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.     

The effect of hyperthyroidism on opiate receptor binding and pain sensitivity

This study was conducted to determine the effect of thyroid hormone on opiate receptor ligand-binding and pain sensitivity. Specific opiate receptor-binding was performed on brain homogenates of Swiss-Webster mice. There was a significant increase in 3H-naloxone-binding in thyroxine-fed subjects (hyperthyroid). Scatchard analysis revealed that the number of opiate receptors was increased in hyperthyroid mice (Bmax = 0.238 nM for hyperthyroid samples vs. 0.174 nM for controls). Binding affinity was unaffected (Kd = 1.54 nM for hyperthyroid and 1.58 nM for control samples). When mice were subjected to hotplate stimulation, the hyperthyroid mice were noted to be more sensitive as judged by pain aversion response latencies which were half that of control animals. After morphine administration, the hyperthyroid animals demonstrated a shorter duration of analgesia. These findings demonstrate that thyroxine increases opiate receptor number and native pain sensitivity but decreases the duration of analgesia from morphine.     

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.     

Lack of opiate receptor involvement in centrally induced calcitonin analgesia.

Calcitonin (CT) injected into the brain ventricles (ICV) of conscious rabbits induced an analgesia not reversable by naloxone which could be repeatedly elicited (for 5 days), while tolerance to morphine developed. CT and morphine synergized $\text{in vivo}$ when administered ICV in combination. CT did not alter electrically-induced contractions of guinea-pig myenteric plexus-longitudinal muscle and no displace of ³H-dihydromorphine by CT was observed in brain opiate receptor preparations. We have concluded that the mechanism of centrally induced CT analgesia may be opiate-independent.     

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 $\text{in vitro}$.     

Evidence for opiate receptor binding in rat small intestine

The influence of ethanol consumption during pregnancy on maternal-fetal transfer of amino acid was studied. Pregnant rats were fed a liquid diet containing 30% ethanol-derived calories from gestation-day 6 to 21; control rats were pair-fed identical diets, except that sucrose substituted isocalorically for ethanol. On gestation-day 21, 2 uCi/100 g body weight of ¹⁴C-alpha-aminoisobutyric acid (¹⁴C-AIB) was injected into the maternal circulation, and 90 minutes later maternal blood and liver, placentas and fetuses were removed for radioactivity measurement. No differences between ethanol-fed and control rats in the distribution of ¹⁴C-AIB in maternal plasma or the uptake of ¹⁴C-AIB by the maternal liver were observed. However, the radioactivities in placenta and fetal tissues suffered a significant 20 to 40% reduction in the ethanol-fed group, suggesting that ethanol feeding during pregnancy impairs placental function.     

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.     

Effect of the position of the phenolic group in morphinans on their affinity for opiate receptor binding

Homologous series of N-methyl, N-allyl and N-cyclopropylmethylmorphinans, differing only in the position of the plenolic hydroxy group, were examined with respect to their binding affinities for the opiate receptor. IC₅₀'s were determined for competition with ³H-naltrexone in the presence and absence of 100 mM NaCl. While the compounds with the hydroxy in the 3-position had, as expected, by far the highest affinity, the corresponding molecules with the hydroxy in the 2- or 4- position had significant binding affinity ranging from 30 nM in the cyclopropyl- methyl series to 400 nM for the 2-hydroxy N-methyl morphinan. The sodium indices were also very similar to those of the corresponding 3-hydroxy compounds. The only 1-hydroxy derivative available was about 5-fold weaker than the corresponding 2- and 4-hydroxy compounds. Covering or removing the hydroxy group greatly weakened the binding but did not totally destroy it. There was good correlation between binding affinity and pharmacological potency for all except the methoxy compounds. Their high potency is consonant with $\text{in vivo}$ hydrolysis of the methyl ether.     

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.     

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.     

Materials with opiate receptor binding activity in bovine testis and ovine pancreas

Two groups of opiate-like materials, one with a molecular weight equal to or greater than 5000 daltons and another with a molecular weight smaller than 5000 daltons as judged by gel filtration on Sephadex G-25, were detected in bovine testes. The existence of opiate-like materials with a molecular weight smaller than 5000 daltons was demonstrated in ovine pancreas. The pancreatic fraction most strongly adsorbed on CM-cellulose possessed the highest opiate receptor binding activity. Bovine testis contained corticotropin-like material(s) which stimulated corticosterone production by isolated rat adrenal cells.     

Effect of opiate receptor agonists and antagonists on maternal aggression in rats

The behavioral effects of opiate agonists and antagonists were studied on the female aggression model. Mu-agonist buprenorphine more selectively decreased maternal aggression than kappa-agonist tifluadom. Kappa-agonists (bremazocine, tifluadom) increased passive defence in lactating female rats. Ethopharmacological data shows predominant involvement of brain mu-opiate receptor system in the integrative processes of maternal behavior and maternal aggression in particular.     

Monoclonal antibody to enkephalins with binding characteristics similar to opiate receptor

Monoclonal antibodies to enkephalins were established by immunization of mice with met-enkephalin, leu-enkephalin or both. Twenty-three clones with a high titer were classified into 6 types according to the binding properties to enkephalins and their derivatives. Antibody LM 239 showed binding characteristics similar to opiate receptor. It has a very high affinity to enkephalins and their derivatives which have a potent opioid activity, but a low affinity to enkephalin derivatives which devoid of opioid activity. The binding of 3H-met-enkephalin to the antibody was inhibited by naloxone and morphine, although the ID50 values were considerably higher than the Ka values of the alkaloids to opiate receptor.     

Effect of picrotoxinin on benzodiazepine receptor binding

The effect of picrotoxinin on [³H]flunitrazepam binding to benzodiazepine receptors was investigated. In mouse forebrain membranes, picrotoxinin inhibited basal, GABA- and pentobarbital-stimulated [³H]flunitrazepam binding; this inhibitory activity was temperature- and chloride ion-dependent. Scatchard analysis of the data indicates that picrotoxinin decreases the number of binding sites without alterating binding affinity. In cerebellar membranes, picrotoxinin did not alter [³H]flunitrazepam receptor binding.     

Developmental differences between high and low affinity opiate binding sites: Their relationship to analgesia and respiratory depression

Saturation studies of rats aged 2 days and 14 days demonstrated marked differences between high and low affinity opiate receptor binding. Whereas the density of low affinity ³H-morphine binding was virtually unchanged between days 2 and 14 (72 and 88% of adult levels, respectively), the density of high affinity binding increased 2.8-fold (22 to 61% of adult levels, respectively; p < 0.002). Similar results were seen in spinal cord binding. The similar sensitivity of 2 day old and 14 day old rats to morphine's depression of respiratory rates and lethality was contrasted by the 40-fold decreased analgesic sensitivity of 2 day old compared to 14 day old rats. These results suggest a correlation between high affinity binding and analgesia and between low affinity binding and respiratory effects.     

Effect of opiate receptor agonists on the course of hemorrhagic shock in rats

The experiments have been performed on 93 male rats, weighing 200-250 g. In acute blood loss various arterial pressure (AP) changes have been demonstrated--the marked hypertension is being changed by gradual AP increase. The injection of m-receptors' agonist DAGO prevents systolic and diastolic AP increase, agonist DADL prevents diastolic AP increase in acute momentary blood loss. In gradual blood loss DAGO (more than DADL) slows down both the decrease and the subsequent AP increase in rats. DAGO is determined to decrease, and DADL--to increase the minute blood volume. The mechanisms of opioids' action and their significance in pathogenesis of hemodynamic disturbances in shock are being discussed.     

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.     

beta-Endorphin: structure-activity relationships in the guinea pig ileum and opiate receptor binding assays.

The opiate activities of some derivatives and enzymatic digests of camel and human β-endorphin were determined in the guinea pig ileum and rat brain opiate receptor binding assays. Derivatives of β-endorphins altered within the amino-terminal five residues showed pronounced losses in activity. Anisylation of the C-terminal glutamic acid residue of β_h-endorphin produced only small reductions in activity. Chymotryptic digestion greatly weakened the opiate activities of β_h-endorphin, whereas carboxypeptidase A, tryptic and leucine aminopeptidase digests showed only small losses in potency. The C-terminus of β-endorphin appears to contribute little directly to opiate activity. Amino acid analysis and assay of the leucine aminopeptidase digests suggest that the larger potency of β-endorphin relative to Met-enkephalin may be a consequence of its greater resistance to exopeptidase attack.     

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.     

Cell-free synthesis of opiate binding sites

A procedure is described for the detection of opiate binding sites synthesized during in vitro translation of various mRNA preparations. RNA were isolated from membrane bound polysomes which were prepared from NG 108-15 hybridoma, C6BU1 glioma cells, as well as from N18TG2, NB2aAg and NB41A3 neuroblastoma cells. Polyadenylated [poly(A)⁺] RNA were purified, translated in vitro in a rabbit reticulocyte lysate and the translation products assayed for their ability to bind [³H] bremazocine. Bound and free ligands were separated by column chromatography. After translation of poly(A)⁺ RNA obtained from NG 108-15 cells we demonstrated a stereospecific, saturable binding of [³H]bremazocine (displaced by levorphanol and not by dextrorphan) with a K_d of 2.4 ± 1.0 nM. The total amount of opiate binding sites synthesized was 6.2 ± 0.5 fmol per μg of poly(A)⁺ RNA. Opiate binding sites were undetectable at zero time and a plateau was reached after translation had proceeded for 20 min. Five time less opiate binding sites were synthesized when the poly(A)⁺ RNA purified from N18TG2 neuroblastoma cells were used under the same experimental conditions. There was no detectable binding of opiate ligands with poly(A)⁺ RNA obtained from C6BU1 glioma cells, NB2aAg or NB41A3 neuroblastoma cells.