Mu and kappa opiate binding sites in the rabbit CNS

We have examined the ability of various opiates to compete with the binding of 3H-etorphine (0.5 nM) in membranes from the rabbit cerebellum and thalamus. Our data suggest that greater than 80% of 3H-etorphine binding occurs at mu receptor sites in cerebellum membranes. In thalamus membranes, D-Ala2, D-Leu5-enkephalin (DADL) resolves binding of 3H-etorphine into two components. The first component accounts for about 50% of binding and may represent interaction of the radioligand with mu receptor sites. The second component is unaffected in the presence of high (1-5 microM) concentrations of DADL. The ranking of potency for opiate inhibition of the second component is ethylketocyclazocine greater than naloxone much greater than morphine much greater than DADL, suggesting it represents binding of 3H-etorphine to a kappa-opiate binding site. In the rabbit brain, the kappa-opiate binding site is particularly abundant in the thalamus followed by frontal cortex and caudate nucleus.     

Different types of opiate agonists interact distinguishably with mu, delta and kappa opiate binding sites

The present studies were undertaken to evaluate whether different types of opiate agonists interact in a distinguishable manner with mu, delta and kappa opiate binding sites. Two approaches were employed: (a) the well known effects of metal ions on opiate agonist binding affinities of subsite selective ligands were studied at mu, delta and kappa sites in rat brain homogenates. Binding parameters were obtained by simultaneous computeranalysis of displacement curves using the prototypic ligands dihydromorphine (DHM), (D-Ala2, D-Leu5) enkephalin (DADL) and ethylketocyclazocine (EKC) of the mu, delta and kappa binding sites respectively. The results show that the effects of metal ions depend not only on the binding site, but also on the ligand under investigation. (b) The interaction of the delta agonist DADL with the mu agonist DHM was investigated at mu binding sites by characterizing the type of competition occurring between the two ligands. The interaction was of the noncompetitive type. It therefore appears that the various opiate agonists either interact preferentially with different parts of a larger receptor site area or bind to topographically distinct sites on a single receptor molecule which are coupled allosterically.     

Affinity of beta-carbolines on rat brain benzodiazepine and opiate binding sites

The affinity of beta-carbolines, which may be formed in the body, to benzodiazepine and opiate receptors was studied by measuring their ability to inhibit the binding of [3H]-flunitrazepam and [3H]-dihydromorphine on rat brain synaptosomal membranes. All "aromatized" beta-carbolines studied (norharmane, harmane and 6-methoxyharmane) inhibited the specific binding of [3H]-flunitrazepam in micromolar concentrations, dihydro-beta-carbolines (6-methoxyharmalan, harmalol) were less potent, while all tetrahydro-beta-carbolines showed very low affinity. 6-Hydroxytetrahydroharmane, which is formed by condensation 5HT with acetaldehyde, inhibited [3H]-dihydromorphine binding in micromolar concentration, while norharmane and tetrahydro-beta-carbolines without OH-group showed little affinity. beta-Carbolines are the most potent known natural benzodiazepine receptor ligands. Because they are formed after alcohol drinking, their effects on benzodiazepine and opiate receptors may be connected with alcohol dependence although some beta-carbolines may inhibit 5HT uptake in still lower concentrations.     

Differential effects of sodium on two types of opiate binding sites.

Effects of Na⁺ on saturable naloxone binding were studied $\text{in vitro}$ using particulate fractions obtained from rat brain homogenates. Na⁺ stimulated the saturable naloxone binding in thalamus-hypothalamus regions, but inhibited it in cerebellum. These findings strongly support the hypothesis that two types of naloxone binding sites exist in brain tissues.     

Relation of cation binding sites on synaptic vesicles to opiate action.

The protein-sensitized fluorenscence of Tb³+ has been used to characterize cation binding sites on synaptic vesicles. In the presence of 100 mM KC1, the vesicles display a single class of sites having affinities for Ca²⁺ and Mg²⁺ in the mM range. Of subcellular fractions synaptic vesicles have the greatest number of sites, which involve tyrosine residues. Morphine, which is known to deplete Ca from nerve terminals $\text{in vivo}$ by a mechanism possibly involving low affinity sites on synaptic vesicles, does not compete $\text{in vitro}$ for the sites monitored by Tb fluorescence. In addition, acute morphine treatment does not alter the binding of Tb to the synaptic vesicle fraction. It is suggested that the depletion of brain Ca induced by morphine is not mediated by a direct action of opiates on these membrane sites.     

Solubilization in good yield of active opiate binding sites from mammalian brain

Active opiate binding sites have been solubilized from mammalian brain cell membranes. The presence of 0.5-0.1 M NaCl during treatment of membranes from rat brain, human frontal cortex, and bovine corpus striatum with glycodeoxycholate or digitonin resulted in the extraction of active opiate binding sites in yields ranging up to 43%. The criteria for solubility of the sites were their inability to sediment at 10(5) x g after 2 hr and their apparent molecular weight of 3- 4 x 10(5) as determined by gel filtration. The receptors in solution resemble the membrane-bound sites with respect to saturability, stereo-specificity, sensitivity to heat and reagents, and high affinity for opioid ligands. The interaction of solubilized sites with immobilized lectins was used to demonstrate the glycoprotein nature of the opiate receptor. Soluble receptors from all species studied were retained by wheat germ agglutinin(WGA)-agarose and could be specifically eluted with N-acetylglucosamine. No retention of solubilized material was observed with eight other lectins examined, including horseshoe crab lectin, a sialic acid specific agglutinin. The receptor protein eluted from WGA columns was enriched 25-50-fold over the crude soluble fraction.     

Testosterone does not influence opiate binding sites in the male rat brain

It has been reported previously that castration produces testosterone-reversible increases in the density of 3H-naltrexone binding sites in the male rat brain. Unfortunately, we were unable to replicate these observations in a comprehensive series of studies. Specifically, we found that castration failed to produce changes in the Kd or Bmax of opiate binding sites in whole male rat brain, or in the hypothalamus, utilizing 3H-dihydromorphine (a mu receptor ligand), 3H-D-alanine, D-leucine enkephalin (delta) or 3H-naltrexone (ubiquitous). Furthermore, we found that the relative proportion of mu and delta binding sites in brain was unchanged by castration. The reasons for the discrepancy between the present results and those previously reported are unclear, but it appears that the provocative hypothesis that testosterone influences opioid receptors in brain must be carefully reevaluated.     

Gonadal steroid-induced modification of opiate binding sites in anterior hypothalamus of female rats

It has been previously shown that treatment with estradiol valerate (EV) or 17 beta-estradiol produces enhanced tritiated naloxone binding in the hypothalamus of the rat. In the present study, the following questions were addressed: Does this effect occur in the preoptic and anterior hypothalamic areas (regions associated with the cyclic gonadotropin surge)? Is chronic estradiol (E2) exposure essential in producing a long-term elevation in opiate binding? Is the effect steroid specific? The results indicated that chronic, but not acute, exposure to estradiol induces and maintains enhanced binding in the anterior hypothalamus. E2 exposure was most effective in this regard, testosterone (T) significantly less so, and 5 alpha-dihydrotestosterone (DHT) caused no elevation in binding above castrate control levels. Furthermore, both T and DHT significantly inhibited the estradiol effect. In addition, intact animals showed significantly less binding than their ovariectomized counterparts, suggesting that the ovary normally produces a factor that suppresses opiate binding. Enhancement of anterior hypothalamic opiate binding is therefore dependent on chronic exposure to E2, and the marginal effects of T are probably due to in situ aromatization. E2 enhancement of opiate binding may be causal in producing the hypothalamic aberration that results in the EV-induced polycystic ovarian condition.     

NMB: a human neuroblastoma cell line with specific opiate binding sites

The human neuroblastoma cell line designated NMB (Brodeur et al., 1977, Cancer 40: 2256) has been shown to have specific opiate binding sites. These sites are highly stereospecific. Two characteristic delta specific peptides, D-Ala2-D-Leu5 enkephalin and D-Thr2-D-Thr6 enkephalin, have high affinity for the binding sites. Morphine binds specifically but with a much lower affinity. Dextrorphan and the mu specific peptide morphiceptin (Tyr-Pro-Phe-Pro-CO-NH2) do not bind to the site. The binding sites are heat and trypsin sensitive. Sodium ions specifically lower agonist binding to the sites. Approximately 14,000 binding sites per cell are found. The binding characteristics of these sites are very similar to those of the delta sites characterized on mouse neuroblastoma cell lines.     

Quantitative autoradiography of the development of mu opiate binding sites in rat brain

The regional developmental appearance of mu binding sites in rat brain was examined by quantitative autoradiography of 3H-dihydromorphine binding in rats 2, 14, 21, and 28 days old. Labeling with 3H-dihydromorphine was heterogeneous in adult rat brains, as previously reported by other laboratories. Levels of 3H-dihydromorphine binding ranged from approximately 250 nCi/g tissue in the interpeduncular nucleus and 100 nCi/g tissue in the habenula to 40 nCi/g tissue in the hypothalamus and periaqueductal gray. Some areas, particularly white matter regions, had no detectable specific binding. The density of 3H-dihydromorphine binding increased in all regions between 2 and 28 days of age. The increases in 3H-dihydromorphine binding in various regions of rat brain developed at different rates. Maximal densities were seen by 14 days of age in most regions examined, including the caudate, hippocampus, amygdala, and hypothalamus. Binding in the medial thalamus and quadrigeminal plate, however, did not reach maximal levels until 21 days. Although quantitative autoradiography offers major advantages in the examination of the regional distribution of opiate binding sites, variability both between sections from the same brain and between sections from different brains demonstrate some of the difficulties associated with this type of experimental approach.     

Localization of human placental opiate binding sites on the syncitial brush border membrane

Opiate binding sites were measured in different placental membrane fractions which were characterized by marker enzyme analysis and electron microscopic examination. The distribution pattern of opiate binding sites in the different fractions closely parallels that of placental alkaline phosphatase. These results clearly show thatopiate binding sites are mainly located on the syncitial brush border membrane. The opiate binding sites found on microvillus membrane fraction have the same pharmacological characteristics as the Kappa opiate binding site previously characterized on placental crude membrane fraction.     

Cell-free protein synthesis systems

Cell-free protein synthesis systems enable the direct in vitro expression of proteins from template DNA or RNA. Use of biochemical and bioengineering techniques has greatly improved the yields and productivities of cell-free systems. In some cases, the yields approach the in vivo levels. Moreover, in vitro systems are capable of rapidly providing artificial polypeptides that greatly facilitate protein engineering. Post-translational modification steps in cell-free systems also offer exciting possibilities as reviewed here.     

Cell-free synthesis of phytochrome apoprotein

A single polypeptide is immunospecifically precipitated by monospecific antiphytochrome from the total translation products of both wheat-germ and rabbit-reticulocyte cell-free protein synthesizing systems programmed with oat (Avena sativa L.) poly(A) RNA. The mobility of this polypeptide is slightly lower on sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis than that of immunoaffinity-purified, 118 kdalton phytochrome and corresponds to an apparent molecular weight of 124 kdalton. Evidence against the possibility that this mobility difference results from intracellular processing of the 124-kdalton protein is provided by extraction of freeze-dried tissue directly into boiling SDS-containing buffer. This procedure yields a phytochrome species with a mobility on SDS polyacrylamide gel electrophoresis indistinguishable from that of the in-vitro translation product. Together the data indicate that the phytochrome polypeptide is synthesized in its mature form in the cell but is subject to modification to a form with lower apparent molecular weight during immunopurification.Abbreviations IgG immunoglobulin G - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

Cell-free synthesis of encephalomyocarditis virus

We developed a system for complete replication of encephalomyocarditis virus (EMCV) in a test tube by using an in vitro translation extract from Krebs-2 cells. Efficient virus synthesis occurred in a narrow range of Mg(2+) and EMCV RNA concentrations. Excess input RNA impaired RNA replication and virus production but not translation. This suggests the existence of a negative-feedback mechanism for regulation of RNA replication by the viral plus-strand RNA or proteins.     

Cell-free synthesis of cod preproinsulin

Poly(A)-containing ribonucleic acid from cod islet greatly stimulated the incorporation of radioactive amino acids into proteins when assayed in a wheat germ translation system. The translation products were examined by specific immunoprecipitation with guinea pig anti cod insulin antibodies and by extraction with acid--ethanol. These measurements revealed at least a fivefold increase in incorporation of labelled amino acid over the nonprogrammed system. Sodium dodecyl sulfate disc gel electrophoresis and gel filtration chromatography showed a product of molecular weight 12 500, a size considerably larger than cod proinsulin (9000). It is concluded that cod proinsulin is synthesized via a larger precursor, preproinsulin.