Nalorphine: actions at an opiate receptor on frog skeletal muscle fibers

Intracellular microelectrode studies were conducted to investigate the actions of the partial agonist-antagonist nalorphine at an opiate receptor on functional frog skeletal muscle fiber membranes. In high bath concentrations (greater than or equal to 10(-4) M), nalorphine alone produces agonist actions similar to the "full" opiate agonists. These actions were (i) to depress both the sodium and potassium (gNa and gK) conductance increases due to electrical stimulation by a nonspecific local anestheticlike mechanism and (ii) to depress gNa by a specific opiate receptor mediated mechanism. In a much lower bath concentration (1 X 10(-8) M) nalorphine acts to antagonize the specific opiate receptor mediated depression of gNa produced by the "full" agonist meperidine. Thus in this preparation nalorphine, "the partial antagonist," has the same actions as naloxone, which is often considered to be a full antagonist. The quantitative differences observed in the effects of these two opiate antagonists are discussed.     

The effects of partial opiate agonists on plasma prolactin and growth hormone levels in the rat.

Opiate agonists, partial agonists, and antagonists differed in their effects on release of prolactin and growth hormone. Agonists (morphine, methadone or meperidine) elevated plasma levels of both hormones. An antagonist (naloxone) lowered levels of prolactin but not growth hormone. All partial agonists studied raised growth hormone levels; among these, levallorphan, nalorphine, and ciramadol lowered prolactin levels while pentazocine and meptazinol did not. Naloxone blocked morphine-induced release of prolactin and growth hormone. The partial agonists suppressed morphine-induced prolactin release, and several suppressed the elevated growth hormone levels as well. Data from the opiate radioreceptor assay (displacement of ³H-naloxone) in the presence and absence of sodium agrees with the above placement of agents into three classes. These results suggest that classification of opioid compounds into agonists, partial agonists and antagonists may be made by their effects on prolactin and growth hormone release.     

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.     

The effect of nalorphine on analgesia induced by peripheral stimulation]

The blockade of effects induced by percutaneous peripheral stimulation were abolished by injection of an opiate antagonist as nalorphine. Our results lead to the hypothesis that central and peripheral stimulations act by the same mechanism in producing blockade of noxious impulses. One may suggest that peripheral stimulations induce release of endogenous morphine-like substances which in turn give descending inhibition.     

NMR studies of flexible opiate conformations at monoclonal antibody binding sites. I. Transferred nuclear Overhauser effects show bound conformations

500 MHz H, homonuclear, intra-molecular, transferred Nuclear Overhauser Effect measurements have been performed on the bound forms of a classical opiate antagonist, nalorphine and an agonist, levorphanol at their respective binding sites in two different specific anti-opiate monoclonal antibody fragments. Based upon previous studies of opiate conformations in solution the results clearly show without extensive interpretation that one of these flexible haptens has the opposite (from solution) isomeric conformation in its bound form. For nalorphine the axial isomer of the N-allyl substituent is the bound form whereas in solution the equatorial isomer dominates at a ratio of 5:1. For levorphanol the bound form is that of equatorial N-methyl in accord with the low energy conformation in solution. In this preliminary report we discuss the initial measurements and results and their implications with respect to the conformations of flexible ligands at macromolecular binding sites including opiate receptors.     

Receptor binding, antagonist, and withdrawal precipitating properties of opiate antagonists

A number of opiate antagonists and the dextro isomers of some of these drugs were studied for antagonism of acute opiate effects on ilea isolated from opiate-naive guinea pigs, precipitation of a withdrawal contraction of ilea isolated from morphine-dependent guinea pigs, precipitation of withdrawal in morphine-dependent rhesus monkeys and stereospecific displacement of 3H-etorphine binding to rat-brain membranes. With the exception of d-naloxone, all of the compounds displaced 3H-etorphine. With the exception of d-naloxone, nalorphine, and quaternary nalorphine, all of the antagonists caused a contraction of ilea isolated from morphine-dependent guinea pigs. Moreover, the IC 50 values of the compounds for displacing 3H-etorphine binding were well correlated with both their Ke values for antagonism in the ileum (r = 0.95) and with their EC 50 values for precipitating a contraction in this preparation (r = 0.92). Generally, the concentration of antagonist necessary to precipitate half maximal contracture was 30-fold greater than the Ke value of the antagonist. Most of the opiate antagonists also precipitated withdrawal when administered to morphine-dependent rhesus monkeys and their in vivo potencies were well correlated with their in vitro potencies in ileum (with Ke: r = 0.95; with EC 50: r = 0.99) and in displacing 3H-etorphine (r = 0.95). The quaternary derivative of naltrexone, however, was an effective opiate antagonist only in vitro, and was ineffective in precipitating withdrawal in morphine-dependent rhesus monkeys. These results suggest that the receptor sites labeled by 3H-etorphine are the same as those involved in antagonism of acute opiate actions and in precipitation of withdrawal.     

Beneficial actions of nalorphine during hemorrhagic shock in cats

Endogenous opiates have been reported to have detrimental effects on the circulatory system during hemorrhagic shock. However, the specific opiate receptor subtype which mediates these actions has not been defined. In the present study, we have utilized the mixed agonist/antagonist, nalorphine (N-allylnormorphine), which exhibits kappa (kappa) and sigma (sigma) receptor agonism as well as mu (mu) receptor antagonism, to investigate the role of the mu receptor in hemorrhagic shock. Nalorphine (2 mg/kg) produced no significant changes in any observed experimental variable in sham-shocked animals. Shocked animals treated with nalorphine (2 mg/kg) maintained significantly higher final mean arterial blood pressures (MABP) than animals which received only vehicle (102 +/- 3.8 vs 61 +/- 6.6 mm Hg, respectively, p less than 0.001). In addition, nalorphine significantly reduced the rise in plasma MDF activity observed in untreated hemorrhaged animals (42 +/- 3.0 vs 59 +/- 4 U/ml, p less than 0.02). Our results support a significant role for the mu receptor in the deleterious actions of endogenous opioids during hemorrhagic shock.     

Reinforcing, but no analgesic, effect of opioid stimulation of the ventral tegmental area

Analgesic and secondary reinforcing effects of morphine, bremazocine and phencyclidine microinjections into ventral tegmental area were studied in rats. The drugs under study failed to affect nociceptive reactions produced by thermal, mechanic and electrical stimuli. Morphine and phencyclidine have shown reinforcing properties in place preference paradigm. Ventral tegmental area seems to be a triggering zone of the reinforcing, and not analgesic effect of opioid agonists, with an important role of mu- and sigma-receptors revealed.     

An investigation of the activity of opiate drug receptors located on frog skeletal muscle fibre membrane.

Extracellular and intracellular microelectrode studies were conducted to test the actions and interactions of opiate agonists, antagonists, and procaine on action potentials in frog sartorius muscles. Extracellular studies showed that morphine, methadone, propoxyphene, and procaine all depressed action potential production. Low concentrations of naloxone or naltrexone antagonized the excitability depression produced by the three opiate agonists but not the depression produced by procaine. Intracellular studies revealed that certain concentrations of the opiate agonists produced a biphasic decline in the stimulus-induced increase in sodium conductance (gNa). Naloxone or naltrexone antagonized only the second phase of this decline. These results show that part of the excitability depression produced by opiate agonists is due to an action on opiate drug receptors.     

Neurogenic component in the effect of papaverine, drotaverine and bencyclane

Papaverine, drotaverine and bencyclane, drugs considered to have direct action on the smooth muscle, inhibited synaptic transmission in the isolated a sympathetic ganglion of the frog. Their effect depended upon the concentration applied. The ganglionic blocking effect of papaverine and drotaverine in the concentration range from 10(-8) to 10(-6) mol/l was partially antagonized by naloxone and nalorphine as well as by increasing the Ca2+ concentration in the incubation medium. This refers to an activation of specific opiate receptors in the mechanism of ganglionic action of these drugs. The ganglionic effect of bencyclane may be due to its local anaesthetic property, since it was prevented by neither naloxone nor nalorphine, and an increase in the Ca2+ concentration in the medium had no influence on it.     

Actions of opiate agonists, naloxone, and paraben preservatives in the rat lung circulation

Previous studies have documented direct vascular effects of opiate substances in the systemic circulation. Because opiate receptors have been identified in the lung, we wondered whether opiate substances might affect vasoreactivity in the lung circulation. We studied the pulmonary vascular effects of three opiate agonists: morphine, leucine-enkephalin, and dynorphin, as well as the opiate receptor antagonist naloxone, in isolated rat lungs perfused with a cell- and plasma-free salt solution. Because of previous reports of the smooth muscle effects of the methyl- and propylparaben preservatives in the naloxone preparation, we also studied the pulmonary vascular effects of these preservatives in the rat lung circulation. We found that morphine, a mu-receptor agonist, leucine-enkephalin, a delta-receptor agonist, and dynorphin, a kappa-receptor agonist, caused no immediate vascular effect when injected into the pulmonary artery. In addition, morphine did not affect the pulmonary vasoconstrictions induced by hypoxia, angiotensin II, or potassium chloride. The commercial preparation of naloxone, Narcan, caused a marked vasodilation during hypoxic pulmonary vasoconstriction. However, this effect was entirely attributable to the preservatives methyl- and propylparaben, as pure naloxone had no effect on either the baseline pulmonary vascular tone or the vasoconstrictive response to hypoxia. We conclude that opiate receptor agonists and antagonists do not affect vasoreactivity in the rat lung circulation and that the methyl- and propylparaben preservatives in Narcan are pulmonary vasodilators.     

Inhibition of ganglionic transmission by narcotic analgesics; an effect antagonized by naloxone, nalorphine and calcium

Methadone, azidomorphine, oxycodone and fentanyl inhibit synaptic transmission in isolated sympathetic ganglia of the frog and rat, just as did morphine and pethidine in our previous investigations. This inhibitory effect can be antagonized not only by naloxone and nalorphine but also by increasing calcium concentration of the perfusion fluid of the ganglia. The inhibitory effect on transmission of narcotic analgesics takes place on specific opiate receptors of the peripheral ganglia.     

Mechanism of opiate of oxidative phosphorylation in mitochondria]

Effect of morphine, codeine, dionine and nalorphine on the oxidative phosphorylation in rat liver mitochondria was studied. Morphine is found to inhibit both ATP-synthetase and ATP-ase activities in mitochondria, but not in submitochondrial particles. Morphine-suppressed oxidative phosphorylation was competitively reversed with high concentrations of ADP, but not of inorganic phosphate. The effect of other opiates (i.e. codeine, dionine, nalorphine) was similar. It is suggested, that opiates inhibit the transport of adenine nucleotides through inner mitochondrial membrane, as it does atractyloside. A significance of the hydrophobic interaction between the inhibitor and adenine nucleotide translocase is outlined, since the degree of the inhibition of oxidative phosphorylation is increased with the increase in the number of non-ionized opiate molecules (at alkaline pH values) and in the length of the carbon chain of narcotic molecule as follows: morphine--codeine--dionine--nalorphine.     

Effects of 5,5''-Dithiobis-(2-Nitrobenzoic Acid) on Opiate Binding to Both the Membrane-Bound Receptor and the Partially Purified Opiate Receptor

Pretreatment of partially purified opiate receptor from rat brains with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) decreased opiate agonist binding more effectively than that of antagonist. This agent, at a concentration that inhibits only 3H-agonist binding, increases the IC50 values of agonists but not those of antagonists. We also observed similar effects of DTNB on opiate binding to the membrane-bound receptor that are in good agreement with the published data. Moreover, there was an excellent correlation between the IC50 values of the two different preparations. However, opiate binding to the partially purified receptor was about a thousandfold more sensitive to DTNB than binding to this membrane-bound receptor. Dithiothreitol, a sulfide bond reducing agent, reversed the effects of DTNB on the opiate binding.     

Evidence for a central but not adrenal, opioid mediation in hypertension induced by brief isolation in the rat

Naloxone was found to provoke a hypotensive effect related to the dose on high blood pressure (BP) induced by short-term isolation in young rats. Another opiate antagonist, nalorphine, also reduced the arterial pressure of socially deprived rats. In contrast, naltrexone methylbromide that selectively blocked peripheral opiate receptors did not alter the elevated BP. To investigate whether adrenomedullary opioids were somehow implicated in the development of isolation-induced hypertension, bilaterally adrenalectomized rats were kept under social deprivation for 7 consecutive days. The data obtained indicated that high systolic BP developed in the same manner as in intact rats run in parallel. In conclusion, central opioids appear to be involved in BP elevation due to the stress generated by brief social deprivation in young rats.     

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.     

Antagonism of N-methyl-D-aspartate (NMDA) evoked increases in cerebellar cGMP and striatal ACh release by phencyclidine (PCP) receptor agonists: evidence for possible allosteric coupling of NMDA and PCP receptors

A comparison was made of the actions of phencyclidine receptor agonists and N-methyl-D-aspartate (NMDA) receptor antagonists in two well-defined neurochemical test systems. These included (i) [3H]acetylcholine release from striatal cholinergic interneurons in vitro, a system known to be positively modulated by corticostriatal excitatory amino acid inputs in vivo; and (ii) cerebellar cGMP levels in vivo, an indicator of cerebellar Purkinje cell activity, which is also modulated by excitatory amino acid inputs. Using these neuronal systems, we report that phencyclidine receptor agonists demonstrated a noncompetitive antagonism of NMDA receptor agonist actions.     

Effect of met-enkephalin and morphine on gastric secretion and blood flow

The effects of met-enkephalin and morphine on gastric acid and pepsin secretion and gastric mucosal and total blood flow were studied in anaesthetized dogs with an in vivo chambered secretion stomach preparation. It was found that both agents infused intraarterially caused an increase in histamine-induced acid and pepsin secretion and mucosal and total blood flow. The above responses were significantly blocked by naloxone and nalorphine. In the resting stomach both opiates did not induce secretory changes but they increased mucosal and total blood flow. Met-enkephalin and morphine were also effective after intravenous administration. Met-enkephalin but not morphine fails to stimulate acid secretion if given into the portal vein. The likely mechanism of action of opiates on gastric secretion is discussed and a hypothesis of existence of opiate receptors in the gastric wall is presented.     

Evidence that opiate receptors mediate suppression of hypertonic saline-induced drinking in the mouse by narcotic antagonists

The effects of naloxone, its $\text{dextro}$-stereisomer, and five other narcotic antagonists were determined on water intake induced by intracellular dehydration in the mouse. The intraperitoneal administration of a 2M sodium chloride solution served as the model for intracellular dehydration. $\text{1}$-Naloxone (0.01-10 mg/kg) reduced drinking in a dose-dependent fashion with an ED₅₀ of 0.55 mg/kg. In contrast, $\text{d}$-naloxone failed to suppress water consumption at doses up to 10 mg/kg. The other narcotic antagonists tested --- naltrexone, diprenorphine, levallorphan, oxilorphan, and nalorphine --- also produced dose-dependent decreases in water consumption. The order of potency of these narcotic antagonists in suppressing water intake was highly correlated with their orders of potency in other procedures involving the opiate receptor. The stereoselectivity and order of potency suggest that the suppressant effects of the narcotic antagonists on drinking induced by hypertonic saline administration in the mouse are mediated through an opiate receptor-dependent mechanism.     

The role of monocytes in the effects of β-endorphin and selective agonists of μ-and δ-opiate receptors on the proliferative activity of peripheral blood lymphocytes

The effects of β-endorphin under the conditions of naloxone hydrochloride blockade of opiate receptors, as well as the effects of the selective agonists of μ-and δ-receptors DAGO and DADLE and the effects of melanocyte-potentiating factor (MPF), on the in vitro proliferative response of lymphocytes were studied. The dose-effect dependence indicated stimulating effects of β-endorphin, DAGO, and DADLE on the proliferative response in the presence of phytohemagglutinin (PHA). The tetrapeptide MPF, which is the C-terminal sequence of β-endorphin, had almost no effect on the proliferative activity of lymphocytes. β-Endorphin, naloxone, and the μ-and δ-receptor selective agonists enhanced the proliferative response of lymphocytes in an unfractionated cell culture, whereas β-endorphin, naloxone, and DAGO suppressed the proliferative activity of lymphocytes in the mononuclear fraction purified of monocytes. In both cases, the naloxone blockade of opiate receptors enhanced rather than eliminated the β-endorphin effect.