enkephalin--ala(2)-mephe(4)-gly(5)- and beta-funaltrexamine

G-protein-coupled receptors (GPCRs) are key signaling proteins that mostly function as monomers, but for several receptors constitutive dimer formation has been described and in some cases is essential for function. Using single-molecule microscopy combined with super-resolution techniques on intact cells, we describe here a dynamic monomer-dimer equilibrium of µ-opioid receptors (µORs), where dimer formation is driven by specific agonists. The agonist DAMGO, but not morphine, induces dimer formation in a process that correlates both temporally and in its agonist- and phosphorylation-dependence with β-arrestin2 binding to the receptors. This dimerization is independent from, but may precede, µOR internalization. These data suggest a new level of GPCR regulation that links dimer formation to specific agonists and their downstream signals.

Topics: Animals; beta-Arrestins; CHO Cells; Cricetulus; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Fluorescence Resonance Energy Transfer; Morphine; Mutation; Naloxone; Naltrexone; Narcotic Antagonists; Phosphorylation; Protein Multimerization; Receptors, Opioid, mu; Single Molecule Imaging

Neuropeptide FF (NPFF) is known to be an endogenous opioid-modulating peptide. Nevertheless, very few researches focused on the interaction between NPFF and endogenous opioid peptides. In the present study, we have investigated the effects of NPFF system on the supraspinal antinociceptive effects induced by the endogenous µ-opioid receptor agonists, endomorphin-1 (EM-1) and endomorphin-2 (EM-2). In the mouse tail-flick assay, intracerebroventricular injection of EM-1 induced antinociception via µ-opioid receptor while the antinociception of intracerebroventricular injected EM-2 was mediated by both µ- and κ-opioid receptors. In addition, central administration of NPFF significantly reduced EM-1-induced central antinociception, but enhanced EM-2-induced central antinociception. The results using the selective NPFF1 and NPFF2 receptor agonists indicated that the EM-1-modulating action of NPFF was mainly mediated by NPFF2 receptor, while NPFF potentiated EM-2-induecd antinociception via both NPFF1 and NPFF2 receptors. To further investigate the roles of µ- and κ-opioid systems in the opposite effects of NPFF on central antinociception of endomprphins, the µ- and κ-opioid receptors selective agonists DAMGO and U69593, respectively, were used. Our results showed that NPFF could reduce the central antinociception of DAMGO via NPFF2 receptor and enhance the central antinociception of U69593 via both NPFF1 and NPFF2 receptors. Taken together, our data demonstrate that NPFF exerts opposite effects on central antinociception of endomorphins and provide the first evidence that NPFF potentiate antinociception of EM-2, which might result from the interaction between NPFF and κ-opioid systems.

Topics: Adamantane; Animals; Benzeneacetamides; Dipeptides; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Injections, Intraventricular; Male; Mice; Naltrexone; Nociception; Oligopeptides; Pyrrolidines

Neuropathic pain alters opioid self-administration in rats. The brain regions altered in the presence of neuropathic pain mediating these differences have not been identified, but likely involve ascending pain pathways interacting with the limbic system. The amygdala is a brain region that integrates noxious stimulation with limbic activity.. μ-Opioid receptors were blocked in the amygdala using the irreversible antagonist, β-funaltrexamine, and the antiallodynic and reinforcing effects of heroin were determined in spinal nerve-ligated rats. In addition, the effect of β-funaltrexamine was determined on heroin self-administration in sham-operated rats.. β-Funaltrexamine decreased functional activity of μ-opioid receptors by 60 ± 5% (mean ± SD). Irreversible inhibition of μ-opioid receptors in the amygdala significantly attenuated the ability of doses of heroin up to 100 μg/kg to reverse hypersensitivity after spinal nerve ligation. Heroin intake by self-administration in spinal nerve-ligated rats was increased from 5.0 ± 0.3 to 9.9 ± 2.1 infusions/h after administration of 2.5 nmol of β-funaltrexamine in the lateral amygdala, while having no effect in sham-operated animals (5.8 ± 1.6 before, 6.7 ± 0.9 after). The antiallodynic effects of 60 μg/kg heroin were decreased up to 4 days, but self-administration was affected for up to 14 days.. μ-Opioid receptors in the lateral amygdala partially meditate heroin's antiallodynic effects and self-administration after peripheral nerve injury. The lack of effect of β-funaltrexamine on heroin self-administration in sham-operated subjects suggests that opioids maintain self-administration through a distinct mechanism in the presence of pain.

Topics: Amygdala; Analgesics, Opioid; Animals; Behavior, Animal; Brain; Conditioning, Operant; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Guanosine 5'-O-(3-Thiotriphosphate); Heroin; Hyperalgesia; Infusions, Intravenous; Male; Naltrexone; Narcotic Antagonists; Peripheral Nerve Injuries; Rats; Rats, Inbred F344; Reinforcement, Psychology; Self Administration; Spinal Nerves

It has been demonstrated that the antinociception induced by i.t. or i.c.v. administration of endomorphins is mediated through mu-opioid receptors. Moreover, though endomorphins do not have appreciable affinity for kappa-opioid receptors, pretreatment with the kappa-opioid receptor antagonist nor-binaltorphimine markedly blocks the antinociception induced by i.c.v.- or i.t.-injected endomorphin-2, but not endomorphin-1. These evidences propose the hypothesis that endomorphin-2 may initially stimulate the mu-opioid receptors, which subsequently induces the release of dynorphins acting on kappa-opioid receptors to produce antinociception. The present study was performed to determine whether the release of dynorphins by i.c.v.-administered endomorphin-2 is mediated through mu-opioid receptors for producing antinociception. Intracerebroventricular pretreatment with an antiserum against dynorphin A, but not dynorphin B or alpha-neo-endorphin, and s.c. pretreatment with kappa-opioid receptor antagonist nor-binaltorphimine dose-dependently attenuated the antinociception induced by i.c.v.-administered endomorphin-2, but not endomorphin-1 and DAMGO. The attenuation of endomorphin-2-induced antinociception by pretreatment with antiserum against dynorphin A or nor-binaltorphimine was dose-dependently eliminated by additional s.c. pretreatment with a selective mu-opioid receptor antagonist beta-funaltrexamine or a selective mu1-opioid receptor antagonist naloxonazine at ultra low doses, which are inactive against micro-opioid receptor agonists in antinociception, suggesting that endomorphin-2 stimulates distinct subclass of micro1-opioid receptor that induces the release of dynorphin A acting on kappa-opioid receptors in the brain. It concludes that the antinociception induced by supraspinally administered endomorphin-2 is in part mediated through the release of endogenous kappa-opioid peptide dynorphin A, which is caused by the stimulation of distinct subclass of micro1-opioid receptor.

Topics: Analgesics; Animals; Dynorphins; Endorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Immune Sera; Injections, Intraventricular; Male; Mice; Naloxone; Naltrexone; Oligopeptides; Protein Precursors; Receptors, Opioid, kappa; Receptors, Opioid, mu

Acute pharmacological studies have implicated mu-opioid receptors (MORs) in the shell of the nucleus accumbens (NAC) in mediating responses for palatable food and other natural and drug-induced rewards. However, the long-term behavioral effects of inactivating signal transduction via accumbal MORs, as quantified by an anatomically defined loss of cellular activity, have never been analysed. We combined microinfusion of the irreversible MOR antagonist, beta-funaltrexamine (beta-FNA; 8.0 nmol/0.8 microL, n=9; controls, n=6) with mapping by [35S]GTPgammaS autoradiography to demonstrate an anatomically specific loss of the coupling of MORs to their G-proteins in the dorsal caudomedial shell of the NAC in rabbits. beta-FNA did not alter the stimulated coupling of kappa-opioid receptors. This selective blockade of the cellular function of MORs persistently decreased consumption of a palatable sucrose solution by 40% during a daily 4-h test conducted 2, 3 and 4 days after infusion. beta-FNA did not alter body weight or 20-h consumption of standard chow or water. In 10 different rabbits, infusion of the selective, competitive MOR antagonist, CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2) into the same locus produced a reversible decrease in sucrose consumption, with normal intakes returning on the next day. Together, these data appear to establish that MORs in this accumbal subregion support responding for orosensory reward. Overall, these results visualize a discrete brain locus where cellular actions of endogenous opioids mediate behaviors involved in self-administration of foods and perhaps other hedonically valued substances, such as ethanol and drugs of abuse.

Topics: Analgesics, Opioid; Animals; Autoradiography; Eating; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Food Preferences; Guanosine 5'-O-(3-Thiotriphosphate); Male; Microinjections; Naltrexone; Nucleus Accumbens; Peptide Fragments; Peptides; Rabbits; Receptors, G-Protein-Coupled; Receptors, Opioid, mu; Somatostatin; Sucrose

Acute pharmacological studies implicate mu-opioid receptors (MORs) in the parabrachial nucleus (PBN) of the brainstem in modulating eating. The long-term effects of preventing the cellular function of parabrachial MORs on food consumption remain to be elucidated.. To determine whether (1) chronic inhibition of MOR-mediated G-protein coupling in the PBN of rats would persistently reduce eating and (2) food properties dictate the effects of MOR blockade.. We microinfused the irreversible MOR antagonist, beta-funaltrexamine (beta-FNA) into the lateral PBN and measured the intake of standard and calorically dense palatable chow for 1 week. First, rats were given standard chow for 20 h daily and a calorically dense palatable chow for 4 h during the day. We infused the agonist, [D: -Ala(2), N-Me-Phe(4), Glycinol(5)]-Enkephalin (DAMGO), 1 week after beta-FNA to probe the acute effects of exogenous stimulation of MORs on palatable food intake. [(35)S]GTPgammaS autoradiography quantified regional loss of MOR cellular function. Next, we measured the actions of beta-FNA on food intake in rats given only standard or palatable chow for 1 week.. One infusion of beta-FNA persistently decreased consumption of standard but not palatable chow, regardless of feeding regimen. beta-FNA also blocked DAMGO-stimulated palatable chow intake, prevented DAMGO-stimulated G-protein coupling in the central and external lateral subnuclei of the PBN, and decreased coupling in the medial PBN. beta-FNA did not affect kappa-opioid receptors.. MORs in the lateral PBN serve a physiological role in stimulating consumption of standard food. Properties of the diet, such as high palatability or caloric density, may override the influence of inhibiting MOR function.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics, Non-Narcotic; Analgesics, Opioid; Animals; Behavior, Animal; Circadian Rhythm; Eating; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Feeding Behavior; Food Preferences; GTP-Binding Proteins; Male; Naltrexone; Narcotic Antagonists; Pons; Rats; Rats, Sprague-Dawley; Receptors, G-Protein-Coupled; Receptors, Opioid, mu; Time Factors

The antagonistic properties of Tyr-d-Pro-Trp-Gly-NH(2) (d-Pro(2)-Tyr-W-MIF-1), a Tyr-Pro-Trp-Gly-NH(2)(Tyr-W-MIF-1) analog, on the antinociception induced by the mu-opioid receptor agonists Tyr-W-MIF-1, [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO), Tyr-Pro-Trp-Phe-NH(2) (endomorphin-1), and Tyr-Pro-Phe-Phe-NH(2) (endomorphin-2) were studied in the mouse paw-withdrawal test. d-Pro(2)-Tyr-W-MIF-1 injected intrathecally (i.t.) had no apparent effect on the thermal nociceptive threshold. d-Pro(2)-Tyr-W-MIF-1 (0.1-0.4 nmol) coadministered i.t. showed a dose-dependent attenuation of the antinociception induced by Tyr-W-MIF-1 without affecting endomorphin- or DAMGO-induced antinociception. However, higher doses of d-Pro(2)-Tyr-W-MIF-1 (0.8-1.2 nmol) significantly attenuated endomorphin-1- or DAMGO-induced antinociception, whereas the antinociception induced by endomorphin-2 was still not affected by d-Pro(2)-Tyr-W-MIF-1. Pretreatment i.t. with various doses of naloxonazine, a mu(1)-opioid receptor antagonist, attenuated the antinociception induced by Tyr-W-MIF-1, endomorphin-1, endomorphin-2, or DAMGO. Judging from the ID(50) values for naloxonazine against the antinociception induced by the mu-opioid receptor agonists, the antinociceptive effect of Tyr-W-MIF-1 is extremely less sensitive to naloxonazine than those of endomorphin-1 or DAMGO. In contrast, endomorphin-2-induced antinociception is extremely sensitive to naloxonazine. The present results clearly suggest that d-Pro(2)-Tyr-W-MIF-1 is the selective antagonist to be identified for the mu(2)-opioid receptor in the mouse spinal cord. d-Pro(2)-Tyr-W-MIF-1 may also discriminate between Tyr-W-MIF-1-induced antinociception and the antinociception induced by endomorphin-1 or DAMGO, all of which show a preference for the mu(2)-opioid receptor in the spinal cord.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics, Opioid; Animals; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Male; Mice; MSH Release-Inhibiting Hormone; Naloxone; Naltrexone; Narcotic Antagonists; Oligopeptides; Receptors, Opioid, mu

Striatal cholinergic interneurons play a crucial role in the control of movement as well as in motivational and learning aspects of behaviour. Neuropeptides regulate striatal cholinergic transmission and particularly activation of mu opioid receptor (MOR) inhibits acetylcholine (ACh) release in the dorsal striatum. In the present study we investigated whether this cholinergic transmission could be modulated by an enkephalin/MOR direct process. We show that mRNA and protein of MORs are expressed by cholinergic interneurons in the limbic/prefrontal territory but not by those in the sensorimotor territory of the dorsal striatum. These MORs are functional because potassium-evoked release of ACh from striatal synaptosomes was dose-dependently reduced by a selective MOR agonist, this effect being suppressed by a MOR antagonist. The MOR regulation of cholinergic interneurons presented a diurnal variation. (i) The percentage of cholinergic interneurons containing MORs that was 32% at the beginning of the light period (morning) increased to 80% in the afternoon. (ii) The MOR-mediated inhibition of synaptosomal ACh release was higher in the afternoon than in the morning. (iii) While preproenkephalin mRNA levels remained stable, enkephalin tissue content was the lowest (-32%) in the afternoon when the spontaneous (+35%) and the N-methyl-d-aspartate-evoked (+140%) releases of enkephalin (from microsuperfused slices) were the highest. Therefore, by acting on MORs present on cholinergic interneurons, endogenously released enkephalin reduces ACh release. This direct enkephalin/MOR regulation of cholinergic transmission that operates only in the limbic/prefrontal territory of the dorsal striatum might contribute to information processing in fronto-cortico-basal ganglia circuits.

Topics: Acetylcholine; Analgesics, Opioid; Animals; Blotting, Northern; Choline O-Acetyltransferase; Circadian Rhythm; Corpus Striatum; Drug Interactions; Electric Stimulation; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Excitatory Amino Acid Agonists; Immunohistochemistry; In Vitro Techniques; Interneurons; Limbic System; Membrane Potentials; N-Methylaspartate; Naltrexone; Narcotic Antagonists; Patch-Clamp Techniques; Potassium; Protein Precursors; Radioimmunoassay; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Synaptosomes; Tritium

Antagonistic properties of buprenorphine for epsilon- and micro -opioid receptors were characterized in beta-endorphin- and [d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO)-induced antinociception, respectively, with the tail-flick test in male ICR mice. epsilon-Opioid receptor agonist beta-endorphin (0.1-1 micro g), micro -opioid receptor agonist DAMGO (0.5-20 ng), or buprenorphine (0.1-20 micro g) administered i.c.v. dose dependently produced antinociception. The antinociception induced by 10 micro g of buprenorphine given i.c.v. was completely blocked by the pretreatment with beta-funaltrexamine (beta-FNA) (0.3 micro g i.c.v.), indicating that the buprenophine-induced antinociception is mediated by the stimulation of the micro -opioid receptor. The antinociceptive effects induced by beta-endorphin (1 micro g i.c.v.) and DAMGO (16 ng i.c.v.) were dose dependently blocked by pretreatment with smaller doses of buprenorphine (0.001-1 micro g i.c.v.), but not by a higher dose of buprenorphine (10 micro g i.c.v.). beta-FNA at a dose (0.3 micro g i.c.v.) that strongly attenuated DAMGO-induced antinociception had no effect on the antinociception produced by beta-endorphin (1 micro g i.c.v.). However, pretreatment with buprenorphine (0.1-10 micro g) in mice pretreated with this same dose of beta-FNA was effective in blocking beta-endorphin-induced antinociception. beta-FNA was 226-fold more effective at antagonizing the antinociception induced by DAMGO (16 ng i.c.v.) than by beta-endorphin (1 micro g i.c.v.). The antinociception induced by delta-opioid receptor agonist [d-Ala2]deltorphin II (10 micro g i.c.v.) or kappa1-opioid receptor agonist trans-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl]cyclohexyl)benzeneacetamine methanesulfonate salt [(-)-U50,488H] (75 micro g i.c.v.) was not affected by pretreatment with buprenorphine (0.1-1.0 micro g i.c.v.). It is concluded that buprenorphine, at small doses, blocks epsilon-opioid receptor-mediated beta-endorphin-induced antinociception and micro -opioid receptor-mediated DAMGO-induced antinociception, and at high doses produces a micro -opioid receptor-mediated antinociception.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics; Analgesics, Non-Narcotic; Analgesics, Opioid; Animals; beta-Endorphin; Buprenorphine; Disease Models, Animal; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Injections, Intraventricular; Male; Mice; Mice, Inbred ICR; Naltrexone; Narcotic Antagonists; Oligopeptides; Pain; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Time Factors

1. Whole-cell patch recordings were made from substantia gelatinosa (SG) neurons in transverse lumbar spinal cord slices of 15- to 30-day-old rats. 2. Endomorphin 1 (EM-1) or EM-2 (

Topics: Animals; Bicuculline; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Excitatory Postsynaptic Potentials; Female; Male; Membrane Potentials; Naloxone; Naltrexone; Narcotic Antagonists; Neurons; Oligopeptides; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu; Spinal Cord; Substantia Gelatinosa; Tetrodotoxin; Time Factors

Paradoxically, the potencies (EC(50)) of agonists stimulating [35S]GTPgammaS binding are several orders of magnitude lower than their affinities in receptor binding assays. We have investigated the quantitative stoichiometry of mu-opioid receptor-G-protein coupling in postmortem human brain. [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO) displaced [3H]naloxone binding in a biphasic pattern. The ratio between K(i-low) and EC(50) of DAMGO stimulating [35S]GTPgammaS binding was lower than one. The K(A) of DAMGO was calculated following mu-opioid receptor alkylation by beta-funaltrexamine from [35S]GTPgammaS binding data using the "nested hyperbolic method", yielding K(A)/EC(50)>1. Thus, only 1.2 +/- 0.2% of mu-opioid receptors was needed to be occupied to achieve the half-maximal effect of DAMGO. The estimated ratio between the G-proteins activated by 10 microM DAMGO (determined by isotopic dilution curves) and the occupied-mu-opioid receptors was 1304. In conclusion, we have determined the stoichiometric and the kinetic parameters in the mu-opioid receptor-G-protein system.

Topics: Alkylation; Analgesics, Opioid; Baclofen; Binding, Competitive; Brain Chemistry; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enzyme Inhibitors; Ethylmaleimide; GABA Agonists; GTP-Binding Protein Regulators; GTP-Binding Proteins; Humans; In Vitro Techniques; Kinetics; Membranes; Naloxone; Naltrexone; Narcotic Antagonists; Receptors, Opioid, mu; Signal Transduction

Opioid receptor subtypes may have site-specific effects and play different roles in modulating serotonergic neurotransmission in the mammalian central nervous system. To test this hypothesis, we used in vivo microdialysis to measure changes in extracellular serotonin (5-hydroxytryptamine; 5-HT) in response to local infusion of mu-, delta-, and kappa-opioid receptor ligands into the dorsal raphe nucleus (DRN), median raphe nucleus (MRN), and nucleus accumbens (NAcc) of freely behaving rats. The mu-opioids [D-Ala(2)-N-Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO), endomorphin-1, and endomorphin-2 were administered by reverse dialysis infusion into the DRN. In response, extracellular 5-HT was increased in the DRN, an effect that was blocked by the selective mu-receptor antagonist beta-funaltrexamine, but not by the delta-receptor antagonist N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH (ICI-174,864). Infusion of delta-receptor agonists, [D-Ala(2),D-Len(5)]enkephalin (DADLE), [D-Pen(2,5)]enkephalin (DPDPE), and deltophin-II into the DRN also increased extracellular 5-HT, an effect that was blocked by selective delta-receptor antagonists. In contrast to the DRN, local infusion of mu- and delta-opioids had no effect on 5-HT in the MRN or NAcc. These data indicate that mu- and delta-opioid ligands have a selective influence on serotonergic neurons in the DRN. Finally, the kappa-receptor agonist U-50,488 [trans-(+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide] produced similar decreases in 5-HT during local infusion into the DRN, MRN, and NAcc. These results provide evidence of differential regulation of 5-HT release by opioid receptor subtypes in the midbrain raphe and forebrain.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics, Opioid; Animals; Central Nervous System; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine-2-Alanine; Male; Microdialysis; Microinjections; Naltrexone; Nucleus Accumbens; Raphe Nuclei; Rats; Rats, Sprague-Dawley; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Serotonin

The present study examined opioid receptor(s) mediation of feeding elicited by mu opioid agonists in the ventral tegmental area using general or selective opioid antagonist pretreatment. Naltrexone as well as equimolar doses of selective mu and kappa, but not delta opioid antagonists in the ventral tegmental area significantly reduced mu agonist-induced feeding, indicating a pivotal role for these receptor subtypes in the full expression of this response.

Topics: Animals; Appetite Regulation; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Feeding Behavior; Male; Naltrexone; Narcotic Antagonists; Narcotics; Neural Pathways; Neurons; Nucleus Accumbens; Rats; Rats, Sprague-Dawley; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Synaptic Transmission; Ventral Tegmental Area

The guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding assay for the determination of relative opioid efficacy has been adapted to measure G protein activation in digitonin-permeabilized C6 rat glioma cells expressing a cloned mu-opioid receptor. The mu-agonist [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) caused a 3-fold increase in [35S]GTPgammaS binding over basal in a naloxone-sensitive manner. Relative mu-agonist efficacy was DAMGO > fentanyl > or = morphine > buprenorphine. Nalbuphine showed no efficacy. G protein activation by receptors has been predicted to occur by random encounter. In this model a reduction in the number of receptors will decrease the rate of G protein activation but not the maximum number of G proteins activated. To test this model C6 mu cells were treated with the irreversible mu-antagonist beta-funaltrexamine (10 nM) prior to permeabilization. This reduced the number of mu-opioid receptors determined with [3H]diprenorphine to 23 +/- 3% of control with no change in affinity. A commensurate reduction (to 29 +/- 10% of control) in the level of [35S]GTPgammaS binding stimulated by DAMGO was observed, but the t(1/2) for [35S]GTPgammaS binding remained unchanged. Thus, random encounters of receptor and G protein failed to occur in this permeabilized cell preparation. A model that assumes an organized association of G proteins with receptors better describes the activation of G proteins by opioid mu-receptors.

Topics: Analgesics, Opioid; Animals; Digitonin; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Glioma; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Indicators and Reagents; Naloxone; Naltrexone; Narcotic Antagonists; Rats; Receptors, Opioid, mu; Tumor Cells, Cultured

Food intake is significantly increased by administration of mu-selective opioid agonists into the nucleus accumbens, particularly its shell region. Pretreatment with either opioid (mu, delta(1), delta(2) or kappa(1)) or dopaminergic (D(1)) receptor antagonists in the nucleus accumbens shell reduce mu opioid agonist-induced feeding. Selective GABA(A) (muscimol) and GABA(B) (baclofen) agonists administered into the nucleus accumbens shell each stimulate feeding which is respectively and selectively blocked by GABA(A) (bicuculline) and GABA(B) (saclofen) antagonists. The present study investigated whether feeding elicited by the mu-selective opioid agonist, [D-Ala(2),NMe(4),Gly-ol(5)]-enkephalin in the nucleus accumbens shell was decreased by intra-accumbens pretreatment with an equimolar dose range of either GABA(A) or GABA(B) antagonists, and further, whether general opioid or selective GABA antagonists decreased feeding elicited by GABA(A) or GABA(B) agonists in the nucleus accumbens shell. Feeding elicited by the mu-selective opioid agonist was dose-dependently increased following intra-accumbens pretreatment with GABA(A) (bicuculline) antagonism; this enhancement was significantly blocked by pretreatment with general or mu-selective opioid antagonists. In contrast, mu opioid agonist-induced feeding elicited from the nucleus accumbens shell was dose-dependently decreased by GABA(B) (saclofen) antagonism. Neither bicuculline nor saclofen in the nucleus accumbens shell altered baseline food intake. Whereas muscimol-induced feeding elicited from the nucleus accumbens shell was reduced by bicuculline and naltrexone, but not saclofen pretreatment, baclofen-induced feeding elicited from the nucleus accumbens shell was reduced by saclofen, but not by bicuculline or naltrexone. These data indicate that GABA(A) and GABA(B) receptor subtype antagonists differentially affect feeding elicited by mu opioid receptor agonists within the nucleus accumbens shell in rats.

Topics: Animals; Baclofen; Bicuculline; Eating; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; GABA Agonists; GABA Antagonists; GABA-A Receptor Antagonists; GABA-B Receptor Antagonists; gamma-Aminobutyric Acid; Male; Naltrexone; Narcotic Antagonists; Narcotics; Neurons; Nucleus Accumbens; Rats; Rats, Sprague-Dawley; Receptors, GABA; Receptors, GABA-A; Receptors, GABA-B; Receptors, Opioid

The most convincing evidence demonstrating constitutive activation of mu-opioid receptors is the observation that putative inverse agonists decrease basal G-protein activity in membrane preparations. However, it is not clear whether constitutively active receptors in isolated membranes have any physiological relevance in intact cells. GH3 cells expressing mu-opioid receptors (GH3MOR) exhibit higher basal G-protein activity and lower basal cAMP levels than wild-type GH3 cells, indicative of constitutively active receptors. This study determined whether alkylation of mu-opioid receptors by the irreversible antagonist beta-funaltrexamine would decrease spontaneous receptor activity in intact cells, revealing constitutive activity. GH3MOR cells were pretreated with increasing concentrations of beta-funaltrexamine followed by functional testing after removal of unbound drug. beta-Funaltrexamine pretreatment produced a concentration-dependent decrease in mu-opioid receptor binding with an IC50 of 0.98 nm and an Emax of 77%. Similar concentrations of beta-funaltrexamine pretreatment produced a half-maximal reduction in basal [35S]GTPgammaS binding, a decrease in basal photolabeling of G-proteins with azidoanilido-[alpha-32P]GTP, and an increase in basal adenylyl cyclase activity in intact cells. Therefore, mu-opioid receptors are constitutively active in intact cells, producing stimulation of G-proteins and inhibition of adenylyl cyclase. Importantly, photolabeling of Galpha-subunits with azidoanilido-[alpha-32P]GTP demonstrated that constitutively active mu-opioid receptors activate individual G-proteins differently than the agonist [d-Ala2,N-MePhe4,Gly-ol5]enkephalin.

Topics: Adenylate Cyclase Toxin; Adenylyl Cyclase Inhibitors; Cell Line; D-Ala(2),MePhe(4),Met(0)-ol-enkephalin; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; GTP-Binding Proteins; Naltrexone; Potassium Chloride; Protein Binding; Receptors, Opioid, mu; Virulence Factors, Bordetella

The inhibitory effect of intracerebroventricularly-administered [D-Arg(2), beta-Ala(4)]-dermorphin (1-4) (TAPA), a highly selective mu(1)-opioid receptor agonist, on mouse gastrointestinal transit was compared with that of morphine and [D-Ala(2), N-methyl-Phe(4), Gly(5)-ol]-enkephalin (DAMGO). When administered intracerebroventricularly 5 min before the oral injection of charcoal meal, TAPA (10-100 pmol), morphine (0.25-4 nmol), and DAMGO (20-80 pmol) dose-dependently inhibited gastrointestinal transit of charcoal. The inhibitory effect of each mu-opioid receptor agonist was completely antagonized by naloxone, a nonselective opioid receptor antagonist. The inhibitory effects of morphine and DAMGO were significantly antagonized by both beta-funaltrexamine, a selective mu-opioid receptor antagonist, and naloxonazine, a selective mu(1)-opioid receptor antagonist. In contrast, the inhibitory effect of TAPA was not affected at all by beta-funaltrexamine, naloxonazine, nor-binaltorphimine (a selective kappa-opioid receptor antagonist), or naltrindole (a selective delta-opioid receptor antagonist). These results suggest that the inhibitory effect of TAPA on gastrointestinal transit may be mediated through an opioid receptor mechanism different from that of morphine and DAMGO.

Topics: Analgesics; Animals; Charcoal; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Female; Gastrointestinal Transit; Injections, Intraventricular; Mice; Mice, Inbred Strains; Morphine; Naloxone; Naltrexone; Narcotic Antagonists; Oligopeptides

To examine the role of mu-opioid receptor subtypes, we assessed the antinociceptive effect of H-Tyr-D-Arg-Phe-beta-Ala-OH (TAPA), an analogue of dermorphin N-terminal peptide in mice, using the tail-flick test. Intracerebroventricularly (i.c.v.) or intrathecally (i.t.) injected TAPA produced potent antinociception with tail-flick as a thermal noxious stimulus. The selective mu(1)-opioid receptor antagonist, naloxonazine (35 mg/kg, s.c.), or the selective mu-opioid receptor antagonist, beta-funaltrexamine, 24 h before testing antagonized the antinociceptive effect of i.t. or i.c.v. TAPA on the response to noxious stimuli. Pretreatment with beta-funaltrexamine completely antagonized the antinociception by both i.c.v. and i.t. administered TAPA and [D-Ala(2), Me-Phe(4), Gly(ol)(5)]enkephalin (DAMGO). Especially in the tail-flick test, pretreatment with naloxonazine produced a marked rightward displacement of the i.t. TAPA dose-response curve for antinociception. Though DAMGO is a highly selective mu-opioid receptor agonist, pretreatment with naloxonazine partially blocked the antinociceptive response to DAMGO after i.c.v., but not after i. t. injection. These results indicate that TAPA can act as a highly selective mu(1)-opioid receptor agonist (notable naloxonazine-sensitive receptor agonist) at not only the supraspinal level, but also the spinal level. These data also reveal different antinociceptive mechanisms for DAMGO and for TAPA.

Topics: Analgesics; Analgesics, Opioid; Animals; Drug Antagonism; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Injections, Intraventricular; Male; Mice; Naloxone; Naltrexone; Narcotic Antagonists; Oligopeptides; Opioid Peptides; Pain Measurement; Receptors, Opioid, mu; Time Factors

The potential interactions of natively expressed mu-opioid and opioid receptor-like (ORL1) receptors were studied by exposing intact BE(2)-C cells to agonists or antagonists for 1 h. Pretreatment with the mu-opioid receptor agonist, [D-Ala(2), N-Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO), or the ORL1 receptor agonist, orphanin FQ/nociceptin desensitized both mu-opioid and ORL1 receptor responses. beta-Funaltrexamine (beta-FNA) pretreatment also blocked both mu-opioid and ORL1 receptor responses, but only mu-opioid receptor binding was reduced. Moreover, beta-FNA (1 microM) failed to inhibit specific ORL1 receptor binding.

Topics: Brain Neoplasms; Cyclic AMP; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Humans; Morphine; Naltrexone; Narcotic Antagonists; Narcotics; Neuroblastoma; Nociceptin; Nociceptin Receptor; Opioid Peptides; Receptors, Opioid; Receptors, Opioid, mu; Tumor Cells, Cultured

The beta-chemokine RANTES has recently been implicated in the neuropathogenesis of the human immunodeficiency virus. Based upon previous studies of the effects of morphine on microglial cell production of cytokines and chemotaxis towards the activated complement component C5a, we tested the hypothesis that this opiate would alter the production of and migration towards RANTES by human microglia. Treatment of highly purified microglial cell cultures with morphine (10(-8)-10(-6) M) potently inhibited RANTES production by lipopolysaccharide- and interleukin-1beta-stimulated cells. Using a chemotaxis chamber to assess directed migration towards RANTES, treatment of microglial cells with morphine (10(-10)-10(-6) M) was found to suppress chemotaxis. The inhibitory effects of morphine on RANTES production and on chemotaxis were blocked by naloxone and beta-funaltrexamine, indicating that morphine mediated its suppressive effects via activation of microglial p-opioid receptors. Morphine's inhibitory effect on chemotaxis did not appear to be associated with an alteration in RANTES-induced [Ca2+]i mobilization. While the clinical significance of these in-vitro findings is unknown, they suggest that mu-opioid receptor agonists could alter certain neurodegenerative and inflammatory processes within the brain.

Topics: Brain; Calcium; Cells, Cultured; Chemokine CCL5; Chemotaxis; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Fetus; Humans; Interleukin-1; Lipopolysaccharides; Microglia; Morphine; Naloxone; Naltrexone; Narcotic Antagonists; Recombinant Proteins

We investigated the role of mu-opioid receptor subtypes in both endomorphin-1 and endomorphin-2 induced antinociception in mice using supraspinally mediated behavior. With tail pressure as a mechanical noxious stimulus, both intracerebroventricularly (i.c.v.) and intrathecally (i.t.) injected-endomorphins produced potent and significant antinociceptive activity. Antinociception induced by i.t. and i.c.v. injection of endomorphin-1 was not reversed by pretreatment with a selective mu1-opioid receptor antagonist, naloxonazine (35 mg/kg, s.c.). By contrast, antinociception induced by i.t. and i.c.v. endomorphin-2 was significantly decreased by mu1-opioid receptor antagonist. Antinociception of both i.t. and i.c.v. endomorphin-1 and -2 was completely reversed by pretreatment with beta-funaltrexamine (40 mg/kg, s.c.). The results indicate that endomorphins may produce antinociception through the distinct mu1 and mu2 subtypes of mu-opioid receptor.

Topics: Analgesics, Opioid; Animals; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Injections, Intraventricular; Injections, Spinal; Male; Mice; Naloxone; Naltrexone; Narcotic Antagonists; Nociceptors; Oligopeptides; Pain; Receptors, Opioid, mu; Time Factors

Our laboratory has previously shown that delta-opioid receptors are involved in the cardioprotective effect of ischemic preconditioning in the rat heart. However, this class of receptors consists of two subtypes, delta1, and delta2, and mu- or kappa-opioid receptors may also exist in the heart. Therefore, the purpose of the present study was to test the hypothesis that ischemic preconditioning is mediated through stimulation of one or both delta-opioid receptor subtypes.. Anesthetized, open chest, male Wistar rats were assigned to 1 of 14 groups. All animals were subjected to 30 minutes of occlusion and 2 hours of reperfusion. Ischemic preconditioning was elicited by three 5-minute occlusion periods interspersed with 5 minutes of reperfusion. Two doses of 7-benzylidenenaltrexone (BNTX; 1 and 3 mg/kg i.v.), a selective delta1-opioid receptor antagonist, or naltriben (NTB; 1 and 3 mg/kg i.v.), a selective delta2-opioid receptor antagonist, were given before ischemic preconditioning. To test for a role of mu-opioid receptors, rats were pretreated with beta-funaltrexamine (beta-FNA; 15 mg/kg s.c), an irreversible mu-opioid receptor antagonist, 24 hours before ischemic preconditioning or given the mu-opioid receptor agonist D-Ala,2N-Me-Phe,4glycerol5-enkephalin (DAMGO) as three 5-minute infusions (1, 10, and 100 microg/kg per infusion i.v., respectively) interspersed with 5-minute drug-free periods before the prolonged ischemic and reperfusion periods (lowDAMGO, medDAMGO, and hiDAMGO, respectively). The involvement of kappa-opioid receptors was tested by administering one of two doses of nor-binaltorphimine (nor-BNI; 1 and 5 mg/kg i.v.) before ischemic preconditioning. Infarct size (IS) as a percent of the area at risk (AAR) was measured by triphenyltetrazolium stain. Ischemic preconditioning markedly reduced IS/AAR (14+/-4%, P<.05) compared with control (55+/-4%). NTB, beta-FNA, and nor-BNI were unable to block the cardioprotective effect of ischemic preconditioning. In addition, DAMGO had no effect on IS/AAR. However, the high dose of BNTX (3 mg/kg i.v.) significantly attenuated the cardioprotective effect of ischemic preconditioning (39+/-5%; P<.05 versus control and ischemic preconditioning).. These results indicate that delta1-opioid receptors play an important role in the cardioprotective effect of ischemic preconditioning in the rat heart.

Topics: Animals; Benzylidene Compounds; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Hemodynamics; Ischemic Preconditioning, Myocardial; Male; Myocardial Infarction; Naltrexone; Narcotic Antagonists; Rats; Rats, Wistar; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu

These studies were undertaken to determine the effects of mu-opioid receptor depletion through irreversible alkylation on the dose-effect curve for heroin self-administration. Heroin maintained responding in rats with an inverted U-shaped dose-effect curve and administration of 10 nmol of beta-funaltrexamine i.c.v. (beta-FNA) significantly increased the ED50 on the ascending limb from 1.9 to 5.3 micrograms/infusion, and from 24.3 to 211.8 micrograms/infusion on the descending limb. Administration of saline i.c.v. produced no effect on heroin self-administration. Administration of 40 nmol of beta-FNA increased the ED50S from 5.1 to 33.9 and from 14.4 to 502.8 micrograms/infusion on the ascending and descending portions of heroin's dose-effect curve, respectively. beta-FNA (40 nmol, i.c.v.) had no effect on cocaine self-administration. [3H]DAMGO binding density was decreased in the caudate and nucleus accumbens by 29 or 54% 24 h after administration of 10 or 40 nmol of beta-FNA i.c.v., respectively. The effects of beta-FNA on heroin self-administration were completely overcome by increasing the dose of heroin however, as the shape and slope of the self-administration dose-effect curve was not different when higher doses of heroin were made available for self-administration compared to control data or saline administration. Therefore, there appear to be spare mu-opioid receptors for heroin for the production of its reinforcing effects in rats. Furthermore, the self-administration dose-effect curves returned to control values prior to the return of [3H]DAMGO binding, further suggesting that the full complement of mu-opioid receptors is not necessary for heroin to produce its reinforcing effects. These findings support the existence of spare mu-opioid receptors for heroin in maintaining self-administration in rats.

Topics: Analgesics, Opioid; Animals; Brain; Caudate Nucleus; Cocaine; Culture Techniques; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Heroin; Heroin Dependence; Motivation; Naltrexone; Narcotic Antagonists; Nucleus Accumbens; Radioligand Assay; Rats; Receptors, Opioid, mu; Self Administration

Endogenous opioids exert a variety of functions outwith the central nervous system, including modulation of some murine lymphocyte functions. The results of this study indicate that mu-, delta- and kappa-receptor selective agonists are potent in vitro stimulators of mitogen-induced proliferation of murine T-lymphocytes. Moreover, the observed enhancement of mitogen-induced proliferation was reversed by mu-, delta- and kappa-receptor class selective antagonists, beta-funaltrexamine, ICI 174,864 and nor-binaltorphimine, respectively. An additional study has revealed that repeated administration (four injections) of the opioid receptor selective agonists DAGO, DPDPE and U-50488 also enhanced the concanavalin A-induced proliferation of lymphocytes. These results suggest that there are three classes of opioid receptors on T-lymphocytes and that all these receptor classes are involved in the stimulation of concanavalin A-induced proliferation.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine; Enkephalins; Immunity; Lymphocyte Activation; Male; Mice; Mice, Inbred C57BL; Mitogens; Naltrexone; Narcotic Antagonists; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; T-Lymphocytes

Microglia are important immune effector cells within the brain. The phagocytosis of nonopsonized Cryptococcus neoformans by swine microglia was used as an in vitro model for studies on cellular mechanisms of opiate-mediated immunomodulation in the brain. Morphine inhibited potently (IC50 approximately 10(-16) M) the phagocytosis of C. neoformans by primary cultures of neonatal pig microglia. The mu opioid agonist Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol (DAMGO) also suppressed phagocytosis but with a much lower potency than morphine (IC50 approximately 10(-8) M). The inhibitory effects of morphine and DAMGO were blocked by equimolar concentrations of naloxone and by the selective mu opiate receptor antagonist beta-funaltrexamine. Pertussis toxin but not cholera toxin reversed the inhibitory effects of both morphine and DAMGO. Our data suggest that morphine inhibits phagocytosis of C. neoformans by swine microglia via a mechanism involving mu opiate receptors coupled to a pertussis toxin-sensitive Gi/G(o) protein signaling pathway.

Topics: Animals; Cryptococcus neoformans; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; GTP-Binding Proteins; Microglia; Morphine; Naltrexone; Narcotics; Phagocytosis; Swine

The mu opioid receptor mediates ingestive behavior: mu-selective agonists stimulate food intake and antagonists reduce intake in many ingestive situations. Antisense oligodeoxynucleotides directed against each of the four exons of the MOR-1 clone were equally effective in reducing spontaneous food intake and body weight in rats. However, antisense probes directed against only exon 1 or 4 of the MOR-1 clone reduced mu-mediated analgesia. The present study examined whether central administration of antisense probes directed against each of the four exons of the MOR-1 clone or a missense control altered hyperphagia elicited by the mu agonist DAMGO across a range of doses. Antisense probes directed against only exon 1 or 4 blocked hyperphagia at agonist doses of 0.5 and 1.0 microg; this pattern was identical to that observed for mu-mediated analgesia. A missense control failed to exert significant effects, which suggests specificity of antisense actions. The effective antisense probes failed to reduce hyperphagia at a higher (5 microg) agonist dose, a result consistent with limitations in down-regulation of receptor proteins by antisense. The mu antagonist beta-funaltrexamine produced a similar pattern of effects on mu-mediated hyperphagia. The selective actions of antisense probes directed against different exons of the MOR-1 clone in reducing hyperphagia induced by DAMGO suggest that multiple splice variants of the MOR-1 clone exist and raise the possibility of further opioid receptor subclassifications.

Topics: Animals; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Hyperphagia; Male; Naltrexone; Oligonucleotides, Antisense; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu

Intracerebroventricular administration of beta-funaltrexamine (beta-FNA) reduces the density of mu opioid receptors as measured by in situ autoradiography by 40-50% throughout the brain, with little regional variation [Martin et al. (1993) J. Pharmacol. Exp. Ther. 267:506-514] Recently an assay has been developed to study opioid stimulation of [35S]GTP-gamma-S binding autoradiographically in situ using slide-mounted brain sections [Sim et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92:7242-7246]. The present study was undertaken to determine the effect of mu opioid receptor alkylation on G protein activation by the mu opioid agonist DAMGO. Animals were injected intracerebroventricularly with 40 nmol of beta-FNA or saline and sacrificed 24 hours later. DAMGO stimulated [35S]GTP-gamma-S binding with an anatomical specificity consistent with the localization of mu opioid receptors. The percent stimulation by DAMGO ranged from approximately 50 to 100% in the regions studied. beta-FNA significantly decreased G protein activation by DAMGO in regions that are consistent with its reported long-lasting and insurmountable antagonism of the antinociceptive (medial thalamus, central gray) and reinforcing (nucleus accumbens) effects of mu opioid agonists [Adams et al. (1990) J. Pharmacol. Exp. Ther. 255:1027-1032; Martin et al. (1995) J. Pharmacol. Exp. Ther. 272:1135-1140]. However, the effects of beta-FNA were not equal in all brain regions. This may indicate regional differences in the coupling efficiency of mu opioid receptors with G proteins, or in the effects of beta-FNA on mu opioid receptor binding or on mu opioid receptor-stimulated G protein activity.

Topics: Animals; Brain; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Guanosine 5'-O-(3-Thiotriphosphate); Injections, Intraventricular; Male; Naltrexone; Narcotic Antagonists; Rats; Rats, Inbred F344; Receptors, Opioid, mu; Sulfur Radioisotopes

The present experiments explored the role of endogenous opioids in the behavioral response to a formalin-induced nociceptive stimulus in the rat. Flinching was taken as a measure of the intensity of the nociceptive stimulus after the administration of formalin into the dorsal surface of the paw of control animals, or in animals receiving i.p. administration of receptor-selective doses of opioid antagonists including naloxone, naltrindole (delta opioid antagonist), nor-binaltorphimine (kappa opioid antagonist) or beta-funaltrexamine (mu opioid antagonist). Additionally, antisera to [Leu5]enkephalin, [Met5]enkephalin and dynorphin A (1-13) (dynorphin) were administered intrathecally before formalin to explore the contribution of endogenous opioids in modulation of the flinching response. Formalin-induced flinching was increased significantly by naloxone, and receptor selective doses of naltrindole and nor-binaltorphimine, but not beta-funaltrexamine. Additionally, antisera to [Leu5]enkephalin and dynorphin also resulted in a significant increase in formalin-induced flinching, whereas antisera to [Met5]enkephalin had no effect. On the basis of significant increases in formalin-induced flinching produced by 1) receptor-selective doses of delta and kappa, but not mu, opioid antagonists and 2) antisera to [Leu5]enkephalin and dynorphin A, but not [Met5]enkephalin, these data suggest the presence of an opioid inhibitory tone which acts to limit the intensity of the pain signal. This tone appears to be mediated via activation of delta and kappa receptors, possibly by a [Leu5]enkephalin- and dynorphin-like substance, respectively.

Topics: Animals; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Formaldehyde; Immune Sera; Male; Naloxone; Naltrexone; Narcotic Antagonists; Opioid Peptides; Pain; Rats; Rats, Sprague-Dawley; Receptors, Opioid, delta; Receptors, Opioid, kappa

To elucidate further the role of mu-opioid receptors in mediating analgesia and cardiovascular function at rest and during stress, rats were pretreated ICV with either saline (5 microliters) or beta-funaltrexamine (beta-FNA, 5 nmol/5 microliters), a noncompetitive opioid receptor antagonist that inactivates irreversibly mu receptors, 2 days prior to [D-Ala2, N MePhe4, Gly5-ol]enkephalin (DAMPGO, 1 nmol, ICV) administration. Pretreatment with beta-FNA blocked DAMPGO-induced analgesia as measured by the tail-flick test. DAMPGO also produced an increase in blood pressure (BP), sympathoadrenal outflow, and a bradycardia. Pretreatment with beta-FNA converted the DAMPGO-induced bradycardia to a tachycardia, significantly reduced the DAMPGO-induced increase in epinephrine by 60%, and the norepinephrine response by 45%, and attenuated mildly the increase in BP due to DAMPGO. In saline-treated rats, restraint stress evoked an increase in HR, BP, and plasma catecholamines. Pretreatment with beta-FNA partially attenuated the increase in HR in response to stress. In the presence of DAMPGO, restraint stress resulted in a further bradycardia, which was significantly blocked by pretreatment with beta-FNA. Stress also produced increases in BP and plasma catecholamines, which were not prevented by pretreating rats with beta-FNA. These results indicate that beta-FNA may not have inactivated all the receptors accessible to DAMPGO which control BP, or alternatively, beta-FNA may selectively inactivate a subtype of mu receptors. In addition, brain mu opioid receptors appear to be significantly involved in mediating supraspinal analgesia and regulating parasympathetic outflow to the heart and sympathoadrenal release of catecholamines.

Topics: Adrenal Glands; Analgesics; Animals; Blood Pressure; Catecholamines; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Heart Rate; Hemodynamics; Male; Naltrexone; Narcotic Antagonists; Organ Size; Pain Measurement; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu; Stress, Psychological; Sympathetic Nervous System

In the Xenopus oocytes expressing mu- or kappa-opioid receptors, agonist-induced currents were observed only when the oocyte was coinjected with Gi1 alpha RNA and pretreated with K-252a, a potent inhibitor of protein kinases. The evoked currents were abolished by intracellular injection of EGTA or inositol 1,4,5-trisphosphate and the current-voltage relationship revealed that they are mediated through typical calcium-dependent chloride channels. These findings suggest that the mu- and kappa-receptors mediate phospholipase C activation through Gi1 alpha, and that these receptor mechanisms including downstream signalings might be inhibited by phosphorylation in vivo in the Xenopus oocyte.

Topics: Animals; Benzeneacetamides; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Enzyme Activation; Female; GTP-Binding Proteins; Naltrexone; Oocytes; Pyrrolidines; Receptors, Opioid, kappa; Receptors, Opioid, mu; Type C Phospholipases; Xenopus laevis

The present study was designed to investigate the contribution of opioids and nitric oxide (NO) to hypoxia-induced pial vasodilation. Newborn pigs equipped with a closed cranial window were used to measure pial arteriolar diameter and to collect cortical periarachnoid cerebrospinal fluid (CSF) for assay of opioids and guanosine 3',5'-cyclic monophosphate (cGMP). Hypoxia-induced pial dilation was potentiated by norbinaltorphimine, 10(-6) M, a kappa-opioid antagonist (25 +/- 2 vs. 33 +/- 3%, n = 5), but was blunted by beta-funaltrexamine, 10(-8) M, a mu-opioid antagonist (28 +/- 2 vs. 19 +/- 1%, n = 5). Hypoxia-induced vasodilation was associated with increased CSF methionine enkephalin, a mu-opioid agonist (884 +/- 29 vs. 2,638 +/- 387 pg/ml, n = 5). N omega-nitro-L-arginine (L-NNA), an NO synthase inhibitor (10(-6) M), also blunted hypoxia-induced vasodilation that was further diminished by coadministration of L-NNA and beta-funaltrexamine (26 +/- 2, 14 +/- 1, and 9 +/- 1%, respectively, n = 5). Reversal of the above order of antagonist administration resulted in similar inhibition of hypoxia-induced pial dilation. Hypoxia-induced vasodilation was also associated with an increase in CSF cGMP that was attenuated by L-NNA (2.1 +/- 0.1- vs. 1.1 +/- 0.2-fold change in CSF cGMP, n = 5). Sodium nitroprusside (10(-6) M) increased CSF cGMP and methionine enkephalin concentration similar to hypoxia. These data suggest that hypoxia-induced pial arterial vasodilation, in part, is due to NO and/or cGMP-induced methionine enkephalin release as well as the direct action of NO.

Topics: Amino Acid Oxidoreductases; Analysis of Variance; Animals; Animals, Newborn; Arginine; Arterioles; Cerebral Arteries; Cyclic GMP; Dynorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Methionine; Enkephalins; Female; Hypoxia; Male; Muscle, Smooth, Vascular; Naltrexone; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Nitroprusside; Receptors, Opioid, kappa; Receptors, Opioid, mu; Swine; Vasodilation

mu-Opioid receptor agonist [D-Ala2,NMe-Phe4,Gly5-ol]enkephalin (DAMGO)-induced peripheral analgesic effects occur early in hindpaws inoculated with Freund's complete adjuvant and increase in parallel to the development of inflammatory signs. Antagonism of these effects by beta-funaltrexamine, an irreversible mu-opioid receptor antagonist, suggests that the effective number of peripheral opioid receptors does not increase during early stages, but does so at later stages of the inflammation. As determined by a ribonuclease protection assay, mu-opioid receptor mRNA in dorsal root ganglia is abundant in untreated animals, but does not significantly increase following inflammation. Thus, peripheral analgesic efficacy of DAMGO is not correlated with transcription or number of mu-opioid receptors at early inflammatory stages. At later stages, however, the number of peripheral mu-opioid receptors appears to increase and may enhance opioid efficacy.

Topics: Amino Acid Sequence; Analgesia; Analgesics; Animals; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Foot; Ganglia, Spinal; Inflammation; Male; Molecular Sequence Data; Naltrexone; Narcotic Antagonists; Pain Measurement; Rats; Rats, Wistar; Receptors, Opioid, mu; Ribonucleases; RNA, Messenger

We examined the effects of an exogenous mu opioid agonist and antagonist on systemic physiology and neurological outcome following TBI in the rat. Experiment I: [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) (0.1 nMol or 0.3 nMol in 5 microliters) (n = 10) or artificial CSF (n = 10) was administered 5 min prior to fluid-percussion brain injury (2.1 atmospheres). Motor performance was assessed on days 1-5 after TBI. The mu receptor agonist, DAMGO significantly reduced both beam-walking latency and body weight loss after injury (p < 0.05). DAMGO-treated rats (n = 5) did not differ from CSF-treated rats (n = 5) on either systemic arterial blood pressure or heart rate responses to injury. Experiment II: Beta-funaltrexamine (beta-FNA) (20.0 nMol in 5.0 microliters) (n = 10) or artificial CSF (n = 10) was administered (icv) to rats 5 min prior to fluid-percussion brain injury (1.8 atmospheres). Motor performance was assessed on days 1-5 after TBI. The mu receptor antagonist, beta-FNA, significantly increased beam-walking latency after injury (p < 0.05). beta-FNA-treated rats (n = 5) did not differ from CSF-treated rats (n = 5) on either systemic arterial blood pressure or heart rate responses to injury. Experiment III: Neither beta-FNA nor DAMGO affected motor performance in uninjured rats. These results suggest that activation of mu opioid receptors by exogenous agonists may provide protection against deficits in motor performance produced by fluid percussion brain injury.

Topics: Animals; Brain Injuries; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Male; Naltrexone; Psychomotor Performance; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu; Treatment Outcome

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

Effects of 24 h pretreatment with intracerebroventricular (icv) beta-funaltrexamine (beta-FNA) on brain opioid receptor binding in rats were examined under various conditions. Agonist binding to mu and delta opioid receptors (with [3H][[cap]dAla2,MePhe4,Gly-ol5]enkephalin (DAMGO)[3H][D-Pen2, D-Pen5]enkephalin (DPDPE), respectively) was performed under three different conditions: i) pretreatment of membranes with GDP and Na+ and binding in the presence of Mg++ in Tris-HCI buffer containing EGTA and leupeptin for 1.5 to 3 h; ii) binding in Tris-HCI buffer containing bacitracin, leupeptin, chymostatin and bestatin for 3 to 4 h; iii) binding in Tris-HCI buffer containing EGTA and leupeptin for 45 min. Condition i was shown to convert opioid receptors to a high affinity state for agonists. beta-FNA (2, 6 or 20 nmol) significantly reduced 1 nM [3H]DAMGO binding in the whole brain with i but not with ii. With iii, 20 nmol beta-FNA reduced [3H]DAMGO binding, but not 2 or 6 nmol. Saturation experiments with i showed that the reduction in [3H]DAMGO binding after 6 or 20 nmol beta-FNA was due to a decrease in Bmax and an increase in KD. For delta binding, there was no significant change in [3H]DPDPE (2 nM) binding with i after 2, 6 or 20 nmol beta-FNA. Thus, under i, icv beta-FNA reduced [3H]DAMGO binding significantly without affecting [3H]DPDPE binding. In addition, mu binding was also conducted with 1 nM [3H]naloxone under three different conditions: iv) in the presence of Na+ and GDP; v), in the presence of Na+, Gpp(NH)p and Mg++; vi) in the presence of Na+. Both iv and v were shown to shift opioid receptors to a low affinity state for agonists. beta-FNA (20 nmol) significantly decreased 1 nM [3H]naloxone binding under each of the three conditions. Competitive inhibition of 1 nM [3H]naloxone binding by DAMGO in the presence of Na+ and GDP showed that receptors existed in a single low affinity state for DAMGO, and that icv beta-FNA caused a reduction in Bmax without affecting the KD of DAMGO. In summary, when all the receptors were converted to a high agonist affinity state i or a low agonist affinity state iv, the changes in mu binding induced by beta-FNA could be revealed with agonist binding. Additionally, changes in mu binding induced by beta-FNA could be detected with [3H]naloxone, which always displayed high affinity regardless of agonist affinity states, under each of the three conditions (iv, v and vi).

Topics: Analgesics; Animals; Binding, Competitive; Brain; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Injections, Intraventricular; Male; Naloxone; Naltrexone; Narcotic Antagonists; Protein Binding; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu; Tritium

Mu opioid receptors are known to be directly involved in the reinforcing effects of opiates; however, little is known regarding the relationship between alteration of mu opioid receptor binding and opiate reinforcement. Intracerebroventricular (i.c.v.) administration of beta-funaltrexamine (beta-FNA) has been shown to reduce the number of mu opioid receptors throughout the brain and can be used to address questions regarding the relationship of the density of these receptors to the pharmacological effects of opiates. The time course of the effects of beta-FNA on heroin self-administration was compared with the effects on mu opioid receptor binding. beta-FNA (40 nmol) or saline was administered i.c.v. to animals trained to self-administer either 18 or 60 micrograms/kg per infusion of heroin. The number of infusions decreased after beta-FNA administration but steadily returned to base-line levels approximately 10 days after beta-FNA treatment. The time course of the effects of beta-FNA on mu opioid receptor binding was determined in separate groups of animals. beta-FNA treatment decreased the number of [3H]D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin binding sites by 34 to 50% in rat brain sections; an effect that persisted for up to 18 days. The affinity was unaffected initially, but decreased in a linear manner from days 9 to 18 after beta-FNA administration. The return of heroin self-administration before the return of mu opioid receptor binding suggests that the recovery of mu opioid receptor function after beta-FNA treatment is more complex than merely synthesis of new receptors.

Topics: Alkylation; Animals; Behavior, Animal; Binding, Competitive; Brain; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Heroin; In Vitro Techniques; Injections, Intraventricular; Naltrexone; Rats; Rats, Inbred F344; Receptors, Opioid, mu; Self Administration

To determine whether the peripheral opioid system participates in hypertension development we studied responses to various opioid receptor agonists in field-stimulated isolated tail artery segments taken from spontaneously hypertensive rats (SHR), normotensive Wistar-Kyoto (WKY) and Sprague-Dawley (SD) rats at different ages. The mu-selective agonist (DAGO) and the delta-selective D-Ala2-D-Leu5-enkephalin (DADLE) both suppressed the electrically stimulated vasoconstriction (EIC), but only in SHR arteries. The mu-selective antagonist beta-funaltrexamine reversed the effects of both DAGO and DADLE. Since the delta-selective antagonist ICI-174864 did not block DADLE inhibition, it is likely that both DAGO and DADLE effects were mu-receptor-mediated. Effects of DAGO and DADLE were qualitatively and quantitatively similar at all ages of SHR tested, and were not temporally related to hypertension development. Dynorphin (1-13) (DYN), a kappa-agonist, increased basal tone and EIC in all three rat strains. These responses were not blocked by nor-binaltorphimine, a selective kappa-opioid antagonist, suggesting that they may not involve kappa-receptor activation. There was a greater sensitivity to DYN at younger ages in all three rat strains and the sensitivity decreased with age. At 16 weeks when SHR hypertension was fully developed, SHR tail artery became almost totally insensitive to DYN in contrast to the continued responsiveness of 16-week-old WKY and SD arteries. The diminished effects to DYN in 16-week-old SHR tail arteries is suggestive of a compensatory mechanism to the hypertensive state. Collectively, the results establish that opioid receptor responses in SHR tail artery differ from those of normotensive rats. The significance of these differences to hypertension development in SHR remains to be determined.

Topics: Age Factors; Animals; Dose-Response Relationship, Drug; Dynorphins; Electric Stimulation; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine-2-Alanine; Enkephalins; Hypertension; In Vitro Techniques; Male; Naltrexone; Narcotic Antagonists; Phenylephrine; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Rats, Sprague-Dawley; Receptors, Opioid; Tail; Vasoconstriction

The effects of intrastriatal (i.st.) injections of mu-, delta-, and kappa-selective opioid receptor agonists on the augmentation of apomorphine-induced behaviors were determined in 6-hydroxydopamine-treated mice by using multidimensional behavioral analyses. 6-Hydroxydopamine (16 mu g/mu l, i.st.) was unilaterally injected into the striatum 30 min after pretreatment with desipramine (25 mg/kg, s.c). Mice were tested 14 days after injection of 6-hydroxydopamine. Apomorphine (0.5 mg/kg, s.c.) produced a marked increase in linear locomotion, contralateral circling and/or rearing behavior in 6-hydroxydopamine- but not vehicle-treated mice. Although the mu-selective opioid receptor agonist [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAMGO) (0.1 and 0.3 ng, i.st.) or the kappa-selective opioid agonist dynorphin A-(1-13) (0.1 and 0.3 mu g, i.st.) did not produce any significant effects on behavior, these peptides had an inhibitory effect on the apomorphine (0.5 mg/kg, s.c.)-induced increase in behavioral responses such as linear locomotion, contralateral circling and/or rearing behavior in 6-hydroxydopamine-treated mice. The inhibitory effects of DAMGO (0.3 ng, i.st.) and dynorphin A-(1-13) (0.3 mu g, i.st.) were fully reversed by selective opioid receptor antagonists such as beta-funaltrexamine (5 mu g, i.c.v.) and (--)-(1R,5R,9R)-5,9-diethyl-2-(3-furyl-methyl)-2'-hydroxy-6,7-benzomorph an (Mr2266) (10 mg/kg, s.c.), respectively. In contrast, the delta-selective opioid receptor agonist [D-Pen2,L-Pen5]enkephalin (DPLPE) (0.03, 0.1 or 0.3 mu g, i.st.) had no marked effects on the apomorphine (0.5 mg/kg, s.c.)-induced behavior in 6-hydroxydopamine-treated mice. These results suggest that the stimulation of mu- and kappa- but not delta-opioid receptors plays an inhibitory role in the behavioral augmentation induced by the activation of postsynaptic dopamine receptors in the striatum sensitized with 6-hydroxydopamine.

Topics: Animals; Apomorphine; Behavior, Animal; Corpus Striatum; Dopamine; Dynorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Male; Mice; Naltrexone; Oxidopamine; Peptide Fragments; Receptors, Opioid

Whole-cell voltage-clamp technique was used to examine the effects of a mu-opioid receptor agonist DAGO (Tyr-D-Ala-Gly-Me-Phe-Gly-ol-enkephalin) on GABA-induced currents in acutely isolated spinal dorsal horn (DH) neurons from laminae I-IV of young rats. We found that a bicuculline-sensitive GABA-induced current was potentiated by DAGO (0.5-500 nM), in a dose-dependent manner, in approximately 62% of the tested cells. The elevated GABA responses outlasted the period of DAGO application, and either recovered within 10 min after the removal of the peptide or persisted for up to 50 min. The potentiating effect of DAGO was reduced or prevented by naloxone and the mu-opioid receptor-selective antagonist beta-funaltrexamine. A similar enhancing effect on the membrane currents activated by administration of muscimol, a GABAA receptor-specific agonist, was produced by DAGO. In addition, a transient depression of GABA responses was observed in approximately 25% of the cells tested. These results indicate that the mu-opioid agonist DAGO modulates the sensitivity of postsynaptic GABAA receptors in a large proportion of spinal neurons from laminae I-IV, with the major effect being facilitation. The DAGO action could contribute to the regulation of the strength of primary afferent neurotransmission, including nociception.

Topics: Action Potentials; Amino Acid Sequence; Animals; Bicuculline; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; GABA-A Receptor Agonists; GABA-A Receptor Antagonists; Molecular Sequence Data; Muscimol; Naloxone; Naltrexone; Neurons, Afferent; Rats; Rats, Sprague-Dawley; Receptors, GABA-A; Receptors, Opioid, mu; Spinal Cord

The irreversible opioid receptor antagonists naloxonazine and beta-funaltrexamine have been used to determine whether multiple mu-opioid receptors exist on undifferentiated SH-SY5Y human neuroblastoma cells. Naloxonazine binds irreversibly to the mu 1-opioid receptor subtype and reversibly to the mu 2-opioid receptor subtype. On SH-SY5Y cells naloxonazine afforded a Ki of 3.4 +/- 0.7 nM, and was fully reversible, indicating the mu-opioid receptor population on SH-SY5Y cells was solely of the mu 2-opioid receptor subtype. The alkylating agent beta-funaltrexamine was maximally able to alkylate only 60% of the mu-opioid receptor sites on SH-SY5Y cells, labelled with [3H]diprenorphine or [3H][D-Ala2,MePhe4,Gly(ol)5]enkephalin (DAMGO). The reversible binding of naloxonazine and the insensitivity of a percentage of the mu-opioid receptor sites to alkylation by beta-funaltrexamine suggests that differences do exist in the mu 2-opioid receptor population on undifferentiated SH-SY5Y cells. This may indicate further heterogeneity or the inability of beta-funaltrexamine to alkylate all relevant nucleophilic groups in a single population of receptors.

Topics: Amino Acid Sequence; Animals; Brain Neoplasms; Cerebral Cortex; Diprenorphine; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Humans; Ligands; Molecular Sequence Data; Naloxone; Naltrexone; Neuroblastoma; Rats; Receptors, Opioid, mu; Somatostatin; Tumor Cells, Cultured

The effects of intracerebroventricular injection of mu-, kappa- and delta-selective opioid agonists on cocaine-induced behavior were investigated in mice using multidimensional behavioral analysis. Cocaine (3.0 mg/kg) produced a marked increase in linear locomotion, circling, rearing and/or grooming, although the mu-opioid agonist [D-Ala2, NMePhe4, Gly-ol] enkephalin (DAMGO) (0.003 and 0.01 microgram), the kappa-opioid agonist dynorphin A- (1-13) (3.0 and 12.5 micrograms) or the delta-opioid agonist [D-Pen2, L-Pen5]enkephalin (DPLPE) (0.3 and 1.0 micrograms) did not significantly affect behavioral responses. DAMGO (0.003 and 0.01 microgram) and dynorphin A- (1-13) (12.5 micrograms) inhibited the cocaine (3.0 mg/kg)-induced increase in linear locomotion, circling and/or rearing. In contrast, DPLPE (1.0 micrograms) enhanced the cocaine (3.0 mg/kg)-induced increase in circling. The effects of DAMGO (0.003 microgram), dynorphin A- (1-13) (12.5 micrograms) and DPLPE (1.0 micrograms) were fully reversed by receptor-selective opioid antagonists, such as beta-funaltrexamine (5.0 micrograms), Mr2266 (5.6 mg/kg) and naltrindole (10.0 mg/kg), respectively. These results suggest that the activation of mu- and kappa-opioid receptors inhibits cocaine-induced behavior, while that of delta-opioid receptors enhances the behavior.

Topics: Animals; Behavior, Animal; Cocaine; Dynorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Injections, Intraventricular; Male; Mice; Mice, Inbred Strains; Naltrexone; Opioid Peptides

The effects of [D-Ala2, NMePhe4, Gly-ol]enkephalin (DAMGO), a mu-selective opioid receptor agonist, [D-Ala2]deltorphin II (DELT) and [D-Pen2, L-Pen5]enkephalin (DPLPE), delta-selective opioid receptor agonists, on different behavioral responses were investigated in mice by multidimensional behavioral analyses. DAMGO (0.1 and/or 0.3 microgram) produced a marked increase in circling, rearing and grooming within 15-30 min after the start of behavioral measurements. DELT (0.3, 1.0 and/or 3.0 micrograms) produced a significant increase in linear locomotion and circling within 15-30 min after the start of behavioral measurements, whereas DPLPE (10.0 micrograms) increased only circling. The behavioral effects of DAMGO, DELT or DPLPE were almost completely antagonized by beta-funaltrexamine (2.5 and 5.0 micrograms) or naltrindole (3.0 and 10.0 mg/kg). These results suggest that opioid peptides selective for receptor types elicit different behavioral responses in mice. Moreover, the different behavioral effects between DPLPE and DELT may be due to the delta opioid receptor subtypes such as delta-1 and delta-2.

Topics: Analysis of Variance; Animals; Behavior, Animal; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Male; Mice; Mice, Inbred Strains; Naltrexone; Oligopeptides

Opioid peptides are potent inhibitors of gastric somatostatin secretion. In the current investigation the effect of mu-opioid receptor blockade on responses to [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAGO) was studied. Gastric inhibitory polypeptide (GIP; 1 nM) -stimulated secretion of immunoreactive somatostatin was almost completely inhibited by DAGO (1 microM). The mu-receptor antagonists, beta-funaltrexamine and naloxonazine, blocked the effect of DAGO. Pretreatment of rats with beta-funaltrexamine, 24 h prior to perfusion, reduced the percentage inhibition by DAGO from 88.6 +/- 5.2% to 50.7 +/- 9.3%. These studies support the involvement of mu-opioid inhibitory receptors in the regulation of gastric somatostatin secretion.

Topics: Amino Acid Sequence; Analgesics; Animals; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Gastric Inhibitory Polypeptide; Gastric Mucosa; In Vitro Techniques; Male; Molecular Sequence Data; Naloxone; Naltrexone; Narcotic Antagonists; Radioimmunoassay; Rats; Rats, Wistar; Somatostatin

Previous work in our laboratory has shown that DAMGO (ICV) will cause an elevation in plasma corticosterone (CS). The effect was blocked by pretreatment with beta-FNA but not by naloxonazine, suggesting indirectly that DAMGO's effect was via a mu 2-opioid receptor. TRIMU-5, a mu 2 agonist/mu 1 antagonist, was tested in a similar series of experiments to show more directly that the effect of DAMGO to increase plasma CS was via the mu 2 receptor. Experiments were conducted on conscious, unrestrained, male Sprague-Dawley rats with chronic IV catheters and ICV cannula guides allowing for serial blood sampling and drug injection into the right lateral ventricle. During this process, animals remained isolated in sound-attenuated one-way vision boxes. TRIMU-5, 50 micrograms, produced a sustained increase in plasma CS for a 3-h period. The response peaked at 30 min, showing a plasma CS level of 19.7 +/- 1.4 micrograms/dl. A lower dose, 10 micrograms, did not produce a significant response. A higher dose, 100 micrograms, produced an elevated hormone response in a pilot study but was lethal in half the animals. The plasma CS increase was blocked by pretreatment with beta-FNA, 20 micrograms ICV, given 18 h before TRIMU-5, but was unaffected by naloxonazine pretreatment, 20 mg/kg i.v., also administered 18 h before TRIMU-5. These data confirm our earlier conclusion that the effect of DAMGO to elevate plasma CS was through a mu 2-opioid receptor.

Topics: Animals; Corticosterone; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Hypothalamo-Hypophyseal System; Kinetics; Male; Naloxone; Naltrexone; Oligopeptides; Pituitary-Adrenal System; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu

The regulation of adenylyl cyclase by opioid receptor types was characterized in the rat nucleus accumbens, a brain region that is involved in the reinforcing effects of drugs of abuse, and in the caudate putamen, a region not implicated in drug reinforcement. Both mu and delta opioid ligands inhibited adenylyl cyclase activity in the nucleus accumbens and in the caudate putamen of rat, whereas the kappa agonist, U69,593 (5 alpha, 7 alpha, 8 alpha)-(+)-N-methyl-N-[7-(pyrrolidinyl)-1-oxaspiro [4,5]dec-8-yl]-benzeneacetamide, was ineffective. The mu agonists, DAMGO and Tyr-D-Arg-Phe-Sar, were more potent inhibitors of the enzyme in caudate putamen than in nucleus accumbens. The delta-selective agonists, DSLET and [D-Ala2]-deltorphin II more potently inhibited adenylyl cyclase in nucleus accumbens than in caudate putamen. Inhibition of the enzyme by DAMGO and Tyr-D-Arg-Phe-Sar was antagonized by the mu-selective competitive antagonist, CTOP D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2, and the noncompetitive mu antagonists, beta-funaltrexamine and naloxonazine. Inhibition of adenylyl cyclase activity by the delta-selective ligands, DPDPE, DSLET and [D-Ala2]-deltorphin II was unaffected by these antagonists. Conversely, the delta-selective antagonists, ICI 174,864 N-allyl2-Tyr-(alpha-aminisobutyric acid)2-Phe-Leu-OH and naltrindole, blocked the effects of the delta but not the mu opioid ligands. Adenylyl cyclase activity in nucleus accumbens and in caudate putamen is subject to regulation by both mu and delta opioid receptors.

Topics: Adenylyl Cyclases; Amino Acid Sequence; Animals; Benzeneacetamides; Caudate Nucleus; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine; Enkephalins; Male; Molecular Sequence Data; Naloxone; Naltrexone; Nucleus Accumbens; Oligopeptides; Putamen; Pyrrolidines; Rats; Rats, Sprague-Dawley; Receptors, Opioid, delta; Receptors, Opioid, mu; Signal Transduction; Somatostatin

Intravenous (i.v.) administration of morphine produces a dose-dependent inhibition of the tail-flick (TF) reflex, depressor response, and bradycardia in the rat. Some of these effects depend on interactions of i.v. morphine with peripheral opioid receptors and the integrity of cervical vagal afferents. The present studies used the relatively specific mu, delta, and kappa opioid receptor agonists (DAGO, DPDPE or U-50,488H) and the relatively specific mu, delta, and kappa opioid receptor antagonists (beta-FNA, naloxonazine, naltrindole or nor-BNI) in either intact rats or rats with bilateral cervical vagotomy (CVAG) to delineate the vagal afferent/opioid-mediated components of these effects. I.v. administration of DAGO in intact rats produced a dose-dependent inhibition of the TF reflex, depressor response, and bradycardia virtually identical to those produced by i.v. morphine. All of these effects of either i.v. DAGO or i.v. morphine were significantly attenuated by either bilateral CVAG or pre-treatment with the mu 2 opioid receptor antagonist beta-FNA. Pre-treatment with the mu 1 opioid receptor antagonist naloxonazine affected i.v. DAGO-induced inhibition of the TF reflex and bradycardia, but had no significant effects on i.v. morphine-produced responses. I.v. administration of DPDPE produced a dose-dependent pressor response, but had no marked effects on the either the TF reflex or heart rate (HR). The pressor response was unaffected by either bilateral CVAG or pre-treatment with naltrindole, naloxone, hexamethonium, or bertylium. i.v. administration of U-50,488H produced a depressor response and bradycardia, but had no significant effect on the TF reflex. The depressor response and bradycardia produced by i.v. U-50,488H were unaffected by bilateral CVAG, but could be antagonized by pre-treatment with either nor-BNI or naloxone. These studies suggest that the vagal afferent-mediated antinociceptive and cardiovascular effects of i.v. morphine are primarily mediated by interactions with low affinity mu 2 opioid receptors.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analysis of Variance; Animals; Blood Pressure; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Heart Rate; Hexamethonium; Hexamethonium Compounds; Indoles; Injections, Intravenous; Male; Morphinans; Morphine; Naloxone; Naltrexone; Narcotic Antagonists; Narcotics; Pain; Pyrrolidines; Rats; Rats, Sprague-Dawley; Reference Values; Time Factors; Vagotomy; Vagus Nerve

Topics: Analgesics; Cell Line; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Humans; Kinetics; Naloxone; Naltrexone; Narcotic Antagonists; Neuroblastoma; Receptors, Opioid, mu; Tumor Cells, Cultured

In spite of an extensive body of knowledge regarding the pharmacological effects of centrally administered beta-funaltrexamine (beta-FNA), little is known about the distribution of mu opiate receptor alkylation produced by i.c.v. administration. This study examines the dose relationship between i.c.v. beta-FNA pretreatment and the affinity and density of mu opioid binding sites in discrete brain regions using in situ binding and quantitative receptor autoradiography. [3H]DAMGO binding was determined in coronal sections obtained from the frontal portion of the brain from animals 24 hr after i.c.v. administration of 0, 1, 5, 10, 20 or 40 nmol of beta-FNA. The Kd and Bmax values of [3H]DAMGO binding were unaltered in animals treated with saline or 1 nmol of beta-FNA, whereas treatment with 5, 10 and 20 nmol of beta-FNA increased Kd and decreased Bmax values. The 40 nmol dose did not affect the Kd but decreased the Bmax value. Administration of 40 nmol of beta-FNA i.c.v. was not found to affect either the Kd or Bmax of delta opioid receptors assessed with [3H]DPDPE. Although some brain regions appeared to be affected to a greater degree than others, the autoradiographic localization of [3H]DAMGO binding at 10 different brain levels revealed a generally homogeneous loss of binding after 40 nmol of beta-FNA. beta-FNA appears to alkylate mu opiate receptors throughout the brain after i.c.v. administration.

Topics: Animals; Autoradiography; Brain Mapping; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; In Vitro Techniques; Injections, Intraventricular; Male; Naltrexone; Rats; Rats, Inbred F344; Receptors, Opioid, mu

The effects of intracerebroventricular (i.c.v.) injections of mu- and delta-selective opioid agonists on the methamphetamine-induced behavioral alterations in the mouse were determined by using multi-dimensional behavioral analyses. Methamphetamine (1.0 mg/kg) produced a marked increase in linear locomotion, circling, rearing and grooming behavior. Although the mu-selective opioid agonist [D-Ala2,NMePhe4,Gly-ol]enkephalin (DAMGO) (0.003 and 0.01 microgram) itself did not significantly affect different behavioral responses, DAMGO (0.003 and/or 0.01 microgram) antagonized the methamphetamine (1.0 mg/kg)-induced increase in behavioral responses such as linear locomotion, circling, rearing and grooming. Additionally, the effects of DAMGO (0.01 microgram) on the methamphetamine (1.0 mg/kg)-induced behavioral responses were fully reversed by pretreatment with the mu-selective alkylating agent beta-funaltrexamine (beta-FNA) (5.0 micrograms). In contrast, the delta-selective opioid agonist [D-Pen2,L-Pen5]enkephalin (DPLPE) (0.3 or 1.0 microgram) had no marked effects on the methamphetamine (1.0 mg/kg)-induced behavioral responses. These results suggest that the stimulation of mu but not delta opioid receptors plays an inhibitory role in the methamphetamine-induced behavioral responses.

Topics: Analgesics; Animals; Behavior, Animal; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Injections, Intraventricular; Male; Methamphetamine; Mice; Mice, Inbred Strains; Naltrexone; Narcotic Antagonists

The effect of pertussis toxin (PTX) on the locomotor-enhancing action of systemic and intracerebroventricular (i.c.v.) morphine was investigated in mice. Mice were i.c.v. injected with either PTX (0.25 and 0.5 micrograms) or saline as a control. The s.c. (5-20 mg/kg) and i.c.v. (7-30 nmol) administration of morphine produced a dose-related locomotor-enhancing action in control mice. The peak effect of morphine (30 nmol, i.c.v.)-induced hyperlocomotion was observed 90 min after the morphine injection. At the same time, morphine significantly increased dopamine (DA) metabolism in the limbic forebrain (nucleus accumbens and olfactory tubercle). Similarly, the selective mu-opioid receptor agonist [D-Ala2,N-MePhe4,Gly-ol5]enkephalin (DAGO, 4 nmol, i.c.v.) also significantly increased locomotor activity and DA metabolism in the limbic forebrain. Both morphine- and DAGO-induced hyperlocomotion and elevation of DA turnover were antagonized by pretreatment with the mu antagonist beta-funaltrexamine (beta-FNA). These results suggest that the locomotor-enhancing action of morphine results from the activation of central mu-opioid receptors, and that the activation of the mesolimbic DA system may be involved in the expression of morphine-induced hyperlocomotion in mice. Furthermore, pretreatment with PTX (0.5 micrograms, i.c.v., 6 days prior to the testing) significantly reduced hyperlocomotion and elevation of DA turnover in the limbic forebrain which had been induced by administrations of morphine (30 nmol, i.c.v.) and DAGO (4 nmol, i.c.v.). These findings suggest that the central PTX-sensitive GTP-binding protein (G-protein) mechanism may play an important role in opioids-induced locomotor-enhancing action. Furthermore, the activation of mesolimbic DA transmission by mu-opioid agonists may also be mediated by a PTX-sensitive G-protein mechanism in mice.

Topics: Analgesics; Analysis of Variance; Animals; Cerebral Ventricles; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Injections, Intraventricular; Injections, Subcutaneous; Kinetics; Male; Mice; Mice, Inbred Strains; Morphine; Motor Activity; Naltrexone; Narcotic Antagonists; Pertussis Toxin; Time Factors; Virulence Factors, Bordetella

DAMGO, a highly selective mu opioid agonist, is capable of stimulating the hypothalamo-pituitary-adrenal (HPA) axis to produce a dose-related elevation in plasma corticosterone (CS). The purpose of this study was to confirm that this action was mu receptor selective and to determine which of the mu receptors was involved using naloxonazine, a mu-1 receptor-selective antagonist. Experiments were done in male rats with chronic i.v. catheters and i.c.v. cannula guides. This enabled the withdrawal of serial blood samples in conscious unrestrained animals that were isolated in sound-attenuated one-way vision boxes. DAMGO, 8 and 16 micrograms administered i.c.v. caused significant and prolonged elevation of plasma CS. beta-funaltrexamine (beta-FNA) in progressively increasing doses (i.c.v.), antagonized the effect of DAMGO. The hormone response to DAMGO was unaffected by pretreatment with norbinaltorphimine or naltrindole (both i.c.v.). Naloxonazine, 50 micrograms, administered i.c.v. 18 hr before DAMGO did not antagonize the response to DAMGO. The same dose of naloxonazine given 2 hr before did reduce the response to DAMGO. Naloxonazine, 20 mg/kg i.v., given 18 hr before did not alter DAMGO's effect on plasma CS; however, the analgesic response to DAMGO in the same animals 24 hr later was antagonized by naloxonazine pretreatment. Pretreatment with beta-FNA or naloxonazine i.c.v. did not alter the plasma CS increase after exposure to ether vapor.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Analgesics; Animals; Corticosterone; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Hypothalamo-Hypophyseal System; Male; Naloxone; Naltrexone; Pituitary-Adrenal System; Rats; Rats, Sprague-Dawley; Receptors, Opioid, mu

Human neuroblastoma SK-N-SH cells, which contain both mu- and delta-opioid receptors, were grown under conditions that provided a mu:delta ratio of 1.5:1. Both receptors were down-regulated after 72 hr of exposure to 100 nM etorphine. Selective down-regulation was demonstrated using selective opioid agonists; the mu agonist Tyr-D-Ala2-Gly-(Me)Phe4-Gly-ol down-regulated mu- but not delta-opioid receptors, whereas prolonged exposure to the selective delta agonist D-Pen2,D-Pen5-enkephalin resulted in delta- but not mu-opioid receptor down-regulation. Morphine, which binds mu- as well as delta-opioid receptors, down-regulated both receptor subtypes. NG108-15 cells, which contain delta receptors exclusively, were also tested. NG108-15 cells did not exhibit delta-opioid receptor down-regulation when exposed to morphine. The discrepancy between the effect of chronic morphine treatment on delta receptors in SK-N-SH cells and in NG108-15 cells raised the question of whether the coexistence of mu receptors in the former allowed morphine to down-regulate delta receptors. The role of mu-opioid receptors in morphine-induced delta receptor down-regulation was studied by using the irreversible mu antagonist beta-funaltrexamine. Pretreatment of SK-N-SH cells with beta-funaltrexamine prevented down-regulation of delta receptors in response to chronic exposure to morphine but did not affect down-regulation of delta receptors in response to D-Pen2,D-Pen5-enkephalin. The experimental data indicate that morphine-induced delta-opioid receptor down-regulation is dependent on the presence of functional mu receptors in the same cell.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics; Down-Regulation; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Humans; Morphine; Naltrexone; Neuroblastoma; Pyrrolidines; Receptors, Opioid, delta; Receptors, Opioid, mu; Tumor Cells, Cultured

Receptor binding assays using [3H]DAGO ([D-Ala2,MePhe4-Gly5-ol]enkephalin) (mu), [3H]DPDPE ([D-Pen2,D-Pen5]enkephalin) (delta) and [3H]U-69593 (kappa) were done in guinea pig whole brain membranes. Agonist activity was determined in norbinaltorphimine or beta-funaltrexamine (beta-FNA) treated guinea pig ileum (mu and kappa, respectively) and beta-FNA-treated mouse vas deferens (delta). The compounds with highest affinity were the most potent at the mu-receptor. The selectivity observed in the binding affinities was also found in in vitro activity. No correlation was found between mu-affinity and selectivity; the highest affinity analog, lofentanil, was found to be among the least selective, while another high affinity analog, R30490, was the most mu-selective. The results show that not all fentanyls are highly mu-selective, and could produce actions through delta- and kappa-opiate receptors.

Topics: Analgesics, Opioid; Animals; Benzeneacetamides; Brain; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Fentanyl; Guinea Pigs; Ileum; Male; Mice; Naltrexone; Pyrrolidines; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Vas Deferens

1. The effects of pre-incubation with beta-funaltrexamine (beta-FNA) on the binding of [3H]-[D-Ala2, MePhe4, Gly-ol5]enkephalin ([3H]-DAMGO) to homogenates of guinea-pig brain and myenteric-plexus longitudinal muscle have been studied. 2. beta-FNA pretreatment of brain homogenates in Tris-HCl buffer reduced the amount of [3H]-DAMGO binding. This was principally due to a reduction in the maximal number of binding sites measurable. However, approximately 30% of sites labelled by 1 nM [3H]-DAMGO were insensitive to 1 microM beta-FNA. Similar findings were obtained when the alkylation was performed in brain homogenates prepared in Krebs solution buffered with HEPES. 3. beta-FNA pretreatment of whole myenteric-plexus longitudinal muscle strips caused an increase in the IC50 values of mu-agonists, but not of kappa-agonists. However, the binding of [3H]-DAMGO to homogenates of myenteric-plexus longitudinal muscle was not altered by pre-incubation with beta-FNA in Tris-HCl buffer. On the other hand when the pretreatment was carried out in whole tissue in Krebs solution, or in homogenates in the presence of NaCl and Gpp(NH)p, a marked reduction in [3H]-DAMGO binding was observed. 4. These results suggest that a low affinity form of the mu-opioid receptor is the physiologically relevant site for beta-FNA alkylation in the myenteric-plexus and that differences exist between mu-receptor systems in guinea-pig myenteric plexus and brain.

Topics: Alkylating Agents; Alkylation; Animals; Brain; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Guinea Pigs; In Vitro Techniques; Male; Myenteric Plexus; Naltrexone; Receptors, Opioid, mu

beta-Funaltrexamine (beta-FNA) is an irreversible mu antagonist and a reversible kappa agonist in in vivo and in vitro tests. However, whether it produces irreversible delta antagonism is controversial. In binding studies, it is clear that beta-FNA does not bind irreversibly (it does reversibly) to kappa receptors. Yet there is no consensus as to whether beta-FNA binds irreversibly to mu and/or delta receptors. In this study, irreversible binding of [3H]beta-FNA to opioid receptors was examined in rat forebrain sections in the presence of 200 mM NaCl and its distribution compared with those of mu and delta opioid receptors, labeled by [3H][D-Ala2,MePhe4,Gly-ol5]enkephalin ([3H]DAMGO) and [3H][D-Pen2,D-Pen5]enkephalin ([3H]DPDPE), respectively. Irreversible binding of [3H]beta-FNA was determined as the binding that remained following 5 washes at room temp. for 1, 5, 20, 20, and 20 min each. Non-specific binding was defined by including 10 microM naloxone, beta-chlornaltrexamine (beta-CNA), or beta-FNA in the incubation mixture. At 37 degrees C, specific irreversible binding of [3H]beta-FNA to opioid receptors reached a plateau at 10 nM in 60 min, and constituted 50-70% of total irreversible binding. Series of 4 sections of similar anatomical levels were labeled with [3H]DAMGO, [3H]beta-FNA, [3H]beta-FNA + 10 microM naloxone, beta-CNA, or beta-FNA, and [3H]DPDPE, resp., and exposed to [3H]-Ultrofilm. The distribution of [3H]beta-FNA (5 nM) irreversible labeling is very similar to that of [3H]DAMGO, i.e. patches and subcallosal streaks in caudate-putamen, patches in nucleus accumbens, dense labeling in thalamus, and more binding in the rostral than caudal striatum.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Autoradiography; Diencephalon; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Male; Naltrexone; Rats; Rats, Inbred Strains; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu; Telencephalon; Tritium

Using rats with chronic i.t. catheters, dose-response curves were carried out using the hot plate (HP) test for a number of receptor-preferring opioids. The ordering of activity (i.t. ED50; nmol) on the HP was: (D-Ala2, N-Me-Phe4, Gly5-ol) enkephalin (DAMGO 0.5); sufentanil (0.4); and morphine (6.6). To assess the effects of pretreatment with the irreversible mu ligand beta-funaltrexamine (beta-FNA), rats received saline, 0.2, 2.0 or 20.0 nmol of beta-FNA. After 24 hr, base-line response latencies were not different from control animals. Dose-response curves for morphine, sufentanil and DAMGO were then obtained. Pretreatment with beta-FNA resulted in a concentration-dependent rightward shift in the agonist dose-response curves with the dose-ratio values of the respective doses of beta-FNA being for morphine: 3.5, 15.7, and 37.3; sufentanil: 1.2, 1.9, and 5.3; and DAMGO: 1.9, 4.0 and 7.3. The slopes of the agonist dose-response curves also displayed mild reductions in slope, the magnitude of which was dependent upon beta-FNA pretreatment concentrations. Testing at 6 days after beta-FNA treatment revealed a significant recovery of effect. beta-FNA (20 nmol, i.t.) had no effect on a just maximally effective dose of ST-91 (90 nmol, i.t.), an alpha-2 adrenergic agonist. beta-FNA (20 nmol, i.t.), but not 2.0 nmol resulted in a modest but significant reduction in the effect of the delta agonist DPLPE (120 nmol). These data are interpreted as supportive of the hypothesis that the mu-preferring ligands differ in the number of spinal receptors that must be occupied to produce a given antinociceptive effect (i.e., they differ in intrinsic efficacy).

Topics: Analgesics; Animals; Binding, Competitive; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Fentanyl; Injections, Spinal; Male; Morphine; Naltrexone; Narcotic Antagonists; Rats; Rats, Inbred Strains; Receptors, Opioid; Receptors, Opioid, mu; Spinal Cord; Sufentanil

Morphine has been considered to be primarily a mu opiate receptor agonist. The present study was designed to determine if opiate receptor subtypes in addition to mu contribute to morphine analgesia at the level of the spinal cord. Extracellular activity of single wide dynamic range (WDR) neurons in the feline lumbar spinal cord were studied. Intrathecal administration of DAGO (selective mu agonist) or DPDPE (selective delta agonist) suppressed the noxiously (51 degrees C radiant heat) evoked activity of WDR neurons. Pretreatment with spinal beta-FNA (selective mu antagonist) antagonized the suppressive effects of spinal DAGO, but not that of DPDPE. Two doses of spinal morphine (200 and 400 micrograms) suppressed the noxiously evoked activity of WDR neurons confirming our previous report. Following beta-FNA pretreatment, the suppressive effects of morphine were reduced, however, when ICI 174,864 (selective delta antagonist) was co-administered with morphine on the spinal cord of the animals pretreated by beta-FNA, there was an even greater reduction in the neuronal suppression by morphine. Intravenous ICI 174,864 also reversed the suppressive effects of morphine in beta-FNA pretreated animals. beta-FNA antagonism of spinal morphine is evidence of the well-known mu receptor-mediating antinociception. However, antagonism by ICI 174,864 of morphine suppression in beta-FNA-pretreated animals demonstrates that morphine is capable of suppressing noxiously evoked activity of WDR neurons as a result of an interaction with delta receptors in addition to mu receptors at the level of spinal cord.

Topics: Analgesia; Animals; Cats; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine; Enkephalins; Evoked Potentials; Female; Hot Temperature; Injections, Spinal; Male; Morphine; Naltrexone; Narcotic Antagonists; Neurons; Pain; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu; Spinal Cord

In this study we investigated the development of cross-tolerance among intrathecally (i.t.)- administered mu and delta opioid receptor selective peptides in beta-funaltrexamine (beta-FNA)-treated mice. Tolerance to the antinociceptive effect of i.t. administered DPDPE was accomplished by administration of 16 nmol/mouse of DPDPE, i.t. 3 hr before testing in beta-FNA-treated mice (10 mumol/kg, s.c., 24 hr before the experiment). Cross-tolerance developed to the antinociceptive effect of i.t. administered DADLE but not to those of DSLET or DAMGO. DSLET (0.1 nmol/mouse i.t.) administration in beta-FNA-treated mice resulted in tolerance development to its antinociceptive effect. The same pretreatment resulted in a marginally significant increase in the antinociceptive ED50 value of DPDPE. There was no cross-tolerance to the antinociceptive effect of i.t. administered DADLE or DAMGO. These results provide further evidence for the existence of delta opioid receptor subtypes where DADLE and DPDPE interact with one site and DSLET with a different one.

Topics: Animals; Drug Tolerance; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Injections, Spinal; Male; Mice; Naltrexone; Narcotic Antagonists; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu; Spinal Cord

The effects of intracerebroventricular injections (10 microliters) of the mu-selective opioid peptide DAGO on apomorphine (0.1, 0.56, 1.0 and/or 3.0 mg/kg)-induced motor activity were investigated in the mouse using multi-dimensional behavioral analyses. A lower dose (0.1 mg/kg) of apomorphine failed to significantly affect motor activity, whilst higher doses (0.56, 1.0 and 3.0 mg/kg) of the drug produced a marked increase in linear locomotion, circling, rearing and/or grooming behaviors. DAGO (0.003 and 0.01 micrograms) did not significantly affect different behaviors. DAGO (0.01 micrograms) antagonized the apomorphine (1.0 mg/kg)-induced increase in behaviors such as rearing and grooming. However, DAGO (0.003 or 0.01 micrograms) did not affect behaviors induced by a 3.0 mg/kg dose of apomorphine. Furthermore, the effects of DAG]O on apomorphine-induced behaviors were fully reversed by treatment with the mu-selective alkylating agent beta-FNA (beta-funaltrexamine) (5.0 micrograms). These results suggest that mu opioid receptors play a principal role in the apomorphine-induced increase in rearing and grooming behaviors.

Topics: Animals; Apomorphine; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Female; Grooming; Injections, Intraventricular; Mice; Mice, Inbred Strains; Motor Activity; Naltrexone; Narcotic Antagonists; Receptors, Opioid; Receptors, Opioid, mu

We studied the effect of the mu antagonist, beta-funaltrexamine (beta-FNA) on deprivation and opioid-induced feeding. Intracerebroventricular pre-treatment of 20 h deprived rats with 0.1, 1, 10 and 20 nmol of beta-FNA decreased feeding by 24%, 50%, 50% and 38% during the first hour. Central administration of beta-FNA (0.1, 1 and 10 nmol) also decreased feeding induced by the mu opioid agonist, DAMGO by 57%, 60% and 71%. Feeding induced by the delta agonist, DSLET, was decreased by pre-treatment with beta-FNA; but only during the 1-2 h time points, a time when relatively little food was ingested. Intraventricular injection of beta-FNA failed to alter feeding stimulated by the kappa opioid agonist, U-50,488H. These data further substantiate a role for the opioid receptor in deprivation and opioid-induced feeding.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Feeding Behavior; Food Deprivation; Injections, Intraventricular; Male; Naltrexone; Narcotic Antagonists; Pyrrolidines; Rats; Rats, Inbred Strains; Receptors, Opioid; Receptors, Opioid, kappa

The effects of intracerebroventricular (i.c.v.) beta-funaltrexamine (beta-FNA) pretreatment at -24 or -6 h were studied on mu and delta opioid receptor binding and on antinociception produced by i.c.v. morphine in rats. Mu and delta opioid receptor binding in brain membrane preparations was performed with [3H][D-Ala2,MePhe4,Gly-ol5]enkephalin (DAGO) and [3H][D-Pen2,D-Pen5]enkephalin (DPDPE) as radiolabeled ligands, respectively. Effects of i.c.v. beta-FNA (24 h) on mu and delta binding depended on dosage. For [3H]DAGO binding, 3 micrograms beta-FNA did not affect either the Kd or Bmax, whereas 10 micrograms increased the Kd without changing the Bmax. beta-FNA pretreatment for 24 h did not alter [3H]DPDPE binding at 3 micrograms; at 10 micrograms, the Kd was increased with no change in the Bmax. Pretreatment with 10 micrograms beta-FNA for 6 h gave similar results to the 24-h treatment in mu binding, but did not change delta binding. When mu binding was performed on various brain regions, pretreatment with 10 micrograms beta-FNA for 24 h increased the Kd in all regions studied (the periaqueductal gray, thalamus, striatum and cortex). However, this pretreatment decreased the Bmax only in the periaqueductal gray (by 22%) and cortex (by 14%). Pretreatment of rats with beta-FNA (3 or 10 micrograms at -24 h), which by itself caused some hyperalgesia, greatly antagonized the antinociceptive effect of morphine (10 micrograms i.c.v.) in the hot-plate test. Our work with beta-FNA has revealed an apparent discrepancy between binding and behavioral results. This dichotomy may, in part, be the result of the limited distribution of beta-FNA to the periventricular area.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Brain; Cerebral Cortex; Corpus Striatum; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Injections, Intravenous; Kinetics; Male; Morphine; Naltrexone; Narcotic Antagonists; Nociceptors; Periaqueductal Gray; Rats; Rats, Inbred Strains; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu; Thalamus; Time Factors; Tritium

Four experiments were done to determine which receptor type(s) mediates the effects of third ventricular microinjections of four opioid peptide agonists on blood levels of glucose, free fatty acids, and corticosterone. Tests were performed in unanesthetized adult male albino rats having chronic intraventricular cannulas; blood samples were taken from the tail tip at 0, 15, 30, 60, 90, and 120 min postmicroinjection. In experiment 1, the agonists DAGO (Tyr-D-Ala-Gly-N-methyl-Phe-Gly-ol), beta-endorphin, DSLET (d-Ser2-Leu-enkephalin-Thr), and dynorphin A-(1-17) (0, 0.3, 1, 3, and 10 nmol/rat) produced three distinct patterns of changes in serum glucose, free fatty acid, and corticosterone values. Experiment 2 showed that the effects of DAGO and beta-endorphin were inhibited by prior injection with the opiate-receptor blocker naloxone (1 mg/kg sc) and that the effects of dynorphin were not diminished. Experiment 3 determined that dynorphin effects were also not diminished by naloxone given intraventricularly. Experiment 4 found that blockade of the mu-receptor by intraventricular pretreatment with the specific antagonist beta-funaltrexamine (20 micrograms/rat, 24 h before) completely abolished the effects of DAGO and beta-endorphin on glucose and corticosterone. The mu-receptor is critical to the mediation of the hyperglycemia and hypercorticosteronemia induced by the central administration of opiate agonists. These results imply that mu-opioid binding sites previously identified in central autonomic regions may be involved in the regulation of circulating glucose and corticosterone.

Topics: Animals; beta-Endorphin; Blood Glucose; Cerebral Ventricles; Corticosterone; Dynorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Fatty Acids, Nonesterified; Injections, Intraventricular; Male; Naltrexone; Narcotic Antagonists; Narcotics; Rats; Rats, Inbred Strains; Receptors, Opioid; Receptors, Opioid, mu

Our previous work suggests that opioid peptides modulate sensory nerves in the perfused rat mesentery. Therefore we tested the hypothesis that opioids are involved in the ongoing regulation of sensory nerve activity using selective opioid receptor antagonists. In the presence of guanethidine and methoxamine, transmural nerve stimulation caused a vasodilator response which was potentiated significantly by naloxone (3 x 10(-7) M). However, naloxone did not affect vasodilator responses to exogenous calcitonin gene related peptide. IC1 174.864 (3 x 10(-7) M), a selective delta receptor antagonist, had no effect on vasodilator responses to transmural nerve stimulation. In contrast CTOP (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Phe-Thr-NH2) (3 x 10(-7) M), a selective mu receptor antagonist, significantly inhibited vasodilator responses to transmural nerve stimulation, effects which were abolished by naloxone treatment. In preparations pretreated with beta-FNA (beta-funaltrexamine HCl), an irreversible mu receptor antagonist, naloxone no longer potentiated vasodilator responses to transmural nerve stimulation. These results suggest that potentiation of vasodilator responses to transmural nerve stimulation by naloxone may be due to blockade of mu receptors, resulting in a reduced inhibitory modulation by endogenous opioids. These findings support the contention that prejunctional opioid receptors on sensory nerves may play a role in modulating activity of the cardiovascular system.

Topics: Animals; Calcitonin Gene-Related Peptide; Electric Stimulation; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalins; Guanethidine; In Vitro Techniques; Male; Mesenteric Arteries; Methoxamine; Muscle, Smooth, Vascular; Naloxone; Naltrexone; Narcotic Antagonists; Neurons, Afferent; Perfusion; Rats; Rats, Inbred Strains; Somatostatin; Vasodilation

The effects of intracerebroventricular injection (10 microliters) of mu- and delta-selective opioid peptides on behaviors induced by the dopamine D2-selective agonist RU 24213 were investigated in the mouse, using multi-dimensional behavioral analyses. Fifteen to 30 min after the start of behavioral measurements, a 3.0 mg/kg dose of RU 24213 produced a marked increase in linear locomotion, circling, rearing and grooming behaviors. Although the mu-selective opioid peptide [D-Ala2,NMePhe4,Gly-ol5]enkephalin (DAGO) (0.003 and 0.01 microgram) itself did not significantly affect behaviors, DAGO (0.01 microgram) antagonized the RU 24213 (3.0 mg/kg)-induced increase in behaviors such as linear locomotion, circling, rearing, and grooming. Additionally, the effects of DAGO on RU 24213-induced behaviors were fully reversed by treatment with the mu-selective alkylating agent beta-funaltrexamine (beta-FNA) (5.0 micrograms). In contrast, the delta-selective opioid peptide [D-Pen2,L-Pen5]enkephalin (0.3 or 1.0 micrograms) had no marked effects on RU 24213 (3.0 mg/kg)-induced behaviors. These results suggest that mu- but not delta-opioid receptors play an inhibitory role in the behaviors induced by the selective activation of dopamine D2 receptors.

Topics: Analysis of Variance; Animals; Behavior, Animal; Dopamine Agents; Dose-Response Relationship, Drug; Drug Interactions; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Male; Mice; Motor Activity; Naltrexone; Phenethylamines

Opioids and opiates decrease the duration of action potentials and the amount of neurotransmitter released from sensory neurons. The mu-type opioid receptor, the binding site for morphine, is thought to act exclusively on K+ channels. Here, we show that activation of the mu receptor inhibits Ca2+ channels in rat sensory neurons; the effect is blocked by a mu antagonist and is not mimicked by kappa or delta receptor agonists. Both low-threshold (T-type) and high-threshold Ca2+ currents are partially suppressed. omega-Conotoxin-sensitive and omega-conotoxin-insensitive, high-threshold Ca2+ currents are inhibited. The kinetic effect on high-threshold current is like that caused by diminished rest potential: the transient component is selectively lost, whereas the sustained component is spared.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Amino Acid Sequence; Animals; Calcium Channels; Cells, Cultured; Electric Conductivity; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Molecular Sequence Data; Mollusk Venoms; Naltrexone; Narcotic Antagonists; omega-Conotoxin GVIA; Pyrrolidines; Rats; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu

beta-Funaltrexamine (beta-FNA) irreversibly blocks morphine analgesia, lethality and its inhibition of gastrointestinal transit, confirming that these actions involve mu receptors. In dose-response studies, beta-FNA antagonized all the actions with similar potencies (ID50 values of 12.1, 11.3 and 12.3 mg/kg, respectively). beta-FNA also reduced intra-cerebroventricular and intrathecal DAMGO analgesia equally well (ID50 values of 6.09 and 7.7 mg/kg, respectively). Naloxanazine blocked systemic morphine analgesia (ID50 value 9.5 mg/kg) and supraspinal DAMGO analgesia (ID50 value 6.1 mg/kg) as potently as beta-FNA. However, against spinal DAMGO analgesia, morphine's inhibition of gastro-intestinal transit or lethality, naloxonazine (ID50 values 38.8, 40.7 and 40.9 mg/kg, respectively) was significantly less active than beta-FNA (p less than 0.05). beta-FNA remains a valuable tool in the classification of mu opioid actions. Within the mu category, actions can be defined as either mu 1 (naloxonazine-sensitive) or mu 2 (naloxonazine-insensitive).

Topics: Animals; Drug Administration Schedule; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Injections, Subcutaneous; Male; Mice; Morphine; Naloxone; Naltrexone; Narcotic Antagonists; Receptors, Opioid; Receptors, Opioid, mu

This study examined the effect of beta-funaltrexamine (beta-FNA), an irreversible mu-receptor antagonist, on naltrexone-induced upregulation of mu-(mu cx + mu nex) and delta nex-opioid receptors. [The subscripts 'cx' and 'nex' denote binding sites 'in' (cx) and 'not in' (nex) the opioid receptor complex.] Rats were treated according to the following protocol. Two naltrexone or two placebo pellets were implanted subcutaneously in a nylon mesh on day 1. and were removed intact on day 8. Rats were given either saline or 20 nmol of beta-FNA in 10 microliters of saline (i.c.v.) on days 1, 3, 5 and 6, 60 min prior to implantation of the pellet. On day 9 frozen lysed-P2 membranes were prepared for assay of mu binding sites. In other experiments, membranes were depleted of mu-receptors by pretreatment with the site-directed acylating agent 2-(4-ethoxybenzyl)-l-diethylaminoethyl-5-isothiocyanatobenzimid azole.HCl (BIT) for assay of delta nex binding sites, using [3H] [D-ala2, D-leu5]enkephalin. The results demonstrated that beta-FNA did not upregulate the mu binding sites and also did not prevent naltrexone-induced upregulation of mu binding sites. Both beta-FNA and naltrexone increased the Bmax of delta nex binding sites and their effects were additive. These data suggest that the mechanism(s) responsible for antagonist-induced upregulation of opioid receptors are more complex than previously appreciated.

Topics: Animals; Brain; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine-2-Alanine; Enkephalins; In Vitro Techniques; Isothiocyanates; Kinetics; Naltrexone; Narcotic Antagonists; Oxymorphone; Rats; Rats, Inbred Strains; Receptors, Opioid; Receptors, Opioid, mu; Thiocyanates; Up-Regulation

From previous observations, we have suggested that an endogenous opioid input tonically inhibits the pressor neurons of the rostral ventrolateral medulla (RVLM) of rabbits. In the present studies, the specific opioid receptor subtype(s) which may be activated by such an endogenous innervation were examined using local administration of selective antagonists. The RVLM pressor region of chloralose-anesthetized and artificially ventilated rabbits was functionally identified by local administration of L-glutamate (5 nmol). Selective blockade of neither mu nor kappa receptors in the RVLM after bilateral injections of beta-funaltrexamine (300-900 pmol) or nor-binaltorphimine (1 nmol), respectively, had any effect on either mean arterial pressure or heart rate. Delta receptors were blocked with ICI 174,864 (30-300 pmol). After the highest dose, there was a significant pressor effect (+32 +/- 6 mm Hg, mean +/- S.E.), which was of immediate onset and rapid time course (approximately 15 min), and which was accompanied by a bradycardia. In contrast, vehicle injections or injection of an inactive analog (ICI 178,173) had no effects. These results, together with previous pharmacological and anatomical evidence, suggest that there exists an enkephalinergic input to the RVLM that tonically inhibits the presympathetic pressor neurons via activation of delta-opioid receptors.

Topics: Animals; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; Glutamates; Glutamic Acid; Hemodynamics; Male; Medulla Oblongata; Microinjections; Naltrexone; Narcotic Antagonists; Pressoreceptors; Rabbits; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu

Topics: Animals; Autoradiography; Brain; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; In Vitro Techniques; Male; Naltrexone; Rats; Rats, Inbred Strains; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu

Naloxone enantiomorphs were given intracerebroventricularly (i.c.v.) to rats socially deprived for a brief period of time (7-14 days) in order to ascertain the mediation of central opiate receptors in the reversal effect of opiate antagonists on the high systolic blood pressure induced by this type of stress. While the active enantiomorph ((-)-naloxone, 20 nmol per rat) lowered the elevated blood pressure, the (+)-enantiomorph (which shows a 10,000-fold lower affinity for opiate receptors) had no effect. Additionally, the antihypertensive effect induced by i.c.v. administration of an antagonist of the mu-opiate receptor (beta-funaltrexamine, 20 nmol per rat), but not of the delta-opiate receptor (ICI 174,864, 15 nmol per rat) pointed to the involvement of mu-opiate receptors as the endogenous component of the hypertensive response of rats to stress.

Topics: Animals; Blood Pressure; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; Hypertension; Injections, Intraventricular; Male; Naloxone; Naltrexone; Narcotic Antagonists; Rats; Rats, Inbred Strains; Receptors, Opioid; Social Isolation; Stereoisomerism; Stress, Psychological

A variety of data support the hypothesis of an opiate receptor complex composed of distinct, yet interacting mu and delta binding sites (termed mu cx and delta cx to indicate binding sites 'in the complex'), in addition to independent mu and delta binding sites, termed mu ncx and delta ncx, to indicate binding sites 'not in the complex'. Ligand binding studies using membranes and slide-mounted sections of rat brain support the hypothesis that the irreversible mu-antagonist beta-funaltrexamine (FNA) selectively alkylates the opiate receptor complex, altering the binding of mu agonists to the mu cx binding site and the binding of [3H][D-Ala2,D-Leu5]enkephalin to the delta cx site. Previous studies demonstrated that the chronic administration of morphine to rats selectively 'upregulates' the opiate receptor complex. In contrast, the chronic administration of naltrexone upregulates several types of opioid receptors, including kappa, the delta ncx binding site, and multiple binding sites labeled by mu agonists. A prediction based upon these observations is that, using [3H][D-Ala2,MePhe4,Gly-ol5]enkephalin to label mu binding sites, chronic morphine should upregulate only the mu cx binding site, whereas chronic naltrexone should additionally up-regulate the mu ncx binding site. In this study we test and confirm this hypothesis, using sensitivity to FNA to define the mu cx binding site. The implications of these data for models of the opioid receptors and the mechanism(s) of tolerance and dependence are discussed.

Topics: Animals; Benzomorphans; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; In Vitro Techniques; Male; Morphine; Naltrexone; Narcotic Antagonists; Rats; Rats, Inbred Strains; Receptors, Opioid; Receptors, Opioid, kappa; Receptors, Opioid, mu

beta-Funaltrexamine (beta-FNA) is an alkylating derivative of naltrexone. Considerable data support its use as an irreversible mu receptor antagonist. However, pretreatment of rats with beta-FNA attenuates the ability of delta antagonists and naloxone to reverse delta receptor-mediated physiological effects, suggesting that physically adjacent mu and delta receptors interact in vivo. The purpose of this study was to determine which opiate receptor subtype is altered by i.c.v. injections of beta-FNA, as well as by in vitro incubations with beta-FNA, and then to examine the hypothesis that pretreatment of rats with beta-FNA increases the IC50 for naloxone at the altered binding site. The results demonstrate that beta-FNA alters the conformation of the opiate receptor complex, as evidenced by a decrease in the Bmax of the lower affinity [3H]D-Ala2-D-Leu5-enkephalin binding site and a doubling of the naloxone IC50 for displacing [3H]D-Ala3-D-Leu5-enkephalin from this site. [3H]D-Ala2-MePhe4,Gly-ol5-enkephalin binding sites were not detectably altered by i.c.v. injections of beta-FNA. These data collectively support the concept of coupling among opioid receptor subtypes.

Topics: Animals; Binding Sites; Binding, Competitive; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; Injections, Intraventricular; Male; Naloxone; Naltrexone; Rats; Rats, Inbred Strains; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu

Experiments were performed to test the hypothesis that the field-stimulated rat vas deferens preparation contains opioid receptors, other than of mu-type, which mediate part of the inhibitory effect of beta-endorphin. The Piebald Viral Glaxo strain of rats was used. The reported finding that delta-opioid receptors are present in Sprague-Dawley rat vas deferens, the effects of which are greatly enhanced in reduced calcium concentrations, could not be replicated in the rat strain used. Reducing the calcium concentration from 2.5 to 1.25 mM improved the response to opioid drugs: all full agonists were about 10 times more potent, the partial agonist normorphine became able to inhibit the twitch completely, and morphine (which behaves as a competitive antagonist in 2.5 mM Ca2+) appeared to behave as a partial agonist. The pA2 values for antagonism by naloxone in low calcium of the mu-selective peptide [D-Ala2,MePhe4,Gly(ol)5]enkephalin and other mu- or delta-selective agonists were consistent with an action at mu-receptors only. The value for beta-endorphin was slightly but significantly lower. A similar small discrepancy was found with two other competitive antagonists. The discrepancy remained in the presence of the peptidase inhibitors thiorphan, bestatin and bacitracin. Responses to both [D-Ala2,MePhe4,Gly(ol)5]enkephalin and beta-endorphin were attenuated by the irreversible antagonists beta-funaltrexamine and beta-chlornaltrexamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Calcium; Endorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; In Vitro Techniques; Male; Muscle Contraction; Naloxone; Naltrexone; Rats; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, kappa; Vas Deferens

The possibility that the opioid delta-receptor mediates antinociception in tests where heat is the noxious stimulus was investigated using highly selective mu- and delta-agonist and -antagonists. Antinociceptive dose-response curves were constructed for mu ([D-Ala2,NMePhe4,Gly-ol]enkephalin, DAGO; morphine) and delta ([D-Pen2,D-Pen5]enkephalin, DPDPE)-agonists in the absence, and in the presence of the mu non-surmountable antagonist, beta-funaltrexamine (beta-FNA) or the delta-antagonist ICI 174,864 (N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH, where Aib is alpha-amino-isobutyric acid). Agonists and ICI 174,864 were given alone in the same intracerebroventricular (i.c.v.) or intrathecal (i.th.) injection to mice 20 min prior to testing in the warm-water (55 degrees C) tail-withdrawal test (+10 min for i.th. DPDPE); beta-FNA was given as a single i.c.v. or i.th. pretreatment dose (20 and 0.01 nM, respectively) 4 h prior to testing. I.c.v. pretreatment with beta-FNA resulted in a rightward displacement of the DAGO and morphine antinociceptive dose-response lines, but failed to displace the i.c.v. DPDPE curve. Similarly, i.th. pretreatment with beta-FNA displaced the i.th. morphine dose-response curve to the right without affecting the i.th. DPDPE antinociceptive dose-response line. ICI 174,864 (1 and 3 micrograms) produced a dose-related antagonism of i.c.v. or i.th. DPDPE, but did not alter the antinociceptive effects of DAGO or morphine given by the same routes. Co-administration of ICI 174,864 (3 micrograms) with i.c.v. morphine in beta-FNA pretreated (but not control) mice resulted in a further rightward displacement of the morphine dose-response line.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Brain; Dose-Response Relationship, Drug; Drug Combinations; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine; Enkephalins; Hot Temperature; Injections, Intraventricular; Injections, Spinal; Male; Mice; Mice, Inbred ICR; Morphine; Naltrexone; Nociceptors; Pain; Receptors, Opioid; Receptors, Opioid, delta; Spinal Cord

The opioid receptor types involved in the mediation of enkephalin-induced electroencephalographic (EEG) seizures were studied in unanesthetized, freely moving rats. Four receptor-selective peptide ligands were evaluated for effectiveness in producing nonconvulsive EEG seizures after i.c.v. administration; these included the mu agonist, [D-Ala2-N-methyl-Phe4-Gly5-ol]enkephalin (DAGO), the mixed mu-delta agonist, [D-Ala2-D-Leu5]enkephalin (DADLE), and the selective delta agonists, [D-Pen2-D-Pen5]enkephalin and [D-Pen2-L-Pen5]enkephalin. Only DAGO and DADLE were found to produce EEG seizures, with DAGO being 9 times more potent than DADLE. DAGO produced a greater number of seizure episodes with a greater overall incidence compared with DADLE, reflecting its potent effect to elicit EEG seizure activity in these rats. Injections of [D-Pen2-D-Pen5]enkephalin or [D-Pen2-L-Pen5]enkephalin, even at the highest doses tested, failed to produce seizure activity. Behaviorally, the DAGO and DADLE EEG seizures were nonconvulsive but were temporally associated with episodic bursts of wet-dog shakes. The enkephalin-induced responses were extremely sensitive to antagonism by naloxone and completely blocked by pretreatment with the irreversible mu antagonist beta-funaltrexamine. The selective delta opioid receptor antagonist ICI 174,864 (N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH) was ineffective. The use of the most selective agonists and antagonists for mu and delta opioid receptors suggests that, in rats, enkephalin-induced EEG seizures are mediated exclusively by mu opioid receptors and not by delta opioid systems.

Topics: Animals; Electroencephalography; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; Male; Naloxone; Naltrexone; Rats; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu; Seizures

The morphine-like discriminative stimulus effects of opioid peptides with selectivity for the mu- or delta-opioid receptors were examined in rats trained to discriminate 3.0 mg/kg of morphine (s.c.) from saline in a two-choice discrete-trial avoidance paradigm. The mu-selective peptides D-Ala2-NMePhe4-Gly5(ol)enkephalin, FK 33,824 and morphiceptin, the delta-selective peptides D-Ala2-D-Leu5enkephalin and metkephamid and beta-endorphin (mu- and delta-selective) produced morphine-like stimulus effects after administration into the lateral ventricle. Generalization with the morphine cue was dose-dependent and occurred over a wide range of doses (0.01-30 micrograms), depending upon peptide. On a molar basis, the order of relative potency of the peptides as morphine-like discriminative stimuli was: D-Ala2-NMePhe4-Gly5(ol)enkephalin = FK 33,824 greater than beta-endorphin greater than D-Ala2-D-Leu5enkephalin = metkephamid greater than morphiceptin. The discriminative effects of D-Ala2-NMePhe4-Gly5(ol)enkephalin, D-Ala2-D-Leu5enkephalin and beta-endorphin were antagonized by low doses of s.c. naltrexone (0.01-1.0 mg/kg). Furthermore, the stimulus effects of s.c. morphine were antagonized by 24-hr pretreatment of rats with the irreversible mu-antagonist beta-funaltrexamine (5.0 micrograms i.c.v.). Based upon the order of relative potency of the peptides and the relative potency for antagonism of their discriminative effects by naltrexone and beta-funaltrexamine, mu-opioid receptors in the brain appear to be an important element in the genesis of morphine-like discriminative effects by opioid peptides.

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

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

The effect of beta-funaltrexamine (beta-FNA), an irreversible mu receptor blocker in isolated tissue bioassays, on mu, kappa and delta opioid receptor binding and the binding of beta-[3H]FNA were determined in guinea pig brain membranes. beta-FNA inhibited the binding of mu, kappa and delta opioid ligands to their receptors with Ki values of 2.2, 14 and 78 nM, respectively. Pretreatment of brain membranes with beta-FNA (less than 2 microM) followed by extensive washing inhibited mu binding and to a lesser degree delta binding, without changing kappa binding. The extent of the irreversible inhibition was dependent on the concentration of beta-FNA, and this inhibition on mu binding could be observed with as little as 1 nM beta-FNA. The irreversible inhibition of mu binding by beta-FNA pretreatment was due to a decrease in the number of binding sites with little change in Kd, and was more pronounced in the presence of increasing concentrations of NaCl. Specific binding of beta-[3H]FNA to opioid receptors was demonstrated. The rate of specific binding with 2 nM beta-[3H]FNA was rapid in the initial 10 min and did not reach maximum in 90 min. The dissociation of bound beta-[3H]FNA (5 nM added) by the addition of excess unlabeled naloxone reached maximum at 30 min with approximately 35% of specifically bound beta-[3H]FNA remaining. Mu opioids were most effective in preventing specific binding of beta-[3H]FNA when added before beta-[3H]FNA. Opioids added 1 hr after 2 nM beta-[3H]FNA could displace maximally only 70 to 75% of specific binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Brain; Cyclazocine; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Enkephalins; Ethylketocyclazocine; Ethylmaleimide; Guinea Pigs; Kinetics; Male; Membranes; Naloxone; Naltrexone; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Sodium Chloride