Magnetic fields differentially inhibit mu, delta, kappa and sigma opiate-induced analgesia in mice

An exposure for 60 min to a 0.5 Hz rotating magnetic field (1.5-90 G) significantly attenuated the daytime analgesic effects of the mu and kappa opiate agonists, morphine and U50,488H, respectively, and significantly inhibited the analgesic actions of the delta agonist, D-Ala2-D-Leu5-enkephalin, in mice. The magnetic stimuli had no significant effects on the analgesic effects of the prototypic sigma opiate agonist (+/-) SKF-10,047. These results show that exposure to relatively weak magnetic stimuli has significant and differential inhibitory influences on various opioid systems.     

Motility effects of opioid peptides in dog intestine

Six opioid peptides, like morphine, were found to produce dose-dependent contractions of dog isolated intestine when administered as intraarterial boluses. The increases in incidence and amplitude of intestinal contractions were antagonized by naloxone. The rank order of potency of the opioid agonists tested was D-Ala2-met-enkephalinamide greater than D-Ala2-leu-enkephalinamide greater than met-enkephalin greater than beta-endorphin 1-31 greater than morphine greater than morphiceptin greater than dynorphin 1-13. The contractions induced by two opioid agonists displayed differential sensitivity to blockade by tetrodotoxin (TTX). Met-enkephalin was barely affected by concentrations of TTX that markedly reduced responses to morphiceptin. Some portion of the motility effect of metenkephalin may be exerted directly on intestinal smooth muscle.     

Potentiation of opioid analgesia by cocaine: the role of spinal and supraspinal receptors.

These studies examined the effect of cocaine on the analgesia produced by systemically and centrally administered opioid agonists. Cocaine (50 mg/kg, s.c.) increased the analgesic potency of systemic, ICV and IT morphine; and the ICV and IT analgesic effects of the delta selective peptide, [D-Pen2,D-Pen5]enkephalin (DPDPE). Cocaine also increased the analgesic potency of the mu selective ligand [D-Ala2,NMePhe4,Gly-ol5]enkephalin (DAGO) administered ICV. However, cocaine did not alter the ED50 for IT DAGO. GC-MS studies indicated that brain cocaine concentration was approximately 3.0 micrograms/g wet weight 45 min following s.c. administration. These results suggest that cocaine-induced increases in opioid analgesic potency are mediated at brain mu and delta receptors and spinal mu receptors. Furthermore, there might be functional differences between spinal and supraspinal sites at which DAGO produces analgesia.     

Retention of supraspinal delta-like analgesia and loss of morphine tolerance in delta opioid receptor knockout mice

Gene targeting was used to delete exon 2 of mouse DOR-1, which encodes the delta opioid receptor. Essentially all 3H-[D-Pen2,D-Pen5]enkephalin (3H-DPDPE) and 3H-[D-Ala2,D-Glu4]deltorphin (3H-deltorphin-2) binding is absent from mutant mice, demonstrating that DOR-1 encodes both delta1 and delta2 receptor subtypes. Homozygous mutant mice display markedly reduced spinal delta analgesia, but peptide delta agonists retain supraspinal analgesic potency that is only partially antagonized by naltrindole. Retained DPDPE analgesia is also demonstrated upon formalin testing, while the nonpeptide delta agonist BW373U69 exhibits enhanced activity in DOR-1 mutant mice. Together, these findings suggest the existence of a second delta-like analgesic system. Finally, DOR-1 mutant mice do not develop analgesic tolerance to morphine, genetically demonstrating a central role for DOR-1 in this process.     

Opioid receptors in human neuroblastoma SH-SY5Y cells: evidence for distinct morphine (mu) and enkephalin (delta) binding sites

Human neuroblastoma SH-SY5Y cells exhibited a heterogeneous population of mu and delta types of opioid binding sites. These specific binding sites displayed the characteristic saturability, stereospecificity and reversibility, expected of a receptor. Scatchard analysis of [3H]-D-Ala2-D-Leu5-enkephalin (DADLE) in the presence of 10(-5) M D-Pro4-morphiceptin (to block the mu receptors) and the competitive displacement by various highly selective ligands yielded the binding parameters of delta sites which closely resemble those of the delta receptors in brain and mouse neuroblastoma clones. Similarly, the high affinity binding of [3H]-dihydromorphine, together with the higher potency of morphine analogues to displace [3H]-naloxone binding established the presence of mu sites. Guanine nucleotides and NaCl significantly inhibited the association and increased the dissociation of [3H]-DADLE binding. The observed heterogeneity of opioid receptors in cultured SH-SY5Y cells would serve as an excellent model for the biochemical and pharmacological characterization of brain opiate receptors.     

Characterization of the receptor binding profile of (3H)-dermorphin in the rat brain

Amphibian skin synthesizes a variety of biologically active peptides. Of these, dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2) is an extraordinarily potent opioid peptide up to 1000 times more active than morphine in inducing analgesia after intracerebroventricular administration. Dermorphin has little in common with the sequence of all hitherto known mammalian opioid peptides and is unique in having a D-amino acid residue in position 2. Specific binding properties of tritium labeled dermorphin were characterized in the rat brain. Scatchard or Hill analysis of equilibrium measurements performed over a large range of concentrations revealed a single population of dermorphin binding sites with a Kd value of 0.46 nM. Dermorphin and the selective mu-receptor ligand (D-Ala2, MePhe4, Gly5-ol)-enkephalin (DAGO) had similar high potencies in competing with (3H)-dermorphin binding, whereas the inverse holds for the prototypical delta receptor ligand (D-Pen2, D-Pen5)-enkephalin (DPDPE), which exhibited a potency three orders of magnitude lower. Dermorphin was tested for its relative affinity to mu and delta binding sites by determining its potency in displacing (3H)-DAGO and (3H)-DPDPE from rat brain membrane preparations. Based on these comparisons, dermorphin exhibited a selectivity ratio Ki(DPDPE)/Ki(DAGO) = 100, a value almost identical to that of DAGO, this ligand being considered as the protypical mu-receptor probe. The high affinity and selectivity of (3H)-dermorphin together with its very low nonspecific binding make this peptide a useful tool for dissecting the role(s) of the mu-receptor(s).     

Differential effects of opioid receptor agonists on nociception and cAMP level in the spinal cord of monoarthritic rats.

Changes in functional responsiveness of spinal opioid receptors in monoarthritic rats were investigated at the behavioral and the molecular level. After intrathecal administration of morphine, D-Ala2-D-Leu5-enkephalin (DADLE), D-Pen2-D-Pen5-enkephalin (DPDPE) and dynorphin monoarthritic rats showed an enhanced antinociceptive response as measured by a tail-flick latency. No such changes were observed following administration of the selective kappa agonists U50,488H and U69,593. The opioid mu and delta receptor agonists (0.1-1.0 microM) inhibited the basal, as well as the forskolin-stimulated cAMP formation in spinal cord slices obtained from monoarthritic rats, whereas no significant changes were found in control animals. Higher concentrations of the mu and delta opioid receptor agonists were required to attenuate the cAMP level in spinal cord of control animals. The selective kappa agonists U50,488H and U69,593 did not influence the cAMP formation in monoarthritic or control animals. Additionally, we found that the GppNHp-stimulated level of cAMP was higher in the spinal cord slices of monoarthritic rats, which points to an enhanced responsiveness of the adenylate cyclase effector system to the action of this GTP analog. Our data suggest that the enhanced antinociceptive response to intrathecally administered opioids in monoarthritic rats may be connected with the increased sensitivity of adenylate cyclase to the inhibitory effects of mu and delta agonists.     

The actions of naloxazone on the binding and analgesic properties of morphiceptin (NH2Tyr-Pro-Phe-Pro-CONH2), a selective mu-receptor ligand

Active in both binding and biological assays, morphiceptin (NH₂ Tyr-Pro-Phe-Pro-CONH₂), a potent opioid peptide derivative of β-casamorphine, binds specifically and selectively to mu or morphine-type receptors with little affinity for delta sites. Displacement studies of a variety of ³H-labeled opiates and enkephalins show biphasic curves. Naloxazone, which blocks irreversibly and selectively high affinity opiate and enkephalin binding, abolishes morphiceptin's inhibition of binding at low concentrations, suggesting that the high affinity binding of enkephalins and opiates represents a mu or morphine-type receptor. Unlike the reversible antagonist naloxone, naloxazone treatment $\text{in}$$\text{vivo}$ inhibits for over 24 hours the analgesic activity of morphiceptin like it inhibits morphine, β-endorphin and enkephalin analgesia. Together, these studies imply that opiates and enkephalins bind with highest affinity to a mu receptor which mediates their analgesic activity. The ³H-D-ala²-D-leu⁵-enkephalin binding remaining after naloxazone treatment, representing a lower affinity site (K_D 4 nM), is quite insensitive to morphiceptin inhibition and has the characteristics of a delta receptor. However, the ³H-dihydromorphine binding present after naloxazone treatment, which also represents a lower affinity site (K_D 6 nM), is far more sensitive to both morphine and morphiceptin and may represent a second morphine-like, or mu, receptor.     

Synthesis of [D-Ala2,4'-125I-Phe3,Glu4]deltorphin and characterization of its delta opioid receptor binding properties.

Both [D-Ala2,Glu4]Deltorphin and [D-Ala2,4'-I-Phe3,Glu4]Deltorphin are highly selective ligands for delta, relative to mu, opioid receptors. Radiolabeled [D-Ala2, 4'-125I-Phe3,Glu4]Deltorphin ([125I]Deltorphin) was prepared with a specific activity of 2200 Ci/mmol from [D-Ala2, 4'-NH2-Phe3, Glu4]Deltorphin through a diazonium salt intermediate. The inhibition of [125I]Deltorphin binding to rat brain membranes by ligands selective for mu, delta, and kappa opioid receptors is consistent with binding by the radioligand to a single site having the properties of a delta opioid receptor. The results of these studies are in good agreement with those obtained by structurally different delta opioid receptor ligands. The similarity between the delta receptor site labeled by [125I]Deltorphin and those labeled by other delta receptor agonists, in contrast to differences seen by in vivo studies of their analgesic effects, is discussed.     

Mu1: a very high affinity subtype of enkephalin binding sites in rat brain

Displacement studies of [3H]-[D-Ala2-MePhe4-Gly-ol5]-enkephalin ([3H]-DAGO) and [3H]-[D-Ala2-D-Leu5]-enkephalin ([3H]-DADL) by the corresponding unlabeled ligands show that there are at least three classes of sites which bind these enkephalin analogs with high affinity. Using computer modeling, the introduction of the third site significantly improved the goodness of fit in ten consecutive experiments. These sites appear to correspond to the mu, delta and mu 1 sites, with mean dissociation constants of 11, 1.3 and 0.9 nM for DADL and 2.5, 300 and 0.3 nM for DAGO, respectively.     

Biphasic competition between opiates and enkephalins: does it indicate the existence of a common high affinity ("mu-1") binding site?

Displacement from brain membranes of labeled opiates by low concentrations of enkephalins and of labeled enkephalins by low concentrations of opiates has been previously explained by the existence of a common high affinity site termed mu-1. An alternative interpretation of the same results is that the trough seen in the low concentration zone of the displacement curves represents cross binding of mu and delta opioid ligands to delta and mu receptors, respectively. In three sets of experiments with brain membranes, the size of the trough is shown to be dependent on the labeled ligand used: The ratio between the size of troughs seen with [3H]D-Ala, D-Leu enkephalin and with [3H]morphine varies with experimental conditions (storage of membranes at 4 degrees C for 72 h), with ratio of mu:delta receptors (e.g. in thalamus and cortex which are enriched in mu and delta sites, respectively) and with pretreatment of membranes with naloxonazine. These results can not be explained by a common high affinity site, but rather by binding of [3H]D-Ala, D-Leu enkephalin to mu and of [3H]morphine to delta opioid receptors.     

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.     

Opioid control of the ruminant stomach motility: functional importance of mu, kappa and delta receptors

In sheep, the subcutaneous (SC) or intracerebroventricular (ICV) administration of the mu-type opioid agonists, fentanyl and morphine, evokes a blockade of the cyclic contractions of the reticulum. A similar inhibition of forestomach motility was recorded following the administration of the two enkephalin analogs, D-Ala2-Met5-enkephalinamide (DAMA) and D-Ala2-D-Leu5-enkephalin (DADLE) which are mixed mu - delta opioid agonists. In contrast, the reticular contractions were enhanced by the SC or ICV administration of the kappa type agonist, ethylketazocine (EKC) and U - 50 488 H. The proximal duodenum motor activity was transiently increased resulting in the occurrence of a phase III-like activity by these opioid agonists, regardless of the subtypes. The effects of the opioid agonists on reticular motility were prevented by the injection of naloxone but not by the quaternary parent compound methylnaloxone which does not cross the blood-brain barrier. The duodenal motor effects elicited by the opioid agonists were antagonized by both naloxone and methylnaloxone. The results suggest that the inhibition of the ruminant stomach motility is centrally mediated by mu - delta type opioid agonists and are consistent with opposite effects from kappa type opioid agonists. The stimulatory effect of peptide and non-peptide opioid agonists on the duodenum may result in part from direct opioid receptor-mediated actions on smooth muscle.     

Distinct Subtypes of the Opioid Receptor with Allosteric Interactions in Brain Membranes

The binding isotherms of opioid receptors in rat brain membranes with [3H]D-Ala2-D-Leu5-enkephalin ([3H]DADLE), [3H]dihydromorphine ([3H]DHM), and [3H]etorphine were analysed to show the effects of Mg2+, Na+, and guanine nucleotides. Four opioid receptor subtypes of delta, kappa, mu 1, and mu 2 specificities were differentiated, where necessary with the aid of specific displacing ligands. Both a guanine nucleotide [guanosine-5'-(beta, gamma-imido)triphosphate] and the cations (Na+, Mg2+) affect the affinity state of all four subtypes of the receptor. The opioid binding behaviour is found on detailed inspection to be complex, with cases of "half-of-the-sites" reactivity and of cooperativity. By their behaviour under the various ionic conditions noted, it was concluded that these subtypes are distinct, without the need to assume interconvertibility by such agents. The evidence suggests that the formation of heterologous kappa-delta or mu 1-mu 2 receptor complexes is required for stabilization of the high-affinity conformational state of the receptor. Important effects of cations in increasing the binding and regulating the equilibria of receptor association-dissociation were observed when these studies were conducted, not in the Tris-HCl buffer commonly used in opioid binding assays, but in N-tris[hydroxymethyl]-methyl-2-aminoethanesulphonate (K+) buffer (TES-KOH; 10 mM, pH 7.5): it was found that ionic species of Tris can substitute for divalent cations. Dithiothreitol effects on agonist binding in the presence and absence of the cations suggested that those cation effects involve the exchange of -SH/-SS- bonds between receptor subunits. All of the behaviour is interpreted in terms of a model involving association-dissociation equilibria of homologous and/or heterologous receptor subunits of an oligomeric opioid receptor structure.     

Opioid pharmacology in the rat hippocampal slice

The ability of several opioids in potentiating the synaptic activation of CA1 pyramidal cells in the rat hippocampal slice were compared. Morphine and the opioid peptides, (D-ala2, D-leu5)-enkephalin (DADL), morphiceptin, beta-endorphin, and Tyr-D-Ser-Gly-Phe-Leu-Thr (DSThr) caused a concentration-dependent, naloxone-reversible shift to the left in the input-output (IO) curve constructed by plotting the population spike as a function of the field EPSP. These opioids then produced an increase in the size of the population spike while leaving the EPSP unaffected. In contrast, the kappa agonist prototype, ethylketazocine, had no effect on the IO curve when perfused in concentrations up to 10 microM. The rank order of potency for the opioids in the CA1 region of the hippocampus was DADL greater than DSThr greater than beta-endorphin greater than morphiceptin greater than morphine much greater than ethylketazocine. Thus, opioids that are more specific for delta opiate receptors were the most potent and mu receptor agonists, the least potent in this action. Taken together with previous studies suggesting that morphine and DADL may interact with a common opiate receptor in the CA1 region, the results are consistent with the notion that these epileptiform effects may be primarily mediated by delta opiate receptors in this area although the potency of morphiceptin indicates that mu receptors play some role in this effect.     

Upregulation of opioid receptor subtypes correlates with potency changes of morphine and DADLE

Chronic treatment with opioid antagonists increases the potency of opioid agonists and produces an increase in brain opioid binding sites. In the present study, 8 day treatment with naltrexone blocked morphine and DADLE analgesia for the entire treatment period and increased mu 1, mu 2 and delta opioid receptor binding sites in mouse brain. mu 1 and mu 2 binding were increased by 81 and 67%, respectively, while delta binding was increased by 31%. Consistent with these binding changes, the potency of ICV morphine to produce analgesia was increased by over 3-fold, while the potency of ICV DADLE was increased by only 1.7. These findings indicate that relative increases in opioid receptor subtypes agree with pharmacodynamic studies on potency changes of opioid agonists.     

The occurrence of different kappa opioid receptors (K1 and K2) in frog (Rana esculenta) brain membranes

Opioid receptors of the frog (Rana esculenta) brain are characterized mainly by their relatively high ethylketocyclazocine (EKC) binding properties and by their low affinity to mu and delta ligands when D-Ala2-(Me)Phe4-Gly5-ol enkephalin (DAGO) and D-Ala2-Leu5-enkephalin (DALE) is used. In competition experiments it has been established that EKC and N-cyclopropylmethyl-norazidomorphine (CAM), which are non-selective kappa-ligands, have relatively high affinity to frog brain as well as the kappa 2 (which is DALE sensitive subpopulation of the kappa receptor) ligands etorphine and Metenkephalin-Arg6-Phe7 (1.). The kappa 2 subtype in frog brain resembles more to the mu subtype than to the delta subtype of opioid receptors, but it differs from the mu subtype in displaying low affinity toward beta-endorphin and DAGO.     

An Assessment of the Role of Opioid Receptors in the Response to Cannabimimetic Drugs

Cannabimimetic drugs have been shown to inhibit adenylate cyclase activity in N18TG2 neuroblastoma cells. This investigation examines the possible role of opioid receptors in the cannabimimetic response. Opioid receptors of the delta subtype were found on N18TG2 membranes using [3H]D-Ala2-D-Leu5-enkephalin. No mu or kappa receptors were detected using selective ligands for these sites. The delta binding affinity and capacity were unaltered by cannabimimetic drugs. To test if cannabimimetic drugs may modulate opioid effector mechanisms, cyclic AMP metabolism was determined in intact cells and in membranes. N18TG2 adenylate cyclase was inhibited by the cannabimimetic drugs delta 9-tetrahydrocannabinol and desacetyllevonantradol, and by the opioid agents morphine, etorphine, and D-Ala2-Met5-enkephalinamide. The opioid inhibition was reversed by naloxone and naltrexone; however, the cannabimimetic response was unaffected. Both cannabimimetic and opioid drugs decreased cyclic AMP accumulation in intact cells, but opioid antagonists blocked the response only to the latter. Thus, cannabimimetic effects are observed even though opioid receptors are blocked by antagonist drugs. The interaction between desacetyllevonantradol and etorphine was neither synergistic nor additive at maximal concentrations, suggesting that these two drugs operate via the same effector mechanism. Other neuronal cell lines having an opioid response were also examined. The cannabimimetic inhibition of cyclic AMP accumulation in NG108-15 neuroblastoma X glioma cells was not as great as the response in N18TG2. N4TG1 neuroblastoma cells did not respond to cannabimimetic drugs under any conditions tested. Thus, the cannabimimetic inhibition of adenylate cyclase is not universally observed, and the efficacy of the cannabimimetic response does not correlate with the efficacy of the opioid response.     

Further studies on opioids and hibernation: delta opioid receptor ligand selectively induced hibernation in summer-active ground squirrels

To examine the possible involvement of multiple opioid receptors in animal hibernation, we infused opioids selective for mu, kappa, and delta opioid receptors into summer-active ground squirrels (Citellus tridecemlineatus). The effects of those opioid treatments on the hibernation induced by HIT (Hibernation Induction Trigger) were also examined. Mu opioids morphine (1.50 mg/kg/day) and morphiceptin (0.82 mg/kg/day) and kappa opioid peptide dynorphin A (0.82 mg/kg/day) did not induce hibernation. On the contrary, morphine, morphiceptin and dynorphin A antagonized HIT-induced hibernation in summer-active ground squirrels. Infusion of delta opioid DADLE (D-Ala2-D-Leu5 enkephalin; 1.50 mg/kg/day), however, induced summer hibernation in a manner comparable to that induced by HIT. It is concluded therefore that delta opioid receptor and its ligand may be intimately involved in animal hibernation. In view of the fact that HIT was obtained from winter hibernating animals and might therefore be responsible for natural hibernation, our results also suggest that naturally occurring mu and kappa opioids may play an important role in the arousal state of hibernation.