A peptide-like substance from pituitary that acts like morphine. I. Isolation.

A method is described for extraction from bovine pituitary glands of a substance that shows several properties characteristic of opiates. The material inhibits the twitch tension of the electrically stimulated guinea pig longitudinal muscle-myenteric plexus preparation and of the electrically stimulated mouse vas deferens. This inhibition is reversed and blocked by the opiate antagonist naloxone. The extract also inhibits binding of the opiate agonist etorphine and the opiate antagonist naloxone to stereospecific binding sites in synaptic membranes of guinea pig brain. The inhibition of naloxone binding is decreased by Na⁺ in the manner characteristic of opiate agonists. The physiologic role of the pituitary opioid remains to be investigated.     

Studies on the Inhibitory Action of Opiate Compounds in Isolated Bovine Adrenal Chromaffin Cells: Noninvolvement of Stereospecific Opiate Binding Sites

In isolated bovine adrenal chromaffin cells, beta-endorphin, dynorphin, and levorphanol caused a dose-dependent inhibition of catecholamine (CA) secretion elicited by acetylcholine (ACh), with an ID50 of 50, 1.3, and 4.3 microM, respectively. The inhibition by the opiate compounds was specific for the release evoked by ACh and nicotinic drugs and was noncompetitive with ACh. Stereospecific binding sites for the opiate agonist [3H]etorphine were found in homogenates of bovine adrenal medulla (KD = 0.59 nM). beta-Endorphin, dynorphin, levorphanol, and naloxone were potent inhibitors of the binding of [3H]etorphine with an ID50 of 12, 0.4, 5.2, and 6.2 nM, respectively. However, [3,5-I2Tyr1]-beta-endorphin, [3,5-I2Tyr1]-dynorphin, and dextrorphan, three opiate compounds with no or little activity in the guinea pig ileum assay, were relatively ineffective in inhibiting the binding of [3H]etorphine (ID50 700, 600, and 10,000 nM, respectively). On the other hand, these three compounds were equipotent with beta-endorphin, dynorphin, and levorphanol, respectively, in inhibiting the ACh-evoked release of CA from the adrenal chromaffin cells (ID50 of 10, 1.5, and 6 microM, respectively). Inhibition of CA release was also obtained with naloxone (ID50 = 14) microM) and naltrexone (ID50 greater than 10(-4) M), two classical antagonists of opiate receptors, and this effect was additive to that of beta-endorphin. These data indicate that the opiate modulation of CA release from adrenal chromaffin cells is not related to the stimulation of the high affinity stereospecific opiate binding sites of the adrenal medulla. The physiological function of these sites remains to be determined.     

Opiates Stimulate Low Km GTPase in Brain

Low Km GTP hydrolysis in rat brain is stimulated in a concentration-dependent manner by the opiate alkaloid etorphine, and by the opioid peptide D-Ala2-leucine-enkephalinamide. The opiate antagonist naloxone inhibits the maximal D-Ala2-leucine-enkephalinamide stimulation of the GTPase, also with concentration dependency. The magnitude of maximally stimulated, opioid-sensitive, GTP hydrolysis is differentially distributed across brain regions. Opioid-stimulated GTPase may represent one means of identifying a specific type of opioid receptor.     

CHARACTERISTICS AND REGIONAL DISTRIBUTIONS OF TWO DISTINCT [3H]NALOXONE BINDING SITES IN THE RAT BRAIN

Using [³H]naloxone at a concentration of 4.5 nm , the potent opiate agonist etorphine as well as the potent antagonist diprenorphine displace only about 75% of specific naloxone binding P₂ fractions from rat whole forebrain, without additive effect. Several other opiates and antagonists completely displace specific naloxone binding. This indicates that etorphine and diprenorphine specifically bind to one and the same naloxone binding site (type I) while leaving another naloxone binding site (type II) unaffected. Type I binding sites are much more thermo-labile than type II. [³H]Naloxone binding to type I sites is unaffected by incubation temperature in the range 10 to 25°C. while binding type II sites decreases rapidly with increasing incubation temperature, no specific type II binding being detectable at or above 20°C. The two naloxone receptor types also differ with respect to pH dependence, and affinity for naloxone with types I and II having affinity constants (K_d) of 2 and 16 nm , respectively, at 0°C. The two binding sites have different regional distributions with high relative levels of type II receptors in cerebellum and low relative levels in pons-medulla and striatum. In whole rat brain there are about 4 times as many type II receptors as type I. These results suggest that naloxone and several other opiate agonists and antagonists bind to two distinct receptor types which are probably not agonist/antagonist aspects of the same receptor.     

Decrease in the number of high-affinity opiate binding sites during the aging process inMytilus edulis (Bivalvia)

As Mytilus edulis(Bivalvia) ages, the number of stereospecific opiate binding sites in the visceral ganglia significantly decreases. The number of high-affinity etorphine sites decreased by 30%; the number of naloxone sites decreased by 23%. The affinity of the ligands did not vary with age. In addition, the augmentation by opioids of dopamine levels in the visceral ganglia was less in older animals and was accompanied by a reduced sensitivity of the ganglia to etorphine and DAMA (d-Ala ²,Met⁵-enkephalinamide).     

Comparison of the behavioral effects of β-endorphin and enkephalin analogs

The behavioral effects of β-endorphin, enkephalin analogs, morphine and etorphine were briefly compared. In the tail-flick test in mice and in the wet shake test in rats, β-endorphin and D-Ala²-D-Leu⁵-enkephalin had equal antinociceptive activity; D-Ala² -Met-enkephalinamide and D-Leu⁵-enkephalin were less active. The order of activity of the enkephalin analogs and opiate alkaloids for stimulating locomotor activity in mice paralleled their analgesic activities; β-endorphin, however, had only minimal stimulatory actions. Morphine sulfate, 50 μg injected into the periaqueductal gray, produced hyperactivity but this effect was not observed with etorphine or opioid peptides. By contrast, “wet dog” shakes was observed with the opioid peptides but not with either opiate alkaloid. These heterogenous behavioral responses, which were all antagonized by naloxone, indicate that multiple types of receptors mediate the effects of opiates in the central nervous system.     

Failure of opiates to increase the hydrolysis of GTP in neuroblastoma-glioma 108-15 cells

It has been repeatedly demonstrated that the neuroblastoma-glioma (NG 108-15) cell line has opiate receptors that inhibit adenylate cyclase and it has been proposed that this inhibition is mediated by a naloxone reversible stimulation of a low K_m GTPase (Koski and Klee, Proc. Natl. Acad. Sci. 78:4185, 1981). The guanine nucleotides of NG cells were labeled with [³H]guanine followed by incubation with 10^?6M guanine. Etorphine (10^?6M] or vehicle were added and the incubations continued for 1–4 min. The reaction was stopped with 5 percent TCA containing nucleotides as carriers and markers for the HPLC. Marker nucleotides were detected at 254 nm and the labeled nucleotides by liquid scintillation spectrometry. In several experiments, etorphine failed to produce any measurable change in the labeled nucleotides or in the GTP/GDP ratios. To verify that the opiate receptors were functional we measured its capacity to inhibit the formation of cAMP induced by PGE₁. We also studied the effects of naloxone and PGE₁ on the formation of cAMP in opiate tolerant cells. Tolerant cells responded to naloxone with a 50 percent increase in cAMP, indicating again that the opiate receptors were functional. Our results are consistent with the idea that in intact NG108-15 cells the opiate-mediated hydrolysis of GTP observed in cell membrane preparations is of very small magnitude.     

Involvement of Both Inhibitory and Stimulatory Guanine Nucleotide Binding Proteins in the Expression of Chronic Opiate Regulation of Adenylate Cyclase Activity in NG108-15 Cells

Abstract: Chronic etorphine treatment of neuroblastoma × glioma NG108-15 cells results in both an increase in adenylate cyclase activity (upon addition of the opiate antagonist naloxone) as well as an homologous desensitization of the opiate receptor. The continued ability of opiate agonists to regulate adenylate cyclase activity following opiate receptor desensitization can be understood by proposing that the catalytic subunit of adenylate cyclase in NG108-15 cells is under tonic regulation by both guanine nucleotide regulatory (N_i) and stimulatory (N_s) components. Inactivation of N_i by pertussis toxin (PT) treatment resulted in elevated adenylate cyclase activities comparable to those observed in control cells following chronic opiate treatment. This increased enzymatic activity could not be further induced by PT treatment of cells exposed to opiate previously. In addition, procedures that prevented receptor-mediated activation of N_s, i.e., treatment with NaF or desensitization of the stimulatory receptors (prostaglandin E₁, adenosine) eliminated the increase in adenylate cyclase activity induced by naloxone following chronic opiate exposure. Hence, the increase in enzymatic activity observed following chronic opiate treatment may be due to a loss in tonic inhibitory regulation of adenylate cyclase mediated through N_i resulting in the unimpeded expression of N_s activity. This tonic inhibition of adenylate cyclase activity is one of the multiple mechanisms by which N_i regulates adenylate cyclase in this cell line.     

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.     

Inhibition of analgesia by C-terminal deletion analogs of human beta-endorphin

Human beta-endorphin (beta h-EP) analogs of variable chain lengths have been investigated for their potency in inhibiting analgesia induced by beta h-EP or by the potent opiate etorphine. It was found that beta h-EP-(1-28) inhibits the analgesic effect of beta h-EP and etorphine when co-injected intracerebroventricularly into mice. Antagonism by competition at same opioid receptor subtypes is suggested from parallel shifts of the dose-response curve of etorphine or beta h-EP in the presence of increasing doses of beta h-EP-(1-28). On a molar basis, beta h-EP-(1-28) is nearly 10 times more potent than naloxone. The reduction of the chain length from residues 1-28 to 1-27 lowered the antagonist potency while further reduction of the peptide chain led to a complete loss of inhibitory activity. From comparison of the opioid-receptor binding affinity, analgesic activity and antagonist potency, it is concluded that the C-terminus of beta-EP is critical to the biological efficacy of the molecule and that the antagonist activity of C-terminal deletion analogs is probably mediated through residues 27 and 28.     

Opiate binding to cerebroside sulfate: a model system for opiate-receptor interaction.

Cerebroside sulfate was shown to bind etorphine and levorphanol with high affinity. The relative potency of narcotic analgesics in preventing the binding of levorphanol to cerebroside sulfate correlated well with their reported analgetic activity. The data indicate similarities between cerebroside sulfate and a purified opiate receptor from mouse brain which has been reported to be a proteolipid. Some preliminary animal data also imply the involvement of CS in opiate action We, therefore, propose that CS may serve as a useful “receptor” model for the study of opiate-receptor interaction $\text{in vitro}$.     

Use of naloxone in the assessment of opiate dependence

All subjects participating in an outpatient study comparing treatments for opiate dependence were given a naloxone challenge to document their level of dependence. Subjects were assessed at 0, 10, 20, and 30 minutes following the administration of intramuscular naloxone (0.4 mg) using an opiate withdrawal assessment scale and measurements of pupillary diameter. Subjects' self reports of daily dollar amounts of opiate use and time since last use were also examined for possible correlation with withdrawal scale scores and pupillary measurements. A significant negative correlation was obtained between pupil diameter and time since last reported use of an opiate. Results indicated that the scale was a reliable indicator of opiate dependence. Ways in which it might be improved are discussed.     

Chronic naloxone increases opiate binding in SHR and WKY rats

We have previously reported that chronic administration of naloxone to SHR and WKY rats results in a significant increase in their systolic blood pressure relative to control animals. In the present study we show that chronic naloxone is also accompanied by a marked increase in the number of brain opiate receptors. Although the relative difference in blood pressure diminishes with increasing maturity, the elevation in brain opiate receptors remains in the treated animals. Mechanisms for these differential effects are discussed.     

The anticonvulsant effects of DADLE are primarily mediated by activation of delta opioid receptors: interactions between delta and mu receptor antagonists

Dose-response comparisons of the ability of the selective delta antagonist ICI 154,129 (12.5-50 nmol), the nonselective antagonist naloxone (29-290 nmol), and the irreversible selective mu antagonist beta-fNA (1.3-21 nmol) to alter the threshold response to DADLE or etorphine was studied in the rat flurothyl seizure test. DADLE (35 nmol, i.c.v.) and etorphine (122 nmol/kg, s.c.) both caused increases in seizure threshold which were differentially antagonized by pretreatment (i.c.v.) with the respective antagonists. For DADLE, only ICI 154,129 and naloxone produced a dose-related blockade of the increase in seizure threshold, with ICI 154,129 being more potent than naloxone. In contrast, the anticonvulsant action of etorphine was not antagonized by ICI 154,129 (50 nmol), but was blocked by a low dose of naloxone (29 nmol) or beta-fNA (21 nmol). In addition, prior occupancy of mu-sites with beta-fNA (21 nmol) significantly diminished the abilities of either ICI 154, 129 (50 nmol) or naloxone (290 nmol) to antagonize the anticonvulsant action of DADLE. The results of this study demonstrated that the effects of DADLE to increase seizure threshold in the rat were primarily mediated by activation of a delta-opioid receptor system. Furthermore, evidence has been provided for a functional interaction between delta and mu receptors in the opioid regulation of seizure threshold.     

5'-Guanylylimidodiphosphate decreases affinity for agonists and apparent molecular size of a frog brain opioid receptor in digitonin solution

When assayed for specific opiate binding in the presence of 120 mM NaCl, digitonin extracts from frog (Rana ridibunda) brain membranes were found to contain about the same quantity (0.5 pmol/mg of protein) of high (Kdh = 0.4 nM) and of lower (Kdl = 15-20 nM) affinity sites for the opiate agonist [3H]etorphine. The two classes of [3H]etorphine binding sites displayed equally high (Kd = 0.3 nM) affinity for the opiate antagonist [3H]diprenorphine. 5'-Guanylylimidodiphosphate (GppNHp) selectively and potently (IC50 = 0.1 microM) inhibited high affinity binding of the tritiated agonist, and this inhibition resulted from the GppNHp-induced conversion of the high into the lower affinity sites for [3H]etorphine. Following centrifugation of the digitonin extract in sucrose gradients, opioid binding activity was found to be associated with two clearly separated macromolecular components of apparent sedimentation coefficients 11.5 and 9.7 S, respectively. The two components bound [3H]diprenorphine equally well, whereas the fast sedimented component bound [3H]etorphine better than did the slower sedimented one. In addition, labeling of the component of bigger apparent size with [3H]etorphine was considerably reduced in the presence of 50 microM GppNHp. Finally, in soluble extracts which had been (i) preincubated with and (ii) centrifuged in the presence of GppNHp, the fast sedimented component was no longer observed while there was about twice as much of the component of smaller apparent size as in control (no GppNHp) extracts. Together, these results demonstrated the existence of an opioid receptor-G protein complex which, in digitonin solution, was still amenable to regulation (dissociation) by guanine nucleotides.     

Opioid binding properties of the purified kappa receptor from human placenta

A glycoprotein with a molecular weight of 63,000 has been purified, in an active form, from human placental villus tissue membranes. The binding properties of this glycoprotein to opioid alkaloids and peptides indicates that it is the kappa opiate receptor of human placenta. The receptor binds the tritiated ligands etorphine, bremazocine, ethylketocyclazocine and naloxone specifically and reversibly with Kd values of 3.3, 4.4, 5.1 and 7.0nM, respectively. The binding of 3H-Bremazocine to the purified receptor is inhibited by the following compounds with the corresponding Ki values EKC, 1.3 x 10(-8)M; Dynorphin 1-8, 3.03 x 10(-7); U50,488H, 4.48 x 10(-9); U69-593,2.28 x 10(-8), morphine, 4.05 x 10(-6) DADLE, 6.47 x 10(-6) and naloxone, 2.64 x 10(-8). The purified receptor binds 8 nmole of 3H-Etorphine and 1.7 nmole 3H-BZC per mg protein. The theoretical binding capacity of a protein of this molecular weight is 15.8. Although the iodinated purified receptor appears by autoradiography as one band on SDS-PAGE, yet homogeneity of the preparation is not claimed.     

Effect of the opiate antagonist naloxone on the electrical activity of identified neurons in the edible snail

The opiate antagonist naloxone modifies the electric activity of some identified neurons of the Helix lucorum which have not been preliminary exposed to the effect of exogenous opioids. Some neurons are excited while others are inhibited by naloxone, and in both cases the reaction may have both a short and long latent period. The reactions depend on naloxone dose and become less expressed or are blocked when naloxone is administered together with the agonists of opiate receptor (morphine, D-Ala2, D-Leu5-enkephalin, bremazocine and beta-endorphin). Opioids alone do not produce any effect on neurons. The effect of naloxone on neurons is assumed to be a result of the elimination by the opiate antagonist of the tonic effect of endogenous opioids by their replacing on opiate receptors which are constantly stimulated by endogenous ligands.     

Antiarrhythmic action of naloxone: direct, non-opiate effect on the rat heart

The opiate antagonist naloxone reduced the incidence and severity of cardiac arrhythmia induced in rats by intracarotid administration of adrenaline. Naloxone also reversed the adrenaline-induced arrhythmia in isolated heart preparations, suggesting a local antiarrhythmic action of the opiate antagonist. Similar effects were obtained with the (+) stereoisomer of naloxone which is inactive as an opiate antagonist. Thus, the direct action of naloxone at the rat heart is probably not mediated by opiate receptors.     

Evidence for nuclear sites of stereospecific opiate binding in neuroblastoma cells in continuous culture.

A class of stereospecific, saturable receptors for narcotic drugs has been found in a clone of mouse neuroblastoma cells in continuous culture. Cells grown in the presence of 10(-8) M etorphine, a synthetic opiate, had an increased doubling time. The neuroblastoma cell nucleus was found to be the sole site for the high affinity binding of etorphine. It was found that these nuclear receptors (1) became saturated at a concentration of 2 X 10(-9) M etorphine, (2) had a dissociation constant of 5 X 10(-11) M etorphine, (3) were capable of binding etorphine stereospecifically at 37 degrees C but not at 4 degree C, and t4) were protein in nature as evidenced by treatment with proteolytic enzymes. More importantly, the receptor activity appeared to be chromatin-associated.