Biochemical characterization and regional quantification of mu, delta and kappa opioid binding sites in rat spinal cord.

The spinal cord contains mu, delta and kappa opioid receptors which mediate the antinociceptive effects of opioid agonists administered onto the spinal cord. In this study, we characterized the binding sites for highly-selective mu, delta and kappa opioid radioligands and quantified the distribution of opioid binding sites in rat lumbosacral spinal cord using autoradiography. In sections of rat brain mounted on glass slides, the mu ligand, [3H]sufentanil, bound with high affinity with an apparent Kd of 0.46 nM. The delta ligand, [3H]DPDPE [( D-Pen2.5]-enkephalin), bound with a Kd of 4.31 nM, and the kappa-ligand, [3H]U69593, bound with a Kd of 2.27 nM. Three regions of the spinal gray were targeted for quantification of binding sites by autoradiography. The data indicate that when considered as a percentage of the total opioid binding capacity within a region, the contribution of mu sites in laminae I-II was about 90%, with delta and kappa sites 7% and 3%, respectively. In lamina V, the mu sites comprised about 70% of the total opioid sites, with delta and kappa sites comprising 28% and 2%, respectively. In the area adjacent to the central canal, mu sites contributed about 65% of the total opioid sites followed by delta sites at 33% and kappa sites at 2% of total opioid sites. These results demonstrate a differential distribution of mu, delta and kappa binding sites with respect to the organization of the spinal gray matter. The preferential occurrence of all 3 opioid binding sites in the superficial dorsal horn is noteworthy since many fine caliber primary afferent fibers mediating nociception establish synaptic contact in this region.     

Differential ontogeny of multiple opioid receptors (mu, delta, and kappa)

We investigated the postnatal ontogeny of opioid receptors in rat brain under assay conditions which, when combined with computerized analysis, effectively reflect the developmental profile of high affinity binding to mu, delta, and kappa subpopulations. Concentrations of mu sites were assessed with the selective ligand 3H-[D-ala2,mePhe4,gly-ol5]enkephalin (DAGO). The other two sites were analyzed in binding assays with less selective radioligands but in the presence of specific unlabeled ligands which suppress cross-reactivity. We utilized 3H-[D-ala2,D-leu5]enkephalin (DADL) in the presence of 10 nM DAGO to label delta sites and 3H-ethylketocyclazocine (EKC) in the presence of 100 nM DADL + 100 nM [D-ala2,mePhe4,Met(0)ol5]enkephalin to detect kappa receptors. After birth, the density (femtomoles per milligram of wet weight) of mu sites declined for several days and then rose sharply over the next 2 weeks, increasing 2-fold by adulthood. Delta (delta) sites appeared in the second week postnatal and increased more than 8-fold in the next 2 weeks. Levels of kappa receptors were relatively low at birth and increased slowly (2-fold, overall). Computerized analyses of binding data revealed that DAGO and DADL were binding to single populations of sites throughout the postnatal period. DAGO and EKC affinities did not fluctuate in this period, whereas DADL affinities were low for the first week and then rose to adult levels. In summary, mu, kappa, and delta receptors exhibit differential postnatal developmental profiles. The former two are present at birth, whereas the latter appears in the second week. The postnatal increase for all three sites appear to be preceded by the previously demonstrated emergence of opioid peptides.     

Autoradiographic differentiation of mu, delta, and kappa opioid receptors in the rat forebrain and midbrain

While there is an abundance of pharmacological and biochemical evidence to suggest the existence of multiple opioid receptors, their precise localization within the brain is unclear. To help clarify this issue, the present study examined the distributions of the mu, delta, and kappa opioid receptor subtypes in the rat forebrain and midbrain using in vitro autoradiography. Mu and delta receptors were labeled with the selective ligands 3H-DAGO (Tyr- D-Ala-Gly-MePhe-Gly-ol), and 3H-DPDPE (D-Pen2, D-Pen5-enkephalin), respectively, while the kappa receptors were labeled with 3H-(-)bremazocine in the presence of unlabeled DAGO and DPDPE. Based on previous findings in our laboratory, the labeling conditions were such that each ligand selectively occupied approximately 75% of each of the opioid sites. The results demonstrated that all 3 opioid receptor subtypes were differentially distributed in the rat brain. Mu binding was dense in anterior cingulate cortex, neocortex, amygdala, hippocampus, ventral dentate gyrus, presubiculum, nucleus accumbens, caudate putamen, thalamus, habenula, interpeduncular nucleus, pars compacta of the substantia nigra, superior and inferior colliculi, and raphe nuclei. In contrast, delta binding was restricted to only a few brain areas, including anterior cingulate cortex, neocortex, amygdala, olfactory tubercle, nucleus accumbens, and caudate putamen. Kappa binding, while not as widespread as observed with mu binding, was densely distributed in the amygdala, olfactory tubercle, nucleus accumbens, caudate putamen, medial preoptic area, hypothalamus, median eminence, periventricular thalamus, and interpeduncular nucleus. While all 3 opioid receptor subtypes could sometimes be localized within the same brain area, their precise distribution within the region often varied widely. For example, in the caudate putamen, mu binding had a patchy distribution, while delta and kappa sites were diffusely distributed, with delta sites being particularly dense ventrolaterally and kappa sites being concentrated ventromedially. These results support the existence of at least 3 distinct opioid receptors with possibly separate functional roles.     

Pharmacological and anatomical evidence of selective mu, delta, and kappa opioid receptor binding in rat brain

While the distribution of opioid receptors can be differentiated in the rat central nervous system, their precise localization has remained controversial, due, in part, to the previous lack of selective ligands and insensitive assaying conditions. The present study analyzed this issue further by examining the receptor selectivity of [3H]DAGO (Tyr-D-Ala-Gly-MePhe-Gly-ol), [3H]DPDPE (2-D-penicillamine-5-D-penicillamine-enkephalin), [3H]DSLET (Tyr-D-Ser-Gly-Phe-Leu-Thr) and [3H](-)bremazocine, and their suitability in autoradiographically labelling selective subpopulations of opioid receptors in rat brain. The results from saturation, competition, and autoradiographic experiments indicated that the three opioid receptor subtypes can be differentiated in the rat brain and that [3H]DAGO and [3H]DPDPE selectively labelled mu and delta binding sites, respectively. In contrast, [3H]DSLET was found to be relatively non-selective, and labelled both mu and delta sites. [3H]Bremazocine was similarly non-selective in the absence of mu and delta ligands and labelled all three opioid receptor subtypes. However, in the presence of 100 nM DAGO and DPDPE, concentrations sufficient to saturate the mu and delta sites, [3H]bremazocine did label kappa sites selectively. The high affinity [3H]bremazocine binding sites showed a unique distribution with relatively dense kappa labelling in the hypothalamus and median eminence, areas with extremely low mu and delta binding. These results point to the selectivity, under appropriate conditions, of [3H]DAGO, [3H]DPDPE and [3H]bremazocine and provide evidence for the differential distribution of mu, delta, and kappa opioid receptors in rat brain.     

Relative affinities of the quaternary narcotic antagonist, N-methyl levallorphan (SR 58002), for different types of opioid receptors

The relative affinities for different subtypes of opioid receptors (mu, kappa and delta) of the peripheral narcotic antagonist N-methyl levallorphan (SR 58002) have been studied in two in vitro smooth muscle systems (guinea-pig ileum and rabbit vas deferens) and by binding studies (guinea-pig brain and cerebellum membranes) using selective tritiated ligands. All the evidence obtained indicates that SR 58002 is a pure antagonist with relative affinity for mu receptors vs kappa and delta superior to that of the parent tertiary compound, levallorphan.     

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)     

Naloxone and its quaternary derivative,naloxone methiodide,have differing affinities for mu,delta, and kappa opioid receptors in mouse brain homogenates

Naloxone and naloxone methiodide both act on opioid receptors but naloxone methiodide has limited access to the brain. Naloxone methiodide has been shown to have a lower affinity for opioid receptors than naloxone in the rat and guinea pig but has not been tested in the mouse. We aimed to investigate this by using [3H]DAMGO, [3H]DPDPE and [3H]U-69,593 to compare the ability of naloxone and naloxone methiodide to displace binding to mu, delta and kappa opioid receptors in mouse brain homogenates. Significant binding was observed for each receptor type and the binding affinity for naloxone versus naloxone methiodide was found to be 15:1 for mu, 6:1 for kappa and 330:1 for delta receptors. Therefore, naloxone methiodide does have a lower affinity for opioid receptors than naloxone in mouse brain tissue, which must be taken into consideration in experimental designs.     

Opioid research in amphibians: an alternative pain model yielding insights on the evolution of opioid receptors

This review summarizes the work from our laboratory investigating mechanisms of opioid analgesia using the Northern grass frog, Rana pipiens. Over the last dozen years, we have accumulated data on the characterization of behavioral effects after opioid administration on radioligand binding by using opioid agonist and antagonist ligands in amphibian brain and spinal cord homogenates, and by cloning and sequencing opioid-like receptor cDNA from amphibian central nervous system (CNS) tissues. The relative analgesic potency of mu, delta, and kappa opioids is highly correlated between frogs and other mammals, including humans. Radioligand binding studies using selective opioid agonists show a similar selectivity profile in amphibians and mammals. In contrast, opioid antagonists that are highly selective for mammalian mu, delta, and kappa opioid receptors were not selective in behavioral and binding studies in amphibians. Three opioid-like receptor cDNAs were cloned and sequenced from amphibian brain tissues and are orthologs to mammalian mu, delta, and kappa opioid receptors. Bioinformatics analysis of the three types of opioid receptor cDNAs from all vertebrate species with full datasets gave a pattern of the molecular evolution of opioid receptors marked by the divergence of mu, delta, and kappa opioid receptor sequences during vertebrate evolution. This divergence in receptor amino acid sequence in later-evolved vertebrates underlies the hypothesis that opioid receptors are more type-selective in mammals than in nonmammalian vertebrates. The apparent order of receptor type evolution is kappa, then delta, and, most recently, the mu opioid receptor. Finally, novel bioinformatics analyses suggest that conserved extracellular receptor domains determine the type selectivity of vertebrate opioid receptors.     

Methionine-enkephalin stimulates hydrogen peroxide and nitric oxide production in rat peritoneal macrophages: interaction of mu, delta and kappa opioid receptors

OBJECTIVE: Methionine-enkephalin (MET) modulates various functions of macrophages related to both immune and inflammatory reactions in a naloxone reversible manner, suggesting that opioid receptors are involved in the regulation of macrophage activity. Since an endogenous opioid ligand might interact with more than one type of opioid receptor, the receptor interaction determines its effect on a particular function. METHODS: In the present study we have investigated the involvement of different opioid receptor types/subtypes in MET-induced modulation of H(2)O(2) and NO production in macrophages. Thioglycollate-elicited or resident rat peritoneal macrophages were treated in vitro with MET and/or specific antagonists of delta(1,2), delta(1), delta(2), mu and kappa opioid receptors. RESULTS: MET increased H(2)O(2)production in phorbol myristate acetate-stimulated rat peritoneal macrophages mainly through delta(1) opioid receptor. MET also enhanced NO production in rat peritoneal macrophages stimulated with lipopolysaccharide through delta(1) and mu opioid receptors. The blockade of mu and kappa receptor facilitated a potentiating effect of MET on H(2)O(2) release, and blockade of kappa receptor further raised the MET-induced increase of NO production in macrophages. CONCLUSION: It is concluded that both negative and positive functional interaction between delta, mu and kappa opioid receptors regulate the influence of MET on H(2)O(2) and NO production in rat peritoneal macrophages.     

Selective opioid agonist and antagonist competition for [3H]-naloxone binding in amphibian spinal cord

Opioids elicit antinociception in mammals through three distinct types of receptors designated as mu, kappa and delta. However, it is not clear what type of opioid receptor mediates antinociception in non-mammalian vertebrates. Radioligand binding techniques were employed to characterize the site(s) of opioid action in the amphibian, Rana pipiens. Naloxone is a general opioid antagonist that has not been characterized in Rana pipiens. Using the non-selective opioid antagonist, [3H]-naloxone, opioid binding sites were characterized in amphibian spinal cord. Competitive binding assays were done using selective opioid agonists and highly-selective opioid antagonists. Naloxone bound to a single-site with an affinity of 11.3 nM and 18.7 nM for kinetic and saturation studies, respectively. A B(max) value of 2725 fmol/mg protein in spinal cord was observed. The competition constants (K(i)) of unlabeled mu, kappa and delta ranged from 2.58 nM to 84 microM. The highly-selective opioid antagonists yielded similar K(i) values ranging from 5.37 to 31.1 nM. These studies are the first to examine opioid binding in amphibian spinal cord. In conjunction with previous behavioral data, these results suggest that non-mammalian vertebrates express a unique opioid receptor which mediates the action of selective mu, kappa and delta opioid agonists.     

Distribution of mu, delta, and kappa opiate receptor types in the forebrain and midbrain of pigeons

Ligands that are highly specific for the mu, delta, and kappa opiate receptor binding sites in mammalian brains have been identified and used to map the distribution of these receptor types in the brains of various mammalian species. In the present study, the selectivity and binding characteristics in the pigeon brain of three such ligands were examined by in vitro receptor binding techniques and found to be similar to those reported in previous studies on mammalian species. These ligands were then used in conjunction with autoradiographic receptor binding techniques to study the distribution of mu, delta, and kappa opiate receptor binding sites in the forebrain and midbrain of pigeons. The autoradiographic results indicated that the three opiate receptor types showed similar but not identical distributions. For example, mu, delta, and kappa receptors were all abundant within several parts of the cortical-equivalent region of the telencephalon, particularly the hyperstriatum ventrale and the medial neostriatum. In contrast, in other parts of the cortical-equivalent region of the avian telencephalon, such as the dorsal archistriatum and caudal neostriatum, only kappa receptors appeared to be abundant. Within the basal ganglia, all three types of opiate receptors were abundant in the striatum and low in the pallidum. Within the diencephalon, kappa and delta binding was high in the dorsal and dorsomedial thalamic nuclei, but the levels of all three receptor types were generally low in the specific sensory relay nuclei of the thalamus. Kappa binding and delta binding were high, but mu was low in the hypothalamus. Within the midbrain, all three receptor types were abundant in both the superficial and deep tectal layers, in periventricular areas, and in the tegmental dopaminergic cell groups. In many cases, the distribution of opiate receptors in the pigeon forebrain generally showed considerable overlap with the distribution of opioid peptide-containing fiber systems (for example, in the striatal portion of the basal ganglia), but there were some clear examples of receptor-ligand mismatch. For example, although all three receptor types are very abundant in the hyperstriatum ventrale, opioid peptide-containing fibers are sparse in this region. Conversely, within the pallidal portion of the basal ganglia, opioid peptide-containing fibers are abundant, but the levels of opiate receptors appear to be considerably lower than would be expected. Thus, receptor-ligand mismatches are not restricted to the mammalian brain, since they are a prominent feature of the organization of the brain opiate systems in pigeons.(ABSTRACT TRUNCATED AT 400 WORDS)     

Selective changes in mu, delta and kappa opioid receptor binding in certain limbic regions of the brain in Alzheimer's disease patients

Total opioid binding and levels of the three major types of opioid binding sites were measured in homogenates of various limbic structures from post-mortem brains of Alzheimer's disease patients and age-matched control individuals. The most consistent finding in Alzheimer's disease brains was an increase in kappa binding in all 6 areas of the limbic system examined, with the putamen and caudate regions showing significant increases of 114% and 53%, respectively. In addition, the Alzheimer's disease putamen showed a significantly higher level of total binding (85% increase). The amygdala of Alzheimer's disease patients exhibited significantly lower levels of mu and delta binding (41% and 55% decrease, respectively). Total binding and binding to mu and delta receptors in frontal cortex, caudate and hippocampus of Alzheimer's disease brains was indistinguishable from levels seen in these brain areas from control individuals.     

Pre- and postnatal development of opiate receptor subtypes in rat spinal cord

We have studied the developmental expression of opiate binding sites in the rat spinal cord at various prenatal and postnatal stages. For each developmental stage, we have compared the expression pattern of kappa receptors with that of mu and delta receptor subtypes. Both mu and kappa receptors appear relatively early during spinal cord ontogeny (from the 15th prenatal day), while delta sites are expressed later at the postnatal period (starting at the 1st postnatal day). The number of kappa sites predominates throughout the development (55-80% of total opiate sites) with two peaks of binding activity: one at the 20th gestational day, and the other around the 7th postnatal day. mu sites represent 20-38% of the total opiate receptor population with one peak of binding activity appearing at the 1st postnatal day. The densities of mu and kappa receptors at the adult stage are lower by 40-50% than the peak values observed at the early postnatal periods. The relative amounts of delta sites remain low throughout the ontogeny (4-8% of the total opiate sites). The binding properties of neonatal (1 day after birth) kappa sites (ligand binding affinities, regulation of agonist binding by guanosine triphosphate and various cations) are similar to those displayed by kappa receptors in adult spinal cord.     

Solubilization and characterization of kappa opioid binding sites from guinea pig cerebellum

Solubilization of opioid binding sites from guinea pig cerebellum by digitonin, in the absence and presence of NaCl, resulted in very similar yields (25-30%) of [3H]bremazocine binding. Saturation curves of [3H]bremazocine binding give linear Scatchard plots for both soluble and membrane-bound binding sites yielding similar Kd's and Bmax's. Soluble kappa sites seem to resemble closely their membrane-bound counterparts and retain high affinity and selectivity for various kappa opioid ligands. The apparent molecular weight of soluble kappa sites is ca. 4 X 10(5). Results from this study, along with our previous findings with toad and guinea pig brain, indicate that kappa sites (unlike mu and delta) can be solubilized in good yield by digitonin even in the absence of NaCl. This supports the hypothesis that kappa sites may represent molecular species different from those of mu and delta sites.     

Characterization of kappa opiate receptors in rat spinal cord-dorsal root ganglion cocultures and their regulation by chronic opiate treatment

We have investigated the expression and regulation of kappa opiate receptors in rat spinal cord-dorsal root ganglion primary cocultures. The density of opiate receptors increased markedly during the differentiation of the cultures; after 10 days in vitro the number of [3H]diprenorphine binding sites reached 244 +/- 47 fmol/mg protein. Most of the binding sites were of the kappa type, representing about 65-80% of total opiate receptors, while mu sites were expressed at a lower density (ca. 20% of total opiate sites). Following this period of development, the number of kappa and mu receptors did not change significantly. No detectable delta sites were observed at any time of culture (up to 4 weeks in vitro). Chronic opiate agonist treatment (24 h) of the cultured cells with either 10 microM U50488 (a selective kappa agonist), or 1 microM etorphine (a nonselective opiate agonist), did not change the number of kappa receptors and their binding affinity to [3H]diprenorphine. On the other hand, 50% of the mu receptor sites down-regulated following 24 h treatment with 1 microM etorphine. Chronic antagonist exposure (5 days) with 10 microM naloxone, markedly up-regulated the mu receptors (261% of control), whereas kappa sites exhibited a much weaker upregulation (164% of control). These data demonstrate that kappa opiate receptors are expressed at high concentration in spinal cord-dorsal root ganglion cocultures and that contrary to mu sites, kappa receptor density is less susceptible to modulation by chronic opiate treatment. The results also suggest that postreceptor components are important in regulating the kappa receptor function following prolonged opiate exposure.     

Autoradiographic distribution of multiple classes of opioid receptor binding sites in human forebrain

Receptor binding parameters and autoradiographic distribution of various opioid receptor sites have been investigated in normal human brain, post-mortem. [3H]DAGO, a highly selective mu ligand, binds to a single class of high affinity (Kd = 1.1 nM), low capacity (Bmax = 160 fmol/mg protein) sites in membrane preparations of frontal cortex. These sites show a ligand selectivity profile that resembles that of the mu opioid receptor. On the other hand, [3H]bremazocine, in presence of saturating concentrations of mu and delta blockers, appears to selectively bind to a single population of kappa opioid sites (Kd = 0.13 nM; Bmax = 93.0 fmol/mg protein) in human frontal cortex. Whole hemisphere in vitro receptor autoradiography reveals that [3H]DAGO-mu, [3H]DSLET-delta and [3H]bremazocine (plus blockers)-kappa binding sites are discretely and differentially distributed in human forebrain. In the cortex, mu sites are concentrated in laminae I and IV, delta sites in laminae I and II while kappa sites are found in deeper layers (laminae V and VI). In subcortical nuclei, high densities of mu and delta sites are seen in the caudate and putamen while high amounts of kappa sites are present in the claustrum and amygdala. The nucleus basalis of Meynert is enriched in all three classes of sites while the globus pallidus only contains moderate densities of kappa sites. Thus, the possible alterations of these various classes of opioid receptors in neurological and psychiatric diseases certainly deserve further investigation.     

Time-related decreases in mu and delta opioid receptors in the superficial dorsal horn of the rat spinal cord following a large unilateral dorsal rhizotomy.

The aim of the present study was to measure the time-related modifications of mu and delta opioid binding sites in the superficial layers of the dorsal horn of the rat spinal cord after a C4-T2 unilateral dorsal rhizotomy. Using specific ligands, namely [3H]DAMGO for mu sites and [3H]DTLET for delta sites, and a quantitative autoradiographic analysis, we have observed: (a) a decrease in binding on the ipsilateral side to the lesion as early as the first day postrhizotomy, the maximal loss being attained at 8 days postlesion, (b) after 8 days postlesion, the residual binding remains stable over the period of analysis (90 days), (c) the loss of mu receptors (71-74%) is significantly more pronounced than the loss of delta receptors (57-62%) and (d) affinities of postsynaptic mu and delta receptors are similar to those of the total receptor population in the superficial layers of the dorsal horn. Comparison of these results with the degeneration of primary afferent fibers reported in literature favors the localization of the majority of mu and delta opioid binding sites on fine diameter primary afferent fibers.     

Chronic administration of cholecystokinin antagonists reverses the enhancement of spinal morphine analgesia induced by acute pretreatment

The effects of acute and chronic (22 days) treatment with the cholecystokinin (CCK) antagonists proglumide and lorglumide on antinociception induced by intrathecal (i.t.) morphine were determined at weekly intervals with the rat tail-flick assay. On day 1, acute pretreatment with either proglumide (20 ng, i.t.) or lorglumide (7 ng, i.t.) enhanced morphine (1 μg, i.t.) analgesia compared to saline (1 μl, i.t.) pretreatment, but this facilitation was absent on days 8 and 15 of CCK antagonist treatment and was replaced by attenuation of opioid antinociception on day 22. Following termination of daily proglumide or lorglumide injections, normal (control) morphine response was observed after pretreatment with either CCK antagonist on days 29 and 36. Weekly co-administration of either drug with morphine had similar effects: opioid antinociception was initially enhanced on day 1, but this amplification was lost by day 8 and remained absent for the duration of the study (i.e., up to day 36). Inhibition of morphine analgesia, however, was not observed with this treatment paradigm. Chronic daily administration of either CCK antagonist alone did not lower nociceptive thresholds; further, normal opioid response was retained throughout the study in saline treated controls receiving morphine weekly. This study demonstrates that whereas acute i.t. administration of CCK antagonists enhances i.t. morphine antinociception, chronic treatment causes loss of facilitation or attenuation of opioid antinuaciception, suggesting that (1) compensatory alterations in CCK-opioid interactions develop during chronic CCK blockade and (2) CCK antagonists may not be useful adjuncts to opioid in the management of chronic pain in man.     

Distribution of mu, delta, and kappa opioid receptors in the hypothalamus of the rat

The radioautographic distribution of mu, delta and kappa opioid binding sites was examined by in vitro radioautography in the rat hypothalamus using the highly selective ligands [125I]-FK 33-824, [125I]azidoDTLET and [125I]DPDYN, respectively. Levels of mu opioid binding sites varied considerably amongst hypothalamic nuclei. mu Opioid labeling was dense in the medial preoptic area, medial preoptic nucleus, suprachiasmatic nucleus and ventromedial nucleus, whilst the supraoptic nucleus, paraventricular nucleus, arcuate nucleus and dorsomedial nucleus were devoid of labeling. Delta opioid labeling was sparse throughout most of the hypothalamus; however, moderate binding densities were detected in the suprachiasmatic and ventromedial nucleus. kappa Opioid labeling was also scant throughout the hypothalamus with the exception of the suprachiasmatic nucleus which was very densely labeled. Our results indicate that the 3 opioid receptors types are differentially distributed within the hypothalamus, although a significant overlap exists. In general, the distribution of hypothalamic opioid receptors correlates well with that of opioid-containing terminal fibers and may represent the anatomical substrate for opioid involvement in the hypothalamic regulation of autonomic, behavioral and neuroendocrine functions.     

A brief study of the selectivity of norbinaltorphimine, (-)-cyclofoxy, and (+)-cyclofoxy among opioid receptor subtypes in vitro

Norbinaltorphimine (nor-BNI) is a bifunctional reagent developed as a selective antagonist of the kappa opioid receptor. In this paper we examined the in vitro selectivity of nor-BNI, 6-desoxy-6 beta-fluoronaltrexone (cycloFOXY), and the enantiomer of cycloFOXY, among opioid receptor subtypes. Nor BNI exhibited the highest affinity for kappa binding sites labeled by 3H-U69593 (Ki = 1.8nM), and was 27- to 29-fold less potent at mu and delta binding sites. In contrast, cycloFOXY had the highest affinity for mu binding sites (Ki = 2.62 nM), and bound to kappa and delta binding sites with Ki's of 9.3 nM and 89 nM, respectively. The enantiomer of cycloFOXY, did not inhibit binding even at concentrations greater than 10 microM, validating in part the use of 18F-labeled (+)-cycloFOXY to estimate "non-specific binding" in positron emission tomography. Additionally, we report that (S,S)-U50 488 and (R.R)-U50 488 bind to kappa binding sites labeled by 3H-U69 593 with Ki's of 0.89 nM and 299 nM, respectively.