Characterization of opioid binding sites in murine neuroblastoma

The binding of [3H] [D-Ala2, MePhe4, Gly-ol5]enkephalin ([3H]DAGO), [3H]D-Ala2,D-Leu5]enkephalin ([3H]DADLE) and (+/-)-[3H]ethylketocyclazocine ([3H]EKC) to neurotumor tissues derived from S20Y neuroblastoma cells transplanted into A/Jax mice was examined. Specific and saturable binding to [3H]DADLE and [3H]EKC was detected, and the data fit a single homogeneous binding site for each ligand. Scatchard analysis for [3H]DADLE and [3H]EKC yielded Kd values of 0.65 and 0.45 nM, respectively, and Bmax values of 9.2 and 116 fmol/mg protein. Binding was dependent on time, temperature, and pH, and was sensitive to Na+ and guanine nucleotides. Pretreatment of the tumor homogenates with trypsin markedly reduced binding to both ligands, suggesting that the binding sites were proteinaceous in character. Displacement experiments indicated that delta (delta) receptor related compounds (e.g. DPDPE, ICI 174,864) avidly displaced [3H]DADLE, whereas kappa (kappa) related compounds (e.g. U50,488, dynorphin) markedly competed with [3H]EKC. Mu (mu) receptor drugs (e.g. DAGO, beta-FNA, morphine) were not potent in displacing either [3H]DADLE or [3H]EKC. These results are the first to characterize opioid binding sites in tumor tissue. The function of these sites is unclear, but previous evidence as to the growth regulatory properties of endogenous opioid systems may suggest that either one, or both, binding sites may be involved in carcinogenic events.     

Opioid binding sites in different levels of rat spinal cord

Opioid receptor binding sites were analyzed in various segments of rat spinal cord. Mu and delta types were labelled with [3H]-DHM or [3H]-DAGO and [3H]-DADLE or [3H]-DSLET respectively. Kappa 1 (kappa) and kappa 2 (benzomorphan) binding sites were individually detected by the overall labeling of opioid binding sites with [3H]-etorphine followed by the elimination of binding to particular sites by the use of appropriate selective unlabelled ligands. Whereas lumbo sacral region contained mainly the kappa 2 site, thoracic membranes had a proportion of approximately 20% mu, 20% kappa 1 and 60% kappa 2 and cervical region contained much less kappa 2 sites (25% mu, 20% delta, 28% kappa 1 and 32% kappa 2).     

Interaction of dynorphin with kappa opioid receptors in bovine adrenal medulla

Dynorphin (Dyn) and various prototypic kappa opioid ligands were tested for their ability to bind to opioid receptors in a membrane preparation of bovine adrenal medulla and to modulate the release of catecholamines (CA) from isolated adrenal chromaffin cells. Saturation binding studies with [3H]-ethylketocyclazocine ([3H]-EKC) were performed at 37 degrees C for 30 min in the presence of [D-Ala2,Me-Phe4,Gly-ol5]-enkephalin (DAGO) and [D-Ser2,Thr6]-Leu-enkephalin (DSLET), two specific ligands for crossreacting mu and delta opioid receptors, respectively. Scatchard plot analysis of the data revealed the presence of two receptor sites: a high affinity binding site (kappa) with a KD of 0.66 nM and a Bmax of 12 pmoles/g protein and a low affinity binding site (kappa 2) with a KD of 11.1 nM and a Bmax of 56 pmoles/g protein. The presence of kappa opioid receptors in the membrane preparation was also supported by competition studies. U-50, 488H and Dyn-(1-13), two selective kappa opioid ligands, were potent inhibitors of [3H]-EKC binding with Ki (high affinity binding sites) of 2.5 and 2.3 nM, respectively. Among the various ligands tested for each class of opioid receptors (mu, delta, kappa), U-50, 488H and Dyn-(1-13) were the most potent inhibitors of the acetylcholine-evoked CA secretions from isolated adrenal chromaffin cells with IC50 of 0.31 and 1.14 microM, respectively. The inhibitory effect of U-50, 488H was significantly antagonized by diprenorphine and MR-2266, two opioid antagonists with a high affinity for the kappa opioid receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding characteristics of [3H]opioid ligands to active opioid binding sites solubilized from rat brain membranes by glycodeoxycholate and NaCl: the recovery of binding activity by dilution

This paper describes the binding properties of [3H]peptidergic opioid ligands to binding sites solubilized from rat brain membranes by the treatment with 0.125% sodium glycodeoxycholate and 1 M NaCl. The highest amount of the specific binding of [3H]-[D-Ala2-, Met5]enkephalinamide was obtainable when 10-fold diluted solubilized preparations were incubated in the presence of 0.1 mM MnCl2 and 100 mM NaCl at 0 degree C (on ice) for 3 h. With this assay condition, the significant binding of following [3H]opioid ligands, which have been thought to be selective for receptor types, was also observed: [3H]-[D-Ala2, MePhe4, Gly-ol5]enkephalin (mu-type), [3H]-[D-Ala2, D-Leu5]enkephalin (delta-type) and [3H]dynorphin1-9 (kappa-type). The number of binding sites in solubilized preparations for each [3H]ligand corresponded to 40-50% recovery of original membrane-bound binding sites. The Scatchard plot of the concentration-saturation binding curve showed only one class of binding sites, with a high affinity for each [3H]ligand. Apparent dissociation constants between solubilized receptors and [3H]ligands were the same as membrane-bound ones, but the ligand specificity for each receptor-type, which was examined by binding inhibition tests with unlabeled ligands, decreased. Present results indicate that heterogeneous opioid receptors in rat brain membranes seem to be transformed into less heterogeneous forms through the treatment with glycodeoxycholate and NaCl and the dilution process.     

Opiate binding sites in the chick, rabbit and goldfish retina

The characteristics of opiate binding sites in the retina of the chick, rabbit and goldfish have been investigated. In the newly hatched chick retina, 131 fmol/mg of binding sites for [D-Ala2-D-Leu5]-[3H]enkephalin are present; competition studies with the delta selective peptide [D-Thr-Leu5]-enkephalin (DTLET) and the mu selective peptide morphiceptin show that all of the [D-Ala2-D-Leu5]-[3H]-enkephalin binding sites are of the delta subtype. Dihydro[3H]morphine binds poorly to the chick retina; 13.2 fmol/mg of this binding is displaceable by morphiceptin and corresponds to mu binding sites. Benzomorphan sites are defined as sites occupied by [3H]diprenorphine which is displaceable by low concentrations of ethylketocyclozacine but not by high concentrations of D-Ala2-D-Leu5-enkephalin and morphiceptin. At least 88 fmol/mg of benzomorphan sites are present in the chick retina. [3H]diprenorphine binding to the rabbit and fish retina was measured. The rabbit retina bound 60 fmol/mg, and the fish retina 42 fmol/mg of [3H]diprenorphine. These findings are discussed in the light of the studies on the localization and physiological effects of enkephalin in the retina.     

Characterization of dermorphin binding to membranes of rat brain and heart

Binding of dermorphin to the two major opioid receptor types, mu and delta, in rat brain membranes was examined by displacement of [3H] [D-Ala2, MePhe4, Gly-(ol)5]enkephalin (DAGO) and [3H]-[D-Ala2,D-Leu5]-enkephalin (DADLE) binding. Affinity of dermorphin binding to mu sites, Kd = 1.24 nM, was almost 3 times greater than that of DAGO, Kd = 3.35 nM. In contrast, the Kd value of dermorphin binding to delta sites was 78 nM only, as compared to Kd = 2.27 nM for DADLE. Dermorphin was ineffective in displacing [3H]ethylketocyclazocine (EKC) binding to kappa receptors after prior blocking of [3H]EKC binding to mu and delta sites. Studies of dermorphin binding to mu sites revealed that the potency of dermorphin increased in the presence of Na+ (+31%) but decreased in the presence of Mn2+ (-81%) or Gpp(NH)p (-44%). Displacement of bound [3H]diprenorphine (DPN) by dermorphin from atrial membranes of the rat heart, left side, was detectable, suggesting the presence of mu sites in this section of the heart.     

Kappa opioid receptors in human lumbo-sacral spinal cord

[3H]Etorphine and [3H]ethylketocyclazocine bind with high affinity (Kd between 0.25-2.0 nM) to a single class of sites in human lumbo-sacral spinal cord. Other ligands such as [3H]morphine, [3H]dihydromorphine and [3H]D-Ala2, D-Leu5-enkephalin (DADLE) did not bind to significant number of sites under our incubation conditions. Ligand selectivity pattern strongly suggests that [3H]etorphine labels kappa opioid binding sites in the human lumbo-sacral spinal cord since benzomorphans and oripavines are much more potent than mu and delta agonists. Furthermore, [3H]etorphine and [3H]ethylketocyclazocine binding is sensitive to high concentrations of DADLE suggesting that these sites are of the kappa 2 sub-type. Finally, the visualization of these sites by receptor autoradiography demonstrates that they are mainly concentrated in lamina II and III of the dorsal horn. Moderate densities of sites are present around the central canal. Thus, it is possible that kappa opioid binding sites could be involved in the control of sensory and autonomic functions in the human lumbo-sacral spinal cord.     

The binding of kappa- and sigma-opiates in rat brain

Detailed displacements of [3H]dihydromorphine by ketocyclazocine and SKF 10,047, [3H]ethylketocyclazocine by SKF 10,047, and [3H]SKF 10,047 by ketocyclazocine are all multiphasic, suggesting multiple binding sites. After treating brain tissue in vitro with naloxazone, all displacements lose the initial inhibition of 3H-ligand binding by low concentrations of unlabeled drugs. Together with Scatchard analysis of saturation experiments, these studies suggest a common site which binds mu-, kappa, and sigma-opiates and enkephalins equally well and with highest affinity (KD less than 1 nM). The ability of unlabeled drugs to displace the low affinity binding of [3H]dihydromorphine (KD = 3 nM), [3H]ethylketocyclazocine (KD = 4 nM), [3H]SKF 10,047 (KD = 6 nM), and D-Ala2-D-Leu5-[3H]enkephalin (KD = 5 nM) remaining after treating tissue with naloxazone demonstrates unique pharmacological profiles for each. These results suggest the existence of distinct binding sites for kappa- and sigma-opiates which differ from those sites which selectively bind morphine (mu) and enkephalin (delta).     

Traumatic injury alters opiate receptor binding in rat spinal cord

Recent studies with dynorphin (an endogenous ligand for the kappa-opiate receptor) and receptor-selective opiate antagonists have indicated a role for the kappa-receptor in the pathophysiology of spinal cord injury. However, no studies have specifically examined opiate receptor binding in relation to spinal injury. In the present experiments, opiate receptor binding was measured in spinal cord after traumatic injury in rats using the selective radioligands [3H] [D-Ala2, D-Leu5]enkephalin (delta-receptor agonist); [3H] [D-Ala2,MePhe4,Gly-(ol)5]enkephalin (mu-receptor agonist); and [3H]ethylketocyclazocine (kappa-receptor agonist). The specific binding of ethylketocyclazocine, but not the other agonists, showed a significant, time-dependent, and localized increase at the injury site. Since dynorphin, which has been implicated as an injury factor after spinal trauma, shows similar localized increases after spinal injury, the present data are consistent with the hypothesis that up-regulation of the kappa-receptor after injury may contribute to the subsequent secondary injury process.     

Characterisation of the δ-opioid receptor on the hamster vas deferens

It has recently been reported that the hamster vas deferens contains only delta-opioid receptors. We have demonstrated that three delta-receptor agonists [D-Ala2, D-Leu5]enkephalin (DADLE), [D-Pen2, D-Pen5]enkephalin (DPDPE) and [D-Thr2, Leu5, Thr6] enkephalin (DTLET) appear to mediate their effects via the delta-receptor since they are readily reversed by the selective delta-receptor antagonist ICI 174864. In addition, a number of classical mu and k receptor compounds were devoid of activity in this preparation. However it was observed that some compounds such as etorphine and MR 2034 reported to possess delta-receptor affinity in other assay systems were weak or inactive on the hamster vas deferens.     

LY164929: a highly selective ligand for the lower affinity [3H]D-Ala2-D-Leu-5-enkephalin binding site

Most radiolabeled ligands used to label opiate receptors bind to multiple binding sites. Subtype-selective ligands make possible the labeling of single sites by virtue of their ability to "block" binding of the radiolabeled ligand to selected subtypes. This study compares the selectivity of several ligands for the higher and lower affinity [3H]D-Ala2-D-Leu-5-enkephalin binding sites. The results demonstrated that while morphine and D-ala2-MePhe4,Gly-ol5-enkephalin were 80- and 256-fold selective for the lower affinity [3H]D-Ala2-D-Leu-5-enkephalin binding site, LY164929 was 1,986-fold selective. Additional experiments indicated that whereas morphine was a noncompetitive inhibitor at the lower affinity [3H]D-Ala2-D-Leu-5-enkephalin binding site, LY164929 was a competitive inhibitor, suggesting that this peptide might exhibit different properties in vivo than other mu-like ligands.     

Endogenous material in brain inhibiting [3H]nicotine and [3H]acetylcholine binding

The supernatant obtained from mouse brain homogenates contains material that inhibits the saturable binding of [³H]nicotine in mouse cerebral cortex. This inhibitory material was further purified by heat denaturation, ultrafiltration through an Amicon PM-10 membrane filter, and gel chromatography on Sephadex G-10. The material inhibited the binding of [³H]acetylcholine with the same potency as it did that of [³H]nicotine. It also had some affinity for the sites that specifically bind [³H]D-Ala, D-Leu enkephalin, but had much lower affinity for the binding sites for [³H]quinuclidinyl benzilate (QNB), [³H]spiroperidol, [³H]naloxone, or [³H]imipramine. Acid hydrolysis destroyed the activity. These preliminiary results suggest the presence in brain of “nicotinelike” substances, one of which may be the endogenous ligand for the sites that specifically bind [³H]nicotine.     

Effects of guanyl nucleotides and ions on kappa opioid binding

Displacement curve analyses demonstrated that GTP and its nonhydrolyzable analog, GPP(NH)P, inhibited the binding of [3H]dihydromorphine (mu agonist), [3H]D-Ala2-D-Leu5-enkephalin (delta agonist), and [3H]ethylketocyclazocine (general agonist) but not [3H]diprenorphine (general antagonist). Using a paradigm to block mu and delta sites with specific cold ligands, [3H]ethylketocyclazocine labeled kappa sites which were less GTP sensitive than sites labeled by mu and delta agonists. Further, Na+ and Mg++, important in inhibitory adenylate cyclase systems, also decreased both unblocked and mu-/delta-blocked [3H]ethylketocyclazocine binding. Scatchard analyses revealed that the inhibitory effects of GTP result in a decrease in affinity without a significant change in binding capacity, and that the kappa component of [3H]ethylketocyclazocine binding was less sensitive to the effects of GTP than binding sites labeled by mu or delta agonists. In comparison to the effects of GTP, Na+ decreased binding affinity but increased the binding capacity of the kappa component. These data also suggest that the inhibitory effects of Na+ and GTP on binding affinity are not additive. Association and dissociation plots revealed that although both components of binding may be involved in these affinity changes, the dissociation rate represents the more significant factor. These data suggest that [3H]ethylketocyclazocine binding to kappa sites is GTP and Na+ sensitive. However, it should be noted that [3H]ethylketocyclazocine binding to kappa sites is less sensitive to GTP than its binding to other opiate sites, and that this kappa binding is differentially affected by Na+. The significance of these characteristics with regard to the effect of kappa opiates on adenylate cyclase activity remains to be determined.     

Acute and persistent effects of beta-funaltrexamine on the binding and agonist potencies of opioids in the myenteric plexus of the guinea-pig ileum

In binding assays with homogenates of the myenteric plexus-longitudinal muscle of the guinea-pig, beta-funaltrexamine is more potent at displacing mu-binding than kappa-binding. Incubation of homogenates with beta-funaltrexamine (100 or 1000 nM) for 30 min at 37 degrees C followed by repeated washing with drug-free Tris buffer does not alter the binding of either the selective mu-ligand [3H]-[D-Ala2, MePhe4,Gly-ol5]enkephalin or of [3H]-(-)-bremazocine made selective for the kappa-binding site by the addition of unlabelled mu- and delta-ligands. This observation is surprising since, after treatment with beta-funaltrexamine (100 nM), the IC50 values for the inhibition of the contraction of the myenteric plexus-longitudinal muscle by the mu-ligand [D-Ala2,MePhe4, Gly-ol5]enkephalin are increased 12-fold whereas the IC50 values obtained with the selective kappa-ligand U-50, 488H remain unaltered. It is proposed that the irreversible blockade produced by beta-funaltrexamine is not due to inhibition at the mu-site per se but to an interference with the link between the binding and the effector response.     

Dog cerebral cortex contains μ-, δ- and κ-opioid receptors at different densities: apparent lack of evidence for subtypes of the κ-receptor using selective radioligands

The biochemical and pharmacological properties of mu (mu), kappa (kappa) and delta (delta) opioid receptors were ascertained in dog cerebral cortex homogenates. The selective peptides, [3H]D-Pen2-D-Pen5enkephalin [( 3H]DPDPE) and [3H]D-Ala2-MePhe4-Glyol5-enkephalin [3H]Glyol; [3H]DAMGO), bound to delta- and mu-opioid receptors with high affinity (dissociation constants, Kd values = 4.7 and 1.6 nM) but to different densities of binding sites (Bmax values of 49.2 and 6.6 fmol/mg protein, respectively) in washed homogenates of dog cerebral cortex. In contrast, the non-peptides, [3H]U69593 [( 3H]U69) and [3H]etorphine [( 3H]ET), labeled a high concentration of kappa-opioid receptors (respective Bmax values of 67.2 and 76.6 fmol/mg protein) of high affinity (respective Kds of 1.4 and 0.47 nM) in the same tissue homogenates. Thus, the relative rank order of opioid receptor densities was: kappa greater than delta much greater than mu. The selective labeling of the kappa-receptors with two different drugs [( 3H]U69 and [3H]ET) failed to reveal the possible existence of multiple kappa-sites based on the relative Bmax values of the two radioligands. This conclusion was further supported by the similarity of the pharmacological specificity of both [3H]U69 and [3H]ET binding, where all the opioids tested produced 100% inhibition of these labels and where the rank order of potency of opioids at inhibiting the binding of these probes was: U50488 greater than U69593 greater than dynorphin-(1-8) greater than naloxone much greater than morphine much greater than Glyol (DAMGO) greater than DPDPE.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tyr-D-Ala-Gly-(Me)Phe-chloromethyl ketone: a mu specific affinity label for the opioid receptor

An alkylating tetrapeptide enkephalin derivative, Tyr-D-Ala-Gly-(Me)Phe-chloromethyl ketone (DAMK) was synthesized, and its binding characteristics on rat brain membranes were evaluated. In competition experiments, the product shows high affinity for the mu opioid binding site of the rat brain membranes, whereas its binding to the delta and kappa subtypes is weak. Micromolar concentrations of this ligand produce a dose-dependent, apparently irreversible inhibition of /3H/-naloxone binding, with apparent IC50 value of 1-5 uM. Neither reversibly binding opioids nor tosyl-amino acid chloromethyl ketones show these effects. Saturation binding analysis with /3H/-naloxone of membranes preincubated with Tyr-D-Ala-Gly-(Me)Phe-CH2Cl reveal a selective and irreversible inhibition of the high affinity /3H/-naloxone binding site. Irreversible blockade of mu-selective /3H/-ligand binding by Tyr-D-Ala-Gly-(Me)Phe-CH2Cl is much more effective than that of the binding of /3H/-enkephalin or /3H/-ethylketocyclazocine. The mu-selective binding properties of this new irreversible enkephalin analogue suggest that it could serve as an affinity label for the mu opioid receptor subtype.     

Sodium ions increase the binding of the antagonist peptide ICI 174864 to the delta-opiate receptor

The ability of N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH (ICI 174864) to displace [3H]-[D-Ala2, D-Leu5]enkephalin bound to the delta-opioid site is increased 8-16 fold by addition of 25mM NaCl. A smaller shift is observed for the related N,N-diallyl-Tyr-Gly-Gly-psi-(CH2S)-Phe-Leu-OH (ICI 154129) but no significant shift is seen with either naloxone or diprenorphine. The results stress the importance of using the correct medium for binding assays, and suggest the changes in delta-receptor conformation induced by Na+ ions also increase peptide antagonist binding.     

3H]-beta-endorphin binding in rat brain

The binding of [3H]-beta-endorphin to rat brain homogenates is complex. Although Scatchard analysis of saturation studies yields a straight line, detailed competition studies are multiphasic, suggesting that even at low concentrations of the compound, the 3H-ligand is binding to more than one class of site. A portion of [3H]-beta-endorphin binding is sensitive to low concentrations of morphine or D-Ala2-Leu5-enkephalin (less than 5 nM). The inhibition observed with each compound alone (5 nM) is the same as that seen with both together (each at 5 nM). Thus, the binding remaining in the presence of both morphine and the enkephalin does not correspond to either mu or delta sites. The portion of [3H]-beta-endorphin binding that is inhibited under these conditions appears to be equally sensitive to both morphine and the enkephalin and may correspond to mu1 sites. Treating membrane homogenates with naloxonazine, a mu1 selective antagonist, lowers [3H]-beta-endorphin binding to the same degree as morphine and D-Ala2-Leu5-enkephalin alone or together. This possible binding of [3H]-beta-endorphin to mu1 sites is consistent with the role of mu1 sites in beta-endorphin analgesia and catalepsy in vivo.     

Purification of a kappa-opioid receptor subtype from frog brain

A kappa-opioid receptor subtype was purified from a digitonin solubilized preparation of frog brain membranes using affinity chromatography. The affinity resin was prepared by coupling D-Ala2-Leu5-enkephalin to Sepharose-6B matrix. After elution of the receptor by 50 mumol naloxone, the kappa-subtype was separated from the mu- and delta-subtypes by gel permeation chromatography on Sepharose-6B. The purified receptor binds 3,900 pmol [3H]-ethylketocyclazocine per mg protein (a 4,300-fold purification over the membrane-bound receptor) with a KD of 8.3 nM. The purified receptor protein exhibits high affinity for kappa-selective ligands. The purified fraction shows two bands (Mr 65,000 and 58,000) in sodium dodecyl sulfate gel electrophoresis.     

Irreversible labelling of rat brain opioid receptors by enkephalin chloromethyl ketones

Chloromethyl ketone derivatives of leucine enkephalin (LE), D-Ala2-Leu5-enkephalin (DALE) and D-Ala2-D-Leu5-enkephalin (DADLE) were synthesized. They all show high affinity for rat brain opioid binding sites. Preincubation of the membrane fraction with enkephalin chloromethyl ketones causes a significant inhibition of /3H/-naloxone binding which cannot be reversed by extensive washing. It was found that the irreversible inhibition is selective for the high affinity (KD less than 1 nM) /3H/-naloxone binding site (putative mu-1 site). The irreversible blockade of opioid binding was partially protected by opiate alkaloids and opioid peptides, suggesting that non-specific labelling also occurs. Affinity of enkephalin chloromethyl ketones toward the mu sites is greater than that of the parent compounds. It was also found that the covalent inhibition of mu sites (/3H/-dihydromorphine and /3H/-DAGO binding) is more effective than that of delta sites (/3H/-DALE binding). We conclude that these chloromethyl ketone derivatives can be used as affinity labels for the opioid receptors, allowing us to study the structure of the mu receptor subtype.