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.     

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.     

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)     

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.     

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.     

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.     

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.     

Enhanced binding of nor-binaltorphimine to kappa-opioid receptors in rats dependent on butorphanol

Autoradiographic characterization of binding for brain kappa(1) ([(3)H]CI-977) and kappa(2) ([(3)H]bremazocine) in the presence of DAMGO ([D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin), DPDPE ([D-Pen(2), D-Pen(5)]-enkephalin), and U-69,593 opioid receptors, in the presence of different concentrations of a selective unlabeled kappa-opioid receptor antagonist, nor-binaltorphimine (nor-BNI), was performed in rats in which dependence on or withdrawal from butorphanol had been established. Dependence was induced by a 72 hr intracerebroventricular (i.c.v.) infusion with butorphanol (26 nmol/microl/hr; butorphanol dependent). Butorphanol withdrawal was produced by terminating the infusion of butorphanol in dependent animals. Responses were studied 7 hr following termination (butorphanol withdrawal). IC(50) values from competition studies were estimated by fitting inhibition curves for both kappa(1)- and kappa(2)-opioid receptor assays. In both dependent and withdrawal groups, the IC(50) values obtained against [(3)H]CI-977 or [(3)H]bremazocine with nor-BNI were decreased (ratios of approximately 0.03-0.21 and approximately 0.05-0.42 vs. control, respectively) in brain regions, including frontal cortex, nucleus accumbens, claustrum, dorsal endopiriform nucleus, caudate putamen, parietal cortex, posterior basolateral amygdaloid nucleus, dorsomedial hypothalamus, hippocampus, posterior paraventricular thalamic nucleus, periaqueductal gray, substantia nigra, superficial gray layer of the superior colliculus, ventral tegmental area, and locus coeruleus, compared with control. These results indicate that, in butorphanol-dependent and butorphanol-withdrawal rats, the brain kappa(1)- and kappa(2)-opioid receptors developed a supersensitivity to antagonist binding.     

Tritiated-6-beta-fluoro-6-desoxy-oxymorphone ([3H]FOXY): a new ligand and photoaffinity probe for the mu opioid receptors

6-Beta-fluoro-6-desoxy-oxymorphone (FOXY) is a fluorinated derivative of oxymorphone originally developed as a potential PET scanning ligand. Preliminary work (Rothman et al., Neuropeptides 4: 311-317, 1984) demonstrated that [3H]FOXY selectively labeled mu opioid binding sites with low levels of nonspecific binding. In this study the opiate receptor subtypes labeled by [3H]FOXY and [3H]D-ala2-MePhe4, Gly-ol5-enkephalin ([3H] DAGO) were compared using site directed acylating agents and binding surface analysis. Although the data indicated that both ligands selectively label mu opiate receptors, other experiments demonstrated that [3H]DAGO and [3H]FOXY labeled mu binding sites differently. Additional experiments demonstrated that [3H]FOXY can be used as a high yield photoaffinity label for the mu opiate receptor subtype.     

Autoradiographic determination of changes in opioid receptor binding in the limbic system of the Columbian ground squirrel at different hibernation states

To localize and quantify the state-dependent changes in various opioid receptor subtypes in the limbic system of non-hibernating and hibernating Columbian ground squirrels, quantitative receptor-binding autoradiography was used. Compared to the non-hibernating animals, the binding density of [³H]-[d-Pen^2,5]-enkephalin (DPDPE) to the δ receptor in the lateral septum, CA3, and the hippocampal fissure of the hippocampal formation was significantly decreased in the hibernating ground squirrels. A significant reduction in the binding density of [³H]-[d-Ala²,N-Me-Phe⁴,Gly-ol⁵]-enkephalin (DAGO) to μ receptor was also observed in the medial septum and the CA3 region of the hippocampus of the hibernating animals. In contrast, a decrease in [³H]ethylketocyclazocine (EKC) binding to the κ receptor was only observed in the claustrum and CA3 of the hippocampus during hibernation. The differential changes in binding to various opioid receptors suggest that different opioid subtypes may exert different physiological roles in regulating the specific states (entrance, maintenance and arousal) of a hibernation bout.     

Glial growth is regulated by agonists selective for multiple opioid receptor types in vitro.

To determine whether one or more opioid receptor types might be preferentially involved in gliogenesis, primary mixed glial cultures derived from mouse cerebra were continuously treated with varying concentrations of opioid agonists selective for mu (mu), i.e., DAGO ([D-Ala2, MePhe4, Gly(ol)5]enkephalin), delta (delta), i.e., DPDPE ([D-PEN2,D-PEN5]enkephalin), or kappa (kappa), i.e., U69,593, opioid receptor types. In addition, a group of cultures was treated with [Met5]-enkephalin, an agonist for delta opioid receptors as well as putative zeta (zeta) opioid receptors. Opioid-dependent changes in growth were assessed by examining alterations in (1) the number of cells in mixed glial cultures at 3, 6, and 8 days in vitro (DIV), (2) [3H]thymidine incorporation by glial fibrillary acidic protein (GFAP) immunoreactive, flat (type 1) astrocytes at 6 DIV, and (3) the area and form factor of GFAP-immunoreactive, flat (type 1) astrocytes. DPDPE at 10(-8) or 10(-10) M, as well as [Met5]-enkephalin at 10(-6), 10(-8), or 10(-10) M, significantly reduced the total number of glial cells in culture; but this effect was not observed with DAGO or U69,593 (both at 10(-6), 10(-8), or 10(-10) M). Equimolar concentrations (i.e., 10(-6) M) of [Met5]enkephalin or U69,593, but not DPDPE or DAGO, suppressed the rate of [3H]thymidine incorporation by GFAP-immunoreactive, flat (type 1) astrocytes. DAGO had no effect on growth, although in previous studies morphine was found to inhibit glial numbers and astrocyte DNA synthesis. [Met5]enkephalin (10(-6) M) was the only agonist to significantly influence astrocyte area. Collectively, these results indicate that delta (and perhaps mu) opioid receptor agonists reduce the total number of cells in mixed glial cultures; while [Met5]enkephalin-responsive (and perhaps kappa-responsive) opioid receptors mediate DNA synthesis in astrocytes. This implies that delta opioid receptors, as well as [Met5]enkephalin-sensitive, non-delta opioid receptors, mediate opioid-dependent regulation of astrocyte and astrocyte progenitor growth. These data support the concept that opioid-dependent changes in central nervous system growth are the result of endogenous opioid peptides acting through multiple opioid receptor types.     

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.     

Distribution of mu, delta, and kappa opioid receptor binding sites in the brain of the one-day-old domestic chick (Gallus domesticus): an in vitro quantitative autoradiographic study

Three highly specific opioid ligands--[D-Ala2,Gly-ol]-enkephalin (DAGO) for mu (mu) receptor sites, [D-Pen2,D-Pen5]-enkephalin (DPDPE) for delta (delta) sites, and U-69593 for kappa (kappa) sites--were used to determine the regional distribution of the three major subtypes of opioid receptor binding sites in the brains of 1-day-old domestic chicks by the technique of quantitative receptor autoradiography. Whilst there was a degree of heterogeneity in the binding levels of each of the ligands, some notable similarities existed in the binding of the mu and kappa ligands in several forebrain regions, and in the optic tectum of the midbrain where mu and delta binding was very high. In the forebrain there was a high level of binding of mu and kappa ligands in the hyperstriatum, and for the mu ligand there was a very distinct lamination of binding sites in hyperstriatum accessorium, intercalatum supremum, dorsale and ventrale. Levels of binding of the mu and kappa ligands were also high in nucleus basalis, and (for mu only) in the neostriatum. The distribution of binding of the delta specific ligand in the forebrain showed marked differences to that of mu and kappa, being particularly low in the hyperstriatum and neostriatum. Very high levels of labelling of delta binding sites were, however, found in the nucleus rotundus. Binding of the three ligands was generally low or absent in the cerebellum and medulla, apart from a distinct labelling of the granule cell layer by the mu-ligand. A kinetic analysis was made of the binding of the three ligands to whole forebrain sections using scintillation counting methods.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endogenous opioid systems regulate growth of neural tumor cells in culture

Endogenous opioid systems (i.e., opioids and opioid receptors) play a role in neural cancer. Using a tissue culture system of S20Y murine neuroblastoma to assess the effects of opioids on growth, [Met5]-enkephalin was the most potent compound to influence cell replication. With a median effective concentration of 10(-10) M, this peptide inhibited cell proliferation in a stereospecific and naloxone-reversible manner. [Met5]-Enkephalin depressed both DNA synthesis and mitosis. [Met5]-Enkephalin was detected in neuroblastoma cells by radioimmunoassay, and was found to increase in concentration in culture media over time, suggesting that these cells produced the peptide. Immunocytochemistry showed [Met5]-enkephalin-like activity in the cortical cytoplasm, but not the cell nucleus, of neuroblastoma cells. Binding of [3H]-[Met5]-enkephalin specific and saturable, and Scatchard analysis yielded a Kd of 1.2 +/- 0.1 nM and a binding capacity of 50.2 +/- 4.3 fmol/mg protein. [Met5]-Enkephalin also depressed the growth of N115 murine neuroblastoma, SK-N-MC human neuroblastoma, and HT-1080 human fibrosarcoma. These results indicate that [Met5]-enkephalin, a naturally occurring pentapeptide that is derived from proenkephalin A, is a potent inhibitor of cell growth. Since cancer cells produce [Met5]-enkephalin, and contain a binding site to this ligand, endogenous opioid systems appear to control cell proliferation by an autocrine mechanism.     

Activation of MU opioid receptors in the pre-optic-anterior hypothalamus releases prolactin in the conscious male rat

Microinjection of opioid agonists into the pre-optic-anterior hypothalamus (PO/AHA) was used to determine the identity of the opioid receptor subtype(s) involved in the stimulation of prolactin release. The mu agonist DAGO [(D-Ala2, NMe-Phe4, Gly-o15)-enkephalin] was the only opioid agonist to show dose-dependent release of prolactin, the lowest significant dose being 0.001 nmoles. Neither the specific delta agonist DPDPE [(D-Pen2, D-Pen5)-enkephalin] nor the specific kappa agonist U50,488H [(trans-3,4-dichloro-N-methyl-N-(2-(1-pyrrodinyl)-cyclohexyl)-benz ene acetamide] showed dose-dependent increase of prolactin secretion, or indeed any significant increase in prolactin secretion in the dose range 0.01-1 nmoles and 0.01-10 nmoles respectively. We suggest that mu (and not kappa or delta) opioid receptors in the PO/AHA are involved in the opioid stimulated release of prolactin in the conscious male rat.     

Kappa opioid receptors in human lumbo-sacral spinal cord

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

Modulation of [3H]DAGO binding by substance P (SP) and SP fragments in the mouse brain and spinal cord via MU1 interactions

Binding of [3H]DAGO to fresh, frozen or beta-funaltrexamine (beta-FNA) pretreated membranes of mouse brain and spinal cord was extensively studied using substance P (SP) or SP fragments as potential competitors and/or modulators. The objective was to determine whether SP exerts its analgesic effect by interacting with mu opioid receptors. The affinity of DAGO was reduced and binding capacity was increased in the presence of SP or the N-terminal SP fragments SP(1-9) and SP(1-4) but not the C-terminal SP fragment SP(5-11). Because sub-nanomolar concentrations of SP or N-terminal SP fragments displaced [3H] DAGO binding to a minor but detectable degree, it is suggested that SP interacts with mu 1 sites through its N-terminus portion. The effect of SP on DAGO binding was less in the spinal cord compared to the rest of the brain. Modulation of DAGO binding by SP was enhanced in the brain after pretreatment of membranes with the narcotic antagonist beta-FNA. These results suggest a novel mechanism for the analgesic action of SP.     

Differential radioligand binding properties of [3H]5-hydroxytryptamine and [3H]mesulergine in a clonal 5-hydroxytryptamine1C cell line.

[3H]5-Hydroxytryptamine ([3H]5-HT) and [3H]mesulergine were used to label 5-HT1C receptors expressed in NIH 3T3 mouse fibroblast cells. Using a rapid filtration assay, saturation analysis of the [3H]5-HT radioligand data indicate that the binding is biphasic. Based on computerized analysis of the data, a 2-site model of radioligand binding is significantly more consistent with the data than a one-site model (P less than 0.01). The KD values of [3H]5-HT for the 2 populations are 0.5 +/- 0.1 nM and 31 +/- 15 nM, while the Bmax values are 400 +/- 90 pmol/g protein and 3,000 +/- 600 pmol/g protein, respectively. A biphasic binding pattern is also observed with [3H]5-HT using a centrifugation assay (KD1 = 0.6 +/- 0.06 nM, KD2 = 60 +/- 10 nM; Bmax1 = 740 +/- 90 pmol/g, Bmax2 = 4,000 +/- 700 pmol/g). By contrast, saturation analysis of [3H]mesulergine binding is monophasic (KD = 4.7 +/- 0.7 nM) with a Bmax value (6,800 +/- 1,000 pmol/g protein) that is significantly greater than that obtained using [3H]5-HT (P less than 0.01). Drug competition studies confirm that both [3H]5-HT and [3H]mesulergine label at least 2 subpopulations of expressed 5-HT1C receptors in NIH 3T3 cells. 10(-4) M GTP eliminates the high affinity [3H]5-HT-labeled binding sites with minimal effect on the low affinity [3H]5-HT-labeled sites and no effect on [3H]mesulergine-labeled sites. These data demonstrate that at least 2 distinct subpopulations of 5-HT1C receptors in NIH 3T3 cells can be differentiated using radioligand binding techniques.     

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

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

Autoradiographic distribution of μ, δ and κ opioid binding sites in the superficial dorsal horn, over the rostrocaudal axis of the rat spinal cord

The purpose of this study was to use [3H]DAMGO, [3H]DTLET and [3H]EKC in the presence of 100 nM DAMGO and 100 nM DTLET, combined with a quantitative autoradiography to analyse the different proportions and the rostrocaudal distribution of mu, delta and kappa opioid binding sites in the superficial layers (laminae I and II) of the cervical (C6-C8), thoracic (T5-T7), lumbar (L3-L5) and sacral (S2-S3) dorsal horn of the rat. The proportions of the three main types of opioid binding sites, assessed by autoradiography in laminae I and II, were found homogeneous at each segmental level considered: 70.4-74.3%, 18.4-20.3% and 7.3-9.5% for mu, delta, kappa sites, respectively. The physiological relevance of these data is discussed.