NMB: a human neuroblastoma cell line with specific opiate binding sites

The human neuroblastoma cell line designated NMB (Brodeur et al., 1977, Cancer 40: 2256) has been shown to have specific opiate binding sites. These sites are highly stereospecific. Two characteristic delta specific peptides, D-Ala2-D-Leu5 enkephalin and D-Thr2-D-Thr6 enkephalin, have high affinity for the binding sites. Morphine binds specifically but with a much lower affinity. Dextrorphan and the mu specific peptide morphiceptin (Tyr-Pro-Phe-Pro-CO-NH2) do not bind to the site. The binding sites are heat and trypsin sensitive. Sodium ions specifically lower agonist binding to the sites. Approximately 14,000 binding sites per cell are found. The binding characteristics of these sites are very similar to those of the delta sites characterized on mouse neuroblastoma cell lines.     

Opiates and enkephalins: a common binding site mediates their analgesic actions in rats

³H-Labelled opiate and enkephalin ligands appear to bind with highest affinity to a single site responsible for their analgesic properties. Administered $\text{in vivo}$, naloxazone, an irreversible opiate, selectively inhibits for over 24 hours the high affinity binding of ³H-labelled mu, and kappa opiates and enkephalins. This inhibition of binding gradually resolves over 3 days, perhaps correlating with receptor turnover. Naloxazone treatment also abolishes morphine, D-ala²-met⁵-enkephalinamide and betah-endorphin analgesia. Although morphine and D-ala²-met⁵-enkephalinamide bind with similar potencies to the high affinity site, morphine's potency for the low affinity D-ala²-met⁵-enkephalinamide site is far less than the enkephalin analog. These results imply that all ³H-ligands examined bind with highest affinity to a mu-like receptor while low affinity D-ala²-met⁵-enkephalinamide binding, with a K_D of 6 nM, represents a delta-like receptor.     

Biphasic competition between opiates and enkephalins: does it indicate the existence of a common high affinity ("mu-1") binding site?

Displacement from brain membranes of labeled opiates by low concentrations of enkephalins and of labeled enkephalins by low concentrations of opiates has been previously explained by the existence of a common high affinity site termed mu-1. An alternative interpretation of the same results is that the trough seen in the low concentration zone of the displacement curves represents cross binding of mu and delta opioid ligands to delta and mu receptors, respectively. In three sets of experiments with brain membranes, the size of the trough is shown to be dependent on the labeled ligand used: The ratio between the size of troughs seen with [3H]D-Ala, D-Leu enkephalin and with [3H]morphine varies with experimental conditions (storage of membranes at 4 degrees C for 72 h), with ratio of mu:delta receptors (e.g. in thalamus and cortex which are enriched in mu and delta sites, respectively) and with pretreatment of membranes with naloxonazine. These results can not be explained by a common high affinity site, but rather by binding of [3H]D-Ala, D-Leu enkephalin to mu and of [3H]morphine to delta opioid receptors.     

The actions of naloxazone on the binding and analgesic properties of morphiceptin (NH2Tyr-Pro-Phe-Pro-CONH2), a selective mu-receptor ligand

Active in both binding and biological assays, morphiceptin (NH₂ Tyr-Pro-Phe-Pro-CONH₂), a potent opioid peptide derivative of β-casamorphine, binds specifically and selectively to mu or morphine-type receptors with little affinity for delta sites. Displacement studies of a variety of ³H-labeled opiates and enkephalins show biphasic curves. Naloxazone, which blocks irreversibly and selectively high affinity opiate and enkephalin binding, abolishes morphiceptin's inhibition of binding at low concentrations, suggesting that the high affinity binding of enkephalins and opiates represents a mu or morphine-type receptor. Unlike the reversible antagonist naloxone, naloxazone treatment $\text{in}$$\text{vivo}$ inhibits for over 24 hours the analgesic activity of morphiceptin like it inhibits morphine, β-endorphin and enkephalin analgesia. Together, these studies imply that opiates and enkephalins bind with highest affinity to a mu receptor which mediates their analgesic activity. The ³H-D-ala²-D-leu⁵-enkephalin binding remaining after naloxazone treatment, representing a lower affinity site (K_D 4 nM), is quite insensitive to morphiceptin inhibition and has the characteristics of a delta receptor. However, the ³H-dihydromorphine binding present after naloxazone treatment, which also represents a lower affinity site (K_D 6 nM), is far more sensitive to both morphine and morphiceptin and may represent a second morphine-like, or mu, receptor.     

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.     

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-Ser2,Leu5]enkephalin-Thr6 (DSLET), a relatively selective delta ligand, with mu1 opioid binding sites

Using binding approaches, we have confirmed the high selectivity of [D-Ser2,Leu5]enkephalin-Thr6 (DSLET) to delta, as opposed to morphine-preferring (mu2) sites in rat brain. However, detailed experiments studies indicate that this ligand also labels mu1 sites with very high affinity. Saturation studies of 3H-DSLET binding reveal curvilinear plots. Treating tissue with naloxonazine to block mu1 sites, eliminates the higher affinity binding component. Competition studies of the other peptides against 3H-DSLET and 3H[D-Ala2,MePhe4,Gly(ol)5]enkephalin (3H-DAMPGO) binding also implied high affinity binding of these peptides to mu1 sites. The ability of these peptides to interact with mu1 sites may help explain some of their pharmacological actions.     

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.     

Mu-opiate binding and morphine antagonism by octapeptide analogs of somatostatin

A series of cyclic conformationally restrained octapeptide analogs of somatostatin were examined for their ability to inhibit the binding of tritiated mu, kappa, and delta opiate receptor ligands. Several of these substances were found to have high affinity for mu opiate receptors while having very low affinity for both kappa and delta receptors. Previous suggestions that somatostatin analogs exhibit opiate antagonist activity led to a study of the ability of the two most potent compounds to inhibit morphine analgesia in rats after intracerebroventricular injection. One of the compounds significantly antagonized morphine analgesia although the other displayed severe toxicity. These two compounds differed in that the very toxic compound had previously been found to possess significant somatostatin activity. It thus appears that the structural requirements for toxicity and somatostatin activity can be differentiated from those for opiate activity.     

Quantitative autoradiographic distribution of meptazinol-sensitive binding sites in rat brain

1. Meptazinol is an interesting opioid-producing naloxone-reversible analgesia with few cardiovascular and respiratory effects. Recent studies indicate that mu 1 opioid receptors mediate meptazinol analgesia. Using a computerized autoradiographic subtraction technique, we have examined the regional distribution of meptazinol-sensitive [3H][D-Ala2,MePhe4,Gly(ol)5]enkephalin (DAGO) binding and compared this with the distribution of mu 1 binding determined by competition with low [D-Ala2,D-Leu5]enkephalin (DADL) concentrations. 2. Meptazinol and DADL lowered [3H]DAGO to similar extents in most brain regions studied. The greatest levels of inhibition were observed in the periaqueductal gray, interpeduncular nucleus, thalamus, hypothalamus, and hippocampus. Low levels of inhibition were found in the temporal and frontal cortex. The correlation between the inhibition of [3H]DAGO binding by meptazinol and that by DADL was high (r = 0.83), consistent with the binding of meptazinol to mu 1 sites.     

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 [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.     

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.     

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.     

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.     

Role of steric interactions in the delta opioid receptor selectivity of [D-Pen2, D-Pen5]enkephalin

In order to assess the individual effects of each of the 3-methyl groups in residue 2 of [D-Pen2, D-Pen5]enkephalin on binding affinity to mu and delta opioid receptors, (2S,3S)methylcysteine ((3S)Me-D-Cys) and (2S,3R)methylcysteine ((3R)Me-D-Cys) were synthesized and incorporated into the analogs, [(3S)Me-D-Cys2, D-Pen5] enkephalin and [(3R)Me-D-Cys2, D-Pen5]enkephalin. Of these analogs, [(3S)Me-D-Cys2, D-Pen5]enkephalin appears from 1H n.m.r. spectra to assume a conformation similar to those of [D-Pen2, D-Pen5]enkephalin and the less delta receptor-selective, but more potent, [D-Cys2, D-Pen5]enkephalin. Assessment of binding affinity to mu and delta receptors revealed that [(3S)Me-D-Cys2, D-Pen5]enkephalin exhibits delta receptor affinity intermediate between [D-Pen2, D-Pen5]enkephalin and [D-Cys2, D-Pen5]enkephalin while its mu receptor affinity is similar to that of [D-Cys2, D-Pen5]enkephalin. These results suggest that, for [D-Pen2, D-Pen5]enkephalin, adverse steric interactions between the D-Pen2 pro-R methyl group and the mu receptor binding site lead to the low mu receptor binding affinity observed for this analog. By contrast, both the pro-R and pro-S D-Pen2 methyl groups lead to minor steric interactions which contribute to the somewhat lower delta receptor affinity of this compound.     

Regional interactions of opioid peptides at μ and δ sites in rat brain

Opiate binding sites in five brain regions were labeled with the μ and δ markers, ³H-morphine and ³H-[D-Ala²,D-leu⁵]enkephalin, respectively. The highest densities of both ³H-morphine and ³H-DADLE labeled sites are found in striatum and frontal cortex. Hypothalamus and midbrain contain predominantly ³H-morphine labeled sites. The selectivity of the opioid peptides [D-Ala²,D-leu⁵]enkephalin, β-endorphin and dynorphin(1–13) for the two opiate sites was investigated by comparing the potency of these unlabeled compounds against the μ and δ markers in different brain regions. This determination has the effect of controlling for the breakdown of peptides within each region. While the enkephalin analogue shows a preference for the δ binding site and β-endorphin is more nearly equipotent towards the two binding sites, dynorphin(1–13) shows a high affinity and selective preference for the μ binding site over the δ site. The potency of the opioid peptides in displacing the μ and δ markers varies from region to region according to the relative densities of the two opiate binding site populations.     

High and low frequency electroacupuncture analgesia are mediated by different types of opioid receptors at spinal level: a cross tolerance study

Previous studies have shown that rats subjected to low or high frequency electroacupuncture (EA) stimulation release enkephalins or dynorphins respectively to produce analgesia. This conclusion was tested in the present study by using cross tolerance technique for further analysing their receptor mechanisms. The main results were as follows: (1) In rats subjected to 2 Hz EA for 6 h, there was a gradual decrease in the analgesic effect, leading to a state of tolerance to 2 Hz EA analgesia. These rats, however, still responded to 100 Hz EA. Likewise, rats made tolerant to 100 Hz EA were still effective to 2 Hz EA stimulation, showing not significant cross tolerance between 2 Hz and 100 Hz EA analgesia. (2) Rats made-tolerant to 100 Hz EA analgesia showed a diminished response to intrathecal dynorphin A (1-13), a kappa agonist, whereas the analgesic effect of the delta agonist [D-Pen2, D-pen5] enkephalin (DPDPE) remained intact. (3) Rats made tolerant to 2 Hz EA analgesia showed a cross tolerance to DPDPE, but not to dynorphin A (1-13). Results obtained from aforementioned cross tolerance studies suggest that 2 Hz and 100 Hz EA analgesia are mediated by delta and kappa opioid receptors, respectively, at the spinal cord of the rat.     

Functional dissociation of mu opioid receptor signaling and endocytosis: implications for the biology of opiate tolerance and addiction.

Opiate analgesia, tolerance, and addiction are mediated by drug-induced activation of the mu opioid receptor. A fundamental question in addiction biology is why exogenous opiate drugs have a high liability for inducing tolerance and addiction while native ligands do not. Studies indicate that highly addictive opiate drugs such as morphine are deficient in their ability to induce the desensitization and endocytosis of receptors. Here, we demonstrate that this regulatory mechanism reveals an independent functional property of opiate drugs that can be distinguished from previously established agonist properties. Moreover, this property correlates with agonist propensity to promote physiological tolerance, suggesting a fundamental revision of our understanding of the role of receptor endocytosis in the biology of opiate drug action and addiction.     

Hemorphins, cytochrophins, and human beta-casomorphins bind to antiopiate (TYR-MIE-1) as well as opiate binding sites in rat brain

Novel peptides with opiate activity, derived from endogenous sources (human and bovine casomorphins from milk, hemorphins from hemoglobin, and cytochrophins from mitochondrial cytochrome b), were tested for their ability to inhibit binding of the brain peptide Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) to its high affinity sites in rat brain. The order of potency in inhibiting binding of 125I-Tyr-MIF-1 was: hemorphin and bovine casomorphins greater than Tyr-MIF-1 greater than cytochrophins greater than human casomorphins. Naloxone and DAMGO were ineffective at inhibiting Tyr-MIF-1 binding. The results provide evidence that, in addition to their ability to bind to mu opiate receptors, these novel endogenous peptides with opiate activity and a peptide (Tyr-MIF-1) with antiopiate properties also bind to a non-opiate site labeled by Tyr-MIF-1. These sites could be involved in a balance between opiate and antiopiate peptides.