Simultaneous Differentiation of Three Opiate Receptor Subpopulations by Computer Modelling

Abstract

[³H]-(?)-bremazocine was displaced from guinea-pig brain membrane homogenates by three compounds having different specificity to opiate receptor subpopulations. A three site receptor model showed the best fit of the calculated to the measured value for the opiate mu (DAla², MePhe⁴, Gly(ol)⁵-enkephalin) and the delta specific compound (DAla², DLeu⁵-enkephalin). Computer modelling of data from displacement curves with the opiate kappa specific compound U-50.488H favored a two site receptor model.     

Decrease of cyclic GMP in cerebellum by intrastriatal D-Ala2-met-enkephalinamide.

The bilateral intrastriatal injection of D-Ala²-Met-Enkephalinamide (DALA) at doses of ranging from 12 to 50 μg decreased cyclic-GMP content in the cerebellum and produced catalepsy. These effects were prevented by naltrexone, an opiate receptor antagonist but not by apomorphine, a dopamine agonist. The bilateral injection of DALA in the cerebellum, and substantia nigra neither decreased cerebellar cyclic GMP content nor produced catalepsy. The bilateral injection of DALA (20 ug) into the ventromedial thalamic nuclei caused marked catalepsy but failed to decrease cerebellar cyclic GMP. The results suggest that the effect of DALA on cGMP can be differentiated from the cataleptic response and that it is mediated by an action on opioid receptors located in the striatum, beyond DA receptors.     

A radioreceptor assay for opiate drugs in human cerebrospinal fluid and plasma

We have developed a radioreceptor assay for opiates based on the ability of the plasma and CSF content of these drugs to compete for the binding of ³H-buprenorphine to opiate receptors in rat forebrain membranes. Since plasma proteins significantly inhibit total ³H-buprenorphine binding, and sodium ions reduce the affinity of opiate agonists for the receptor, it was necessary to extract opiates into an organic solvent (ether). The radioreceptor assay is particularly sensitive to buprenorphine and morphine, detecting these compounds at low picogram levels. The assay is simple to perform since 50 samples can be processed in a day, and is specific in that other drugs employed during anaesthesia such as benzodiazepines do not compete with ³H-buprenorphine for the opiate receptor. The extraction and binding techniques described should be applicable to other ³H-ligands which have high affinity for opiate receptors.     

Solubilization of membrane bound opiate receptor from rat brain

Sonication of rat brain membranes for 9 minutes solubilized 35% of their stereospecific opiate binding activity; a second 9 minute sonication of the insoluble residue released an additional 21% of the original binding. The opiate binding properties of the solubilized material were highly similar to those of membrane bound receptor by a number of criteria, including affinity, effect of sodium, and the IC₅₀ of unlabeled opiates in displacing ³H-etorphine binding. Moreover, storage of the solubilized receptor fraction for two weeks at ?20°C did not significantly change the receptor binding. Sonication thus appears to be a useful first step in purifying the opiate receptor.     

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.     

Opiate Receptor: Multiple Effects of Metal Ions

Abstract: The opiate antagonist [³H]diprenorphine ([³H]dip), a universal ligand at the μ, δ, and k opiate receptor subtypes, was used to study the effects of Ca-II, Cu-II, Mg-II, Mn-II, and Na⁺ on the rat cerebral opiate receptor. Two categories of effects were observed: (a) those on the binding rate constants and (b) those on binding capacity. (a) Sodium ions increased on- and off-rates on [³H]dip with a rather small net change in receptor affinity. The effects of Na⁺ and the divalent ions Ca-II, Mg-II, and Mn-II were antagonistic to each other. Ca-II, Mg-II, and the more effective Mn-II decreased receptor association and dissociation rates, again with minimal changes in the overall binding affinity in washed membrane homogenates. Previous studies using equilibrium binding analysis alone failed to detect changes in [³H]dip binding kinetics caused by these metal ions. In untreated rat brain homogenates, however, Ca-II (and to a lesser extent Mg-II) decreased [³H]dip binding, an effect distinct from that on the binding rate constants in washed membrane homogenates. (b) In untreated, Tris-buffer homogenates not containing external metal ions, a gradual decline in [³H]dip binding was observed. Cu-II or an equivalent endogenous divalent metal ion was identified as a causative factor, and Mn-II partially reversed this effect. Moreover, the addition of Mn-II stabilized the [³H]dip binding sites at very low concentrations of the metal (nM to μM range) that did not change the binding rate constants and that were in the physiological range of Mn-II in rat brain. This unique effect of Mn-II may represent a physiological function in the regulation of the opiate receptor that is not shared by Mg-II and Ca-II. The opposite effects of Cu-II and Mn-II on the in vitro receptor stability may be related to their opposite pharmacological effect in vivo. Finally, multiple changes of the effects of the tested metal ions on [³H]dip binding were observed during in vitro membrane homogenate dilution, centrifugation, and washing. These changes indicate that the opiate receptor complex as it exists in vivo may lose some of its functions and control mechanisms in vitro.     

A novel analogue of clonidine with opiate-receptor agonist activity

A new analogue of the α₂-adrenergic receptor ligand clonidine, N-(4-hydroxphenacetyl)-4-aminoclonidine, was synthesized. The analogue possesses opiate-receptor agonist activity in addition to α-adrenergic partial agonist activity. The analogue elicits inhibition of adenylate cyclase of NG108-15 neuroblastoma × glioma hybrid cells; most of the inhibition is reversed by the opiate-receptor antagonist naloxone. The analogue also inhibits the binding of [³H]D-Ala²-Met⁵-enkephalinamide and [³H]dihydromorphine to rat brain opiate receptors. The structure of the analogue suggests common elements in the ligand binding sites of α- and opiate receptors and may lead to a new class of opiate analgesics.     

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 $\text{in vitro}$.     

Down regulation of enkephalin (δ) receptors Demonstration in membrane-bound and solubilized receptors

The pentapeptide leucine enkephalin induced down-regulation of enkephalin receptors in neuroblastoma-glioma NG108-15 hybrid cells in a reversible fashion, whereas the stable enkephalin analogue, d-Ala²-Met-enkephalinamide (AMEA), and the potent opiate alkaloid, etorphine, had a prolonged effect. The opiate alkaloid, morphine, which has low affinity to δ-type enkephalin receptors of these cells did not induce down-regulation, whereas AMEA decreased the binding of both opiate agonists and antagonists but had no effect on the binding of the α₂-adrenergic ligand, [³H]yohimbine. From several experiments that were designed to remove the tightly bound AMEA, and from experiments with solubilized receptor we ruled out the possibility that the decreased binding capacity of enkephalin-treated cells reflects only receptor masking. The study suggests that down-regulation of enkephalin receptors that may also occur in vivo can account for some of the abnormal physiological responses of subjects treated chromically with opiates. However, since opiates from the morphine type can induce opiate tolerance in vivo, but not down-regulation of enkephalin receptors in the cultured cells, we suggest that down-regulation of δ-type opiate receptors may not be prerequisite for the development of the physiological tolerance/dependence on these alkaloids.     

Opiates and enkephalins: a common binding site mediates their analgesic actions in rats

³H-Labelled opiate and enkephalin ligands appear to bind with highest affinity to a single site responsible for their analgesic properties. Administered $\text{in vivo}$, naloxazone, an irreversible opiate, selectively inhibits for over 24 hours the high affinity binding of ³H-labelled mu, and kappa opiates and enkephalins. This inhibition of binding gradually resolves over 3 days, perhaps correlating with receptor turnover. Naloxazone treatment also abolishes morphine, D-ala²-met⁵-enkephalinamide and betah-endorphin analgesia. Although morphine and D-ala²-met⁵-enkephalinamide bind with similar potencies to the high affinity site, morphine's potency for the low affinity D-ala²-met⁵-enkephalinamide site is far less than the enkephalin analog. These results imply that all ³H-ligands examined bind with highest affinity to a mu-like receptor while low affinity D-ala²-met⁵-enkephalinamide binding, with a K_D of 6 nM, represents a delta-like receptor.     

Enkephalinergic Markers in Substantia Nigra and Caudate Nucleus from Parkinsonian Subjects

Marked reductions in opiate receptor binding (-42%), "enkephalinase" activity (-39%), and Met5-enkephalin levels (-72%) accompanied the well-established dopamine depletion in the substantia nigra pars compacta of Parkinsonian subjects. In contrast, enkephalinergic markers were not significantly modified in caudate nucleus.     

Radioactive labeling of alpha-bungarotoxin with tritium.

α-[³H] bungarotoxin (Bgt) was prepared by catalytic reduction of ¹²⁵I-labeled α-Bgt with tritium. Specific activities of 10–15 Ci/mmol were attained. The radioactive label was found in tyrosine. Tritiated α-Bgt appears to bind specifically to the cholinergic receptor of diaphragm and to a similar component of cerebral cortex. This specificity and the high specific radioactivity attained provide a useful tool for the study of acetylcholine receptor in brain and other tissues with low receptor concentration.     

Interaction of iodinated enkephalin analogues with opiate receptors.

Radioiodinated derivatives of the metabolically stable enkephalin analogues, [DAla²,Leu⁵]- and [DAla²,DLeu⁵]-enkephalin, have been prepared. Such derivatives show sterospecific binding to receptors in brain homogenates and some neuroblastoma cell lines such as NG108-15 and N4TG1. The relative effects of levorphanol and dextrorphan and Na⁺ and Mn⁺⁺ ions on enkephalin binding in brain and cells indicate that the iodinated derivatives are interacting with opiate receptors. Levorphanol is considerably more potent in displacing specifically bound enkephalin than dextrorphan. Sodium ions at physiological concentrations decrease enkephalin binding whereas manganese ions enhance it. Unlabelled monoiodo derivatives retain high potency in the guinea-pig ileum, mouse vas deferens and receptor binding assays. Unlabelled diiodo derivatives show far lower potency in these assays. It is concluded that radio-iodinated derivatives containing one iodine per molecule retain high affinity for the opiate receptor but diiodo derivatives do not.     

The interaction of ketamine with the opiate receptor

The analgesic effect of the anesthetic agent ketamine HCl is inhibited in rats by the narcotic receptor antagonist naloxone. Racemic (±) ketamine HCl also displaced ³H-naloxone in an opiate receptor binding-assay. The potency of ketamine in the assay was reduced nearly six-fold by sodium suggesting that the drug interacts as an agonist. However, some activity as an antagonist was not ruled out. The interaction of ketamine HCl with the opiate receptor was stereospecific with the (+) salt being more effective than the (-) salt. The stereoselective nature of the interaction is consistent with other studies (1) demonstrating that (+) ketamine HCl has a greater analgesic effect than the (-) salt.     

Effects of prolyl-leucyl-glycinamide and cyclo(leucyl-glycine) on morphine-induced antinociception and brain μ, δ and κ opiate receptors

The effects of prolyl-leucyl-glycinamide and cyclo (leucyl-glycine) on morphine-induced antinociception in mice and on $\text{in vitro}$ binding of ³H-ligands for opiate receptor subtypes (μ, δ and κ) the mouse brain homogenate were determined. Subcutaneous administration of either of the above peptides (1, 2, and 4 mg/kg) 10 min prior to the injection of morphine did not affect morphine-induced antinociception as evidenced by the identical ED₅₀ values of morphine in vehicle and peptide treated groups. The binding of ³H-dihydromorphine and ³H-naloxone ( μ receptors), ³HDAla²DLeu⁵-enkephalin (δ receptors), and ³H-ethylketocyclazocine (κ receptors) to opiate receptors in the mouse brain homogenate was also unaffected by both the peptides over a large concentration range. It is concluded that these peptides do not interact with brain opiate receptors.     

Evaluation of cetuximab as a candidate for targeted α-particle radiation therapy of HER1-positive disseminated intraperitoneal disease

Although the epidermal growth factor receptor (EGFR), also known as HER1, has been studied for over a decade, it continues to be a molecule of great interest and focus of investigators for development of targeted therapies. The marketed monoclonal antibody cetuximab binds to HER1, and thus might serve as the basis for creation of imaging or therapies that target this receptor. The potential of cetuximab as a vehicle for the delivery of α-particle radiation was investigated in an intraperitoneal tumor mouse model. The effective working dose of 10 μCi of ²¹²Pb-cetuximab was determined from a dose (10–50 μCi) escalation study. Toxicity, as indicated by the lack of animal weight loss, was not evident at the 10 μCi dose of ²¹²Pb-cetuximab. A subsequent study demonstrated ²¹²Pb-cetuximab had a therapeutic efficacy similar to that of ²¹²Pb-trastuzumab (p = 0.588). Gemcitabine given 24 h prior to ²¹²Pb-cetuximab increased the median survival from 174 d to 283 d, but carboplatin suppressed the effectiveness of ²¹²Pb-cetuximab. Notably, concurrent treatment of tumor-bearing mice with ²¹²Pb-labeled cetuximab and trastuzumab provided therapeutic benefit that was greater than either antibody alone. In conclusion, cetuximab proved to be an effective vehicle for targeting HER1-expressing tumors with α-radiation for the treatment of disseminated intraperitoneal disease. These studies provide further evidence that the multimodality therapy regimens may have greater efficacy and benefit in the treatment of cancer patients.     

Solubilization of the opiate receptor

The opiate receptor is solubilized from rat neural membranes by treating the membranes with Triton X-100, followed by centrifugation. Removal of the Triton X-100 was accomplished with Bio-beads SM-2, and the resulting supernatant was capable of stereospecifically binding opiates at 10^?13 moles/mg protein under saturating conditions. Stereospecific binding was measured by equilibrium dialysis and gel filtration using a Sephadex G-25 column, equilibrated with [³H] -ligand and either dextrorphan or levorphanol. The solubilized receptor has affinities for the opiates similar to those observed in membrane preparations and $\text{in vivo}$ experiments. The addition of phosphatidylserine to the supernatant enhances stereospecific binding of etorphine slightly. Phospholipase A₂, trypsin and chymotrypsin completely inhibit binding. The addition of albumin prevents, but does not reverse the inhibition caused by low concentrations of phospholipase A₂. Phosphatidylserine decarboxylase inhibits stereospecific binding by 95%, despite the fact only 10% of the phosphatidylserine present in the supernatant is converted to phosphatidylethanolamine. The solubilized opiate receptor, like the receptor in neural membranes, appears to consist of both protein and lipid moieties.     

Binding characteristics of a potent enkephalin analog

A radioiodinated form of the highly potent enkephalin analog FK 33-824 has been characterized with respect to its binding properties $\text{in vitro}$. ¹²⁵I-FK 33-824 is distinctive among the short opioid peptides in three ways. First, ¹²⁵I-FK 33-824 binds stereospecifically to rat brain homogenates with very high affinity (K_d = 0.42 nM). Secondly, dissociation of the ¹²⁵l-labelled peptide from membrane-bound opiate receptors occurs with a relatively long $\text{τ}\text{1}\text{2}$ of 25 min at 4° in contrast to other enkephalins which dissociate more rapidly. Third, competitive binding analyses reveal that the ¹²⁵l-FK 33-824 binds equally well to both enkephalin (δ) and morphine (μ) classes of opiate receptors. These characteristics distinguish the ¹²⁵l-labelled peptide as a particularly suitable probe for molecular studies and purification of the opiate receptor.     

INORGANIC CARBON REPLETION DISRUPTS PHOTOSYNTHETIC ACCLIMATION TO LOW TEMPERATURE IN THE CYANOBACTERIUM SYNECHOCOCCUS ELONGATUS1

Acclimation of cyanobacteria to ambient fluctuations in inorganic carbon (Ci) and temperature requires reorganization of the major protein complexes involved in photosynthesis. We grew cultures of the picoplanktonic cyanobacterium Synechococcus elongatus Naegeli across most of its range of tolerable temperatures from 23 to 35°C at both low (<0.1 mM) and high Ci (approximately 4 mM). Over that range of temperatures, the chl‐based doubling time did not differ between low and high Ci grown cells but did increase with decreasing temperature. Cells grown at 23°C high Ci showed an elongated morphology, which was not present in 23°C low Ci cells nor at 35°C high and low Ci. Furthermore, 23°C high Ci cells showed premature senescence and death compared with all other treatments. Phycocyanin per cell was greater in high Ci grown cells at all temperatures but showed a characteristic decrease with decreasing temperature. Functional PSII determination showed that 23°C high Ci cells had 1.5 × 10⁵ PSII·cell^–1 compared with only 6.9 × 10⁴ PSII·cell^–1 for 23°C low Ci. The 35°C high and low Ci cells had 7.7 × 10⁴ and 6.4 × 10⁴ PSII·cell^–1, respectively. These data were supported by immunoblot determinations of PsbA content·cell^–1. As a result of their high PSII·cell^–1, 23°C high Ci cells generated more reductant from PSII than could be accommodated by downstream assimilative metabolism, resulting in early senescence and death of 23°C high Ci cells, probably as a result of the generation of reactive byproducts of electron transport.     

Opiate Receptors from Different Tissue Sources: Solubilization and Characterization

Abstract: Membrane-bound opiate receptors from neuroblastoma-glioma hybrid cells and from different parts of the rat brain (whole brain minus cerebellum, cortex, thalamus-hypothalamus and cerebellum) were labeled with the methionine-enkephalin analogue, D-[³H]Ala²-Met-enkephalinamide, and solubilized with the nonionic detergent Brij 36T. The protease inhibitors bacitracin, phenylmethylsulfonyl fluoride, Trasylol, and leupeptin were included in the solubilization buffer to minimize proteolysis. Two simple techniques, ammonium sulfate precipitation and activated charcoal absorbence, were adapted to separate the free and the macromolecule-bound ligands. The solubilized receptor-[³H]enkephalin complexes were partially purified by consecutive passages through Sephadex G-75 and Sepharose 6B columns. Of the three peaks of radioactivity that were observed in the effluent of the Sepharose column, two contained proteins, and one of them, with a Stokes radius of 59 Å, seemed to contain the specific opiate receptor, as evidenced by additional experiments. This peak was further purified on thiol-Sepharose or diethylaminoethanol-Sephadex columns that were eluted with a gradient of 0–50 mM dithiothreitol or with 1.0 M KCI, respectively. The receptor-[³H]enkephalin complex from neuroblastoma-glioma cells (apparent δ-type receptors) binds less to the thiol-Sepharose beads than receptor-(³H]enkephalin prepared from the hypothalamus-thalamus, which is rich in μ receptors. The [³H]enkephalin receptor complexes of the various sources also differed in their stability. The dissociation of the ligand from the neuroblastoma-glioma receptor was monophasic, with a half- life of 250 min, whereas that of two brain regions was biphasic, with half-lives of 195–330 min and 10,000 min. The methods described may be of use for further purification of soluble opiate receptors, either active or cross-linked to the ligand.