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.     

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.     

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.     

Differential postnatal development of mu and delta opiate receptors

We found a differential postnatal development of mu and delta opiate receptors. Mu receptors labelled with low concentrations of [3H]naloxone appeared to develop earlier than did delta receptors labelled with [3H]D-Ala2-D-Leu5-enkephalin (0.5 nM). Competition binding studies also revealed a delayed appearance of delta receptors (day 12 postnatal).     

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.     

Increased delta, but not mu, opiate receptor binding in the medulla oblongata of Long-Evans rats following 5-day water deprivation

Opiate receptors of the mu type were labeled with [125I]D-Ala2,N-Me-Phe4,Met-(O)5-ol-enkephalin (FK-33824). delta receptors were labeled with [125I]D-Ala2-D-Leu5-enkephalin (DADLE) in the presence of excess (N-Me-Phe3,D-Pro4)-morphiceptin (PL017). Since DADLE binds mu and delta receptor sites, and PL017 blocks mu receptors, this protocol improves specific labeling of delta receptors. Quantitative autoradiography showed that chronic dehydration causes no changes in mu receptor binding in the medulla oblongata of Long-Evans rats. However, there is increased delta receptor binding in the solitary, hypoglossal and gracilis nuclei, and the spinal nucleus of trigeminal system of dehydrated animals, suggesting that delta opiate receptors participate in the physiological response to dehydration.     

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)     

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).     

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.     

Spinal and supraspinal opioid analgesia in the mouse: the role of subpopulations of opioid binding sites

The selective in vivo blockade of high affinity (mu1) opioid binding sites in mice by naloxazone reduced the analgesic potency of opiates and opioid peptides, evidenced by a shift of their analgesic ED50 values. However, the extent of these shifts varied significantly between a series of opioid drugs, ranging from 12-fold for morphine to 4-fold for D-Ala2-D-Leu5-enkephalin. These findings suggested that analgesia in naloxazone-treated animals is mediated through a different subpopulation of receptors than in normal controls. Correlating these analgesic shifts for a series of opioids with their affinity for different [3H]opioid binding sites suggested an analgesic role for delta sites. Additional studies in mice with spinal transections suggested that mu1 analgesia was primarily supraspinal.     

A topographic quantitative method for measuring brain tissue pH under physiological and pathophysiological conditions

The autoradiographic regional localization of [3H]beta-endorphin binding in rat brain differed from that of either [3H]dihydromorphine or [3H]D-Ala2-D-Leu5-enkephalin. Comparisons were made from sequential sections through 3 regions of rat brain: striatum, hypothalamus/thalamus, and brainstem. [3H]beta-endorphin labeled some clusters as well as the subcallosal streak in the striatum, the nucleus accumbens, lamina IV of the cortex, medial regions of the thalamus, hippocampus, inferior colliculus, dorsal raphe, median raphe and pontine nuclei. White matter regions had little binding. Although many of these structures were also labeled with either [3H]dihydromorphine or [3H]D-Ala2-D-Leu5-enkephalin, the overall pattern of [3H]beta-endorphin labeling appeared unique, consistent with the proposal of central epsilon receptors.     

Zeta (zeta), a growth-related opioid receptor in developing rat cerebellum: identification and characterization.

Endogenous opioids and opioid receptors (i.e. endogenous opioid systems) are expressed during neural ontogeny, and play a role in the development of the nervous system. Using [3H][Met5]-enkephalin, a potent ligand involved in neural growth, particularly cell proliferation, specific and saturable binding was detected in homogenates of 6-day-old rat cerebellum; the data were consistent with a single binding site. Scatchard analysis yielded a binding affinity (Kd) of 2.2 nM and a binding capacity (Bmax) of 22.3 fmol/mg protein. Binding was linear with protein concentration, dependent on time, temperature, and pH, and was sensitive to Na+, Mg2+, and guanyl nucleotides. Optimal binding required protease inhibitors, and pretreatment of the homogenates with trypsin markedly reduced binding, suggesting that the binding site was proteinaceous in character. The [Met5]-enkephalin binding site was an integral membrane protein located in the nuclear fraction. Competition experiments indicated that [Met5] enkephalin was the most potent displacer of [3H][Met5]-enkephalin, and that binding was stereospecific. In the adult rat cerebellum, non-opioid receptor binding of [3H][Met5]-enkephalin was recorded, mu and kappa receptors were also found in the developing rat cerebellum, while mu, delta, and kappa receptors were recorded in adult cerebellar tissue. The function, pharmacological and biochemical characteristics, subcellular distribution, and temporal expression of the [Met5]-enkephalin binding site suggest the presence of a unique opioid receptor, termed zeta (zeta), in the developing nervous system.     

Increased affinity of dimeric enkephalins is not dependent on receptor density

The equilibrium binding and dissociation kinetics of the enkephalin dimer bis-(D-Ala2-D-Leu5-enkephalin)-ethylenediamide (designated DPE2) to neuroblastoma glioma NG108-15 cells were investigated and compared with the monomers D-Ala2-D-Leu5-enkephalin (DADL) and D-Ala2-Leu5-enkephalinamide (DALEA). Binding was studied after exposure of the membrane to increasing concentrations of the irreversible delta receptor selective ligand FIT in order to decrease the density of binding sites on the cell membrane. The increased affinity of DPE2 did not revert to that of the monomer DADL by this reduction of binding sites. Similarly, the dissociation of DPE2 did not approach that of the monomer DALEA in the presence of 1 microM DALEA. These data strongly suggest that crosslinking does not occur, and fail to confirm the hypothesis that dimers with short spanning chain length aid the clustering of receptors. We postulate: 1) If the dimer binds to a bivalent binding site, the monovalent binding state of our bivalent ligand may not exist to an appreciable extent, and 2) the bivalent ligand cannot bind when the binding site is irreversibly blocked by a monovalent ligand.     

Evidence for multiple "Kappa" binding sites by use of opioid peptides in the guinea-pig lumbo-sacral spinal cord

Binding properties of [3H]-etorphine and [3H]-ethylketocyclazocine have been studied in the lumbo-sacral spinal cord of guinea-pig which does not contain mu or delta binding sites. [3H]-etorphine binds to a single class of high affinity sites, whereas [3H]-ethylketocyclazocine interacts with a high and a low affinity component. Using a discriminative procedure, 5 microM (D-Ala2, D-Leu5) enkephalin (DAL), the high affinity component of [3H]-ethylketocyclazocine can be resolved in two classes of sites, (D-Ala2, D-Leu5) enkephalin sensitive sites (DALS sites) and (D-Ala2, D-Leu5) enkephalin insensitive sites (DALI sites). In these conditions, there is a total loss of [3H]-etorphine sites, whose binding capacity and properties strictly correspond to the DALS sites labelled by [3H]-ethylketocyclazocine. Pharmacological investigations indicate that DALI sites for which dynorphin (1 leads to 17) is the best ligand, can be related to kappa sites previously described in guinea-pig brain, whereas DALS sites for which (Arg6, Phe7) Met-enkephalin possesses a good affinity, closely correspond to benzomorphan sites recently characterized in rat brain and spinal cord. [3H]-ethylketocyclazocine interacts additionally with "non opiate" low affinity sites, for which only benzomorphan drugs exhibit a good affinity, whereas morphine, naloxone, phencyclidine or endogenous opioid peptides do not present any affinity for them. On the basis of these data, a new subdivision of "kappa" sites is discussed.     

Properties of dopamine agonist and antagonist binding sites in mammalian retina

Retinal homogenates of calf, rat, rabbit and Cebus appella and Macaca mulata monkeys were found to contain stereospecific binding sites for the dopamine antagonist [3H]spiroperidol. In further studies with calf and rat retina, stereospecific binding sites were also found for the dopamine agonist [3H]ADTN (2-amino-6,7,-dihydroxy-1,2,3,4-tetrahydronapththalene). The [3H]spiroperidol binding sites in calf retina were pharmacologically similar to the dopaminergic spiroperidol binding sites previously demonstrated to be present in striatum. However, calf and rabbit retina contained less than 1/10 the concentration of [3H]spiroperidol binding sites found in striatum. Saturation studies and Scatchard analyses showed a single class [3H]spiroperidol binding sites with Kd (apparent dissociation constant) = 0.3 and 0.2 nM and Bmax (binding site number) = 38 and 24 fmol/mg protein in calf retina and rabbit retina respectively. Rates of [3H]spiroperidol association and dissociation were also evaluated in calf retina. Drug specificity for [3H]ADTN binding in calf retina resembled that previously reported for striatal [3H]ADTN binding and thus differed from retinal [3H]spiroperidol binding. Calf retinal [3H]ADTN binding sites had a Kd = 9 nM and Bmax = 113 +/- 12 fmol/mg protein. Thus, the total number of [3H]ADTN sites in retina was at least twice that of [3H]spiroperidol sites. Guanine nucleotides (GTP and Gpp (NH)p) but not ATP reduced the affinity of the dopamine agonist ADTN for [3H]spiroperidol binding, and also reduced the specific binding of [3H]ADTN itself up to a maximal value of about 50% of control binding. Saturation studies of calf retinal [3H]ADTN binding confirmed that Gpp(NH)p-displaceable sites were a discrete saturable subset of stereospecific [3H]ADTN sites with Kd = 9 nM and Bmax = 50 +/- 6 fmol/mg protein. The Gpp(NH)p insensitive sites had a Kd = 9 nM and Bmax = 63 +/- 7 fmol/mg protein. It is proposed that although [3H]ADTN sites differ pharmacologically from [3H]spiroperidol sites, since [3H]spiroperidol sites are guanine nucleotide-sensitive and similar in number to the guanine nucleotide-sensitive class of [3H]ADTN sites, they may possibly be related to these sites as well as to adenylate cyclase. In addition, retina contains guanine nucleotide-insenstive [3H]ADTN sites, possibly presynaptic and probably not coupled to adenylate cyclase.     

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)     

Decrease in mu-opioid receptor binding capacity in rat brain after chronic PL017 treatment

In previous studies, we have demonstrated that chronic treatment of rats with either etorphine or D-Ala2, D-Leu5-enkephalin (DADLE) resulted in the reduction of opioid receptor binding activities during the course of tolerance development. In both cases, mu-opioid receptor binding capacity was attenuated together with the delta-opioid receptor binding capacity. Because both etorphine and DADLE are relatively non-specific opioid ligands, interacting with both mu and delta receptors, these studies could not determine whether down-regulation of a specific receptor type is possible. Therefore, in the current studies, animals were rendered tolerant to the mu-opioid receptor-selective ligand PL017 and the receptor binding capacity was measured afterwards. Treating Sprague-Dawley rats with increasing doses of PL017 (2.5-20 micrograms/kg) i.c.v. for 5 days resulted in a 30- to 40-fold increase in the AD50 of the peptide to elicit the antinociceptive response and about 14-fold increase in the ED50 of the peptide to elicit the catatonic effect. When mu- and delta-binding was determined using [3H]diprenorphine in the presence of morphiceptin or DPDPE respectively, a significant decrease (20-30%) in the mu-opioid receptor binding but not in delta-opioid receptor binding was observed in all the brain areas tested after 5 days of PL017 treatment. Scatchard analysis of the [3H]DAMGO saturation binding data revealed a decrease in Bmax values and no change in the Kd values. Hence, mu-opioid receptors can be specifically regulated by ligand in the brain as delta-receptors are in neuroblastoma x glioma NG 108-15 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vivo occupation of mouse brain opioid receptors by endogenous enkephalins: blockade of enkephalin degrading enzymes by RB 101 inhibits [3H]diprenorphine binding.

With the aim of possibly studying the local activity of brain enkephalinergic pathways by autoradiography and positron emission tomography, preliminary competition experiments of [3H]diprenorphine binding in mouse brain were carried out after i.v. administration of the first systemically-active mixed inhibitor of enkephalin degrading enzymes RB 101 (N(R,S)-2-benzyl-3[(S)-(2-amino-4-methylthiobutyldithio]-1-oxoprop yl]- L-phenylalanine benzyl ester). Although devoid of affinity for the opioid binding sites, RB 101 inhibited the [3H]diprenorphine binding to the opioid receptors in a dose-dependent manner. This effect, very likely due to an RB 101-induced increase in extracellular levels of enkephalins, reached a plateau at a dose of 10 mg/kg, where almost 30% displacement was observed. Intravenous administration of either 5 or 20 mg/kg of RB 101 in mice submitted to warm-swim stress led to an additional [3H]diprenorphine displacement, which reached 45% compared to unstressed controls. This ceiling effect could account for the reported minimal morphine-like side effects induced by mixed inhibitors. A large increase in endogenous enkephalin levels induced by RB 101, associated or not with stress, was also indirectly demonstrated by the analgesic responses elicited by i.v. injection of the mixed inhibitor. This effect was blocked by naloxone but not by the delta antagonist naltrindole (NTI), supporting a preferential implication of mu receptors in supraspinal analgesia. Taken together, these results suggest that RB 101 could be used to determine the precise in vivo localization of enkephalinergic pathways recruited by various stimuli.     

Characterization and distribution of [3H]ohmefentanyl binding sites in the human brain

Binding properties and localization of [3H]ohmefentanyl, a new ligand for mu opioid receptors, were investigated on normal human brain sections. Binding assays performed at the level of the basal ganglia revealed: (1) a steady-state binding reached after 60 min incubation at room temperature, (2) the presence, in saturation experiments, of an apparent single class of binding sites with a Kd = 1.68 +/- 0.45 nM and a Bmax = 162 +/- 9 fmol/mg protein, (3) an order of potency to inhibit [3H]ohmefentanyl binding as follows: ohmefentanyl greater than [D-Ala2, MePhe4, Gly-ol5] enkephalin (DAGO) greater than ethylketocyclazocine (EKC) much greater than Tyr-D-Ser(OtBu)-Gly-Phe-Leu-Thr(OtBu) (BUBU) and U-50,488H. Quantitative autoradiography showed an heterogeneous distribution of [3H]ohmefentanyl binding sites with the highest densities in amygdala, medical geniculate body, thalamus, and caudate nucleus. Binding characteristics and anatomical distribution also show that [3H]ohmefentanyl may bind to a small proportion of additional sites called "DAGO-inaccessible [3H]ohmefentanyl specific binding sites." [3H]Ohmefentanyl binding to these sites can be partly inhibited by sigma ligands such as 1,3-di-o-tolylguanidine (DTG) and haloperidol. However, unlabeled DAGO inhibited more than 80% of [3H]ohmefentanyl specific binding in most of the human brain regions studied, suggesting that the major population of sites labeled by [3H]ohmefentanyl represented mu opioid receptors.     

Opiate receptor localization in rat cerebral cortex

The differential distributions of [3H]naloxone-labeled and [3H]D-Ala-D-Leu-enkephalin-labeled opiate receptors in rat cerebral cortex were localized autoradiographically and quantified by grain counting and computerized densitometry. In addition, receptor distributions were compared to terminal patterns of thalamocortical projections labeled by axoplasmic transport of [3H]amino acids. Opiate receptors labeled with [3H]naloxone in a mu ligand selectivity pattern show striking laminar heterogeneity and are densest in limbic cortical areas, intermediate in the motor cortex, and fewest in the primary sensory areas. By contrast, opiate receptors labeled with [3H]D-Ala2-D-Leu5-enkephalin in a delta ligand selectivity pattern are much more homogeneously distributed across both regions and laminae within regions. Mu receptors in most cortical areas have density peaks in layers I and VI and each peak shows a density gradient that is sloped within the layer so that the highest densities are at the most superficial and the deepest portions of cortex. In addition, there is an intermediate peak whose laminar position varies depending on the area in which it is found. In rostral agranular cortex, including limbic and motor areas, the [3H]naloxone binding peaks are in layers I, III, and VI. In primary somatosensory cortex, the intermediate peak is in layer Va and in most of remaining homotypical cortex it is in layer IV. Some areas have only bilaminar labeling, in superficial and deep layers; these include portions of the sulcal and retrosplenial cortices. Piriform and entorhinal cortices have dense [3H]naloxone binding only in the deepest layer and show a descending gradient of density toward the superficial layer. The positions of the mu receptor peaks were compared with termination patterns of projections originating in the thalamus. Close correspondence was found between receptor binding in the prelimbic, primary somatosensory, and entorhinal areas and projection terminations arising from the thalamic mediodorsal, posterior, and central medial nuclei, respectively. Although regional variations in [3H]D-Ala2-D-Leu5-enkephalin-labeled receptor density are uncommon, a gradual decrease in the number of sites along the dorsomedial wall of the cortex from anterior cingulate to caudal retrosplenial limbic cortex can be observed. Laminar variations in binding density are small as well; higher concentrations of the peptide binding sites are usually found in the deep cortical layers. These findings emphasize aspects of opiate receptor architecture which may be relevant to identifying cortical "opiatergic" neurocircuitry and raise the possibility of opiate modulation of thalamocortical transmission.