ring retained or improved MOR selectivity over the kappa opioid receptor while still possessing above 20-fold MOR selectivity over the delta opioid receptor. In contrast, substitutions at the 6'- and/or 7'-position of the isoquinoline ring reduced MOR selectivity as well as MOR efficacy. Among this series of ligands, compound 11 acted as an antagonist when challenged with morphine in warm-water tail immersion assay and produced less significant withdrawal symptoms compared to naltrexone in morphine-pelleted mice. Compound 11 also antagonized the intracellular Ca(2+) increase induced by DAMGO. Molecular dynamics simulation studies of 11 in three opioid receptors indicated orientation of the 6'-nitro group varied significantly in the different 'address' domains of the receptors and played a crucial role in the observed binding affinities and selectivity. Collectively, the current findings provide valuable insights for future development of NAQ-based MOR selective ligands.

Topics: Animals; CHO Cells; Cricetulus; Drug Design; Humans; Isoquinolines; Ligands; Mice; Molecular Dynamics Simulation; Morphinans; Morphine Dependence; Narcotic Antagonists; Receptors, Opioid, mu; Structure-Activity Relationship

The best-established mechanism of opioid dependence is the up-regulation of adenylate cyclase (AC)/cAMP pathway, which was reported to be negatively regulated by hydrogen sulfide (H2S), a novel endogenous neuromodulator. The present study was, therefore, designed to determine whether H2S is able to attenuate the development of opioid dependence via down-regulating AC/cAMP pathway.. We demonstrated that application of sodium hydrosulphide (NaHS) and GYY4137, two donors of H2S, significantly alleviated naloxone-induced robust withdrawal jumping (the most sensitive and reliable index of opioid physical dependence) in morphine-treated mice. Repeated treatment with NaHS inhibited the up-regulated protein expression of AC in the striatum of morphine-dependent mice. Furthermore, NaHS also attenuated morphine/naloxone-elevated mRNA levels of AC isoform 1 and 8, production of cAMP, and phosphorylation of cAMP response element-binding protein (CREB) in mice striatum. These effects were mimicked by the application of exogenous H2S or over-expression of cystathione-β-synthase, an H2S -producing enzyme, in SH-SY5Y neuronal cells on treatment with [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-Enkephalin, a selective μ-opioid receptor agonist. Blockade of extracellular-regulated protein kinase 1/2 (ERK1/2) with its specific inhibitor attenuated naloxone-induced CREB phosphorylation. Pretreatment with NaHS or stimulation of endogenous H2S production also significantly suppressed opioid withdrawal-induced ERK1/2 activation in mice striatum or SH-SY5Y cells.. H2S treatment is important in prevention of the development of opioid dependence via suppression of cAMP pathway in both animal and cellular models.. Our data suggest a potential role of H2S in attenuating the development of opioid dependence, and the underlying mechanism is closely related to the inhibition of AC/cAMP pathway.

Topics: Adenylyl Cyclases; Analgesics, Opioid; Animals; Behavior, Animal; Cell Line; Corpus Striatum; Cyclic AMP; Cyclic AMP Response Element-Binding Protein; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Gene Expression Regulation; Humans; Hydrogen Sulfide; Male; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Morphine; Morphine Dependence; Naloxone; Phosphorylation; Signal Transduction

In state-dependency, information retrieval is most efficient when the animal is in the same state as it was during the information acquisition. State-dependency has been implicated in a variety of learning and memory processes, but its mechanisms remain to be resolved. Here, mice deficient in AMPA-type glutamate receptor GluA1 subunits were first conditioned to morphine (10 or 20 mg/kg s.c. during eight sessions over four days) using an unbiased procedure, followed by testing for conditioned place preference at morphine states that were the same as or different from the one the mice were conditioned to. In GluA1 wildtype littermate mice the same-state morphine dose produced the greatest expression of place preference, while in the knockout mice no place preference was then detected. Both wildtype and knockout mice expressed moderate morphine-induced place preference when not at the morphine state (saline treatment at the test); in this case, place preference was weaker than that in the same-state test in wildtype mice. No correlation between place preference scores and locomotor activity during testing was found. Additionally, as compared to the controls, the knockout mice showed unchanged sensitization to morphine, morphine drug discrimination and brain regional μ-opioid receptor signal transduction at the G-protein level. However, the knockout mice failed to show increased AMPA/NMDA receptor current ratios in the ventral tegmental area dopamine neurons of midbrain slices after a single injection of morphine (10 mg/kg, s.c., sliced prepared 24 h afterwards), in contrast to the wildtype mice. The results indicate impaired drug-induced state-dependency in GluA1 knockout mice, correlating with impaired opioid-induced glutamate receptor neuroplasticity.

Topics: Analgesics, Opioid; Animals; Dopaminergic Neurons; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Female; Male; Mice; Mice, 129 Strain; Mice, Inbred C57BL; Mice, Knockout; Morphine; Morphine Dependence; Motor Activity; Narcotics; Receptors, AMPA; Receptors, Opioid, mu

Based on an established 3D pharmacophore, a series of quinoline derivatives were synthesized. The opioidergic properties of these compounds were determined by a competitive binding assay using (125)I-Dynorphine, (3)H-DAMGO and (125)I-DADLE for kappa, mu, and delta receptors, respectively. Results showed varying degree of activities of the compounds to kappa and mu opioid receptors with negligible interactions at the delta receptor. The compound, S4 was the most successful in inhibiting the two most prominent quantitative features of naloxone precipitated withdrawal symptoms - stereotyped jumping and body weight loss. Determination of IC(50) of S4 revealed a greater affinity towards mu compared to kappa receptor. In conclusion, quinoline derivatives of S4 like structure offer potential tool for treatment of narcotic addictions.

Topics: Acetanilides; Aminoquinolines; Animals; Cell Line, Tumor; Competitive Bidding; Humans; Ligands; Mice; Mice, Inbred BALB C; Morphine Dependence; Narcotic Antagonists; Quinolines; Rats; Receptors, Opioid, kappa; Receptors, Opioid, mu

We have recently demonstrated that the combination of methadone and morphine enhances the ability of morphine to induce mu-opioid peptide (MOP) receptor endocytosis. As a result, rats receiving both drugs show reduced morphine tolerance and dependence. In the present study, we identify the biochemical basis for the protective effect of the drug combination. In rats treated with morphine alone, the inhibitory effect of DAMGO on forskolin-stimulated adenylyl cyclase activity was significantly reduced in a brain-region-selective manner. Importantly, these reductions were prevented in animals receiving the drug combination. We found that these changes were not due to alterations in MOP receptor density, or MOP receptor-G protein coupling, as no significant change in these parameters was observed. Together these data demonstrate that neither changes in receptor number nor function are required for morphine tolerance and dependence. Rather, brain-region-selective changes in adenylyl cyclase signal transduction are critical, and both these biochemical changes and the behavioral effects are prevented by facilitating endocytosis of the MOP receptor.

Topics: Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Analgesics, Opioid; Animals; Brain; Dose-Response Relationship, Drug; Drug Interactions; Drug Tolerance; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Male; Methadone; Morphine; Morphine Dependence; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu

Although the expression of the morphine (MOR) withdrawal syndrome is more marked in male mice than in females, we have demonstrated that the GABAB agonist baclofen (BAC) is able to attenuate MOR withdrawal signs in either sex. In order to extend these previous observations, the aim of the present study was to evaluate the mu-opioid receptor labeling in various brain areas in mice of either sex, during MOR withdrawal and its prevention with BAC. Prepubertal Swiss-Webster mice were rendered dependent by intraperitonial (i.p.) injection of MOR (2 mg/kg) twice daily for 9 days. On the 10th day, dependent animals received naloxone (NAL; 6 mg/kg, i.p.) 60 min after MOR, and another pool of dependent mice received BAC (2 mg/kg, i.p.) previous to NAL. Thirty minutes after NAL, mice were sacrificed and autoradiography with [3H]-[D-Ala2, N-Me-Phe4, -glycol5] enkephalin (DAMGO) was carried out on mice brains at five different anatomical levels. Autoradiographic mapping showed a significant increase of mu-opioid receptor labeling during MOR withdrawal in nucleus accumbens core (NAcC), caudate putamen (CPu), mediodorsal thalamic nucleus (MDTh), basolateral and basomedial amygdala, and ventral tegmental area vs. respective control groups in male mice. In contrast, opiate receptor labeling was not significantly modified in any of the brain areas studied in withdrawn females. BAC reestablished mu-opioid receptor binding sites during MOR withdrawal only in NAcC of males, and a similar tendency was observed in CPu and MDTh, even when it was not statistically significant. The sexual dimorphism observed in the present study confirms previous reports indicating a greater sensitivity of males in response to MOR pharmacological properties. The present results suggest that the effect of BAC in preventing the expression of MOR withdrawal signs could be related with the ability of BAC to reestablish the mu-opioid receptor labeling in certain brain areas.

Topics: Aging; Animals; Autoradiography; Baclofen; Binding Sites; Brain; Disease Models, Animal; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Female; GABA Agonists; Male; Mice; Morphine; Morphine Dependence; Narcotic Antagonists; Narcotics; Receptors, Opioid, mu; Sex Characteristics; Substance Withdrawal Syndrome

Maternal deprivation can trigger long-lasting molecular and cellular modifications in brain functions and might facilitate the appearance of pathogenic behaviors. This study focuses on the vulnerability to develop morphine dependence in adult rats that were separated from their mother and littermates for 3 h per day for 14 d after birth and examines the adaptive changes in the enkephalinergic pathways. Place-preference conditioning was observed with 2 mg/kg morphine in deprived rats, whereas 5 mg/kg morphine was necessary to induce conditioning in nondeprived animals. A prolonged morphine conditioning was shown in deprived rats. A strong increase in oral morphine self-administration behavior and preference was observed in deprived rats. Only a very slight increase in preference for sucrose solution, a more ethological reinforcer known to interact with the opioid system, was shown in deprived rats. These results indicate that this postnatal environment change leads to a hypersensitivity to the reinforcing properties of morphine and to the development of morphine dependence. A significant decrease in preproenkephalin mRNA expression was observed in the nucleus accumbens and the caudate-putamen nucleus of deprived rats. The basal extracellular levels of the Met-enkephalin-like immunoreactivity in the nucleus accumbens were significantly lower in deprived rats when compared with nondeprived animals, whereas no change in mu-opioid receptor binding occurred. These results strongly support that maternal deprivation leads to a basal hypoactivity of the enkephalinergic system and hypersensitivity to morphine effects. Together, our results suggest that maternal deprivation in pups likely represents a risk factor for morphine dependence in adult rats.

Topics: Analysis of Variance; Animals; Animals, Newborn; Autoradiography; Behavior, Animal; Brain; Choice Behavior; Conditioning, Psychological; Dialysis; Dose-Response Relationship, Drug; Drinking Behavior; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Methionine; Enkephalins; Female; In Situ Hybridization; Maternal Deprivation; Morphine; Morphine Dependence; Narcotics; Nucleus Accumbens; Pregnancy; Protein Precursors; Radioimmunoassay; Rats; Rats, Long-Evans; Receptors, Opioid, mu; RNA, Messenger; Self Administration; Sucrose; Time Factors; Tritium

Morphine was provided to rats in drinking water for 21 days. Profound analgesic tolerance was detected both in hot-plate and tail-flick tests. The density of [3H]DAMGO binding sites increased by 76% in spinal cord membranes due to morphine exposure compared to those in opioid naive animals. Slightly augmented [3H]DAMGO binding was measured in the synaptic plasma membranes, with a concomitant decrease in the microsomal membranes, of morphine tolerant/dependent brains. These observations suggest that the regulation of spinal mu opioid receptors might be different from those in the brain. It is emphasized that the molecular changes underlying tolerance/dependence are influenced by several factors, such as the tissue or subcellular fractions used, besides the obvious importance of the route of drug administration. Results obtained after voluntary morphine intake further support the growing number of experimental data that chronic morphine does not internalize/downregulate the mu opioid receptors in the central nervous system.

Topics: Animals; Binding Sites; Brain; Drinking; Drug Tolerance; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Female; Morphine; Morphine Dependence; Pain Measurement; Rats; Rats, Wistar; Receptors, Opioid, mu; Spinal Cord; Synaptic Membranes; Up-Regulation

Prolonged exposure to opioid agonists can induce adaptive changes resulting in tolerance and dependence. Here, rats were rendered tolerant by subcutaneous injections of increasing doses of morphine from 10 to 60 mg/kg for 3, 5, or 10 consecutive days. Binding parameters of the mu-opioid receptor in subcellular fractions were measured with [(3)H]DAMGO ([D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin). Although the density of surface mu-sites did not change after the 5-day morphine treatment, up-regulation of synaptic plasma membrane binding was detected after the 10-day drug administration. In contrast, the number of mu-binding sites in a light vesicle or microsomal fraction (MI) was elevated by 68 and 30% after 5 and 10 days of morphine exposure, respectively. The up-regulated MI mu-sites displayed enhanced coupling to G proteins compared with those detected in saline-treated controls. Pertussis toxin catalyzed ADP ribosylation, and Western blotting with specific antisera was used to quantitate chronic morphine-induced changes in levels of various G protein alpha-subunits. Morphine treatment of 5 days and longer induced significant increases in levels of Galpha(o), Galpha(i1), and Galpha(i2) in MI fractions that are part of an adaptation process. Up-regulation of intracellular mu-sites may be the result of post-translational changes and in part de novo synthesis. The results provide the first evidence that distinct regulation of intracellular mu-opioid receptor G protein coupling and G protein levels may accompany the development of morphine tolerance.

Topics: Analgesics, Opioid; Animals; Brain; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; GTP-Binding Proteins; Morphine; Morphine Dependence; Organ Specificity; Pain; Rats; Receptors, Opioid, mu; Subcellular Fractions

Repeated exposure to mu-opioid analgesics produces unwanted side effects, including tolerance and physical dependence. delta-Opioid antagonists attenuate development of morphine tolerance and physical dependence. We recently reported that SoRI 9409, a mixed mu-agonist/delta-antagonist, produces antinociception with limited development of tolerance after repeated i.c.v. injections. The current studies report on a more complete characterization of the compound in male ICR mice. SoRI 9409 produced limited antinociceptive effects in the 55 degrees C tail-flick test and full agonist effects in the acetic acid writhing assay after i.c.v. or i.p. administration. Repeated i.p. administration of A(90) doses of SoRI 9409 did not produce tolerance. The agonist effects of the compound were preferentially blocked by the mu-selective antagonist beta-funaltrexamine. The kappa-antagonist nor-binaltorphimine produced partial antagonism, whereas the delta-antagonist naltrindole had no effect on SoRI 9409 antinociception. Intraperitoneal administration of SoRI 9409 preferentially antagonized the antinociceptive actions of the delta-2 agonist [D-Ala(2),Glu(4)]deltorphin over the delta-1 agonist cyclic[D-Pen(2),D-Pen(5)]-enkephalin and the mu-agonist [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin. SoRI 9409 did not antagonize the antinociceptive effects of the kappa-agonist U69,593 (doses up to 60 mg/kg). SoRI 9409 (10 mg/kg i.p.) elicited much less vertical jumping than naloxone (10 mg/kg i.p.) in acute and chronic morphine dependence models. SoRI 9409 also suppressed withdrawal jumping when coadministered with naloxone. These studies indicate that SoRI 9409 acts primarily as a partial mu-agonist/delta-antagonist and supports the hypothesis that this type of compound may have a better therapeutic profile than currently available mu-agonists.

Topics: Analgesics, Opioid; Animals; Dose-Response Relationship, Drug; Drug Tolerance; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Injections, Intraventricular; Male; Mice; Mice, Inbred ICR; Morphine; Morphine Dependence; Morphine Derivatives; Naloxone; Narcotic Antagonists; Oligopeptides; Pain Measurement; Receptors, Opioid, delta; Receptors, Opioid, mu

Somatic symptoms and aversion of opiate withdrawal, regulated by noradrenergic signaling, were attenuated in mice with a CNS-wide conditional ablation of neurotrophin-3. This occurred in conjunction with altered cAMP-mediated excitation and reduced upregulation of tyrosine hydroxylase in A6 (locus coeruleus) without loss of neurons. Transgene-derived NT-3 expressed by noradrenergic neurons of conditional mutants restored opiate withdrawal symptoms. Endogenous NT-3 expression, strikingly absent in noradrenergic neurons of postnatal and adult brain, is present in afferent sources of the dorsal medulla and is upregulated after chronic morphine exposure in noradrenergic projection areas of the ventral forebrain. NT-3 expressed by non-catecholaminergic neurons may modulate opiate withdrawal and noradrenergic signalling.

Topics: Aging; Animals; Avoidance Learning; Brain; Colforsin; Cyclic AMP; Electric Stimulation; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Gene Expression Regulation, Enzymologic; In Vitro Techniques; Intermediate Filament Proteins; Locus Coeruleus; Mice; Mice, Knockout; Mice, Transgenic; Morphine; Morphine Dependence; Nerve Tissue Proteins; Nestin; Neurons; Neurotrophin 3; Signal Transduction; Substance Withdrawal Syndrome; Tyrosine 3-Monooxygenase

Withdrawal from repeated exposure to morphine causes a long-lasting increase in the reactivity of nucleus accumbens nerve terminals towards excitation. The resulting increase in action potential-induced exocytotic release of neurotransmitters, associated with behavioral sensitization, is thought to contribute to its addictive properties. We recently showed that activation of N-methyl-D-aspartate (NMDA) as well as dopamine (DA) D1 receptors in rat striatum causes tetrodotoxin-insensitive transporter-dependent GABA release. Since sustained changes in extracellular GABA levels may play a role in drug-induced neuronal hyperresponsiveness, we examined the acute and long-lasting effect of morphine on this nonvesicular GABA release in rat nucleus accumbens slices. The present study shows that morphine, through activation of mu-opioid receptors, reduces nonvesicular NMDA-induced [(3)H]GABA release in superfused nucleus accumbens slices. Moreover, prior repeated morphine treatment of rats (10 mg/kg, sc, 14 days) caused a reduction in NMDA-stimulated [(3)H]GABA release in vitro until at least 3 weeks after morphine withdrawal. This persistent neuroadaptive effect was not observed studying dopamine D1 receptor-mediated [(3)H]GABA release in nucleus accumbens slices. Moreover, this phenomenon appeared to be absent in slices of the caudate putamen. Interestingly, even a single exposure of rats to morphine (>2 mg/kg) caused a long-lasting inhibition of NMDA-induced release of GABA in nucleus accumbens slices. These data suggest that a reduction in nonvesicular GABA release within the nucleus accumbens, by enhancing the excitability of input and output neurons of this brain region, may contribute to the acute and persistently enhanced exocytotic release of neurotransmitters from nucleus accumbens neurons in morphine-exposed rats.

Topics: Analgesics; Animals; Benzeneacetamides; Carrier Proteins; Dopamine; Dose-Response Relationship, Drug; Drug Administration Schedule; Drug Interactions; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Excitatory Amino Acid Agonists; gamma-Aminobutyric Acid; Male; Morphine; Morphine Dependence; N-Methylaspartate; Naloxone; Neural Inhibition; Neurons; Nucleus Accumbens; Organ Culture Techniques; Pyrrolidines; Rats; Rats, Wistar; Receptors, Dopamine D1; Receptors, N-Methyl-D-Aspartate; Receptors, Opioid, mu; Substance Withdrawal Syndrome; Tritium

The present study examined the potential for cross-tolerance development between mu-opioid and gamma-aminobutyric acidB receptor agonists, in hypothalamic arcuate neurons, resulting from chronic morphine treatment. Intracellular recordings were made in hypothalamic slices prepared from ovariectomized female guinea pigs. The mu-opioid receptor agonist D-Ala2,N-Me-Phe4,Gly-ol5-enkephalin and the gamma-aminobutyric acidB receptor agonist baclofen produced dose-dependent membrane hyperpolarizations of arcuate neurons. The reversal potential for both agonist-induced hyperpolarizations was near -95 mV, indicative of the activation of an underlying K+ conductance. Coadministration of maximally effective concentrations of D-Ala2,N-Me-Phe4,Gly-ol5-enkephalin and baclofen produced a response that was not additive, indicating a convergence onto a common K+ channel. In arcuate neurons, including a subset that was immunopositive for tyrosine hydroxylase, chronic morphine treatment for 4 to 7 days produced a 3.2-fold reduction in the potency, with no change in the efficacy, of D-Ala2,N-Me-Phe4,Gly-ol5-enkephalin. In contrast, it affected neither the potency nor the efficacy of baclofen. Therefore, chronic morphine exposure does not produce cross-tolerance between mu-opioid and gamma-aminobutyric acidB receptor agonists in A12 dopamine neurons, suggesting that convergence upon a common effector is not a sufficient criterion for the development of cross-tolerance between receptor systems.

Topics: Animals; Arcuate Nucleus of Hypothalamus; Baclofen; Drug Tolerance; Electrophysiology; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Female; GABA-B Receptor Agonists; Guinea Pigs; Hypothalamus; In Vitro Techniques; Membrane Potentials; Morphine; Morphine Dependence; Neurons; Receptors, Opioid, mu; Tetrodotoxin; Tyrosine 3-Monooxygenase

The effect of repeated morphine administration on conditioned place preference (CPP) using a novel treatment schedule, i.e., drug treatment was always contingent with the conditioned environmental stimuli, was investigated. We also examined whether changes in the mu- and kappa-opioid receptor binding occurred in the brain of morphine-treated animals. Intraperitoneal (i.p.) administration of morphine (2 and 10 mg/kg) induced a place preference after 8 daily conditioning trials (4 morphine injections on alternate trials), the level of preference being the same with the two doses of the opiate. No change in place preference was observed in the morphine-treated rats at 2 mg/kg, when animals were further trained up to a total of 32 conditioning trials (16 morphine injections). Conversely, after 20 conditioning trials (10 morphine injections), a stronger CPP response developed in the morphine-treated rats at 10 mg/kg. Signs of morphine withdrawal were never detected in morphine-treated rats during the experiment. Loss of body weight (index of opiate dependence) was not observed either 24 h or 48 h after the last morphine administration. mu- and kappa-opioid receptor density and affinity were not affected by repeated morphine administrations at either dose. The results demonstrate that no tolerance develops to the rewarding properties of morphine. Indeed, a sensitisation effect may occur at increasing doses of the opiate. Furthermore, changes in the rewarding effect of morphine are not dependent upon alterations in opioid receptors involved in the reinforcing mechanisms.

Topics: Analysis of Variance; Animals; Behavior, Animal; Benzeneacetamides; Body Weight; Disease Models, Animal; Drug Tolerance; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Injections, Intraperitoneal; Male; Morphine; Morphine Dependence; Narcotics; Pyrrolidines; Rats; Rats, Sprague-Dawley; Receptors, Opioid, kappa; Receptors, Opioid, mu

1. Morphine produces a plethora of pharmacological effects and its chronic administration induces several side-effects. The cellular mechanisms by which opiates induce these side-effects are not fully understood. Several studies suggest that regulation of adenylyl cyclase activity by opioids and other transmitters plays an important role in the control of neural function. 2. The aim of this study was to evaluate desensitization of mu- and delta- opioid receptors, defined as a reduced ability of opioid agonists to inhibit adenylyl cyclase activity, in four different brain structures known to be involved in opiate drug actions: caudate putamen, nucleus accumbens, thalamus and periaqueductal gray (PAG). Opiate regulation of adenylyl cyclase in these regions has been studied in control and morphine-dependent rats. 3. The chronic morphine treatment used in the present study (subcutaneous administration of 15.4 mg morphine/rat/day for 6 days via osmotic pump) induced significant physical dependence as indicated by naloxone-precipitated withdrawal symptoms. 4. Basal adenylyl cyclase in the four brain regions was not modified by this chronic morphine treatment. In the PAG and the thalamus, a desensitization of mu- and delta-opioid receptors was observed, characterized by a reduced ability of Tyr-D-Ala-Gly-(NMe)Phe-Gly-ol (DAMGO; mu), Tyr-D-Pen-Gly-Phe-D-Pen (DPDPE; delta) and [D-Ala2]-deltorphin-II (DT-II; delta) to inhibit adenylyl cyclase, activity following chronic morphine treatment. 5. The opioid receptor desensitization in PAG and thalamus appeared to be heterologous since the metabotropic glutamate receptor agonists, L-AP4 and glutamate, and the 5-hydroxytryptamine (5-HT)1A receptor agonist, R(+)-8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide (8-OH-DPAT), also showed reduced inhibition of adenylyl cyclase activity following chronic morphine treatment. 6. In the nucleus accumbens and the caudate putamen, desensitization of delta-opioid receptor-mediated inhibition without modification of mu-opioid receptor-mediated inhibition was observed. An indirect mechanism probably involving dopaminergic systems is proposed to explain the desensitization of delta-mediated responses and the lack of mu-opioid receptor desensitization after chronic morphine treatment in caudate putamen and nucleus accumbens. 7. These results suggest that adaptive responses occurring during chronic morphine administration are not identical in all opiate-sensitive neural populations.

Topics: 8-Hydroxy-2-(di-n-propylamino)tetralin; Adenylyl Cyclases; Aminobutyrates; Analgesics; Animals; Brain; Caudate Nucleus; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Glutamic Acid; Male; Morphine; Morphine Dependence; Naloxone; Narcotic Antagonists; Nucleus Accumbens; Oligopeptides; Periaqueductal Gray; Rats; Rats, Sprague-Dawley; Receptors, Opioid, delta; Receptors, Opioid, mu; Serotonin Receptor Agonists; Substance Withdrawal Syndrome; Thalamus

Despite tremendous efforts in the search for safe, efficacious and non-addictive opioids for pain treatment, morphine remains the most valuable painkiller in contemporary medicine. Opioids exert their pharmacological actions through three opioid-receptor classes, mu, delta and kappa, whose genes have been cloned. Genetic approaches are now available to delineate the contribution of each receptor in opioid function in vivo. Here we disrupt the mu-opioid-receptor gene in mice by homologous recombination and find that there are no overt behavioural abnormalities or major compensatory changes within the opioid system in these animals. Investigation of the behavioural effects of morphine reveals that a lack of mu receptors abolishes the analgesic effect of morphine, as well as place-preference activity and physical dependence. We observed no behavioural responses related to delta- or kappa-receptor activation with morphine, although these receptors are present and bind opioid ligands. We conclude that the mu-opioid-receptor gene product is the molecular target of morphine in vivo and that it is a mandatory component of the opioid system for morphine action.

Topics: Analgesics; Animals; Behavior, Animal; Cell Line; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Gene Deletion; Mice; Mice, Inbred C57BL; Mice, Knockout; Morphine; Morphine Dependence; Narcotics; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Reward; Substance Withdrawal Syndrome

We examined the locomotor-enhancing action of mu-opioid receptor agonists, such as morphine and [D-Ala2, N-MePhe4, Gly-ol5]enkephalin (DAMGO), and physical dependence on morphine in diabetic and nondiabetic mice. Morphine (5-20 mg/kg, s.c.) and DAMGO (1-4 nmol, i.c.v.) had a dose-dependent locomotor-enhancing effect in both nondiabetic and diabetic mice. The locomotor-enhancing effects of morphine and DAMGO were significantly less in diabetic mice than in nondiabetic mice, and were significantly reduced after pretreatment with either beta-funaltrexamine (20 mg/kg, s.c.), a selective mu-opioid receptor antagonist, or naloxonazine (35 mg/kg, s.c.), a selective mu1-opioid receptor antagonist. Both diabetic and nondiabetic mice were chronically treated with morphine (8-45 mg/kg, s.c.) for 5 days. During this treatment, neither diabetic nor nondiabetic mice showed any signs of toxicity. After morphine treatment, withdrawal was precipitated by injection of naloxone (0.3-10 mg/kg, s.c.). Several withdrawal signs, such as weight loss, diarrhea, ptosis, jumping and body shakes, were observed after naloxone challenge in morphine-dependent nondiabetic mice. Although morphine-dependent diabetic mice showed greater weight loss than nondiabetic mice, the incidence of jumping and body shakes after naloxone challenge in diabetic mice were lower than that in nondiabetic mice. These results suggest that diabetic mice are selectively hyporesponsive to mu1-opioid receptor-mediated locomotor enhancement. Furthermore, diabetes may affect mu1-opioid receptor-mediated naloxone-precipitated signs of withdrawal from physical dependence on morphine.

Topics: Animals; Diabetes Mellitus, Experimental; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Male; Mice; Mice, Inbred ICR; Morphine; Morphine Dependence; Motor Activity; Naloxone; Naltrexone; Receptors, Opioid, mu; Streptozocin

The goal of this study was to compare the characteristics of mu- and delta-opioid receptors in the cortex of DBA/2 and C57BL/6 mice, which differ in sensitivity to the long- and short-term effects of morphine. The characteristics of mu-opiate receptors were not different in the cortex of both strains. Both high- and low-affinity binding sites of DSLET, a specific ligand of delta-opiate receptors, were present in the cortex of C57BL/6 mice, whereas the high-affinity binding sites were not found in the cortex of DBA/2 mice. The absence of high-affinity DSLET binding sites, which are similar to the delta 2 type of opioid receptors, may explain the less intensive naloxone-precipitated withdrawal reaction of DBA/2 as compared with C57BL/6 mice.

Topics: Amino Acid Sequence; Analgesics; Animals; Drug Implants; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalins; Male; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Molecular Sequence Data; Morphine; Morphine Dependence; Naloxone; Narcotics; Receptors, Opioid, delta; Receptors, Opioid, mu; Substance Withdrawal Syndrome

1. The effect of morphine tolerance and abstinence on the binding of [3H]naltrexone to discrete brain regions and spinal cord of the rat was determined. 2. Male Sprague-Dawley rats were implanted s.c. under light ether anesthesia with six morphine pellets for a 7-day period. Each pellet contained 75 mg of morphine base. Rats implanted with six placebo pellets each served as controls. 3. This procedure resulted in the development of tolerance to morphine as evidenced by decreased analgesic response to various doses of morphine. 4. The binding characteristics (Bmax or Kd values) of [3H]naltrexone, an opiate receptor antagonist, were determined in various tissues of morphine tolerant and abstinent rats. In the tolerant rats, the pellets were left in place at the time of sacrificing, whereas in the abstinent rats, the pellets were removed 18 hr prior to sacrificing. 5. The binding of [3H]naltrexone to opiate receptors on membranes prepared from brain regions (hypothalamus, hippocampus, cortex, pons and medulla, midbrain, corpus striatum and amygdala) and spinal cord of rats from various treatment groups was determined. 6. [3H]Naltrexone bound to tissue membranes at a single high affinity binding sites. The Bmax values of [3H]naltrexone to bind to opiate receptors on the membranes of amygdala and striatum were increased significantly in morphine tolerant rats when compared to the placebo controls, but the Kd values did not differ. 7. The Bmax and Kd values of [3H]naltrexone did not differ in any other brain region or spinal cord of morphine tolerant rats and their placebo controls. The binding constants of [3H]naltrexone were unaffected in morphine abstinent rats. 8. Previously we had shown that the binding of [3H]D-Ala2, MePhe4, Gly-ol5 enkephalin (DAMGO), a highly specific agonist for mu-opiate receptors was decreased in cortex, pons and medulla and spinal cord of morphine tolerant but not in the abstinent rats. In addition, delta and kappa receptors are unaffected in morphine tolerant and abstinent rats. 9. The results suggest that direction of change, as well as, the brain areas for mu-agonist and -antagonist opiate binding sites are affected differentially in morphine tolerant rats.

Topics: Analgesics; Animals; Brain; Drug Implants; Drug Tolerance; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Male; Membranes; Morphine; Morphine Dependence; Naltrexone; Rats; Rats, Sprague-Dawley; Receptors, Opioid; Receptors, Opioid, mu; Spinal Cord; Substance Withdrawal Syndrome

U-50,488, (trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]- benzeneacetamide hydrochloride), is a selective kappa-opioid receptor agonist. In this study, we found that U-50,488 antagonized morphine-induced antinociception in morphine-naive guinea pigs at doses which did not have any antinociceptive effect by themselves (0.01-3 mg/kg). On the other hand, U-50,488 (3 mg/kg) partially restored morphine-induced antinociception in morphine-tolerant guinea pigs (8 mg/kg/day i.p. morphine HCl for 6 days). Furthermore, the development of tolerance to morphine antinociception was completely blocked by coadministration of U-50,488 at a very low dose (0.003 mg/kg i.p.) which neither exerted an antinociceptive effect by itself nor affected the antinociception induced by 8 mg/kg of morphine HCl. The withdrawal signs induced by 8 mg/kg (i.p.) naloxone HCl on the 7th day were also depressed by coadministration of 0.003 mg/kg U-50,488 with morphine HCl (8 mg/kg i.p.) every day for 7 days. These effects of U-50,488 could be applied to humans to prevent morphine tolerance and dependence.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics; Animals; Dose-Response Relationship, Drug; Drug Synergism; Drug Tolerance; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Guinea Pigs; Male; Morphine; Morphine Dependence; Nociceptors; Pain; Pyrrolidines; Receptors, Opioid, kappa

Bilateral injection of naloxone (3.0-30.0 nmol) into the substantia nigra of morphine-dependent rats produced a withdrawal syndrome consisting of wet-dog shakes, teeth chattering, irritability to touch, diarrhea and hypothermia. Intense wet-dog shakes and grooming were observed after intranigral injection of the mu selective antagonist D-Phe-Cys-Try-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP, 3.0-30.0 nmol) in morphine-dependent animals. Body temperature after 30.0 nmol CTOP was significantly increased. A significant positive correlation between body temperature and wet-dog shakes was observed in morphine-dependent animals that received CTOP. Intranigral injection of beta-funaltrexamine (beta-FNA, 10.0 nmol), an irreversible mu antagonist, produced no signs of withdrawal in morphine-dependent animals. However, intranigral injection of beta-FNA (1.0-3.0 nmol) suppressed the antinociceptive effect of the mu-selective agonist, D-Ala2,N-Me-Phe4,Gly5-ol-enkephalin (DAGO, 1.0 nmol). The withdrawal syndrome produced by CTOP (10.0 nmol) was not suppressed by the administration of U50,488H (10.0 nmol), a kappa agonist, suggesting that the absence of an effect of beta-FNA was not due to its kappa agonist activity. Neither the delta-selective antagonist, naltrindole (NTI, 10.0 nmol) nor the kappa-selective antagonist, nor-binaltorphimine (nor-BNI, 10.0 nmol) produced withdrawal. Only wet-dog shakes were observed when CTOP, NTI and nor-BNI (5 nmol each) were administered together into the nigra. These studies suggest an involvement of mu receptors in the nigra in the wet-dog shakes and thermoregulatory dysfunction that occur during withdrawal of morphine. However, the subtypes of opioid receptors in the nigra, that mediate the other signs of morphine withdrawal remain obscure.

Topics: Amino Acid Sequence; Analgesics; Animals; Behavior, Animal; Body Temperature; Brain; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Male; Molecular Sequence Data; Morphine; Morphine Dependence; Naloxone; Rats; Rats, Sprague-Dawley; Receptors, Opioid; Somatostatin; Stereotaxic Techniques; Substance Withdrawal Syndrome; Substantia Nigra

Previously, we demonstrated the enhanced affinity of opioid receptors for naloxone in striatal slices from morphine-dependent mice. In our present study, binding characteristics of the mu opioid receptor agonists, [D-Ala2,MePhe4,Gly-ol5]enkephalin (DAMGO) and dihydromorphine, the delta opioid receptor agonist, [D-Ala2, D-Leu5]enkephalin (DADLE), and the opioid antagonist, naloxone, were examined in striatal slices from morphine-tolerant and -dependent mice. Striatal slices from mice that were implanted with a morphine pellet for 3,7 and 21 days displayed significant decreases in Kd values (5.1, 4.6 and 5.5 nM, respectively) of [3H]DAMGO when compared to those in slices from control animals that were not implanted or implanted with placebo pellets (9.6 and 9.3 nM, respectively). Also, a significant increase in the binding affinity of naloxone, but not that of dihydromorphine, was observed in striatal slices of mice that were implanted with a morphine pellet for 3 days. Significant increases in the Bmax of delta binding sites in striatal slices of mice that were implanted with a morphine pellet for 3, 7 and 21 days (20.7, 18.1 and 17.7 pmol/mg tissue, respectively) were observed when compared to that in slices from control mice that were implanted with placebo pellets (11.4 pmol/mg tissue). The enhancement in the binding affinity of DAMGO and naloxone and the increased density of DADLE binding sites paralleled the development of morphine tolerance and dependence and [D-Pen2,D-Pen5]enkephalin cross-tolerance in whole animals. An antinociceptive potentiation between morphine and DAMGO was observed in morphine-tolerant and -dependent mice whereas in naive animals the effects of the two drugs were additive.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Analgesics; Animals; Binding Sites; Corpus Striatum; Drug Interactions; Drug Tolerance; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine-2-Alanine; Enkephalins; Male; Mice; Morphine; Morphine Dependence; Naloxone; Pain; Receptors, Opioid

Topics: Animals; Brain Chemistry; Drug Tolerance; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Immunoglobulin G; Injections, Intraventricular; Ligands; Male; Morphine; Morphine Dependence; Oligopeptides; Rats; Rats, Inbred Strains; Receptors, Opioid; Up-Regulation

Topics: Animals; Brain Chemistry; Drug Tolerance; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalins; Ethylketocyclazocine; In Vitro Techniques; Male; Morphine; Morphine Dependence; Rats; Rats, Inbred Strains; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Spinal Cord; Substance Withdrawal Syndrome

Topics: Animals; Corpus Striatum; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Kinetics; Male; Mice; Morphine Dependence; Receptors, Opioid; Receptors, Opioid, mu

The cholecystokinin antagonist proglumide potentiates morphine analgesia. To understand more fully the opiate receptor subtypes involved with this effect, we investigated the effect of proglumide on spinal and supraspinal mu and spinal delta analgesia in mice. Proglumide alone had no effect on tailflick latencies, but increased, in a dose-dependent manner, tailflick latencies in morphine-tolerant mice. Proglumide also potentiated morphine analgesia in naive mice in a dose-dependent manner, with a maximal effect at 5-10 mg/kg. Proglumide both shifted the dose-response curve for morphine analgesia to the left and prolonged morphine's duration of action. Proglumide increased the sensitivity of supraspinal mu 1 receptor mechanisms of analgesia without influencing spinal mechanisms. Proglumide administered subcutaneously potentiated the analgesic actions of intracerebroventricular [D-Ala2,MePhe4,Gly(ol)5]enkephalin (DAGO; (mu 1), but not intrathecal DAGO (mu 2) or [D-Pen2,D-Pen5]enkephalin (DPDPE; delta). The selective mu 1 receptor antagonist naloxonazine blocked proglumide-enhanced morphine analgesia.

Topics: Animals; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Glutamine; Male; Mice; Morphine; Morphine Dependence; Narcotics; Proglumide; Reaction Time; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu

The effect of chronic administration of morphine and its withdrawal on the characteristics of mu-opiate receptors was determined in male Sprague-Dawley rats. The ligand used for characterizing the receptors was [3H][D-Ala2,MePhe4,Gly5-ol]enkephalin ([3H]DAMGO). Rats were implanted s.c. under light ether anesthesia with six morphine pellets (each containing 75 mg of morphine free base). Rats serving as controls were implanted similarly with placebo pellets. Two sets of animals were used. In one group of rats, the pellets were left intact (tolerant-dependent) at the time of killing and in the other the pellets had been removed 18 h earlier (abstinent). The spinal cord and brain regions (amygdala, hippocampus, hypothalamus, corpus striatum, midbrain, pons and medulla, and cortex) were dissected. In morphine-abstinent rats, the binding of ligands of mu-opiate receptors to membranes of spinal cord and brain regions did not change. In non-abstinent morphine-tolerant-dependent rats, the binding of [3H]DAMGO to membranes of spinal cord, pons and medulla, and cerebral cortex was found to be decreased. These changes were due to decreases in the Bmax values rather than Kd values for the binding of [3H]DAMGO. The results clearly indicate that morphine-induced tolerance-dependence in the rat is associated with changes in the selected brain regions and spinal cord with mu-opiate receptors being down-regulated in spinal cord, pons and medulla, and cerebral cortex. It is concluded that tolerance to morphine in the rats may be due to down-regulation of central mu-opiate receptors. However, mu-opiate receptors are unaffected in morphine abstinence.

Topics: Animals; Brain; Down-Regulation; Drug Tolerance; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Male; Membranes; Morphine; Morphine Dependence; Rats; Rats, Inbred Strains; Receptors, Opioid; Spinal Cord

The potassium stimulated release of [3H]norepinephrine ([3H]NE) from terminal fields of locus coeruleus projections can be inhibited in a dose-dependent manner by mu and kappa selective opioids. Chronic exposure to morphine for six days decreases the maximum achievable depression by the mu selective agonist Tyr-D-Ala2-Gly-Me(Phe)-Gly-ol (DAGO), but has no effect on the degree of inhibition produced by the kappa selective opioid U50,488H.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics; Animals; Cerebral Cortex; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Guinea Pigs; In Vitro Techniques; Male; Morphine; Morphine Dependence; Norepinephrine; Pyrrolidines; Receptors, Opioid

The i.c.v. administration of opioid peptides having selectivity for the mu receptor (D-Ala2-NMePhe4-Gly5(ol)enkephalin and FK 33,824) produced effects on the locomotor activity of nondependent and morphine-dependent rats that differed both quantitatively and qualitatively from those effects produced by peptides having selectivity for the delta receptor (D-Ala2-D-Leu5enkephalin and metkephamid) and beta-endorphin, which has similar affinity for both receptors. Peptides selective for the mu receptor: had a biphasic effect on locomotor activity of nondependent rats, inducing an increase at low doses and an initial decrease followed by a later increase at higher doses and had an enhanced stimulant effect on locomotor activity with tolerance to the depressant effect in morphine-dependent rats. Peptides selective for the delta receptor and beta-endorphin: induced only a dose-related increase in the locomotor activity of nondependent rats and had effects on the locomotor activity of morphine-dependent rats that did not differ substantially from those in nondependent rats. Naltrexone (0.1 mg/kg s.c.) and beta-funaltrexamine (5.0 micrograms/rat i.c.v.), an irreversible antagonist, each blocked to a comparable extent the effects of D-Ala2-NMePhe4-Gly5(ol)enkephalin and DAla2-D-Leu5enkephalin on the locomotor activity of nondependent rats. Thus, effects of opioid peptides that act predominantly at mu or delta receptors on locomotor activity cannot be differentiated in nondependent rats by antagonists but can be differentiated in morphine-dependent rats. These results suggest that the depressant and stimulant effects of opioid peptides on locomotor activity are mediated by distinct neuronal sites.

Topics: Animals; beta-Endorphin; D-Ala(2),MePhe(4),Met(0)-ol-enkephalin; Dose-Response Relationship, Drug; Endorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; Male; Morphine Dependence; Motor Activity; Naltrexone; Rats; Rats, Inbred Strains; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu