Characterization of zeta (zeta): a new opioid receptor involved in growth

Endogenous opioid systems (i.e., opioids and opioid receptors) are known to play a role in neural cancer. Using [3H]-[Met5]enkephalin, a potent ligand involved in growth, specific and saturable binding was detected in homogenates of S20Y neuroblastoma transplanted into A/Jax mice; the data fit a single binding site. Scatchard analysis yielded a Kd of 0.49 nM and a binding capacity of 5.32 fmol/mg protein. Binding was dependent on protein concentration, time, temperature, and pH, and was sensitive to Na+ and guanine nucleotides. Optimal binding required protease inhibitors, and pretreatment of the tumor homogenates with trypsin markedly reduced [3H]-[Met5]enkephalin binding, suggesting that the binding site was proteinaceous in character. Displacement experiments indicated that [Met5]enkephalin was the most potent displacer of [3H]-[Met5]enkephalin; other ligands selective for mu, delta, kappa, epsilon, and sigma were not highly competitive. Given the functional significance of [Met5]enkephalin as a potent regulator of normal and abnormal growth, and that the receptor recognized by [Met5]enkephalin does not resemble any previously described, the present study has demonstrated the presence of a new opioid receptor termed zeta (zeta) (from the Greek 'Zoe', life) related to the proliferation of cells and tissues.     

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.     

Ontogeny of zeta (zeta), the opioid growth factor receptor, in the rat brain.

Opioid growth factor (OGF), [Met5]enkephalin, serves as an inhibitory influence on the developing nervous system and is especially targeted to cell proliferative events. OGF interacts with the zeta (zeta) opioid receptor to perform its function. Using [3H]-[Met5]enkephalin, the ontogeny of the zeta receptor in the whole brain and cerebellum of rats was explored. Specific and saturable binding was recorded at the earliest time sampled, prenatal day 15 (E15). In the whole brain, binding capacity (Bmax) was two-fold greater at E15 than at E18 and E20. The quantity of zeta receptor appeared to increase in the first postnatal week, reaching a maximum on postnatal day 8. Binding decreased the remainder of the 2nd week and between postnatal days 15 and 25 binding was no longer recorded. In the cerebellum, binding capacity increased from E20 to the 2nd postnatal week, reaching a maximum on postnatal days 8-10. The Bmax of the zeta receptor decreased precipitously on postnatal day 11, being 5.4-fold lower than on postnatal day 10. Between postnatal days 21 and 30, no binding was observed. The binding affinities of the whole brain and cerebellum were 2.3 and 2.7 nM, respectively, and no differences between ages could be detected. Continuous opioid receptor blockade from birth to postnatal day 6 increased body weight, the Bmax of the zeta receptor in the whole brain and cerebellum (but not the Kd), and increased the number of layers of germinal cells in the cerebellum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Demonstration and characterization of zeta (ζ), a growth-related opioid receptor, in a neuroblastoma cell line

Endogenous opioids and opioid receptors (i.e. endogenous opioid systems) are involved in carcinogenesis. Using homogenates of S20Y neuroblastoma (NB) cells grown in culture, the binding of a growth-selective ligand, [Me⁵]enkephalin, was examined to ascertain the zeta (ζ) opioid receptor. Specific and saturable binding of [³H]-[Met⁵]enkephalin was detected in NB cells; the data were consistent with a single binding site. Scatchard analysis yielded a K_d of 1.6 nM and a binding capacity B_max off 48.1 fmol/mg protein; 14,000 receptors per cell were estimated. Binding was dependent on protein concentration, time, temperature, and pH, and was sensitive to 100 nM, but not 5 nM, Na⁺, Ca²⁺, and Mg²⁺; GppNHp at concentrations of 100–500 mM had little effect on binding. Optimal binding required protease inhibitors, and pretreatment of the tumor cell homogenates with trypsin markedly reduced [³H]-[Met⁵]enkphalin winding, suggesting that the binding site was proteinaceous in character. Displacement experiments indicated that [Met⁵]enkephalin was the most potent displacer of [³H]-[Met⁵]enkephalin. Cell density (log, confluence, postconfluence) did not alter the K_d or B_max. This study serves as the first demonstration and characterization of the zeta (ζ) opioid receptor in tissue culture cells. The homogenous nature of NB cell cultures, along with the enrichment in receptor number, provides an excellent model system to isolate and purify the ζ receptor.     

Characterization of zeta (ζ): a new opioid receptor involved in growth

Endogenous opioid systems (i.e., opioids and opioid receptors) are known to play a role in neural cancer. Using [³H]-[Met⁵]enkephalin, a potent ligand involved in growth, specific and saturable binding was detected in homogenates of S20Y neuroblastoma transplanted into A/Jax mice; the data fit a single binding site. Scatchard analysis yielded aK_d of 0.49 nM and a binding capacity of 5.32 fmol/mg protein. Binding was dependent on protein concentration, time, temperature, and pH, and was sensitive to Na⁺ and guanine nucleotides. Optimal binding required protease inhibitors, and pretreatment of the tumor homogenates with trypsin markedly reduced [³H]-[Met⁵]enkephalin binding, suggesting that the binding site was proteinaceous in character. Displacement experiments indicated that [Met⁵]enkephalin was the most potent displacer of [³H]-[Met⁵]enkephalin; other ligands selective for μ, δ, κ, , and σ were not highly competitive. Given the functional significance of [Met⁵]enkephalin as a potent regulator of normal and abnormal growth, and that the receptor recognized by [Met⁵]enkephalin does not resemble any previously described, the present study has demonstrated the presence of a new opioid receptor termed zeta (ζ) (from the Greek ‘Zoe’, life) related to the proliferation of cells and tissues.     

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)     

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.     

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 beta-funaltrexamine with [3H]cycloFOXY binding in rat brain: further evidence that beta-FNA alkylates the opioid receptor complex.

beta-Funaltrexamine (beta-FNA) is an alkylating derivative of naltrexone. In addition to acting as an irreversible inhibitor of mu-receptor-mediated physiological effects, intracerebroventricular (i.c.v.) administration of beta-FNA to rat attenuates the ability of selective delta receptor antagonists and naloxone to reverse delta receptor-mediated effects. Moreover, recent work demonstrated that i.c.v. administration of beta-FNA alters the conformation of the opioid receptor complex, as inferred by a decrease in the Bmax of the lower affinity [3H][D-ala2,D-leu5]enkephalin binding site. Consistent with the decreased potency of naloxone as an inhibitor of delta receptor mediated effects, beta-FNA doubled the naloxone IC50 for displacing [3H][D-ala2,D-leu5]enkephalin from its lower affinity binding site. These data collectively support the hypothesis that the opioid receptor complex postulated to mediate mu-delta interactions in vivo is identical to the opioid receptor complex as defined by vitro ligand binding studies. A direct prediction of this hypothesis is that beta-FNA should increase the Kd of antagonists for the mu binding site (mu cx) of the receptor complex. The data reported in this paper demonstrate that beta-FNA doubled the IC50 of the potent narcotic antagonist, 6-desoxy-6 beta-fluoronaltrexone (cycloFOXY) for displacing [3H][D-ala2,D-leu5]enkephalin from its lower affinity binding site, and doubled the Kd of [3H]cycloFOXY for its mu binding site, providing additional data that the mu binding site labeled by [3H]cycloFOXY is the mu binding site of the opioid receptor complex. beta-FNA also altered the kappa binding site labeled by [3H]cycloFOXY, and when administered intrathecally to mice, beta-FNA produced a longlasting antinociception in the acetic acid writhing test.     

Characterization of NK-1 receptors in guinea pig and rat brain membranes with NK-1 peptides and a non-peptide antagonist.

The major finding of the present investigation is the demonstration of different NK-1 receptors in rat and guinea pig brain membranes with CP 96345 (non-peptide NK-1 antagonist) and R-544 (NK-1 peptide antagonist). We used [3H][Sar9,Met(O2)11]SP, the highly selective ligand for NK-1 receptor to compare NK-1 binding sites in rat and guinea pig brain membranes. Scatchard analysis revealed the existence of a single population of [3H][Sar9,Met(O2)11]SP binding sites in both preparations. The affinity and the maximal number of binding sites were found closely similar in rat (Kd 2 nM, Bmax = 37 fmol/mg protein) and guinea pig brain membranes (Kd = 3 nM, Bmax = 25 fmol/mg of protein). The order of potency of neurokinins to inhibit [3H][Sar9,Met(O2)11]SP binding from rat brain (SP > NKA > NKB) was found different of that observed on guinea pig brain (SP > NKB > NKA). Results obtained with [Sar9,Met(O2)11]SP, [beta Ala8]NKA(4-10) and [MePhe7]NKB suggest that selective agonists cannot discriminate between NK-1 receptors of different species. Using the non-peptide antagonist CP 96345 and the tripeptide R-544, we found that these two NK-1 antagonists discriminate between rat and guinea pig [3H][Sar9,Met(O2)11]SP binding sites.     

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.     

Characterization of benzodiazepine receptors in the bovine pineal gland: evidence for the presence of an atypical binding site

Bovine and rat pineal benzodiazepine receptors were characterized using ligands with high affinities for either 'central-type' (CBR) or 'peripheral-type' (PBR) benzodiazepine receptors. The characteristics (Bmax = 83 +/- 10 fmol/mg protein, Kd = 3.88 +/- 0.46 nM) of benzodiazepine receptors in bovine pineal membranes measured with [3H]flunitrazepam (using flunitrazepam to define non-specific binding) were consistent with previously reported values. However, if non-specific binding was defined using Ro 15-1788 (a selective CBR ligand), the Bmax and Kd of [3H]flunitrazepam decreased 51 and 58%, respectively. In addition, when using PK 11195 to determine non-specific binding, the Bmax of [3H]flunitrazepam binding to bovine pineal decreased further (approximately 80%, Kd decreased approximately 39%). Together, these observations strongly suggested the presence of PBR in the bovine pineal. Bovine pineal PBR characterized with [3H]PK 11195 revealed a high density (relative to CBR) of high affinity binding sites (Kd = 1.08 +/- 0.30, Bmax = 776 +/- 33.0 fmol/mg protein). In contrast, when [3H]Ro 5-4864 (1-20 nM) was used to define PBR, no binding was detectable. These observations are in sharp contrast to the rat pineal gland, in which both [3H]Ro 5-4864 and [3H]PK 11195 bind to a large number of PBR with high affinity (Kd approximately equal to 1.9 nM, Bmax approximately equal to 26 pmol/mg protein). Bovine pineal PBR were further characterized with compounds structurally related to either Ro 5-4864 or PK 11195.(ABSTRACT TRUNCATED AT 250 WORDS)     

Decrease in delta-opioid receptor density in rat brain after chronic [D-Ala2,D-Leu5]enkephalin treatment

Chronic treatment of Sprague-Dawley rats with [D-Ala2,D-Leu5]enkephalin (DADLE) resulted in the development of tolerance to the antinociceptive effect of this opioid peptide. When opioid receptor binding was measured, time-dependent decreases in [3H]diprenorphine binding to the P2 membranes prepared from the cortex, midbrain and striatum were observed. Scatchard analysis of the saturation binding data revealed a decrease in Bmax values and no change in the Kd values of [3H]diprenorphine binding to these brain regions, indicative of down-regulation of the receptor. This reduction in the opioid receptor binding activities could be demonstrated to be due to the DADLE effect on the delta-opioid receptors in these brain regions. When [3H]DADLE binding was carried out in the presence of morphiceptin, a significant reduction in the delta-opioid receptor binding was observed in all brain areas tested. mu-Opioid receptor binding decrease was observed only in the striatum after 5 days of DADLE treatment. Additionally, the onset of delta-opioid receptor decrease in the midbrain area was rapid, within 6 h of the initiation of the chronic DADLE treatment. Thus, analogous to previous observations in which chronic etorphine treatment preferentially reduced mu-opioid receptor binding, chronic DADLE treatment preferentially reduced delta-opioid receptor binding activity.     

Binding sites for [3H][Sar9, Met(O2)11]substance P in rat brain and guinea pig ileum.

[3H][Sar9,Met(O2)11]substance P (SP) with high specific activity (32 Ci/mmol) was used to study neurokinin-1 (NK-1) binding sites on rat brain and smooth muscle membranes of the guinea pig ileum. The specific binding of [3H][Sar9,Met(O2)11]SP was shown to be saturable, reversible and increased in parallel with the protein concentration. Scatchard analyses of equilibrium binding experiments revealed that [3H][Sar9,Met(O2)11]SP binds to a class of non-interacting binding sites in rat brain membranes (Kd = 2 nM, Bmax = 56 fmol/mg of protein) and ileum muscle membranes (Kd = 2 nM, Bmax = 194 fmol/mg of protein). Competition of [3H][Sar9,Met(O2)11]SP, 125I-BH[Sar9,Met(O2)11]SP and 125I-BH.SP with different tachykinin-related peptides gave the following rank order of potencies: SP greater than physalaemin greater than [Sar9,Met(O2)11]SP greater than N-Ac[Arg6,Sar9,Met(O2)11]SP(6-11) greater than neurokinin A (NKA) greater than or equal to eledoisin greater than or equal to neurokinin B (NKB) greater than [MePhe7]NKB (4-10) greater than [beta-Ala8]NKA(4-10). A very similar pattern was observed on ileum muscle membranes. [3H][Sar9,Met(O2)11]SP was found to be highly selective for NK-1 binding sites in rat brain and in the intestinal tissue. Binding showed good correlation with the biological activity of tachykinins and related peptides. From these data it can be suggested that (a) the NK-1 receptor characterized in the central nervous system is identical to the one in the periphery, (b) the NK-1 binding site of the muscle membranes appears to be similar to the contractile receptor of the guinea pig ileum and (c) the functional site mediating relaxation of the dog carotid artery is similar to the contractile receptor of the guinea pig ileum.     

Heterogeneous binding of [3H]4-DAMP to muscarinic cholinergic sites in the rat brain: evidence from membrane binding and autoradiographic studies.

The present study shows that [3H]4-DAMP binds specifically, saturably, and with high affinity to muscarinic receptor sites in the rat brain. In homogenates of hippocampus, cerebral cortex, striatum, and thalamus, [3H]4-DAMP appears to bind two sub-populations of muscarinic sites: one class of high-affinity, low capacity sites (Kd less than 1 nM; Bmax = 45-152 fmol/mg protein) and a second class of lower-affinity, high capacity sites (Kd greater than 50 nM; Bmax = 263-929 fmol/mg protein). In cerebellar homogenates, the Bmax of [3H]4-DAMP binding sites was 20 +/- 2 and 141 +/- 21 fmol/mg protein for the high- and the lower-affinity site, respectively. The ligand selectivity profile for [3H]4-DAMP binding to its sites was similar for both the high- and lower-affinity sites; atropine = (-)QNB = 4-DAMP much greater than pirenzepine greater than AF-DX 116, although pirenzepine was more potent (16-fold) at the lower- than at the high-affinity sites. The autoradiographic distribution of [3H]4-DAMP sites revealed a discrete pattern of labeling in the rat brain, with the highest densities of [3H]4-DAMP sites present in the CA1 sub-field of Ammon's horn of the hippocampus, the dentate gyrus, the olfactory tubercle, the external plexiform layer of the olfactory bulb and layers I-II of the frontoparietal cortex. Although the distribution of [3H]pirenzepine sites was similar to that of [3H]4-DAMP sites in many brain regions, significant distinctions were apparent. Thus, both the ligand selectivity pattern of [3H]4-DAMP binding and the autoradiographic distribution of sites suggest that although the high-affinity [3H]4-DAMP sites may consist primarily of muscarinic-M3 receptors, the lower-affinity [3H]4-DAMP sites may be composed of a large proportion of muscarinic-M1 receptors.     

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.     

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.     

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.     

Differences between sodium-dependent and desipramine-defined [3H]imipramine binding in intact human platelets

Measurements of sodium-dependent [3H]imipramine binding to intact human platelets from 20 human volunteers were made and compared to desipramine-defined binding, a method commonly employed in population studies of platelet [3H]imipramine sites. The density (Bmax) of sodium-dependent [3H]imipramine sides in platelets was significantly lower (449 +/- 36 sites/platelet) and the affinity (Kd) significantly higher (1.15 +/- 0.12 nM) than those obtained when excess desipramine was used to define specific binding (Bmax 654 +/- 33 sites/platelet, p less than 0.001; Kd 1.52 +/- 0.11 nM, p less than 0.001). There was no significant correlation between the density (Bmax) of sodium-dependent and desipramine-defined binding in individual subjects, suggesting that a different proportion of sites are labeled under the two assay conditions. No age-dependent variation was found in either Kd or Bmax values of sodium-dependent or desipramine-defined [3H]imipramine binding. The results suggest determination of sodium-dependent [3H]imipramine binding to intact platelets may be a useful measure for the estimation of [3H]imipramine recognition sites relevant to the serotonin uptake in studies of patients with affective disorders.     

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.