An inhibitor of opiate receptor binding from human erythrocytes identified as a glutathione-copper complex.

A “peptide-like” inhibitor of opiate receptor binding and of N-methyltransferase previously purified by us from rabbit brain was also found in human red blood cells. Boiled extracts of erythrocytes were fractionated on Sephadex followed by chromatography of the active fractions on silica gel layers. Both, a migrating ninhydrin-positive spot and a naturally blue substance which did not migrate from the origin coincided with the active fractions. The blue substance was identified as copper and the ninhydrin-positive material was identified as oxidized glutathione. While glutathione $\text{per se}$ has no effect, copper and other transition metals are potent inhibitors of opiate receptor binding. A mixture of glutathione and copper plus serum albumin in proportions simulating erythrocyte extracts gave results identical to the latter. Several other laboratories have extracted, from various tissues and body fluids, “opiate-like peptides” which are distinct from the β-LPH derived endorphins. In view of our findings it is possible that metal bound to glutathione or to other peptide ligand may be a complicating factor in some of these studies.     

Identification of opiate receptor binding in intact animals.

After intravenous administration of ³H-naloxone to rats, particulate bound radioactivity accumulated in the brain is selectively associated with opiate receptor binding sites, providing a means of labeling the opiate receptor $\text{in vivo}$. The regional distribution of ³H-naloxone bound $\text{in vivo}$ closely parallels regional differences in opiate receptor binding $\text{in vitro}$ with highest levels in the corpus striatum, negligible receptor-associated binding in the cerebellum and intermediate levels in other regions. ³H-Naloxone binding $\text{in vivo}$ is saturable with the same total number of binding sites determined $\text{in vivo}$ as by $\text{in vitro}$ procedures. Nalorphine is markedly more potent than morphine in inhibiting ³H-naloxone binding $\text{in vivo}$ and non-opiates are ineffective. The half-life for dissociation of ³H-naloxone bound to particles $\text{in vivo}$ is the same as its dissociation rate after binding occurs $\text{in vitro}$, and sodium stabilizes ³H-naloxone bound $\text{in vivo}$ from initial rapid dissociation as predicted from the known properties of the opiate receptor $\text{in vitro}$.     

Mammalian alpha 1-adrenergic receptor. Purification and characterization of the native receptor ligand binding subunit

alpha 1-Adrenergic receptors from a cultured smooth muscle cell line (DDT1 MF-2) have been solubilized with digitonin and purified to apparent homogeneity by sequential chromatography on a biospecific affinity support (Sepharose-A55453 (4-amino-6,7-dimethoxy-2-[4-[5-(4-amino-3-phenyl) pentanoyl]-1-piperazinyl]-quinazoline), an alpha 1 receptor-selective antagonist), a wheat germ agglutinin-agarose gel, and a high performance steric exclusion liquid chromatography column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of iodinated purified receptor preparations reveals a peptide with an apparent Mr = 80,000 that co-migrates with the peptide labeled by the specific alpha 1-adrenergic receptor photoaffinity probe 4-amino-6,7-dimethoxy-2-[4-[5-(4-azido-3-[125I]iodophenyl)pentanoyl] -1-piperazinyl] quinazoline. The specific activity (approximately 13,600 pmol of ligand binding/mg of protein) of purified receptor preparations is consistent with that expected for a pure peptide of Mr = 80,000 containing a single ligand binding site. Overall yields approximate 14% of initial crude particulate binding. The purified receptor preparations bind agonist and antagonist ligands with appropriate alpha 1-adrenergic specificity, stereoselectivity, and affinity. Peptide maps of the pure alpha 1-adrenergic receptor and the pure human platelet alpha 2-adrenergic receptor (Regan, J.W., Nakata, H., DeMarinis, R.M., Caron, M.G., and Lefkowitz, R.J. (1986) J. Biol. Chem. 261, 3894-3900) using several different proteases suggest that these two receptors show little if any structural homology.     

Factor from rat small intestine potently affects opiate receptor binding

The contractile response to bradykinin was studied in isolated longitudinal strips of detrusor muscle from rabbit urinary bladder. Strips responded slowly with contractions which were comparable in magnitude to acetylcholine but much greater than those produced by arachidonic acid. The bradykinin dose-response curve was very shallow (Clark's ratio = 10^?5), with an ED50 of 0.2 μM. Bradykinin-induced contractions were unaffected by 0.4 μM atropine or 0.2 μM eserine. This suggests, in contrast to reports on rat bladder, that acetylcholine release does not contribute to the response. However, pretreatment with 10 μM naproxen antagonized bradykinin-induced contractions without affecting acetylcholine. It is concluded that, as in many other tissues, in the urinary bladder at least part of the response to bradykinin is mediated through prostaglandins. Bradykinin probably also has a direct action since higher concentrations are less susceptible to naproxen, and it produces a much greater contraction than the maximum achievable with arachidonic acid.     

Possible involvement of cerebroside sulfate in opiate receptor binding.

Cerebroside sulfate (CS) appears to fulfill most of the structural requirements of a hypothetical opiate receptor. It possesses many of the properties that are thought to be necessary for the identification of an "opiate receptor," exhibiting high affinity and stereoselective binding to a number of narcotic drugs. Although these properties are insufficient to establish identity of the receptor, it is highly significant that the affinity of this binding can be correlated with the analgetic potency of these drugs in both man and rodents. CS is an endogenous component of brain tissue, and a partially purified opiate receptor from mouse brain has been found to be CS. Other experiments indicate that reduced availability of brain CS decreases the analgetic effects of morphine and this is accompanied by a reduction in number of binding sites, suggesting that the interaction of opiates with CS observed in vitro may also have importance in vivo. CS was also found to be a component of the opiate receptor after marking with 125I-labeled diazosulfanilic acid. The possibility that CS or the SO4-2 group of this lipid may be the "anionic site" of the opiate receptor should be considered.     

Discrimination by temperature of opiate agonist and antagonist receptor binding.

Variations in incubation temperature can markedly differentiate opiate receptor binding of agonists and antagonists. In the presence of sodium increasing incubation temperatures from 0° to 30° reduces receptor binding of ³H-naloxone by 50% while tripling the binding of the agonist ³H-dihydromorphine. Lowering incubation temperature from 25° to 0° reduces the potency of morphine in inhibiting ³H-naloxone binding by 9-fold while not affecting the potency of the antagonist nalorphine. At temperatures of 25° and higher the number of binding sites for opiate antagonists is increased by sodium and the number of sites for agonists is decreased by sodium with no changes in affinity. By contrast, in the presence of sodium lowering of incubation temperature to 0° increases opiate receptor binding of the antagonist naloxone by enhancing its affinity for binding sites even though the total number of binding sites are not changed.     

Direct binding properties of conantokins to native N-methyl-d-aspartate receptors.

Conantokin-G (con-G) is a small, gamma-carboxyglutamic acid (Gla)-containing peptide that functions neurophysiologically by inhibiting the N-methyl-d-aspartate receptor (NMDAR). In the current study, the receptor binding properties of an alanine-rich, Gla-deficient con-G variant, Ala-con-G, were assessed following tracer radioiodination with 125I. Direct binding experiments with [125I]Ala-con-G yielded a single site defined by a Kd value of 516 +/- 120 nm. Displacement of [125I]Ala-con-G binding by Ala-con-G resulted in 100% displacement with an IC50 value of 564 +/- 33 nm, while heterologous displacement by con-G[S16Y], con-G, con-T, and con-R[1-17] yielded IC50 values in the range of 15-45 microm. No displacement was observed with d-gamma-con-G or con-G[L5A], analogs that are inactive at NMDARs. Specific [125I]Ala-con-G binding was displaced by NMDA and 2-amino-5-phosphopentanoic acid in a dose-dependent manner, suggesting an interaction at the glutamate binding site. The direct binding of [125I]Ala-con-G to adult rat brain sections revealed an anatomical distribution of binding sites in all regions known to contain the NR2B subunit of the NMDAR. These results constitute the only known demonstration of the direct binding of a radiolabeled conantokin to the NMDARs present in rat brain membrane preparations and rat brain sections, and suggest that radiolabeled Ala-con-G, and similar conantokin derivatives, may find utility as probes of NMDARs in a variety of systems.     

Effect of beta-FNA on opiate delta receptor binding

beta-FNA, the beta-fumaramate methyl ester of naltrexone, has been shown to antagonize irreversibly the actions of morphine on the guinea pig ileum and mouse vas deferens bioassays but does not affect the actions of delta-receptor ligands on the mouse vas deferens bioassay, suggesting that the compound does not irreversibly bind to the delta receptor. In this paper we examine the effect of beta-FNA on the binding of the prototypic delta agonists, Leu-enkephalin and D-Ala2-D-Leu5-enkephalin, its metabolically stable analogue, and show that treatment of membranes with beta-FNA does lead to alterations in the in vitro properties of delta receptors.     

Location and characterization of opiate receptors regulating pituitary secretion

The site at which opiate agonists and antagonists act to alter secretion of prolactin, growth hormone and luteinizing hormone as well as the pharmacological specificity of the opiate receptors mediating these effects were examined in rats. Injection of β-endorphin but not a 10 fold higher dose of the non opiate peptide β-endorphin, increased release of prolactin and growth hormone in male rats while inhibiting luteinizing hormone release in ovariectomized, estrogen primed female rats. Prior treatment with naltrexone i.p. blocked these responses. Injection of naltrexone into the hypothalamus lowered prolactin release. In rats with a surgically formed hypothalamic island systemic administration of morphine or naltrexone altered prolactin release in the same manner as was observed in intact animals. In contrast no effects of β-endorphin or naltrexone were observed on the spontaneous secretion of prolactin $\text{in}$$\text{vitro}$. In addition β-endorphin did not alter the inhibition of prolactin release produced by apomorphine $\text{in}$$\text{vitro}$. The ED₅₀ for stimulation of prolactin release following intraventricular administration of β-endorphin or the synthetic enkephalin analog FK 33-824 was the same, approximately 0.1 ng/rat. However FK 33-824 at 0.2 ng/rat was able to produce much greater analgesia and catatonia than β-endorphin. The metabolism and distribution of β-endorphin was examined but did not account for these differential effects. These results indicate that opiate agonists and antagonists can act at the hypothalamic but not the anterior pituitary level to alter release of prolactin, growth hormone and luteinizing hormone. In addition the data suggest that the opiate receptors mediating release of prolactin may have a different pharmacological specificity from those involved with analgesia and catatonia.     

Synthesis and binding affinity of neuropeptide Y at opiate receptors

Neuropeptide Y and several metabolic fragments were synthesized and evaluated for binding affinity at non-selective opiate receptors. Neuropeptide Y and several C-terminal fragments were shown to bind to non-selective opiate receptors with an affinity similar to that of Leu-enkephalin.     

Studies on brain opiate receptor stability in vitro: inhibition of opiate receptor binding by adenosine-5'-diphosphate

Incubation of rat brain homogenates at 37° causes a time-dependent decrease in opiate receptor binding which does not occur with a washed membrane fraction. The supernatant fraction contains a heat-stable inhibitor which is partially destroyed by apyrase and completely removed by activated charcoal. ADP causes a similar inhibitory effect in homogenates, but not with washed membranes, which is characterized by a decrease in both opiate agonist and antagonist binding in the absence or presence of NaCl. The ADP inhibition is antagonized by ATP, α,β-methyleneADP, β-thioADP and EDTA. It is concluded that ADP, unlike the guanine nucleotides, facilitates the nonspecific degradation of opiate receptors by an endogenous soluble factor.     

Physical characterization of cyclosporine binding sites in lymphocytes.

The binding of cyclosporine to human peripheral blood lymphocytes (PBLs) was studied by measuring the fluorescence emission spectrum and lifetime of the fluorescent and immunosuppressive cyclosporine derivative dansyl-cyclosporine (DCs). The emission maximum and fluorescence lifetime of DCs were characterized in several solvents. The fluorescence emission maximum and lifetime of DCs increased at a high dielectric constant. The fluorescence lifetime decay curve of DCs was a monoexponential function in all solvents tested. Fluorescence micrographs of lipid vesicles and erythrocytes labeled with DCs exhibit uniform staining patterns, whereas PBLs show heterogeneous DCs labeling. DCs exhibits a relatively low emission maximum (490 nm) in erythrocyte membranes. Such an emission maximum is characteristic of a hydrophobic environment. DCs in PBLs also has a low emission maximum (484 nm). The lifetime of DCs in PBLs required two exponential terms to properly fit the lifetime decay curve and could not be attributed to light scattering. One short component (4.7 +/- 1.0 ns) and a second long component (18.5 +/- 1.0 ns) were resolved from the DCs fluorescence decay curves. Time-resolved anisotropy of DCs in PBLs revealed that the labeled drug was present in an anisotropic environment, consistent with at least some DCs being bound to a membrane. These fluorescence studies suggest that DCs interacts with multiple and/or heterogeneous sites in peripheral blood lymphocytes.     

Distribution of stereospecific opiate receptor binding activity between subcellular fractions from ovine corpus striatum

The thermodynamic binding constants of the opiate ligand etorphine to subcellular fractions prepared from ovine central nervous system tissue are reported. Those examined were synaptosomal, microsomal and myelin fractions. The dissociation constant is smallest and the binding capacity greatest in the microsomal fraction. The results have implications concerning future work on opiate receptor heterogeneity and isolation.     

Effect of morphinone on opiate receptor binding and morphine-elicited analgesia

Specific binding of ³H-naloxone to opiate receptors was found to be irreversibly inactivated by morphinone. This inactivation exhibited pseudo-first-order kinetics. The presence of sulfhydryl compounds or morphine during incubation with morphinone proved good protection. Morphinone-pretreated mice blocked the analgesic effect of morphine. The possible mechanism for these observations is proposed as foolows: morphinone binds covalently to sulfhydryl group of opiate receptors, and inactivates irreversibly opiate binding sites, thus blocking the analgesic effect of morphine.     

Streptococcal Fc receptors. I. Isolation and partial characterization of the receptor from a group C streptococcus

A receptor for the Fc region of immunoglobulin G was extracted from a group C streptococcus, purified and physicochemically characterized. The Fc receptor was extracted in high yield by lysis of the bacteria after infection with bacteriophage. The soluble receptor was purified to functional homogeneity by sequential chromatography on cellulose phosphate, DEAE, and selective elution from a column of immobilized human IgG. Four hundred micrograms of the functionally pure protein was obtained per gram (wet weight) of bacteria extracted. The affinity-purified receptor was functionally homogeneous in binding to the Fc region of human IgG; however, the product was heterogeneous on both non-denaturing and SDS polyacrylamide gels. Four major protein bands were observed, with the predominant form of the Fc receptor having an m.w. of 64,000 daltons. Antibody prepared against the major Fc receptor protein ( FcRc -II) was capable of reacting with all the fractions and completely inhibiting functional activity. The results of the competitive binding studies suggest that the purified Fc receptor behaves as a single receptor, and that the differences in charge and size were probably due to covalently bound cell wall constituents.     

Radioautography of binding of tritiated diprenorphine to opiate receptors in the rat

Radioautography of tritiated diprenorphine in rat brain indicates anatomic distribution of receptors with a greater degree of precision than is possible using dissection techniques. The results of this study largely confirm those of others but indicate some differences in receptor distribution in the thalamus. Differential receptor binding in the periaquaductal gray matter with the highest counts lying laterally is an original observation.     

Biotinylated 1012-S conjugate as a probe ligand for benzodiazepine receptors: characterization of receptor binding sites and receptor assay for benzodiazepine drugs.

A nonisotopic receptor assay using the biotin-1012-S conjugate was developed and the usefulness of this conjugate as a probe ligand for the benzodiazepine receptor was evaluated. The conjugate was incubated in a receptor suspension, and then the concentration of free conjugate in the supernatant was determined nonisotopically with a solid-phase avidin-biotin binding assay. Studies on the ligand saturation with the conjugate demonstrated that the conjugate has very high affinity and specificity for the receptors and the biotin labeling does not decrease the affinity of 1012-S. This assay method was applied to the characterization of binding sites of benzodiazepine receptors in cow brain. Competition interactions between the conjugate and benzodiazepine drugs gave well-defined dose-response curves. These results confirm the possibility that this conjugate could serve as a probe for the study of receptor-ligand interactions and provide the basis of a new nonisotopic receptor assay for benzodiazepine drugs.     

Lectin binding of solubilized opiate receptors: evidence for their glycoprotein nature

Lectin affinity chromatography was used to demonstrate that digitonin-solubilized opiate receptors contain a carbohydrate moiety. Receptors solubilized from toad, rat, chicken, bovine and human brains were retained on columns of wheat germ agglutinin (WGA)-agarose and eluted specifically with N-acetylglucosamine. The fraction retained and subsequently eluted ranged from 40–60% of the applied receptors. The eluted receptor was enriched approx. 30-fold. Evidence is presented which shows that the site of lectin interaction is functionally independent of the opiate binding site.