Delta and mu opiate receptor probes: fluorescent enkephalins with high receptor affinity and specificity

The fluorescent amino acid, L-1-pyrenylalanine (Pya) was incorporated into [D-Ala2,Leu5]enkephalin and its methyl ester at position 4 or 5. Pya-enkephalins showed strong fluorescent intensity and displayed high binding affinity for opiate receptors. Pya4-enkephalins showed high specificity for the mu receptors, while Pya5-enkephalins showed high specificity and selectivity for the delta receptors. Particularly, [D-Ala2,Pya5]enkephalin was as potent as the most utilized delta-specific ligand of [D-Ala2,D-Leu5]enkephalin (DADLE), and yet its delta-selectivity was about 5-times greater than that of DADLE. Thus, Pya-enkephalins per se can be utilized as a fluorescent probe or tracer for the opiate receptor-binding assays.     

Synthesis and receptor binding characteristics of [D-Ala2, cysteamine 5] enkephalin, a thiol-containing probe for structural elements of opiate receptors

For the elucidation of structural elements in the opiate receptors, a thiol-containing enkephalin analog [D-Ala2, cysteamine 5]enkephalin, and its dimeric analog were synthesized and evaluated in the radio-ligand receptor binding assays using rat brain membranes. The dimeric analog was very potent in both delta and mu assays. Comparison of receptor affinities of the thiol-containing enkephalin with those of standard mu or delta receptor specific ligands suggested that the mu receptor contains an essential thiol group which may interact with the thiol group at the C-terminus of the enkephalin analog. It also appears that no metal-ion site, postulated for the delta receptors, is present in the delta binding site.     

Cystamine-enkephalin dimer. Syntheses and biological activities of enkephalin analogs containing cystamine and cysteamine

A cystamine-enkephalin dimer, containing two molecules of [D-Ala2, Leu5] enkephalin cross-linked at the COOH-terminal leucine residue with cystamine, (NH2-CH2-CH2-S-)2, has been synthesized in order to examine directly the dimerization effect of an enkephalin molecule on the opiate receptor interactions. In a comparison of potencies against [3H]-[D-Ala2,D-Leu5] enkephalin (3H-DADLE) and [3H]-[D-Ala2,MePhe4,Gly-ol5] enkephalin (3H-DAGO) as delta and mu tracers, respectively, enkephalin dimer showed a very high affinity, especially for the delta opiate receptors. Dimer was almost threefold more potent than DADLE, which is one of the most utilized delta ligand to date. When the binding affinity of cystamine-dimer was compared with that of its reduced thiol-monomer, namely [D-Ala2,Leu5,cysteamine6] enkephalin, the increment in affinity was four to fivefold for both delta and mu receptors. The results strongly indicate that the dimeric enkephalin is more potent presumably due to the simultaneous interaction with the two binding sites of the opiate receptors.     

Interaction of D-Ala2-[Tyr-3,5-3H]enkephalin(5-D-Leu) with opiate receptors of rat brain

Some kinetic features of D-Ala2-[Tyr-3.5-3H]enkephalin (5-D-Leu) binding to opiate receptors of rat brain were studied. It was shown that the Leu-enkephalin D analog interacts with the high and low affinity binding sites of opiate receptors, the equilibrium constants being equal to 0.71 and 8.4 nM, respectively. The rate constant for the label association with the high affinity binding sites in 2 . 10(8) M-1 min-1; those for the label dissociation from the opiate receptor binding sites with high and low affinities are 7.2 . 10(-3) and 0.16 min-1, respectively. Hence, the half-life time of these complexes is 95.7 and 4.3 min, respectively. Na+, K+ and Li+ markedly decrease the specific finding of the label, while Mg2+, Mn2+ and Ca2+ at the concentrations studied markedly increase its specific binding. It is concluded that the Leu-enkephalin D-analog under study acts as a morphine agonist and reveals a much higher affinity for rat brain opiate receptors than does Leu- or Met-enkephalin. This makes it a useful tool for study of the enkephalin reception under normal and pathological conditions.     

Enkephalin and alpha-adrenergic receptors: evidence for both common and differentiable regulatory pathways and down-regulation of the enkephalin receptor

Several clones of neuroblastoma-glioma NG108-15 hybrid cells were used to reveal whether the regulation of opiate receptor density interacts with the regulation of alpha-adrenergic or acetyl-choline receptors. Low density of alpha-adrenergic receptors in 3 selected clones was accompanied with similar reduction in the density of enkephalin receptors but not in muscarinic acetyl-choline receptors. Yet opiate antagonists that increased the number of opiate receptors in the parent NG108-15 cells in a stereospecific manner had no similar effect on the number of alpha-adrenergic receptors. Moreover, the stable enkephalin analogue D-ala-2-methionine enkephalinamide, but not the opiate alkaloid morphine, decreased the binding of 3H-DAMEA to the membranes and induced down-regulation of enkephalin receptors. Yet DAMEA had no effect on the binding of the alpha-adrenergic antagonist 3H-yohimbine. The study suggests that alpha-adrenergic and enkephalin receptors may share some common regulatory pathways but opiate peptides and antagonists selectively decrease or increase the density of enkephalin receptors, respectively, with no effect on alpha-adrenergic receptor density.     

Interaction of [3H](–)-SKF-10,047 with Brain σ Receptors: Characterization and Autoradiographic Visualization

The sigma opiates differ from other opiates in their stimulatory and psychotomimetic actions. The sigma opiate [3H](-)-SKF-10,047 has been used to characterize sigma receptors in rat nervous tissue. Binding of [3H](-)-SKF-10,047 to rat brain membranes was of high affinity, saturable, and reversible. Scatchard analysis revealed the apparent interaction of this drug with two distinct binding sites characterized by affinities of 0.03 and 75 nM (5 mM Tris-HCl buffer, pH 7.4, at 4 degrees C). Competition analyses involving rank order determinations for a series of opiates and other drugs indicate that the high-affinity binding site is the mu opiate receptor. The lower-affinity site (revealed after suppression of mu and delta receptor binding) has been identified as the sigma opiate/phencyclidine receptor. In vitro autoradiography has been used to visualize neuroanatomical patterns of receptors labeled using [3H](-)-SKF-10,047 in the presence of normorphine and [D-Ala2,D-Leu5]enkephalin to block mu and delta interactions, respectively. Labeling patterns differ markedly from those for mu, delta, or kappa receptors. The highest densities (determined by quantitative autoradiography) are found in the medial portion of the nucleus accumbens, amygdaloid nucleus, hippocampal formation, central gray, locus coeruleus, and the parabrachial nuclei. Receptors in these structures could account for the stimulatory, mood-altering, and analgesic properties of the sigma opiates. Although not the most selective sigma opiate ligand, [3H](-)-SKF-10,047 binds to sigma opiate receptors in brain, and this interaction can be readily distinguished from its interactions with other classes of brain opiate receptors.     

The effect of modification of the carboxyl group in short D-arginine 2-containing enkephalin analogs on their affinity and selectivity for opiate receptors

Radioreceptor binding assay using a membrane fraction from the rat brain was applied to study [D-Arg2, Leu5] enkephalin and two series of its analogues truncated at the C-terminus with a free or modified carboxyl group: tetra- and tripeptide amides and ethyl esters. The affinity to mu-specific opiate receptor subtype of the N-terminal [D-Arg2] tetrapeptide ethyl ester was 44 times as high as that of the tripeptide with a free carboxyl, and thus the ester retained up to 10% of leucine-enkephalin binding potency. However, a comparable esterification of the carboxyl group in the N-terminal [D-Arg2] tripeptide led to a 6-fold reduction in its affinity to mu-receptors. Consequently, identical modifications of the C-terminal carboxyl group in enkephalin analogues of various length can have completely different effects. Substitution of the natural glycine residue by D-arginine residue in position 2 of the enkephalin molecule truncated at the C-terminus increased the mu-receptor binding potency of the tetrapeptide, whereas its delta receptor binding potency declined by more than one order of magnitude. Simultaneous replacement of glycine2 by D-arginine2 and carboxyl amidation resulted in the short enkephalin analogue Tyr--D--Arg--Gly--Phe--NH2, whose affinity to mu receptors was four times as high as that of leucine--enkephalin, the tetrapeptide being 284 times more selective for the mu vs. delta opiate receptors.     

Interaction of S-activated enkephalin analogs with opiate receptors

Enkephalin analogs containing a thiol activated by a thiomethyl (SCH3)*** or 3-nitro-2-pyridinesulfenyl (Npys) group were synthesized. Incubation of such S-activated enkephalin analogs as [D-Ala2, Leu(CH2S)SCH(3)5]enkephalin or [D-Ala2,Leu(CH2S)Npys5]enkephalin with guinea pig ileum (GPI) resulted in the continuous stimulation of the mu opiate receptors. This sustained GPI-activity was completely reversed with the antagonist naloxone, while subsequent washings elicited again the full enkephalin activity. When GPI showing full enkephalin activity was incubated with 1 mM dithiothreitol, about 70% of the activity was eliminated. Examination of enkephalin analogs containing Cys(Npys) at position 1, 5, or 6 suggested that no other thiols occur near the enkephalin binding site of the mu receptor. From these results, it is considered that only one thiol group exists near the binding site of the mu receptor in GPI. Similar results were also obtained for the mu receptors in mouse vas deferens.     

Interaction of iodinated human [D-Ala2]beta-endorphin with opitate receptors.

The interaction of beta-endorphin with opiate receptors was studied by using the radioiodinated, metabolically stable D-Ala2 derivative of human beta-endorphin. This analog binds specifically to rat brain membrane preparations with an apparent Kd of about 2.5 x 10-9 M. The ability of various enkephalin analogs, as well as opiate agonists and antagonists, to inhibit the binding of beta-endorphin clearly demonstrates that this peptide can bind to opiate receptors. However, the effects of various cations on the binding of 125I-[D-Ala2]beta-endorphin are markedly different from those found for enkephalin binding. Sodium ion at physiological concentrations decreases substantially the binding of enkephalins but only slightly decreases endorphin binding, whereas manganese enhances enkephalin binding but has no effect on endorphin binding. Moreover, potassium (100 mM) decreases the binding of beta-endorphin but does not affect enkephalin binding. These results suggest that beta-endorphin and enkephalin bind differently to the same receptor or bind to different receptors with overlapping specificity.     

Patterns of product inhibition for bacterial nitrite reductase

PO +Dehydrophenylalanine (delta Phe) having the E-configuration (delta EPhe ; phenyl and C = O cis) was incorporated into [Leu5]-enkephalin in order to restrict its conformation. Compared with the Z-isomer, in the radio-ligand receptor binding assays, [D-Ala2, delta EPhe4 , Leu5] enkephalin showed drastically decreased potency for the delta and mu opiate receptors, i.e., 260- and 150-fold loss of affinity, respectively. The results strongly indicate that the opiate receptors require the Z-configuration (phenyl and C = O, trans) of the delta Phe4 residue and may require a specific interrelationship between the aromatic rings of the Tyr1 and Phe4 residues in the molecule for binding. The conformation of [Leu5]-enkephalin specific for the delta receptors was analyzed and a comparison made with its crystal structure recently elucidated.     

Glycosylation-Dependent Regulation of Opiate (Enkephalin) Receptors in Neurotumor Cells

Abstract: Electron inactivation analysis revealed that the opiate (enkephalin) binding site in neurotumor cell lines NG108-15 and NCB-20 had an apparent target size of 200,000 daltons. Expression of functional opiate receptors in neurotumor cells appeared to require glycosylation, as treatment of such cells with tunicamycin (TM; under conditions where de novo glycosylation of asparagine residues in protein was reduced by 80%, but overall protein and DNA synthesis were inhibited by <10%) resulted in the loss of 50% of the opiate binding sites. The loss of binding sites could not be prevented by addition of protease inhibitors to cell cultures, but binding sites were partially restored 48–60 h after removal of the TM. In addition, the number of enkephalin binding sites in TM-treated cells was also restored to near-normal levels by addition of physiological concentrations (1-10 mM) of manganese ions to the in vitro receptor binding incubation mixture. TM treatment resulted in receptor supersensitivity to manganese ions for both opiate agonists and antagonists, no change in the sodium effect for either agonists or antagonists, and subsensitivity to GTP for both agonists and antagonists. However, opiate binding to cell membranes was not substantially inhibited by either neuraminidase treatment or short-term incubation with lectins such as wheat germ agglutinin, ricin, or concanavalin A. Thus, the data suggest that oligosaccharide units are not directly involved in opiate receptor-ligand interactions, but protein glycosylation is required for functional expression of receptors.     

Down regulation of enkephalin (delta) receptors. Demonstration in membrane-bound and solubilized receptors

The pentapeptide leucine enkephalin induced down-regulation of enkephalin receptors in neuroblastoma-glioma NG108-15 hybrid cells in a reversible fashion, whereas the stable enkephalin analogue D-Ala2-Met-enkephalinamide (AMEA), and the potent opiate alkaloid, etorphine, had a prolonged effect. The opiate alkaloid, morphine, which has low affinity to delta-type enkephalin receptors of these cells did not induce down-regulation, whereas AMEA decreased the binding of both opiate agonists and antagonists but had no effect on the binding of the alpha 2-adrenergic ligand, [3H]yohimbine. From several experiments that were designed to remove the tightly bound AMEA, and from experiments with solubilized receptor we ruled out the possibility that the decreased binding capacity of enkephalin-treated cells reflects only receptor masking. The study suggests that down-regulation of enkephalin receptors that may also occur in vivo can account for some of the abnormal physiological responses of subjects treated chromically with opiates. However, since opiates from the morphine type can induce opiate tolerance in vivo, but not down-regulation of enkephalin receptors in the cultured cells, we suggest that down-regulation of delta-type opiate receptors may not be prerequisite for the development of the physiological tolerance/dependence on these alkaloids.     

Photolabile opioid derivatives of D-Ala2-Leu5-enkephalin and their interactions with the opiate receptor

Photolabile derivatives of D-Ala2-Leu5-enkephalin were prepared by synthetic procedures in which a 2-nitro-4-azidophenyl group is linked to the terminal carboxyl group of the enkephalin by means of an ethylenediamine or ethylenediamine beta-alanine spacer. These peptides bind to opiate receptors with nanomolar affinities and inhibit electrically stimulated contractions of the mouse vas deferens and adenylate cyclase activity of NG108-15 neuroblastoma x glioma hybrid cell membranes. Both inhibitions are reversed by the opiate antagonist naloxone. Photolysis of the ligands bound to rat brain membranes results in the loss of approximately 50% of the receptor sites. This decrease in receptor number is blocked by naloxone and requires light. A photolabile [3H]enkephalin derivative labels an equivalent number of sites under similar irradiation conditions.     

Interaction of iodinated enkephalin analogues with opiate receptors.

Radioiodinated derivatives of the metabolically stable enkephalin analogues, [DAla²,Leu⁵]- and [DAla²,DLeu⁵]-enkephalin, have been prepared. Such derivatives show sterospecific binding to receptors in brain homogenates and some neuroblastoma cell lines such as NG108-15 and N4TG1. The relative effects of levorphanol and dextrorphan and Na⁺ and Mn⁺⁺ ions on enkephalin binding in brain and cells indicate that the iodinated derivatives are interacting with opiate receptors. Levorphanol is considerably more potent in displacing specifically bound enkephalin than dextrorphan. Sodium ions at physiological concentrations decrease enkephalin binding whereas manganese ions enhance it. Unlabelled monoiodo derivatives retain high potency in the guinea-pig ileum, mouse vas deferens and receptor binding assays. Unlabelled diiodo derivatives show far lower potency in these assays. It is concluded that radio-iodinated derivatives containing one iodine per molecule retain high affinity for the opiate receptor but diiodo derivatives do not.     

Synthesis and receptor binding affinity of both E- and Z-dehydrophenylalanine4 enkephalins

The dehydrophenylalanine4-enkephalin having the E-configuration (delta EPhe; phenyl and C = 0, cis) was prepared by photoisomerization of the Z-isomer with 3100 A light, followed by reversed-phase HPLC separation of the resulting mixture of the Z- and E-isomers. In the radioligand receptor binding assays, the E-isomer of [D-Ala2, delta Phe4, Leu5]enkephalin exhibited an extremely diminished affinity as compared with the Z-isomer, namely 150-260-fold loss of affinity for the delta and mu opiate receptors. The results indicate that the interrelationship of the Tyr1 and Phe4 residues in the enkephalin molecule seems to be of great importance in receptor recognition.     

Enkephalins and Opiate Antagonists Control Calmodulin Distribution in Neuroblastoma-Glioma Cells

The calcium binding protein calmodulin and the opiate receptor binding sites are unevenly distributed in various subcellular fractions of neuroblastoma-glioma NG108-15 cells. The crude mitochondrial-membrane fraction of these cells contains two membrane fractions that are separable by sucrose gradient centrifugation. These two differ in the content of both calmodulin and opiate receptors. Leucine enkephalin and D-Ala2-methionine enkephalinamide decrease the amount of membrane-bound calmodulin in the NC108-15 cells in a time- and dose-dependent manner, whereas the opiate antagonists naloxone and levallorphan have an opposite effect. Naloxone blocks the effect of leucine enkephalin and dextrallorphan has no significant effect. The opiate alkaloids entorphine and phenazocine induce changes similar to that of the enkephalins whereas morphine is inactive even at high concentrations. The alteration in the amount of membrane-bound calmodulin after a short incubation (15 min) with the enkephalins or with naloxone is reflected as an opposite change in the amount of calmodulin in the cell cytosol. Naloxone and levallorphan also increase the number of opiate receptors in NG108-15 cells but dextrallorphan has no such effect. Modulation of the intracellular distribution of calmodulin by opioid peptides and alkaloids may control the activity of various membrane-bound and cytosolic systems that are calmodulin- and/or calcium-dependent.     

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.     

Adrenocorticotropin- and opiate-like hormones from pituitaries of the sockeye salmon Oncorhynchus nerka

The pituitaries of vitellogenic sockeye salmon (Oncorhynchus nerka) were extracted with a mixture of acetone, water, and hydrochloric acid. The precipitate which formed upon the addition of a copious volume of acetone to the extract, designated acid acetone powder, was subjected to salt fractionation and desalting, followed by ion-exchange chromatography on CM-cellulose. An unadsorbed fraction (S-1) and four adsorbed fractions (S-2, S-3, S-4 and S-5) were obtained. Adrenocorticotropic activity was detected in the fractions by their ability to stimulate isolated rat adrenal decapsular cells to produce corticosterone and by their immunoreactivities in an adrenocorticotropin-specific radioimmunoassay. The steroidogenic activities of all fractions, except S-4, were blocked by corticotropin inhibiting peptide. Opiate activity was detected in the fractions by their ability to inhibit the binding of either [3H]naloxone or (D-ala2, D-leu5)-[3H]enkephalin to rat brain membranes. There was a discrepancy in the potencies of the five fractions in the two opiate radioreceptor assays, indicating the presence of opiate peptides with different affinities of binding to the micron- and delta-opiate receptors of the rat brain. There was a separation between adrenocorticotropic and opiate receptor binding activities, suggesting that the activities were due to separate molecular entities.     

Down regulation of enkephalin (δ) receptors Demonstration in membrane-bound and solubilized receptors

The pentapeptide leucine enkephalin induced down-regulation of enkephalin receptors in neuroblastoma-glioma NG108-15 hybrid cells in a reversible fashion, whereas the stable enkephalin analogue, d-Ala²-Met-enkephalinamide (AMEA), and the potent opiate alkaloid, etorphine, had a prolonged effect. The opiate alkaloid, morphine, which has low affinity to δ-type enkephalin receptors of these cells did not induce down-regulation, whereas AMEA decreased the binding of both opiate agonists and antagonists but had no effect on the binding of the α₂-adrenergic ligand, [³H]yohimbine. From several experiments that were designed to remove the tightly bound AMEA, and from experiments with solubilized receptor we ruled out the possibility that the decreased binding capacity of enkephalin-treated cells reflects only receptor masking. The study suggests that down-regulation of enkephalin receptors that may also occur in vivo can account for some of the abnormal physiological responses of subjects treated chromically with opiates. However, since opiates from the morphine type can induce opiate tolerance in vivo, but not down-regulation of enkephalin receptors in the cultured cells, we suggest that down-regulation of δ-type opiate receptors may not be prerequisite for the development of the physiological tolerance/dependence on these alkaloids.     

A radiolabeled-ligand-binding technique for the characterization of opioid receptors in the intact mouse vas deferens

The mouse vas deferens has served as a useful bioassay for examining the properties of opiate receptor subtypes. However, recent data indicate that the response of the vas deferens to opiates may be mediated by one or more of the several opiate receptors found in this preparation. Although a number of techniques can be utilized to assess the relative contribution of these receptors to the response of the mouse vas deferens to opiates (e.g., selective tolerance and naloxone antagonism studies), a radiolabeled-binding technique would provide an independent means of more completely characterizing the opiate receptor profiles in this preparation. Up to the present, however, there has been only limited success in developing a binding assay utilizing crude membrane fractions of the mouse vas deferens. To circumvent these problems, we have developed a binding technique utilizing the intact vas deferens. In contrast to results obtained with membrane fractions, we found highly specific (90–95%) and saturable binding of d-[2-³H]alanine, 5-d-leucine enkephalin, a ligand selective for delta opiate receptors, to the intact vas. Scatchard analyses indicated a single class of binding sites with an apparent K_d of 1.5 nm and a B_max of approximately 12 pmol/2 vas. The selectivity of binding was also examined. Naltrexone was 40 times less potent than unlabeled 2-d-alanine, 5-d-leucine enkephalin in displacing binding, whereas morphine and ethylketocyclazocine were 300 and 500 times less effective, respectively. This technique, coupled with the mouse vas deferens bioassay, should provide a more complete characterization of opioid receptor populations than has heretofore been possible.