Reciprocal opioid-opioid interactions between the ventral tegmental area and nucleus accumbens regions in mediating mu agonist-induced feeding in rats

Feeding elicited by the mu-selective agonist, [D-Ala2, M-Phe4, Gly-ol5]-encephalin administered into the nucleus accumbens is blocked by accumbal pre-treatment with mu, delta1, delta2 and kappa, but not mu1 opioid antagonists. Correspondingly, mu-agonist-induced feeding elicited from the ventral tegmental area is blocked by ventral tegmental area pre-treatment with mu and kappa, but not delta opioid antagonists. A bi-directional opioid-opioid feeding interaction has been firmly established such that mu-agonist-induced feeding elicited from the ventral tegmental area is blocked by accumbal naltrexone, and that accumbal mu-agonist-induced feeding is blocked by naltrexone pre-treatment in the ventral tegmental area. To determine which opioid receptor subtypes mediate the regional bi-directional opioid-opioid feeding interactions between these two sites, the present study examined the dose-dependent ability of either general (naltrexone), mu (beta-funaltrexamine), kappa (nor-binaltorphamine) or delta (naltrindole) opioid antagonists administered into one site to block mu-agonist-induced feeding elicited from the other site. General, mu and kappa, but not delta opioid receptor antagonist pre-treatment in the ventral tegmental area dose-dependently reduced mu-agonist-induced feeding elicited from the nucleus accumbens. General, mu and delta, and to a lesser degree kappa, opioid receptor antagonist pre-treatment in the nucleus accumbens dose-dependently reduced mu-agonist-induced feeding elicited from the ventral tegmental area. Thus, multiple, but different opioid receptor subtypes are involved in mediating opioid-opioid feeding interactions between the nucleus accumbens and ventral tegmental area regions.     

Guanine Nucleotide and Cation Regulation of μ, δ, and k Opioid Receptor Binding: Evidence for Differential Postnatal Development in Rat Brain

A study of the onset of cation and guanine nucleotide regulation of delta, mu, and kappa rat brain opioid receptors during postnatal development was undertaken. Site-specific binding assays were utilized for each receptor type and the effects of 0.5 mM MnCl2, 100 mM NaCl, and/or 50 microM guanosine-5'-(beta, gamma-imido) triphosphate [Gpp(NH)p] were assessed. The most pronounced changes of opioid binding were seen in the presence of Mn2+. In adults, agonist binding to delta sites was stimulated by Mn2+, whereas that to mu sites was not affected and kappa binding was inhibited. The postnatal development of Mn2+ regulation for the three receptor subtypes was distinctly different. The largest effects were seen on delta sites detected in the early neonatal period, Mn2+ eliciting a 68% stimulation of binding over controls at day 1. Significant inhibition of kappa site binding by Mn2+ was detected only after the third postnatal week. Mn2+ caused a significant reversal of Gpp(NH)p inhibition of delta binding in the early neonatal period, exceeding that in the absence of regulators. Inhibition of mu and delta receptor binding by Na+ was greater, and the Mn2+ reversal of this effect was smaller, in the first 2 postnatal weeks than in adults. Gpp(NH)p + Na+ regulation did not change appreciably during the postnatal period. However, Mn2+ reversal of the considerable inhibition elicited by the combination of Na+ and Gpp(HN)p was developmental time-dependent. The data are discussed in terms of multiple sites of interaction for guanine nucleotides and cations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solubilization and Characterization of Opioid Binding Sites from Frog (Rana esculenta) Brain

Active opioid receptors were solubilized from frog (Rana esculenta) brain membrane fractions by the use of 1% digitonin. It was found by kinetic as well as by equilibrium measurements that both the membrane and the solubilized fractions contain two binding sites. For the membrane preparations, KD values were 0.9 and 3.6 nM, and Bmax values were 293 and 734 fmol/mg protein. For the solubilized preparations, KD values were 0.4 and 2.6 nM, an Bmax values were 35 and 266 fmol/mg protein. The stereospecificity of the binding did not change during solubilization. Both the membrane-bound and the solubilized receptors showed weak binding of enkephalin and mu-specific drugs, suggesting that they are predominantly of the kappa-type. The membrane-bound and the soluble receptors showed the same distribution of subtypes, i.e., 70% kappa, 13% mu, and 17% delta for the membrane-bound and 71% kappa, 17% mu, and 12% delta for the soluble receptors.     

Mixed kappa agonists and mu agonists/antagonists as potential pharmacotherapeutics for cocaine abuse: synthesis and opioid receptor binding affinity of N-substituted derivatives of morphinan

A series of new N-substituted derivatives of morphinan was synthesized and their binding affinity for the three opioid receptors (mu, delta, and kappa) was determined. A paradoxical effect of N-propargyl (MCL-117) and N-(3-iodoprop-(2E)-enyl) (MCL-118) substituents on the binding affinities for the mu and kappa opioid receptors was observed. All of these novel derivatives showed a preference for the mu and kappa versus delta binding.     

Exploration of universal cysteines in the binding sites of three opioid receptor subtypes by disulfide-bonding affinity labeling with chemically activated thiol-containing dynorphin A analogs.

A ligand containing an SNpys group, i.e. 3-nitro-2-pyridinesulfenyl linked to a mercapto (or thiol) group, can bind covalently to a free mercapto group to form a disulfide bond via the thiol-disulfide exchange reaction. This SNpys chemistry has been successfully applied to the discriminative affinity labeling of mu and delta opioid receptors with SNpys-containing enkephalins [Yasunaga, T. et al. (1996) J. Biochem. 120, 459-465]. In order to explore the mercapto groups conserved at or near the ligand binding sites of three opioid receptor subtypes, we synthesized two Cys(Npys)-containing analogs of dynorphin A, namely, [D-Ala2, Cys(Npys)8]dynorphin A-(1-9) amide (1) and [D-Ala2, Cys(Npys)12]dynorphin A-(1-13) amide (2). When rat (mu and delta) or guinea pig (kappa) brain membranes were incubated with these Cys(Npys)-containing dynorphin A analogs and then assayed for inhibition of the binding of DAGO (mu), deltorphin II (delta), and U-69593 (kappa), the number of receptors decreased sharply, depending upon the concentrations of these Cys(Npys)-containing dynorphin A analogs. It was found that dynorphin A analogs 1 and 2 effectively label mu receptors (EC50 = 27-33 nM), but also label delta receptors fairly well (160-180 nM). However, for kappa receptors they showed drastically different potencies as to affinity labeling; i.e., EC50 = 210 nM for analog 1, but 10,000 nM for analog 2. Analog 2 labeled kappa receptors about 50 times more weakly than analog 1. These results suggested that dynorphin A analog 1 labels the Cys residues conserved in mu, delta, and kappa receptors, whereas analog 2 only labels the Cys residues conserved in mu and delta receptors.     

Characterization of K Opioid Receptors in Neurosecretosomes from Bovine Posterior Pituitary

The binding properties of opioid receptors on isolated nerve terminals (neurosecretosomes) from bovine posterior pituitaries were characterized. Both [3H]etorphine and [3H]ethylketocyclazocine ([3H]EKC) showed high-affinity binding with complex binding isotherms, consistent with the presence of multiple classes of binding sites. [D-Ala2,D-Leu5]enkephalin showed no specific binding and failed to displace [3H]etorphine at high concentrations, indicating the absence of mu, delta, or benzomorphan (kappa 2) sites. Mathematical modelling of the data suggested the presence of three classes of binding sites. The first was of high affinity with Kd values of 0.9 and 2.0 nM for etorphine and EKC, respectively. The second class of sites appeared to bind etorphine with a KD of 150 nM, and EKC with extremely low affinity (unmeasurable binding). The third class of sites was characterized by KD values of 7 and 2 microM for etorphine and EKC, respectively. These results indicate that the nerve terminals of bovine posterior pituitary contain opioid binding sites of the kappa type. Furthermore, these binding sites appear heterogeneous, consisting of at least two and possibly more subtypes or states.     

Inhibition of mu and delta but not kappa opioid binding to membranes by Fab fragments from a monoclonal antibody directed against the opioid receptor

Fab fragments from a monoclonal antibody, OR-689.2.4, directed against the opioid receptor, selectively inhibited opioid binding to rat and guinea pig neural membranes. In a titratable manner, the Fab fragments noncompetitively inhibited the binding of the mu selective peptide [D-Ala2,(Me)Phe4,Gly(OH)5][3H] enkephalin and the delta selective peptide [D-Pen2,D-Pen5] [3H]enkephalin (where Pen represents penicillamine) to neural membranes. In contrast, kappa opioid binding, as measured by the binding of [3H]bremazocine to rat neural membranes and guinea pig cerebellum in the presence of mu and delta blockers, was not significantly altered by the Fab fragments. In addition to blocking the binding of mu and delta ligands, the Fab fragments displaced bound opioids from the membranes. When mu sites were blocked with [D-Ala2,(Me)Phe4,Gly(OH)5]enkephalin, the Fab fragments suppressed the binding of [D-Pen2,D-Pen5][3H]enkephalin to the same degree as when the mu binding site was not blocked. The Fab fragments also inhibited binding to the mu site regardless of whether or not the delta site was blocked with [D-Pen2,D-Pen5]enkephalin. This monoclonal antibody is directed against a 35,000-dalton protein. Since the antibody is able to inhibit mu and delta binding but not kappa opioid binding, it appears that this 35,000-dalton protein is an integral component of mu and delta opioid receptors but not kappa receptors.     

RTI-4614-4: an analog of (+)-cis-3-methylfentanyl with a 27,000-fold binding selectivity for mu versus delta opioid binding sites.

The objective of this study was to determine the binding affinities of (+/-)-cis-N-[1-(2-hydroxy-2-phenylethyl)-3-methyl-4-piperidyl]- N-phenylpropanamide-HCI (RTI-4614-4), which is an analog of (+)-cis-3-methylfentanyl for opioid receptor subtypes. The Ki values (nM) of this agent for opioid receptor subtypes were as follows: mu (0.0055), delta (148), kappa 1 (84.8), kappa 2a (2275), and kappa 2b (22.3). The selectivity of this agent for the mu binding site was 27,000 vs. the delta binding site, 15,400 vs. the kappa 1 binding site, 413,700 vs the kappa 2a and 4,054 vs the kappa 2b binding site. In contrast, two other fentanyl analogs, N-(2-(4-methylpyridinyl))-N-(1-phenethyl-4-piperidinyl) 2-furamide and N-(2-pyrazinyl)-N-(1-phenethyl-4-piperdinyl)2-furamide had considerably higher Ki values at, and were less selective for, the mu binding site. Since RTI-4614-4 is composed of a mixture of four stereoisomers, the resolution of these isomers should permit identification of an extremely potent and selective agent for the opioid mu receptor.     

Kinetics and Physical Parameters of Rat Brain Opioid Receptors Solubilized by Digitonin and CHAPS

Rat brain opioid receptors were solubilized with digitonin and a zwitterionic detergent, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS). The yield of solubilization was 70-75% with digitonin and 30-35% with CHAPS. Kinetic and equilibrium studies performed from digitonin extracts resulted in KD values comparable with those of the membrane fractions. Two [3H]naloxone binding sites were obtained in the extracts similarly to membrane fractions. The rank order potency of drugs used in the competition experiments did not change during solubilization. The distributions of mu, delta, and kappa opioid receptor binding sites were similar in membrane and digitonin-solubilized fractions (48-50% mu, 35-37% kappa, and 13-17% delta subtypes). The hydrodynamic properties of digitonin- and CHAPS-solubilized preparations were studied by sucrose density gradient centrifugation and Sepharose-6B chromatography. In all cases, two receptor populations were identified with the following parameters: sedimentation coefficients for the digitonin extracts were 9.2S and 13.2S and for CHAPS extract 8S and 15.6S; the Stokes radii were 45 A and 65A for the digitonin extract and 31A and 76A for the CHAPS-solubilized preparation.     

Expression of delta, kappa and mu human opioid receptors in Escherichia coli and reconstitution of the high-affinity state for agonist with heterotrimeric G proteins.

Human opioid receptors of the delta, mu and kappa subtypes were successfully expressed in Escherichia coli as fusions to the C-terminus of the periplasmic maltose-binding protein, MBP. Expression levels of correctly folded receptor molecules were comparable for the three subtypes and reached an average of 30 receptors.cell-1 or 0.5 pmol.mg-1 membrane protein. Binding of [3H]diprenorphine to intact cells or membrane preparations was saturatable, with a dissociation constant, KD, of 2.5 nM, 0.66 nM and 0.75 nM for human delta, mu and kappa opioid receptors (hDOR, hMOR and hKOR, respectively). Recombinant receptors of the three subtypes retained selectivity and nanomolar affinity for their specific antagonists. Agonist affinities were decreased by one to three orders of magnitude as compared to values measured for receptors expressed in mammalian cells. The effect of sodium on agonist binding to E. coli-expressed receptors was investigated. Receptor high-affinity state for agonists was reconstituted in the presence of heterotrimeric G proteins. We also report affinity values of endomorphins 1 and 2 for mu opioid receptors expressed both in E. coli and in COS cells. Our results confirm that opioid receptors can be expressed in a functional form in bacteria and point out the advantages of E. coli as an expression system for pharmacological studies.     

Upregulation of opioid receptor subtypes correlates with potency changes of morphine and DADLE

Chronic treatment with opioid antagonists increases the potency of opioid agonists and produces an increase in brain opioid binding sites. In the present study, 8 day treatment with naltrexone blocked morphine and DADLE analgesia for the entire treatment period and increased mu 1, mu 2 and delta opioid receptor binding sites in mouse brain. mu 1 and mu 2 binding were increased by 81 and 67%, respectively, while delta binding was increased by 31%. Consistent with these binding changes, the potency of ICV morphine to produce analgesia was increased by over 3-fold, while the potency of ICV DADLE was increased by only 1.7. These findings indicate that relative increases in opioid receptor subtypes agree with pharmacodynamic studies on potency changes of opioid agonists.     

Subunit Structure and Purification of Opioid Receptors

Abstract

Considerable evidence indicates the existence of multiple types of opioid receptors. The three major types have been named mu, delta and kappa. The earlier evidence was based on pharmacological as well as membrane binding experiments. This paper will emphasize more recent studies using solubilized opioid binding sites.

Several laboratories, including our own, have succeeded in separating kappa receptors from other types. A similar separation of mu from delta receptors has not yet been achieved. By crosslinking experiments with ¹²⁵I- human beta-endorphi we have been able to provide strong evidence for differences in molecular size between the major binding components of mu (65K) and delta (53K) receptors… It is not yet established whether the difference resides in the in the protein or carbohydrate portion of these glycoproteins. These results suggest that the three major types of opioid receptors represent distinct molecular entities.

An active opioid binding protein solubilized from bovine striatal membranes has been purified to apparent homogeneity. The major purification steps involve affinity chromatography and lectin chromatography on immobilized wheat germ agglutinin. The purified material gave a single band of molecular weight 65K Da on SDS-PAGE. Its specific activity for opioid binding was ca. 13000 pmol/mg protein and its properties are those of a component of the mu receptor.     

A human neuroblastoma cell line expresses mu and delta opioid receptor sites

A series of neuroblastoma cell lines were screened for the presence of opioid receptor sites with the tracers [3H]diprenorphine (mu, delta, kappa ligand) and [3H]naloxone (mu-selective ligand). One human neuroblastoma cell line, SK-N-SH, displayed avid binding for both tracers. Binding experiments with multiple tracers revealed the presence of both mu and delta sites. These sites were stereospecific, saturable, and proteinaceous in character. Saturation binding experiments provided an estimate of 50,000 mu and 10,000 delta sites/cell. NaCl (100 mM) and guanine nucleotide, guanylyl imidodiphosphate (50 microM), reduced opioid agonist but not antagonist binding to these sites. Etorphine at 1 nM inhibited prostaglandin E1-stimulated cyclic AMP production by approximately 20%, which was reversible by naloxone. The opioid-binding sites on SK-N-SH cells closely resemble the previously reported mu and delta sites in human and rodent brain. Therefore, the SK-N-SH neuroblastoma cell line represents a useful tool to study the molecular functions of opioid receptors.     

Localization of kappa opioid receptor binding sites in human forebrain using [3H]U69,593: Comparison with [3H]bremazocine

1. The autoradiographic distribution of kappa opioid receptor binding sites in human brain was examined using two radiolabeled probes, namely [3H]U69,593 and [3H]bremazocine. 2. [3H]U69,593 binding was performed in the absence of blockers for other sites, while [3H]bremazocine binding was investigated in the presence of saturating concentrations of mu and delta blockers to ensure selective labeling of kappa opioid receptors. 3. Our results show that the autoradiographic distribution of [3H]U69,593 and [3H]bremazocine (plus blockers) binding sites is identical, with high densities of sites found in deep cortical layers and claustrum. 4. This indicates that [3H]U69,593 is a highly selective ligand of the kappa opioid receptor type.     

Desipramine modulation of sigma and opioid peptide receptor expression in glial cells.

Exposure of C6 glial cell cultures to desipramine induced the appearance of opioid receptors and up-regulated sigma receptors. Opioid binding was demonstrated with 3H-etorphine and 3H-dihydromorphine (DHM), but was not observed with the mu, delta and kappa ligands 3H-DAMGE, 3H-DADLE or 3H-(-)ethylketocyclazocine in the presence of specific blockers, respectively. Competition experiments with 3H-DHM and either (-)naloxone or (+)naloxone indicated the presence of authentic opioid receptors. In similar studies with beta-endorphin, its truncated form (1-27) or their N-acetyl derivatives, beta-endorphin proved to have the highest affinity. Opioid receptors in glial cell aggregates were primarily kappa, with few mu and delta sites. Desipramine increased Bmax values for kappa but not mu and delta.     

Comparison of opioid receptor distributions in the rat central nervous system

The opioid receptors, mu, delta and kappa, conduct the major pharmacological effects of opioid drugs, and exhibit intriguing functional relationships and interactions in the CNS. Previously established hypotheses regarding the mechanisms underlying these phenomena specify theoretical patterns of relative cellular localisation for the different receptor types. In this study, we have used double-label immunohistochemistry to compare the cellular distributions of delta and kappa receptors with those of mu receptors in the rat CNS. Regions of established significance in opioid addiction were examined. Extensive mu/delta co-localisation was observed in neuron-like cells in several regions. mu and kappa receptors were also often co-localised in neuron-like cell bodies in several regions. However, intense kappa immunoreactivity (ir) also appeared in a separate, morphologically distinct population of cells that did not express mu receptors. These small, ovoid cells were often closely apposed against the larger, mu-ir cell bodies. Such cellular appositions were seen in several regions, but were particularly common in the medial thalamus, the periaqueductal grey and brainstem regions. These findings support proposals that functional similarities, synergy and cooperativity between mu and delta receptors arise from widespread co-expression by cells and intracellular molecular interactions. Although co-expression of mu and kappa receptors was also detected, the appearance of a separate population of kappa-expressing cells supports proposals that the contrasting and functionally antagonistic properties of mu and kappa receptors are due to expression in physiologically distinct cell types. Greater understanding of opioid receptor interaction mechanisms may provide possibilities for therapeutic intervention in opioid addiction and other conditions.     

Selective activation of opioid receptors differentially affects lordosis behavior in female rats

The effects of opioid peptides that are highly selective ligands for mu receptors (morphiceptin). delta receptors (delta-receptor peptide), kappa receptors (dynorphin 1-9), and the mu/delta complex (beta-endorphin), were tested on lordosis behavior in ovariectomized rats primed with estrogen and progesterone. Intracerebroventricular infusions of beta-endorphin or morphiceptin both inhibited and facilitated lordosis in a dose-dependent fashion whereas all doses of delta-receptor peptide facilitated lordosis. Dynorphin 1-9 had no significant effect at any dose, although a trend toward increased lordosis quotients was observed 30 min after infusion. The effects of beta-endorphin, morphiceptin, and delta-receptor peptide were reversed with naloxone, although naloxone alone had no effect on lordosis behavior. These results indicate that the specific activation of opioid receptor subtypes differentially affects lordosis behavior. It appears that binding to high-affinity mu 1 receptors exerts an inhibitory influence on lordosis, whereas binding to low-affinity mu 2 receptors or delta receptors exerts a facilitatory influence. Binding to kappa receptors does not appear to affect lordosis behavior.     

Different types of opiate agonists interact distinguishably with mu, delta and kappa opiate binding sites

The present studies were undertaken to evaluate whether different types of opiate agonists interact in a distinguishable manner with mu, delta and kappa opiate binding sites. Two approaches were employed: (a) the well known effects of metal ions on opiate agonist binding affinities of subsite selective ligands were studied at mu, delta and kappa sites in rat brain homogenates. Binding parameters were obtained by simultaneous computeranalysis of displacement curves using the prototypic ligands dihydromorphine (DHM), (D-Ala2, D-Leu5) enkephalin (DADL) and ethylketocyclazocine (EKC) of the mu, delta and kappa binding sites respectively. The results show that the effects of metal ions depend not only on the binding site, but also on the ligand under investigation. (b) The interaction of the delta agonist DADL with the mu agonist DHM was investigated at mu binding sites by characterizing the type of competition occurring between the two ligands. The interaction was of the noncompetitive type. It therefore appears that the various opiate agonists either interact preferentially with different parts of a larger receptor site area or bind to topographically distinct sites on a single receptor molecule which are coupled allosterically.     

NPY-induced feeding: pharmacological characterization using selective opioid antagonists and antisense probes in rats

The ability of neuropeptide Y to potently stimulate food intake is dependent in part upon the functioning of mu and kappa opioid receptors. The combined use of selective opioid antagonists directed against mu, delta or kappa receptors and antisense probes directed against specific exons of the MOR-1, DOR-1, KOR-1 and KOR-3/ORL-1 opioid receptor genes has been successful in characterizing the precise receptor subpopulations mediating feeding elicited by opioid peptides and agonists as well as homeostatic challenges. The present study examined the dose-dependent (5-80 nmol) cerebroventricular actions of general and selective mu, delta, and kappa1 opioid receptor antagonists together with antisense probes directed against each of the four exons of the MOR-1 opioid receptor gene and each of the three exons of the DOR-1, KOR-1, and KOR-3/ORL-1 opioid receptor genes upon feeding elicited by cerebroventricular NPY (0.47 nmol, 2 ug). NPY-induced feeding was dose-dependently decreased and sometimes eliminated following pretreatment with general, mu, delta, and kappa1 opioid receptor antagonists. Moreover, NPY-induced feeding was significantly and markedly reduced by antisense probes directed against exons 1, 2, and 3 of the MOR-1 gene, exons 1 and 2 of the DOR-1 gene, exons 1, 2, and 3 of the KOR-1 gene, and exon 3 of the KOR-3/ORL-1 gene. Thus, whereas the opioid peptides, beta-endorphin and dynorphin A(1-17) elicit feeding responses that are respectively more dependent upon mu and kappa opioid receptors and their genes, the opioid mediation of NPY-induced feeding appears to involve all three major opioid receptor subtypes in a manner similar to that observed for feeding responses following glucoprivation or lipoprivation.