Further evidence that gonadal steroids do not modulate brain opiate receptors in male rats

It is still unclear whether, in the male rat, castration and androgen replacement affect the binding characteristics of brain opiate receptors. To clarify this issue, the effects exerted by orchidectomy and testosterone (T) replacement on the subpopulation of brain mu opiate receptors were studied in male rats; testosterone was administered via subcutaneous Silastic capsules. Utilizing 3H-dihydromorphine (a mu receptor ligand) it has been shown that the affinity constant (Ka) of brain mu opiate binding sites, measured in plasma membrane preparations, is not affected by castration. When mu receptor concentrations were measured in individual brains, it was found that gonadectomy and T replacement failed to produce any change in the number of mu opiate receptors. These data suggest that, in male rats, gonadal steroids do not develop their central feedback effects by affecting brain mu opiate receptors.     

Covalent labeling of opioid receptors with radioiodinated human beta-endorphin. Identification of binding site subunit

125I-beta-Endorphin (human) binds with high affinity, specificity, and saturability to rat brain and neuroblastoma X glioma hybrid cell (NG 108-15) membranes. Dissociation constants and binding capacities were obtained from Scatchard plots and are 2 nM and 0.62 pmol/mg of protein for rat whole brain and 6 nM and 0.8 pmol/mg of protein for NG 108-15 cells. Results from competition experiments also indicate that this ligand interacts with high affinity with both mu and delta opioid binding sites, with a slight preference for mu sites, while exhibiting low affinity at kappa sites. We have demonstrated that human 125I-beta-endorphin is a useful probe for the investigation of the subunit structure of opioid receptors. The specific cross-linking of this ligand has revealed the presence of four reproducible bands or areas after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography at 65, 53, 38, and 25 kDa. All labeled bands seem to be opioid receptor related since they are eliminated when binding is carried out in an excess of various opiates. The evidence we have obtained using rat whole brain (delta congruent to mu), rat thalamus (largely mu), bovine frontal cortex (delta:mu congruent to 2:1), and NG 108-15 cells (delta) demonstrates that different labeling patterns are obtained when mu and delta binding sites are cross-linked. The pattern obtained on sodium dodecyl sulfate-polyacrylamide gel electrophoresis from cross-linked mu sites contains a major (heavily labeled) component of 65 kDa and a minor component of 38 kDa, while patterns from delta sites contain a major labeled component of 53 kDa. This 53-kDa band appears clearly in extracts from NG 108-15 cells and bovine frontal cortex, while in rat whole brain a diffusely labeled region is present between 55 and 41 kDa. In addition, NG 108-15 cells also display a minor labeled component at 25 kDa. The relationship of the minor bands to the major bands is not clear.     

Comparison of mu, delta, and kappa opiate binding sites in rat brain and spinal cord

The binding characteristics of mu, delta, and kappa opiate sites were studied in rat brain and spinal cord membrane homogenates. Scatchard analysis of 3H-Dihydromorphine, 3H-D-Ala2-D-Leu5-Enkephalin (in the presence of morphiceptin), and 3H-Ethylketocyclazocine (in the presence of morphiceptin and D-Ala2-D-Leu5-Enkephalin) binding sites revealed similar high affinities of these ligands for their respective sites in brain and spinal cord. The majority of binding in brain and spinal cord was attributed to mu and delta sites, with only about 10% of the combined total binding capacity being kappa.     

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

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

Differences of binding characteristics of non-selective opiates towards mu and delta receptor types

[3H]ET (etorphine), which is considered either as an "universal" ligand or a mu agonist, interacts with identical affinities KD = 0.33-0.38 nM to hybrid cells and rabbit cerebellum, pure delta and mu-enriched opioid receptor preparations, respectively. In rat brain tissue, [3H]ET binding is inhibited by DAGO (Tyr-D-Ala-Gly-(Me)-Phe-Gly-ol), a mu selective agonist, in a competitive manner without apparent modification of the maximal number of sites. Furthermore, even at a DAGO concentration (300 nM) which should be sufficient to block [3H]ET interaction with mu sites, no variation in the total capacity of the tritiated ligand is observed. In contrast, DTLET (Tyr-D-Thr-Gly-Phe-Leu-Thr), a delta-preferential agonist, blocks [3H]ET binding in rat brain at a concentration able to saturate delta-sites. At higher concentrations, where DTLET cross reacts with mu-sites, this ligand exhibits similar properties to those of DAGO. These data are very different from those obtained with [3H]EKC (ethylketocyclazocine), another "universal" ligand, the binding properties of which are easily explained by the occurrence in rat brain tissue of independent sites exhibiting pharmacological profiles of mu, delta and kappa sites. Our results underline the possible misinterpretation of binding data obtained by using [3H] etorphine as a non selective ligand.     

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.     

Evidence for a mu-delta opioid receptor complex in CHO cells co-expressing mu and delta opioid peptide receptors

Based on non-competitive binding interactions we suggested that mu and delta receptors associate as a mu/delta receptor complex in rat brain. We hypothesized that the same non-competitive binding interactions observed in rat brain will be seen in CHO cells that co-express mu and delta receptors, but not in cells that express just mu or delta receptors. We used CHO cells expressing the cloned human mu receptor, cloned human delta receptor, or cloned mouse delta/human mu ("dimer cell"). Cell membranes were prepared from intact cells pretreated with 100nM SUPERFIT. [(3)H][d-Ala(2),d-Leu(5)]enkephalin binding assays followed published procedures. SUPERFIT, a delta-selective irreversible ligand, decreased [(3)H][d-Ala(2),d-Leu(5)]enkephalin binding to delta receptors by approximately 75% and to mu receptors by approximately 50% in dimer cells. SUPERFIT treatment did not decrease [(3)H][d-Ala(2),d-Leu(5)]enkephalin binding to mu cells. The IC(50) values observed in SUPERFIT-treated dimer cells were: [d-Pen(2),d-Pen(5)]enkephalin (1820nM) and morphine (171nM). Saturation binding experiments with SUPERFIT-treated dimer cells showed that [d-Pen(2),d-Pen(5)]enkephalin (5000nM) was a competitive inhibitor. In contrast, morphine (1000nM) lowered the B(max) from 1944fmol/mg to 1276fmol/mg protein (35% decrease). Both [d-Pen(2),d-Pen(5)]enkephalin and morphine competitively inhibited [(3)H][d-Ala(2),d-Leu(5)]enkephalin binding to SUPERFIT-treated mu cells. The results indicate that the mu-delta opioid receptor complex defined on the basis of non-competitive binding interactions in rat brain over 20 years ago likely occurs as a consequence of the formation of mu-delta heterodimers. SUPERFIT-treated dimer cells may provide a useful model to study the properties of mu-delta heterodimers.     

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.     

Multiple opioid receptors: An examination of the dissociation of [3H]leucine enkephalin from rat brain membranes

The experiments reported in this paper address the hypothesis that [3H]leucine enkephalin labels both mu and delta receptors. As reported by other workers, this peptide dissociates from rat brain membranes in a biphasic manner. This is consistent with a two site binding model which hypothesizes that the peptide labels both opioid mu and delta receptors from which it dissociates at different rates. To test this hypothesis, we determined the dissociation of bound ligand from rat brain membranes incubated to equilibrium with [3H]leucine enkephalin in the absence and presence of 100 nM morphine. The data were not significantly different. We conclude that the biphasic off-kinetics of [3H]leucine enkephalin is not evidence for a two-site binding model.     

Characterization of type 2 opiate receptors

Recent evidence suggests that the Type 1 opiate receptor (in rat striatal patches) is a mobile receptor which is able to adopt a mu, delta, or kappa opiate receptor ligand selectivity pattern under appropriate conditions. In this paper, we have investigated such a possibility for Type 2 opiate receptors which are visualized diffusely over rat striatum. Ligand selectivity analysis suggested that the Type 2 opiate binding site is equivalent to a delta opiate receptor. The auto-radiographic distribution of Type 2 opiate binding sites is diffuse over most areas of rat brain. Thus, Type 2 opiate binding sites are different from Type 1 opiate receptors which are very discretely distributed in rat brain. Our results suggest that Type 2 opiate receptors, unlike Type 1 opiate receptors, are receptors locked in a delta-like ligand selectivity conformation.     

Autoradiographic evidence for two classes of mu opioid binding sites in rat brain using [I]FK33824

Previous studies demonstrated that pretreatment of brain membranes with the irreversible mu antagonist, beta-funaltrexamine (beta-FNA), partially eliminated mu binding sites [25,35], consistent with the existence of two mu binding sites distinguished by beta-FNA. This paper tests the hypothesis that the FNA-sensitive and FNA-insensitive mu binding sites have different anatomical distributions in rat brain. Prior to autoradiographic visualization of mu binding sites, [³H]oxymorphone, [³H]D-ala²-MePhe⁴, Gly-ol⁵-enkephalin (DAGO), and [¹²⁵I]D-ala²-Me-Phe⁴-met(o)-ol]enkephalin (FK33824) were shown to selectively label mu binding sites using slide mounted sections of molded minced rat brain. As found using membranes, beta-FNA eliminated only a portion of mu binding sites. Autoradiographic visualization of mu binding sites using the mu-selective ligand [¹²⁵I]FK33824 in control and FNA-treated sections of rat brain demonstrated that the proportion of mu binding sites sensitive to beta-FNA varied across regions of the brain, particularly the dorsal thalamus, ventrobasal complex and the hypothalamus, providing anatomical data supporting the existence of two classes of mu binding sites in rat brain.     

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.     

3H][D-Ser2(O-tert-butyl),Leu5]enkephalyl-Thr6 and [D-Ser2(O-tert-butyl),Leu5]enkephalyl-Thr6(O-tert-butyl). Two new enkephalin analogs with both a good selectivity and a high affinity toward delta-opioid binding sites

Insertion of bulky tertiobutyl groups into the sequence of [D-Ser2,Leu5]enkephalyl-Thr6 leads to a conformationally induced large increase in selectivity toward rat brain delta-opioid binding sites, as shown by the ratio of apparent affinities for mu and delta receptors of [D-Ser2(O-tert-butyl),Leu5]enkephalyl-Thr6,KI(mu)/KI(delta) = 130, and [D-Ser2(O-tert-butyl),Leu5]enkephalyl-Thr6 (O-tert-butyl),KI(mu)/KI(delta) = 280. In addition to a selectivity similar to that of the cyclic compounds [D-Pen2, D-Pen5]enkephalin and [D-Pen2,L-Pen5]enkephalin, the affinity of [3H][D-Ser2(O-tert-butyl),Leu5]enkephalyl-Thr6 for the delta sites of rat brain membranes is significantly better (KD = 2.2 nM) than that of [3H][D-Pen2,D-Pen5]enkephalin (KD approximately 8.5 nM). Therefore, [3H][D-Ser2(O-tert-butyl),Leu5]enkephalyl-Thr6 seems to be the most appropriate delta-probe currently available for binding studies. Moreover, the lipophilic and protected peptide [D-Ser2(O-tert-butyl),Leu5]enkephalyl-Thr6(O-tert-butyl) behaves as the most specific ligand for the delta-opioid binding sites and appears appropriate for in vivo investigations. The inactive analogue [D-Thr2(O-tert-butyl),Leu5]enkephalyl-Thr6 might serve as a negative control in biochemical or pharmacological studies.     

Biologically active MK-801 and SKF-10,047 binding sites distinct from those in rat brain are expressed on human lung cancer cells.

We have shown previously that cultured human lung cancer cells of different histologic types express multiple opioid receptors that can regulate their growth. In this report, we show that these cells also express specific, saturable, and high-affinity binding sites (Kd approximately 1 nM) for the non-opioid phencyclidine (PCP), [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,b]cyclohepten-5,10-imine hydrogen maleate] (MK-801) and sigma N-allylnormetazocine (SKF-10,047) receptor ligands. Characterization of these binding sites showed them to be protein in nature and sensitive to the guanine nucleotide GTP. Pharmacological studies showed that (+) MK-801 and (+) SKF-10,047 competed with each other for their binding sites and also for the methadone binding site present in these cells. However, the mu and delta opioid ligands did not compete for (+) MK-801 and (+) SKF-10,047 binding sites. In addition, these binding sites on lung cancer cells appear to be distinct from the N-methyl D-aspartate/PCP receptor ionophore complex reported to be present in rat brain. MK-801 and SKF-10,047, at nM concentrations, were found to inhibit the growth of these cells in culture within a few hours of exposure, and this effect was irreversible after 24 h. The growth effects of these ligands could not be reversed by the opioid antagonist naloxone, suggesting involvement of nonopioid type receptors in the actions of these ligands. The abundant expression of biologically active MK-801 and SKF-10 047 binding sites in these cell lines, distinct from those in rat brain, suggests that these cell lines may prove to be a valuable source for further characterization and purification of these binding sites.     

Opiate antagonist binding sites in discrete brain regions of spontaneously hypertensive and normotensive Wistar-Kyoto rats

The binding of 3H-naltrexone, an opiate receptor antagonist, to membranes of discrete brain regions and spinal cord of 10 week old spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats was determined. The brain regions examined were hypothalamus, amygdala, hippocampus, corpus striatum, pons and medulla, midbrain and cortex. 3H-Naltrexone bound to membranes of brain regions and spinal cord at a single high affinity site with an apparent dissociation constant value of 3 nM. The highest density of 3H-naltrexone binding sites were in hippocampus and lowest in the cerebral cortex. The receptor density (Bmax value) and apparent dissociation constant (Kd value) values of 3H-naltrexone to bind to opiate receptors on the membranes of amygdala, hippocampus, corpus striatum, pons and medulla, midbrain, cortex and spinal cord of WKY and SHR rats did not differ. The Bmax value of 3H-naltrexone binding to membranes of hypothalamus of SHR rats was 518% higher than WKY rats but the Kd values in the two strains did not differ. It is concluded that SHR rats have higher density of opiate receptors labeled with 3H-naltrexone in the hypothalamus only, in comparison with WKY rats, and that such a difference in the density of opiate receptors may be related to the elevated blood pressure in SHR rats.     

A photoactivatable naltrexone derivative labels glycoproteins of different molecular weight corresponding to the mu- and kappa-opioid receptors.

The synthesis and characterization of a novel opioid receptor photoaffinity probe [3H]naltrexyl urea phenylazido derivative ([3H]NUPA) is described. In the absence of light, [3H]NUPA binds with high affinity in a reversible and saturable manner to rat brain and guinea pig cerebellum membranes. Dissociation constants and binding capacities (Scatchard plots) are 0.11 nM and 250 fmol/mg of protein for rat brain and 0.24 nM and 135 fmol/mg of protein for guinea pig cerebellum. Competition experiments indicate that this ligand interacts with high affinity at both mu- and kappa-opioid binding sites while exhibiting low affinity at delta sites (Ki = 21 nM). On irradiation, [3H]NUPA incorporates irreversibly into rat brain and guinea pig cerebellum membranes. SDS gel electrophoresis of rat brain membranes reveals specific photolabeling of a 67-kDa molecular mass band. Conversely, a major component of 58 kDa and a minor component of 36 kDa are obtained from [3H]NUPA-labeled guinea pig cerebellum membranes. Different photolabeling patterns are obtained in rat brain (mu/delta/kappa, 4/5/1) and guinea pig cerebellum (mu+delta/kappa, 1,5/8,5) membranes in the presence of selective opioid ligands indicating labeling of mu and kappa sites, respectively. Thus, [3H]NUPA behaves as an efficient photoaffinity probe of mu- and kappa-opioid receptors, which are probably represented by distinct glycoproteins of 67 and 58 kDa, respectively.     

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.     

Opioid-Inhibited Adenylyl Cyclase in Rat Brain Membranes: Lack of Correlation with High-Affinity Opioid Receptor Binding Sites

Opioid agonists bind to GTP-binding (G-protein)-coupled receptors to inhibit adenylyl cyclase. To explore the relationship between opioid receptor binding sites and opioid-inhibited adenylyl cyclase, membranes from rat striatum were incubated with agents that block opioid receptor binding. These agents included irreversible opioid agonists (oxymorphone-p-nitrophenylhydrazone), irreversible antagonists [naloxonazine, beta-funaltrexamine, and beta-chlornaltrexamine (beta-CNA)], and phospholipase A2. After preincubation with these agents, the same membranes were assayed for high-affinity opioid receptor binding [3H-labeled D-alanine-4-N-methylphenylalanine-5-glycine-ol-enkephalin (mu), 3H-labeled 2-D-serine-5-L-leucine-6-L-threonine enkephalin (delta), and [3H]ethylketocylazocine (EKC) sites] and opioid-inhibited adenylyl cyclase. Although most agents produced persistent blockade in binding of ligands to high-affinity mu, delta, and EKC sites, no change in opioid-inhibited adenylyl cyclase was detected. In most treated membranes, both the IC50 and the maximal inhibition of adenylyl cyclase by opioid agonists were identical to values in untreated membranes. Only beta-CNA blocked opioid-inhibited adenylyl cyclase by decreasing maximal inhibition and increasing the IC50 of opioid agonists. This effect of beta-CNA was not due to nonspecific interactions with G(i), Gs, or the catalytic unit of adenylyl cyclase, as neither guanylylimidodiphosphate-inhibited, NaF-stimulated, nor forskolin-stimulated activity was altered by beta-CNA pretreatment. Phospholipase A2 decreased opioid-inhibited adenylyl cyclase only when the enzyme was incubated with brain membranes in the presence of NaCl and GTP. These results confirm that the receptors that inhibit adenylyl cyclase in brain do not correspond to the high-affinity mu, delta, or EKC sites identified in brain by traditional binding studies.     

Discrete mapping of brain Mu and delta opioid receptors using selective peptides: quantitative autoradiography, species differences and comparison with kappa receptors

The opioid peptides, [3H]DAGO and [3H]DPDPE, bound to rat and guinea pig brain homogenates with a high, nanomolar affinity and to a high density of mu and delta receptors, respectively. [3H]DAGO binding to mu receptors was competitively inhibited by unlabelled opioids with the following rank order of potency: DAGO greater than morphine greater than DADLE greater than naloxone greater than etorphine much greater than U50488 much greater than DPDPE. In contrast, [3H]DPDPE binding to delta receptors was inhibited by compounds with the following rank order of potency: DPDPE greater than DADLE greater than etorphine greater than dynorphin(1-8) greater than naloxone much greater than U50488 much greater than DAGO. These profiles were consistent with specific labelling of the mu and delta opioid receptors, respectively. In vitro autoradiographic techniques coupled with computer-assisted image analyses revealed a discrete but differential anatomical localization of mu and delta receptors in the rat and guinea pig brain. In general, mu and delta receptor density in the rat exceeded that in the guinea pig brain and differed markedly from that of kappa receptors in these species. However, while mu receptors were distributed throughout the brain with "hotspots" in the fore-, mid- and hindbrain of the two rodents, the delta sites were relatively diffusely distributed, and were mainly concentrated in the forebrain with particularly high levels within the olfactory bulb (OB), n. accumbens and striatum. Notable regions of high density of mu receptors in the rat and guinea pig brain were the accessory olfactory bulb, striatal "patches" and "streaks," amygdaloid nuclei, ventral hippocampal subiculum and dentate gyrus, numerous thalamic nuclei, geniculate bodies, central grey, superior and inferior colliculi, solitary and pontine nuclei and s. nigra. Tissues of high delta receptor concentration included, OB (external plexiform layer), striatum, n. accumbens, amygdala and cortex (layers I-II and V-VI). Delta receptors in the guinea pig were, in general, similarly distributed to the rat, but in contrast to the latter, the hindbrain regions such as the thalamus, geniculate bodies, central grey and superior and inferior colliculi of the guinea pig were apparently more enriched than the rat. These patterns of mu and delta site distribution differed dramatically from that of the kappa opioid sites in these species studied with the peptide [125I]dynorphin(1-8).     

Affinity labeling of delta opioid receptors by an enkephalin-derivative alkylating agent, DSLET-Mal

Opioid binding properties of Tyr-D-Ser-Gly-Phe-Leu-Thr-NH-NH-Gly-Mal (DSLET-Mal), a novel enkephalin-framed affinity label, was determined in rat brain membranes. In competition studies the ligand showed high affinity for the delta opioid sites, labelled by [(3)H][Ile(5,6)]deltorphin II (K(i) = 8 nM), whereas its binding to the mu ([(3)H]DAMGO) and kappa ([(3)H]EKC) sites was weaker. Preincubation of the rat brain membranes with DSLET-Mal at micromolar concentrations resulted in a wash-resistant and dose-dependent inhibition of the [(3)H][Ile(5,6)]deltorphin II binding sites (96% blocking at 10 microM concentration). Intracerebroventricular (ICV) administration of DSLET-Mal reduced the density of delta opioid receptors and had no effect on mu and kappa receptors, as determined by saturation binding studies. [Ile(5, 6)]deltorphin II-stimulated [(35)S]GTPgammaS binding was determined in membrane preparations of different brain areas of the ICV-treated animals. In both frontal cortex and hippocampus DSLET-Mal significantly decreased G protein activation by the delta agonist, having no effect on DAMGO stimulated [(35)S]GTPgammaS binding. DSLET-Mal had qualitatively similar effects on both receptor binding and G protein activation. These characteristics of the compound studied suggest that DSLET-Mal can serve as an affinity label for further studies of the delta-opioid receptors.