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The efficacy of different opioid agonists to induce acute desensitization of the human delta-opioid receptor-mediated inhibition of cAMP accumulation was investigated in the neuroblastoma cell line SK-N-BE, which endogenously expresses these receptors. While etorphine, a non-selective alkaloid agonist, caused 50% desensitization after a 30-min incubation, the same treatment in the presence of the selective peptide agonists, DPDPE ([D-Pen2,D-Pen5]enkephalin) and deltorphin I (Tyr-D-Ala-Phe-Asp-Val-Val-Gly), almost totally desensitized the delta-opioid receptor-mediated inhibition of adenylyl cyclase. When SK-N-BE cells were prechallenged either with alkaloid or peptide agonist, we observed a cross-desensitization that was less marked when cells were pretreated with peptide agonists and then challenged with etorphine. Taken together, these results demonstrate that human delta-opioid receptors are differentially desensitized by alkaloid and peptide agonists.

The adrenal medulla produces opioids which exert paracrine effects on adrenal cortical and chromaffin cells and on adrenal splanchnic nerves, via specific binding sites. The opioid binding sites in the adrenals are detectable mainly in the medullary part of it and differ in type between species. Thus, the bovine adrenal medulla contains mostly kappa-opioid binding sites and fewer delta- and mu-opioid binding sites while primate adrenals contain mainly delta sites and few kappa-opioid binding sites. Most chromaffin cell tumors, the pheochromocytomas, produce opioids which suppress catecholamine production by the tumor. The aim of the present work was to identify the types of opioid binding sites in human pheochromocytomas. For this purpose, we characterized the opioid binding sites on crude membrane fractions prepared from 14 surgically excised pheohromocytomas and on whole KAT45 cells, a recently characterized human pheochromocytoma cell line. Our data showed that human pheohromocytomas are heterogeneous, as expected, with regard to the production of catecholamines and the distribution and profile of their opioid binding sites. Indeed, only one out of the 14 pheochromocytomas expressed exclusively delta and mu opioid sites, while in the remaining 13 tumors kappa-type binding sites were dominant. The KAT45 cell line possessed a significant number of kappa1 binding sites, fewer kappa2-opioid binding sites and kappa3-opioid binding sites, and minimal binding capacity for delta- and mu-opioid receptor agonists sites. More specifically, the kappa1 sites/cell were approximately 18,000, the kappa2 4500/cell and the kappa3 sites 2000/cell. Our findings for the surgical specimens and the cell line combined with previously published pharmacological data obtained from KAT45 cells suggest that kappa sites appear to be the most prevalent opioid binding sites in pheochromocytomas. Finally, in normal bovine adrenals the profile of opioid binding sites differs in adrenaline and noradrenaline producing chromaffin cells. To test the hypothesis that the type of catecholamine produced by a pheochromocytoma depends on its cell of origin, we compared our binding data with the catecholamine content of each pheochromocytoma examined. We found no correlation between the type of the predominant catecholamine produced and the opioid binding profile of each tumor suggesting that this hypothesis may not be valid.

delta-Opioid receptors belong to the superfamily of G protein-coupled receptors, characterized by seven putative transmembrane domains, and have been shown to interact with a host of effector systems. It has been suggested that the charge on the conserved aspartic acid residue at position 128 in transmembrane domain 3 of the delta-opioid receptor contributes to both the conformation of the receptor binding pocket and the molecular rearrangements which accompany the establishment of high-affinity states of the receptor. In light of this, we used site-directed mutagenesis to determine whether this residue participates in the transmission of signals to adenylyl cyclase, the effector with which opioid receptors have been classically associated. Substitution of this aspartic acid (D128) for the neutral amino acid alanine, or the protonated amino acids lysine and histidine, constitutively couples the receptor to adenylyl cyclase, as evidenced by a curtailed response to forskolin stimulation in transfected cells. In addition, this constitutive activity can be blocked by pretreatment of the transfected cells with pertussis toxin. Interestingly, naloxone blocks this effect in cells expressing the D128A mutant, but acts as an agonist at the D128K mutant. Our findings support the hypothesis that the interaction between agonist and receptor promotes conformational changes that may be mimicked, at least in part, by mutation of the aspartate residue at position 128. Furthermore, these changes appear to be involved not only in receptor activation, but also in the functional discrimination between agonists and antagonists.

D-Ala2-Met5-enkephalinamide (DALA) (1.0 microg/microl) was administered intraventricularly to mice responding for electrical stimulation from the dorsal or ventral aspects of the VTA immediately prior to footshock (Experiment 1). Predictably, footshock reduced self-stimulation from the dorsal but not the ventral VTA immediately, 24, and 168 h following the stressor. Intraventricular DALA administration effected a partial attenuation of stressor-induced self-stimulation reductions from the dorsal VTA immediately and 24 h poststressor. Deficits appeared among DALA-Shocked mice responding for brain stimulation from the ventral VTA during comparable test intervals. The long-term depressant influence of footshock on self-stimulation from the dorsal VTA was abolished among DALA-treated mice and DALA-associated reductions in self-stimulation from the ventral A10 region among stressed mice were not evident 1 week later. Administration of D-Ala2, N-Me-Phe4, Gly-Ol5-enkephalin (DAGO) (0.01 microg/microl) or D-Pen2, D-Pen5-enkephalin (DPDPE) (1.0 microg/microl) intraventricularly prior to footshock effected an immediate and a delayed antagonism, respectively, of the stressor on self-stimulation from the dorsal VTA, which persisted for 1 week. Prophylactic administration of 0.001 microg/microl DAGO or 0.01 microg/microl DPDPE prior to the stressor failed to influence self-stimulation from the ventral VTA (Experiment 2). Administration of 0.01 microg/microl DAGO or 1.0 microg/microl DPDPE among mice responding for brain stimulation from the dorsal VTA following footshock produced a weak therapeutic effect immediately poststressor, but effected protracted amelioration of footshock-induced reductions of self-stimulation from the dorsal VTA (Experiment 3). Taken together, mu, delta, and mu-delta activation influenced self-stimulation differentially from the dorsal and ventral VTA according to the temporal order of opioid peptide challenge relative to stressor imposition. These data are discussed with respect to stressors, motivational alterations, and the putative modulating influence of endogenous enkephalin activity in subareas of the VTA.

Activation of 5-hydroxytryptamine-2A receptors increases the frequency of excitatory postsynaptic currents through a focal action at apical, but not basilar, dendrites of neocortical layer V pyramidal cells. Since mu-, delta- and kappa-opiate receptors are known to inhibit depolarization-induced glutamate release in cerebrocortical slices, we examined the opiate receptor subtype(s) that suppress(es) 5-hydroxytryptamine-induced excitatory postsynaptic currents in the medial prefrontal cortex and whether this suppression was occurring through a presynaptic or a postsynaptic mechanism. Only opioid agonists that act upon mu-receptors (i.e. [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin, the endogenous mu-selective agonist endomorphin-1 and the non-selective opioid agonist [Met]enkephalin) suppressed 5-hydroxytryptamine-induced excitatory postsynaptic currents. The delta-agonist [D-phen(2,5)]enkephalin and the kappa-agonist U50,488 were ineffective. Only the selective mu-antagonist CTOP blocked the suppressant effect of enkephalin, while the selective delta-antagonist naltrindole and the selective kappa-antagonist norbinaltorphimine were ineffective. Since the 5-hydroxytryptamine-induced excitatory postsynaptic currents are mediated by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-type excitatory amino acid receptors, the failure of mu-agonists to either block postsynaptic AMPA responses or induce outward currents in layer V pyramidal cells suggest that mu-agonists are acting at a presynaptic site to block 5-hydroxytryptamine-induced excitatory postsynaptic currents. Strikingly, a regional selectivity in the suppressant effect of mu-receptor activation on 5-hydroxytryptamine-induced excitatory postsynaptic currents exists, as 300 nM [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin suppressed 5-hydroxytryptamine-induced excitatory postsynaptic currents in the medial prefrontal cortex by nearly 100%, while in the frontoparietal cortex 1 microM [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin suppressed 5-hydroxytryptamine-induced excitatory postsynaptic currents by only 58%. This is the first demonstration of a previously unsuspected physiological interaction between 5-hydroxytryptamine-2A and mu-opiate receptors and may be relevant to the relationship between these receptors and both mood and psychotic disorders.

Orphanin FQ/nociceptin (OFQ/N) is a recently identified neuropeptide with high affinity for the orphan opioid receptor. OFQ/N blocked morphine analgesia in mice in a dose-dependent manner, as well as the analgesic actions of [D-Pen2, D-Pen5]enkephalin (DPDPE), morphine-6 beta-glucuronide, trans-3,4-dichloro-N-[2-(1-pyrrolindinyl)-cyclohexyl]-benzeneac eta mide, methane sulfonate hydrate (U50,488H) and naloxone benzoylhydrazone. These actions are anti-analgesic, because OFQ/N also blocked clonidine analgesia and OFQ/N was inactive against the inhibition of gastrointestinal transit by morphine. Although OFQ/N was quite potent in these paradigms, two truncated forms, OFQ/N(1-11) and OFQ/N(1-7), were inactive. An antisense oligodeoxynucleotide targeting the first coding exon of KOR-3, the mouse homolog of the orphan opioid receptor, effectively prevented the anti-opioid actions of OFQ/N, confirming the importance of the orphan opioid receptor in this action.

CATH.a cells are a catecholaminergic cell line of neuronal origin. The opioid receptor complement expressed by CATH.a cells was defined pharmacologically and by reverse transcription-polymerase chain reaction (RT-PCR). CATH.a cells were found to express mRNA encoding all three of the major subtypes of opioid receptors. The relative abundance of CATH.a cell opioid receptor transcripts was delta > kappa> mu. Pharmacological and functional data were in agreement with the results of RT-PCR inasmuch as delta-opioid receptor was identified as the most abundant opioid receptor subtype expressed by CATH.a cells. In addition, at least one of the opioid signalling pathways, inhibition of adenylyl cyclase activity, was found to be operant in this cell line. CATH.a cells should be of general utility for the study of opioid receptor signalling mechanisms in the context of catecholaminergic neurons.

The effects of chronic treatment with naltrexone, an opioid receptor antagonist, on delta1- and delta2-opioid receptor agonist-induced antinociception and ligand binding were investigated in mice. Antinociception by intracerebroventricular (i.c.v.) [D-Pen2,5]enkephalin (DPDPE) and [D-Ala2]deltorphin II, agonists selective for delta1- and delta2-opioid receptors, respectively, was blocked following subcutaneous (s.c.) implantation of a naltrexone pellet (7.5 mg) for 7 days. Removal of the naltrexone pellet was followed 24 h later by a decrease of 7.5-fold in the ED50 value of [D-Ala2]deltorphin II, but not that of DPDPE. In a whole brain homogenate the binding of [3H][D-Ala2]deltorphin II was increased twice as much as that of [3H]DPDPE. Chronic naltrexone treatment also produced an 8.6-fold decrease in the ED50 value of i.c.v. administered morphine. The increase in morphine potency was reversed to a control (placebo-treated mice) value by the selective delta2-opioid receptor antagonist, naltriben (25 pmol, i.c.v.). Thus, chronic naltrexone selectively increases delta2-opioid receptor-mediated antinociception, supporting the existence of delta opioid receptor subtypes with distinct adaptive characteristics. The data also indicate that delta2-opioid receptors are critically involved in the expression of morphine supersensitivity.

We investigated the effects of SNC 80 ((+)-4-[alphaR)-alpha-((2S,5R)-4-ally1-2,5-dimethyl-1-pipera zinyl)-3-methoxybenzyl]-N,N-diethylbenzamide), a new highly selective, non-peptidic and systemically active delta-opioid receptor agonist, on gastrointestinal and colonic propulsion in mice. Intraperitoneally (i.p.) SNC 80 (1, 10 and 30 mg/kg) significantly decreased gastrointestinal propulsion measured as transit of an orally administered charcoal meal. Pretreatment with the delta-opioid receptor antagonist, naltrindole (1 mg/kg) subcutaneously (s.c.), with the non-selective opioid antagonist, naloxone (5 mg/kg, s.c.) or the mu1-opioid receptor antagonist, naloxonazine (10 mg/kg, i.p.), significantly decreased the antitransit effect of SNC 80 but pretreatment with the non-selective opioid antagonist, naloxone methiodide (5 mg/kg, s.c.), a quaternary salt of naloxone that does not cross the blood-brain barrier, did not. SNC 80 (1, 5 and 10 mg/kg, i.p.), produced dose-related inhibition of colonic propulsion measured as the increase in mean expulsion time of a 3 mm glass bead placed in the distal colon. Naloxone (5 mg/kg, s.c.) and naltrindole (1 mg/kg, s.c.), completely antagonized the colonic antipropulsive effect of SNC 80. In contrast, naloxone methiodide (5 mg/kg, s.c.), left the inhibitory effect of i.p. SNC 80 on colonic function unchanged. These results suggest that peripherally injected SNC 80 inhibits gastrointestinal transit and colonic propulsion. It does so mainly through a central mechanism. Although the gastrointestinal antitransit effect of SNC 80 is naltrindole- and naloxonazine-sensitive, we cannot exclude an opioid-independent mechanism. The colonic antipropulsive effect of SNC 80 confirms the inhibitory role of the central delta-opioid receptor system on colonic motility.

The potential effect of inhibition of phospholipase C on the response of Gi-coupled receptors was investigated in neuroblastoma x glioma hybrid (NG108-15) cells. The phospholipase C specific inhibitor 1-[6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H -pyrrole-2,5-dione (U73122), which did not affect basal and forskolin-stimulated adenylyl cyclase activities, time- and dose-dependently blocked delta-opioid receptor-mediated inhibition of adenylyl cyclase activity, the EC50 (0.5 microM) of which was consistent with that for inhibition of bradykinin-dependent phospholipase C activation (EC50 = 1 microM). U73122 treatment also blocked functional responses of m4 muscarinic receptor and alpha2-adrenoceptor in NG108-15 cells and three opioid receptors (mu, delta and opioid receptor-like receptor (ORL1)) in human neuroblastoma SK-N-SH cells. 1-[6-((17Beta-3-Methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-2, 5-pyrrolidinedione (U73343), an inactive analog of U73122, did not show any effect, which suggests that the blockade by U73122 of Gi-coupled receptor-mediated signaling is probably mediated through inhibition of phospholipase C, although a possible direct modification of G proteins can not be excluded. Furthermore, treatment with U73122 but not U73343 blocked the GTP-induced inhibition of adenylyl cyclase, indicating blockade at the level of Gi proteins.

The effects of intracerebroventricular administration of delta1- and delta2-selective opioid receptor agonists on spontaneous alternation performance, elevated plus-maze behavior and passive avoidance learning including step-down and step-through types were examined in mice. Although the delta1-selective opioid receptor agonist, [D-Pen2,L-Pen5]enkephalin (DPLPE) (1-10 microg) or the delta2-selective opioid receptor agonist, [D-Ala2]deltorphin II (deltorphin) (1-10 microg) did not markedly affect spontaneous alternation performance or elevated plus-maze behavior, DPLPE (1, 3 and/or 10 microg) and deltorphin (3 and 10 microg) inhibited passive avoidance learning including step-down and step-through types. The delta1-selective opioid receptor antagonist, 7-benzylidenenaltrexone (3.5 ng), and the delta2-selective opioid receptor antagonist, naltriben (19 ng), significantly antagonized the inhibitory effects of DPLPE (3 microg) and deltorphin (3 microg) on passive avoidance learning, respectively. In contrast, DPLPE (3 microg) or deltorphin (3 microg) did not markedly influence behavioral responses induced by electroshocks during training of passive avoidance learning. Moreover, DPLPE (0.3-3 microg) or deltorphin (0.3-3 microg) failed to significantly affect the radiant heat-induced nociceptive responses. These results suggest that stimulation of delta1- and delta2-opioid receptors produces amnesia, depending on the learning tasks used.

Male Swiss-Webster mice were injected intracerebroventricularly (i.c.v.) with [D-Pen2,D-Pen5]enkephalin (20 microg/mouse) twice a day for 2 days. This procedure resulted in down-regulation of binding sites for [3H][D-Pen2,D-Pen5]enkephalin as evidenced by a 52% decrease in the Bmax value. Twice daily injections of (+)-5-methyl-10,11-dihydro-5H-dibenzo-[a,d]-cyclohepten-5,10-imine (MK-801) (0.1 mg/kg, i.p.) or [(-)3-SR,4a-RS,8a-SR-6-(phosphonomethyl)-1,2,3,4,4a,5,6,7,8,8a-decahy droisoquinoline-3-carboxylic acid] (LY 235959) (2 mg/kg, i.p.), the noncompetitive and competitive antagonist of the N-methyl-D-aspartate (NMDA) receptor, respectively, for 2 days did not alter the Bmax or Kd value of [3H][D-Pen2,D-Pen5]enkephalin binding to the mouse brain. Concurrent treatment of MK-801, but not of LY 235959 with [D-Pen2,D-Pen5]enkephalin, reversed the decreases in Bmax value of [3H][D-Pen2,D-Pen5]enkephalin. Twice daily injections of [D-Ala2,Glu4] deltorphin II (20 microg/mouse) for 2 days caused an increase in the Kd value, but not the Bmax value of [3H][D-Pen2,D-Pen5]enkephalin to bind to brain membranes. Concurrent treatment of [D-Ala2,Glu4]deltorphin II with LY 235959 reversed the increase in Kd value of [3H][D-Pen2,D-Pen5]enkephalin binding induced by multiple injections of [D-Ala2,Glu4]deltorphin II, but MK-801 had no effect. The results suggest that multiple injections of delta1- and delta2-opioid receptor agonists down-regulate delta1-opioid receptors of the brain by modifying Bmax and Kd values of [3H][D-Pen2,D-Pen5]enkephalin binding, respectively. MK-801 and LY 235959 reverse delta1- and delta2-opioid receptor agonists-induced down-regulation of brain delta1-opioid receptor, respectively, apparently by different mechanisms. It is concluded that short term treatment of mice with delta1-opioid receptor agonist down-regulates brain delta1-opioid receptors by decreasing Bmax of the ligand which is partially reversed by concurrent treatment with MK-801 but not by LY 235959. On the other hand, short term treatment of mice with delta2-opioid receptor agonist down-regulates brain delta1-opioid receptors by increasing Kd of the ligand which is partially reversed by concurrent treatment with LY 235959 but not by MK-801.

Previous results using an amphibian model showed that systemic and spinal administration of opioids selective for mu, delta and kappa-opioid receptors produce analgesia. It is not known whether non-mammalian vertebrates also contain supraspinal sites mediating opioid analgesia. Thus, opioid agonists selective for mu (morphine; fentanyl), delta (DADLE, [D-Ala2, D-Leu5]-enkephalin; DPDPE, [D-Pen2, D-Pen5]-enkephalin) and kappa (U50488, trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl] benzeneacetamide methanesulfonate; CI977, (5R)-(544alpha,744alpha,845beta)-N-methyl-N-[7-(1-p yrr olidinyl)-1-oxaspiro[4,5]dec-8yl]-4-benzofuranaceta mide++ + monohydrochloride) opioid receptors were tested for analgesia following i.c.v. administration in the Northern grass frog, Rana pipiens. Morphine, administered at 0.3, 1, 3 and 10 nmol/frog, produced a dose-dependent and long-lasting analgesic effect. Concurrent naltrexone (10 nmol) significantly blocked analgesia produced by i.c.v. morphine (10 nmol). ED50 values for the six opioids ranged from 2.0 for morphine to 63.9 nmol for U50488. The rank order of analgesic potency was morphine > DADLE > DPDPE > CI977 > fentanyl > U50488. These results show that supraspinal sites mediate opioid analgesia in amphibians and suggest that mechanisms of supraspinal opioid analgesia may be common to all vertebrates.

The present study was conducted to determine the relative efficacies of the selective delta-opioid receptor agonists SNC80 ((+)-4-[(alphaR)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl )-3-methoxybenzyl]-N,N-diethylbenzamide), pCl-DPDPE (cyclic[D-Pen2,4'-ClPhe4,D-Pen5]enkephalin) and (-)-TAN67 ((-)-2-methyl-4a alpha-(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12a alpha-octahydro-quinolino-[2,3,3-g]isoquinoline). Experiments compared the abilities of the three drugs to competitively inhibit [3H]naltrindole binding and also stimulate [35S]GTPgammaS binding in membranes prepared from stably transfected Chinese hamster ovary (CHO) cells that express the cloned human delta-opioid receptor. Efficacy was determined according to the formula: efficacy = (E(max-A)/Emax)(A'/A + 1) X 0.5. Results show that SNC80 and pCl-DPDPE had efficacy values that were about 6-7 times greater than that of (-)-TAN67.

Interactions between delta-opioid receptors and morphine-preferring mu-opioid receptor subtypes have been suggested. Availability of transgenic mu-opioid receptor knockout mice allows assessment of mu-opioid receptor roles in the analgesia produced by the classical delta-opioid receptor agonist [D-Pen2,D-Pen5]enkephalin (DPDPE) in hot-plate and tail-flick tests. DPDPE analgesia was dramatically reduced in mu-opioid receptor knockout mice in a gene-dose-dependent fashion. The analgesia induced by this classic delta-opioid receptor agonist depends on intact mu-opioid receptors, suggesting that selective delta-opioid receptor drugs may require mu-opioid receptor occupancies for full efficacy.

The effects of selective mu-, delta- and kappa-opioid receptor agonists on the discriminative stimulus properties of cocaine were examined in rats trained to discriminate between cocaine (10 mg/kg) and saline. Cocaine produced a dose-related increase in cocaine-appropriate responses in all of the rats. In generalization tests, neither morphine (mu-opioid receptor agonist) nor N-methyl-N-7-(1-pyrrolidinyl)-1-oxaspiro[4,5]dec-8-11-4-benzofu ranacetamide (U50,488H: kappa-opioid receptor agonist) generalized to the discriminative stimulus properties of cocaine. On the other hand, the newly synthesized non-peptide selective delta-opioid receptor agonist 2-methyl-4a alpha-(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12a alpha-octahydro-quinolino(2,3,3,-g)isoquinoline (TAN-67) partially generalized (56.7% cocaine-appropriate responses) to the discriminative stimulus properties of cocaine. Intracerebroventricular (i.c.v.) administration of [D-Ala2]deltorphin II (peptide delta 2-opioid receptor agonist) completely generalized, while neither [D-Ala2,MePhe4,Gly-ol5]enkephalin (DAMGO: mu-opioid receptor agonist) nor [D-Pen2,D-Pen5]enkephalin (DPDPE; delta 1-opioid receptor agonist) generalized to the discriminative stimulus properties of cocaine. These results suggest that the discriminative stimulus properties of cocaine may be partially mediated by delta-opioid (especially delta 2-opioid) receptors. In combination tests, pretreatment with morphine (3.0 mg/kg) and TAN-67 (3.0 and 10 mg/kg) significantly potentiated the discriminative stimulus properties cocaine. In contrast, pretreatment with U50,488H (2.0 and 4.0 mg/kg) scarcely shifted the discriminative stimulus properties of cocaine. Furthermore, the potentiating effect of 3.0 mg/kg morphine on the discriminative stimulus properties of cocaine was attenuated by 2.0 mg/kg U50,488H. In contrast, the potentiating effect of 10 mg/kg TAN-67 on the discriminative stimulus properties of cocaine was not reversed by either 2.0 or 4.0 mg/kg U50,488H. These results suggest that mu-, delta- and kappa-opioid receptor agonists modulate the discriminative stimulus properties of cocaine through different mechanisms, perhaps through different effects on the dopaminergic system.

The affinity, selectivity and antinociceptive properties of 5 beta-methyl-14 beta-(p-chlorocinnamoylamino)-7,8-dihydromorphinone (MET-Cl-CAMO) and N-cyclopropyl-methyl-5 beta-methyl-14 beta-(p-chlorocinnamoylamino)-7, 8-dihydronormorphinone (N-CPM-MET-Cl-CAMO) for the multiple opioid receptors were characterized. In competition binding assays using bovine striatal membranes, both compounds inhibited the binding of 0.25 nM [3H][D-Ala2, (Me)-Phe4,Gly(ol)5]enkephalin (DAMGO) with IC 50 values of less than 2 nM. Preincubation of membranes with MET-CI-CAMO and N-CPM-MET-Cl-CAMO produced a concentration-dependent, wash-resistant inhibition of mu-opioid receptor binding. Saturation binding experiments with N-CPM-MET-Cl-CAMO showed a reduction in the number of mu-opioid binding sites without a change in affinity. In the mouse 55 degrees C warm-water tail-flick assay, neither MET-Cl-CAMO nor N-CPM-MET-Cl-CAMO at doses up to 100 nmol produced antinociception after intracerebroventricular administration, but morphine-induced antinociception was antagonized in a time- and dose-dependent manner by both compounds. The antagonism produced by 1 nmol of either MET-Cl-CAMO or N-CPM-MET-Cl-CAMO reached a maximal effect after 24 h, and lasted up to 48 h. Analgesia mediated by delta- or kappa-opioids was not altered by either compound. In summary, the data suggest that MET-Cl-CAMO and N-CPM-MET-Cl-CAMO are long-term, mu-opioid receptor antagonists, devoid of agonist properties in the mouse tail-flick assay, and that N-CPM-MET-Cl-CAMO may produce its antagonistic effects by binding irreversibly to the mu-opioid receptor.

The delta-opioid receptors in mouse neuroblastoma x rat glioma NG108-15 cells were characterized by receptor binding and cAMP assays. Saturation binding assays using [3H][D-Pen5]enkephalin (DPDPE) or [3H][D-Ser2, Leu5, Thr6]enkephalin (DSLET) gave similar binding capacities (Bmax). Competition binding assays showed that DPDPE and DSLET have similar affinity for the [3H]DPDPE or 3[H]DSLET binding sites. The rank order of potency of competition with [3H]DPDPE and [3H]DSLET was similar: naltriben approximately DSLET > or = DPDPE > 7-benzylidenenaltrexone (BNTX). Both DPDPE and DSLET were found to decrease cAMP formation. The action of DSLET was antagonized by naltriben but not BNTX, while the action of DPDPE was reversed by both antagonists. Therefore, the delta-opioid receptor in NG108-15 cells has similar affinity for the agonists DPDPE and DSLET, and a higher affinity for the antagonist naltriben than BNTX.

The goal of this study was to determine the relative contribution of entropy and enthalpy to the free energies of binding to recombinant mouse delta-opioid receptors for the peptide agonist, DPDPE ([D-Pen2,D-Pen5]enkephalin), the peptide antagonist, TIPP(psi) (Tyr-Tic(psi)[CH2NH]Phe-Phe-OH), the nonpeptide agonist, SNC80 ((+)-4-[(alphaR)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl )-3-methoxybenzyl]-N,N-diethylbenzamide), and the nonpeptide antagonist, naltrindole. Competitive binding studies were carried out using [3H]naltrindole at 0 degrees C, 12 degrees C, 25 degrees C and 37 degrees C, the affinities calculated and van't Hoff plots constructed for each ligand. The temperature dependence of binding and van't Hoff plots indicated that the entropy contribution is the major component of the free energy, for all four ligands, independent of its activity or chemical nature.

The mu-, delta- and kappa-opioid receptors have been expressed in Sf9 and 'High Five' insect cells using the baculovirus expression system. In both cell lines highest receptor levels (pmol/mg membrane protein) were observed 48 h after infection. Concomitant exposure to the narcotic antagonist naloxone (1 microM) enhanced the production of each receptor type. However, "High Five' cells differed from Sf9 cells in a 2-3-fold higher receptor density in the cell membrane and were therefore employed for receptor characterization. In membranes of 'High Five' cells opioid receptor levels ranged from 1.0 +/- 0.2 pmol/mg protein for the kappa-opioid receptor, 1.7 +/- 0.2 pmol/mg for the delta-opioid receptor to 2.1 +/- 0.5 pmol/mg for the mu-opioid receptor. The mu-, delta- and kappa-opioid receptor agonists [D-Ala2,N-methyl-Phe4-Gly-ol5]enkephalin ([3H]DAMGO), [D-Pen2,D-Pen5]enkephalin ([3H]DPDPE) and (5 alpha, 7 alpha, 8 beta)-(+)-N-methyl-N-(7-(1-pyrrolidinyl-1-oxaspiro(4,5)dec-8-yl) benzeneacetamide ([3H]U69,563) bound to the opioid receptors with Kd values of 3.4 +/- 0.3 nM, 4.5 +/- 0.1 nM and 1.2 +/- 0.3 nM, respectively, resembling those reported for opioid receptors expressed in mammalian cells. Testing the functionality of the receptors in 'High Five' cells, we found that high affinity agonist binding was strongly reduced in the presence of GTP gamma S/sodium, indicating their coupling to G proteins. Furthermore, activation of the three receptor types inhibited forskolin-stimulated cAMP formation. The results presented here suggest that the 'High Five' cell/baculovirus system provides a convenient method for high level expression of functionally intact opioid receptors as judged by receptor binding studies, their G-protein coupling and inhibition of adenylyl cyclase.

A rate-free method of determining brain-stimulation reward thresholds was used to identify the rewarding effects of the delta-opioid receptor and mu-opioid receptor agonist peptides, [D-Pen2, D-Pen5]enkephalin (DPDPE) and [D-Ala2-MePhe4-Gly(o1)5]enkephalin (DAMGO). The nucleus accumbens-delivered opioid receptor agonists produced marked lowering of the threshold for ventral tegmental area brain-stimulation reward. No change in baseline thresholds was seen after peripheral administration of the nonpeptide delta-opioid receptor antagonist, naltrindole. However, an unexpected finding was that naltrindole blocked the threshold-lowering effects of both DPDPE and DAMGO. These data demonstrate nucleus accumbens activation of delta- and mu-opioid receptors and ventral tegmental area brain-stimulation reward share common brain substrates. In addition, the interference of both delta- and mu-opioid receptor mediated reward by naltrindole may have implications for therapeutic use.

Single units in the medial subnucleus of the nucleus tractus solitarii, responding to electrical stimulation of subdiaphragmatic vagal fibers, were recorded extracellularly in an in vitro neonatal rat brainstem-gastric preparation. Selective opioid receptor agonists were applied only to the gastric compartment of the bath chamber and therefore, the brainstem functions of the preparation were not affected. The peripheral gastric effects of the mu-opioid receptor agonist, [D-Ala2, N-MePhe4, Gly5-ol]enkephalin (DAMGO) and kappa-opioid receptor agonist, ¿trans-3,4-dichloro-N-methyl-N-[2-(l-pyrrolidinyl)cyclohexyl]cyclohexyl] benzeneacetamide methanesulfonate hydrate¿ (U-50, 488H), were evaluated on 69 units that received the subdiaphragmatic vagal input. For approximately 75% of the units observed, DAMGO (1.0 microM; IC70; 80 nM) and U-50, 488H (1.0 microM; IC70:200 nM) induced a concentration-dependent inhibition of 62.7 +/- 8.9% (mean +/- S.D.) and 50.6 +/- 6.2% of the control level of the brainstem neuronal activity, respectively. The mu-opioid selective receptor antagonist, naltrexone and non-selective opioid receptor antagonist, naloxone, respectively, blocked the inhibitory effects by DAMGO and U-50, 488 H. The delta-opioid receptor agonist, [D-Pen2, D-Pen5]enkephalin (DPDPE) (10 microM; IC70:400 nM) produced a lesser extent of inhibition of 21.9 +/- 8.0% in the only 10 out of 51 (20%) neurons tested, and this effect was blocked by naloxone. The area of the stomach where gastric opioid receptors contributed most to brainstem unitary activity was also examined. This was achieved by comparing the opioid effects on a whole-stomach preparation to its effects on a partial-stomach preparation. Our data indicated that the distal stomach containing the pylorus played a key role in the gastric effects of mu- and kappa-opioid receptors on brainstem neuronal activity. These results suggest that the mu- and kappa-opioid receptors of the distal stomach are important in modulation of brainstem neuronal activity and may play a role in regulating the digestive process.

Effects of [Met5]enkephalin and agonists selective for mu-, delta- and kappa-opioid receptors were tested in vitro on neurons of the hypothalamic ventromedial nucleus of ovariectomized, estrogen-primed rats. Brain slices were perfused with artificial cerebrospinal fluid and opioid drugs were applied by bolus injection into the perfusion line. Single unit activity was recorded extracellularly. The majority of ventromedial hypothalamic nucleus neurons tested exhibited marked inhibitory responses to [Met5]enkephalin. The inhibition was blocked by naloxone, by the selective delta-opioid receptor antagonist naltrindole and, to a lesser extent, by the mu-opioid receptor antagonist beta-funaltrexamine. The kappa-opioid receptor antagonist nor-binalmorphimine had virtually no effect on [Met5]enkephalin inhibition. Agonists selective for delta-([D-Pen2,D-Pen5]enkephalin, DPDPE) and for mu-([D-Ala2,MePhe4,Gly-ol5]enkephalin, DAGO) opioid receptors also potently inhibited the ventromedial hypothalamic nucleus neurons while the kappa-opioid receptors agonist U50,488 only produced a small inhibition in a smaller number of units. These results provide functional evidence that [Met5]enkephalin, a potential opioid transmitter in the ventromedial hypothalamic nucleus, can exert an inhibitory effect by acting on delta-and mu-opioid receptors.

To assess the roles of opioid receptor subtypes in voluntary alcohol drinking, alcohol-preferring AA (Alko, Alcohol) rats, non-deprived of food or water, were used in a paradigm where access to 10% alcohol solution was limited to 1-4-h sessions on every 2nd working day. The delta-opioid receptor antagonist naltrindole (1-5 mg/kg i.p. 15 min before the session) had no effect on alcohol drinking, while it attenuated the delta-opioid receptor agonist [D-Pen2, D-Pen5]enkephalin-induced locomotor stimulation. The mu1-opioid receptor antagonist naloxonazine (1-15 mg/kg i.p. 20 h before the session), at the largest dose, decreased alcohol drinking. It also decreased food intake. When naltrindole (1 mg/kg) and naloxonazine (15 mg/kg) were given prior to 3 consecutive sessions, the former had no effects at any session. Naloxonazine decreased alcohol consumption only in the 1st session, although the reduction of daily water intake became stronger during repeated administration. 4 days after the last drug administration, naloxonazine-treated animals consumed alcohol nearly twice as much as in the control session before any drug treatment. These data suggest that delta-opioid receptors are not involved in the regulation of alcohol drinking in AA rats. mu1-Opioid receptors may be involved in alcohol drinking, although the data suggest that even their prolonged blockade alone is insufficient to induce a sustained decrease in alcohol drinking.

When animals encounter difficult conditions, endogenous opioids are often released and may help in coping with the difficulties. In sows, prolonged confinement results in behavior abnormalities: high levels of stereotypies or excessive inactivity and unresponsiveness. The possibilities of causal links between endogenous opioids and these behavioral indicators of poor welfare have been raised. Mu receptor density was found to be greater in tethered sows than in group-housed sows and to be positively correlated with time spent inactive. There were negative correlations between both mu and kappa receptor densities and stereotypy duration. Kappa agonists are associated with aversion, and there are complex links between endorphin levels, dynorphin levels, and dopamine action. These results provide the first demonstration of a relationship between abnormal behavior in pigs and opioid receptor density, and help to clarify the links between behavioral responses and opioid action.

Morphine releases adenosine from the spinal cord and this contributes to spinal antinociception. The present study examined possible interactions between mu- and subclasses of delta-opioid receptors in the release of adenosine. Nanomolar (10(-8), 10(-9) M) concentrations of morphine release adenosine from spinal cord synaptosomes under conditions of partial depolarization with elevated K+, and this component of release is mediated by activation of mu-opioid receptors. Subnanomolar (10(-10), 10(-11) M) concentrations of the mu-opioid receptor agonists morphine, [N-MePhe3,D-Pro4]morphiceptin, and [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAMGO) have minimal effects on the release of adenosine from the spinal cord. However, [D-Pen2,D-Pen5]enkephalin (DPDPE), a delta 1-opioid receptor agonist, and [D-Ala2,Cys4]deltorphin, a delta 2-opioid receptor agonist, at doses which exhibit no intrinsic effects (10(-8) and 10(-7) M), shifted the dose-response curve for mu-opioid receptor-evoked adenosine release to the left in a dose-dependent manner. DPDPE was more potent than [D-Ala2,Cys4]deltorphin when combined with the highly selective mu-opioid receptor agonist [N-MePhe3,D-Pro4]morphiceptin, but these agents showed similar activity with the less selective agonists DAMGO and morphine. Simultaneous activation of mu- and delta-opioid receptors generates a synergistic release of adenosine from spinal cord synaptosomes. Although agonists for both delta 1- and delta 2-opioid receptor subtypes produce this response, the delta 1-opioid receptor agonist is more potent at eliciting this effect when the most selective mu-opioid receptor ligand is used.

Cocaine alters opioid receptor densities in rat brain. To investigate the functional consequences of such opioid receptor changes, adenylyl cyclase activity was measured in rat nucleus accumbens and caudate putamen following continuous cocaine administration (50 mg/kg/day, 7 days). In the nucleus accumbens chronic cocaine led to an increase in both the number of mu-opioid receptors and the maximal inhibition of adenylyl cyclase activity by DAMGO ([D-Ala2,N-methyl-Phe4,Glyol]enkephalin). There was no effect on inhibition of adenylyl cyclase activity by DPDPE ([D-Pen2,D-Pen5]enkephalin). There were no changes in the caudate putamen. Thus, continuous cocaine administration for 7 days results in a selective increase in mu-opioid receptor-mediated effector function in the nucleus accumbens.

Graded caval occlusion in conscious rabbits caused a biphasic haemodynamic response. Phase I was characterized by a fall in systemic vascular conductance so that arterial pressure was maintained. When cardiac output had fallen to 65 +/- 2% of its baseline level, phase II supervened. During phase II, conductance rose abruptly and arterial pressure fell to a life-threatening level (< or = 40 mm Hg). Fourth ventricular administration of either N-nitro-L-arginine methyl ester or naloxone prevented the occurrence of phase II. Fourth ventricular administration of L-arginine had no effect on the response to graded caval occlusion but was able to reverse the phase II blocking action of N-nitro-L-arginine methyl ester and naloxone. It is concluded that central nitrergic and opioid mechanisms interact to cause the vasodilatation characteristic of the decompensatory phase II of the cardiovascular response to acute hypovolaemia.

The effects of intrastriatal (i.st.) injections of mu-, delta-, and kappa-selective opioid receptor agonists on the augmentation of apomorphine-induced behaviors were determined in 6-hydroxydopamine-treated mice by using multidimensional behavioral analyses. 6-Hydroxydopamine (16 mu g/mu l, i.st.) was unilaterally injected into the striatum 30 min after pretreatment with desipramine (25 mg/kg, s.c). Mice were tested 14 days after injection of 6-hydroxydopamine. Apomorphine (0.5 mg/kg, s.c.) produced a marked increase in linear locomotion, contralateral circling and/or rearing behavior in 6-hydroxydopamine- but not vehicle-treated mice. Although the mu-selective opioid receptor agonist [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAMGO) (0.1 and 0.3 ng, i.st.) or the kappa-selective opioid agonist dynorphin A-(1-13) (0.1 and 0.3 mu g, i.st.) did not produce any significant effects on behavior, these peptides had an inhibitory effect on the apomorphine (0.5 mg/kg, s.c.)-induced increase in behavioral responses such as linear locomotion, contralateral circling and/or rearing behavior in 6-hydroxydopamine-treated mice. The inhibitory effects of DAMGO (0.3 ng, i.st.) and dynorphin A-(1-13) (0.3 mu g, i.st.) were fully reversed by selective opioid receptor antagonists such as beta-funaltrexamine (5 mu g, i.c.v.) and (--)-(1R,5R,9R)-5,9-diethyl-2-(3-furyl-methyl)-2'-hydroxy-6,7-benzomorph an (Mr2266) (10 mg/kg, s.c.), respectively. In contrast, the delta-selective opioid receptor agonist [D-Pen2,L-Pen5]enkephalin (DPLPE) (0.03, 0.1 or 0.3 mu g, i.st.) had no marked effects on the apomorphine (0.5 mg/kg, s.c.)-induced behavior in 6-hydroxydopamine-treated mice. These results suggest that the stimulation of mu- and kappa- but not delta-opioid receptors plays an inhibitory role in the behavioral augmentation induced by the activation of postsynaptic dopamine receptors in the striatum sensitized with 6-hydroxydopamine.

Autoradiographic data from transgenic mice carrying the human Cu/Zn-superoxide dismutase gene demonstrate an increase in mu-opioid receptor concentration in dopaminergic-related areas and the central grey area. The relative potencies of mu-, delta- and kappa-opioid receptor agonists to induce antinociception in heterozygous and homozygous superoxide dismutase transgenic mice as well as four inbred strains were assessed to determine the functional significance of the increased receptor concentration. Increased superoxide dismutase activity results in an increased sensitivity to mu-agonists in a gene dosage-dependent manner. SOD/Tg/hom mice were less sensitive to the delta-agonist than were SOD/Tg/het mice. The superoxide dismutase transgene did not affect kappa-opioid receptor agonist sensitivity. These data suggest that delta-opioid receptors are not regulated in the same manner as mu-opioid receptors and that kappa-opioid receptors are unaffected by superoxide dismutase activity.

The binding properties and pharmacological activities of H-Tyr(3'-I)-Tic-Phe-Phe-OH ([Tyr(3'-I)1]TIPP) were studied. Similar to the delta-opioid receptor antagonist H-Tyr-Tic-Phe-Phe-OH (TIPP), [Tyr(3'-I)1]TIPP is a selective and potent ligand at delta-opioid receptors. The displacement curve of [3H]diprenorphine binding by [Tyr(3'-I)1]TIPP was shifted to the right in the presence of Na+ and 5'-guanylylimidodiphosphate, suggesting that it acted as a delta-opioid receptor agonist. [Tyr(3'-I)1]TIPP also behaved as a full agonist in the mouse vas deferens assay and its effect was both naloxone- and TIPP-reversible. These data show that monoiodination at the 3'-position of the N-terminal tyrosine aromatic ring of TIPP converted it from a potent and selective antagonist to a full agonist at delta-opioid receptors.

1. When the analgesic effect of salmon-calcitonin (S-CT) and of eel-calcitonin (E-CT), as well as their influence on the morphine-analgesia were compared, no significant differences were found. 2. While on isolated tissues, E-CT induced a significant increase on the effect of bremazocine, [D-Pen2,D-Pen5]enkephalin and [Met5]enkephalin and no changes were observed on the effect of DAMGO, suggesting that E-CT increases the effects of opioids acting on delta or kappa receptors but not on mu receptors. 3. These findings corroborate the possibility of interactions between calcitonin and the opioid system.

The in vivo effects of [D-Pen2,D-Pen5]enkephalin, a cyclic peptide agonist with high affinity and selectivity for the delta opioid receptors, on the endogenous aluminum content of selected areas of rat brain and spinal cord were studied by means of atomic absorption spectrophotometry. Intracerebroventricular injection of a subanalgesic dose of [D-Pen2,D-Pen5]enkephalin (0.2 microgram/3 microliters) produced a transient, time-dependent reduction of the aluminum content. This effect was statistically significant in the frontal cortex, hippocampus and striatum, but did not reach the level of significance in the medulla and thoracic spinal cord. The partial depleting effect of [D-Pen2,D-Pen5]enkephalin on aluminum content, in the range of 0.2-1.0 micrograms/3 microliters, was dose-dependent and could be reversed by naloxone pretreatment. Serum aluminum levels were unchanged after [D-Pen2,D-Pen5]enkephalin treatment. Chronic (five weeks), systemic AlCl3 treatment increased the endogenous aluminum content in all central nervous system areas examined. Interestingly, [D-Pen2,D-Pen5]enkephalin i.c.v. produced a slight depletion of this elevated metal level in these areas to values not significantly different from those of the respective control values. Chronic in vivo, as well as in vitro, effects of aluminum on opioid receptor binding characteristics were also studied. Neither the specific binding of [3H][D-Pen2,D-Pen5]enkephalin nor [3H]Tyr-D-Ala-Gly-NMePhe-Gly-ol to membranes of frontal or parietal cortices, striatum or hippocampus, prepared from rats chronically treated with AlCl3, were affected.(ABSTRACT TRUNCATED AT 250 WORDS)

The actions of opioid receptor agonists and antagonists were studied in isolated rat aortic strips. Morphine (10(-7)-10(-6) M) had no contractile effect on resting strips but when added during the relaxation of the contractions induced by 10(-9) M noradrenaline, it induced a contractile response which was blocked by naloxone. The selective mu-opioid receptor agonist, [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAMGO, 10(-7)-10(-6) M), induced an increase in basal tension which remained after removal of endothelium or in Ca(2+)-free solution, but was inhibited by beta-flunaltrexamine. beta-Flunaltrexamine also inhibited the contractile response induced by DAMGO added during the relaxation of the contractions induced by noradrenaline. The delta-opioid receptor agonist, [D-Pen2,D-Pen5]enkephalin, had no effect on resting tension but potentiated the contractions induced by noradrenaline; these effects were abolished by naltrindol. The selective kappa-opioid receptor agonist, bremazocine, had no effect on resting tension and did not modify the amplitude of the contractions induced by noradrenaline. These results suggest that, at low concentrations, agonists of mu- and delta-opioid receptors may act as modulators of noradrenaline-induced responses, whereas at higher concentrations, mu-opioid receptor stimulation may have a direct contractile effect in isolated rat aorta.

The effects of local treatment with opioid receptor agonists on the early (0-10 min) and late (20-40 min) behavioural response and extravasation induced by intraplantar injection of 1% formalin in rats were examined. The mu-opioid receptor agonist [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAMGO) depressed pain behaviour in the late phase, and extravasation in both phases. The kappa-opioid receptor agonist trans-(+/-)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl] benzeneacetamide methanesulfonate (U50,488H) suppressed the behavioural response in both phases, but extravasation was enhanced in the early phase and not altered in the late phase. The delta-opioid receptor agonist [D-Pen2,5]enkephalin (DPDPE) enhanced the behavioural response in the late phase, but inhibited extravasation in the both early and late phases. Systemic injection of the agonists had no effects, and pretreatment with s.c. naloxone methiodide reversed the effects of locally administered agonists. These data (1) support the notion that different pathophysiological mechanisms underlie the two phases of the formalin test, and (2) indicate that depending on the receptor specificity, opioid receptor agonists have both pro- and antinociceptive effects, as well as pro- and antiinflammatory activity.

Graded caval occlusion in conscious rabbits caused a biphasic cardiovascular response. Phase I was characterized by a fall in systemic vascular conductance so that arterial pressure was maintained. When cardiac output had fallen to 64 +/- 3% of its baseline level, phase II supervened. During phase II, conductance rose abruptly and arterial pressure fell to a life-threatening level (< 40 mm Hg). Intravenous (i.v.) or central (fourth ventricular) administration of the adrenocorticotrophin (ACTH) fragment ACTH-(1-24) prevented the occurrence of phase II. The central dose of ACTH-(1-24) needed to block the occurrence of phase II was approximately 39 times less than the i.v. dose. Central administration of the delta 1-opioid receptor agonist [D-Pen2,D-Pen5]enkephalin (DPDPE) reversed this effect of both central and i.v. ACTH-(1-24). I.v. ACTH-(1-24) also lowered arterial pressure while raising cardiac output and vascular conductance. These effects were insensitive to propranolol and hyoscine methyl bromide, and were not mimicked by cortisol or adrenaline. It is concluded that ACTH-(1-24) has an acute, adrenal-independent, peripheral vasodilator effect as well as a central, anti-shock, effect.

Analgesic cross-tolerance between alpha 2-adrenoceptor and opioid receptor agonists was studied using the mouse tail-flick assay. Mice tolerant to clonidine (0.3 mg/kg s.c.) or xylazine (7 mg/kg s.c.) were cross-tolerant to morphine (5 mg/kg s.c.), nalorphine (70 mg/kg s.c.) and supraspinal [D-Ala2,MePhe4,Gly(ol)5]enkephalin (DAMGO; 4 ng i.c.v.), but not trans-(+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)- cyclohexyl] benzeneacetamide methanesulfonate (U50,488; 5 mg/kg s.c.), spinal DAMGO (10 ng i.t.), supraspinal [D-Pen2,D-Pen5]enkephalin (DPDPE; 9 micrograms i.c.v.) or spinal DPDPE (700 ng i.t.). In the complimentary studies, mice tolerant to morphine and nalorphine were cross-tolerant to both of the alpha 2-adrenoceptor agonists, but U50,488 tolerant mice were not. The results suggest differential interactions between alpha 2-adrenoceptor and mu 1-, mu 2-, delta-, kappa 1- and kappa 3-opioid analgesic circuitry.

Several opioid compounds were evaluated for an ability to modulate the K(+)-stimulated release of [3H]serotonin ([3H]5-hydroxytryptamine, [3H]5-HT) from rat spinal cord synaptosomal and tissue slice preparations. Selective kappa-opioid receptor agonists depressed K(+)-stimulated release of the radiolabelled transmitter from both tissue preparations, an effect which was reversed by norbinaltorphimine. Conversely, the selective mu- and delta-opioid receptor agonists [D-Ala2,NMePhe4,Gly-ol5]enkephalin (DAMGO) and [D-Pen2,D-Pen5]enkephalin (DPDPE), respectively, enhanced the K(+)-stimulated release of [3H]5-HT. This effect was only seen using the tissue slice preparation. When used at concentrations near its reported Kd for mu-opioid receptors, the selective mu-opioid receptor antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) blocked the action of DAMGO, but had no effect on the action of DPDPE. However, higher concentrations of CTOP, as well as all effective concentrations of selective delta-opioid receptor antagonists, blocked the action of both DAMGO and DPDPE. All agonist effects on spinal 5-HT release, regardless of the tissue preparation, were only seen at high (microM) concentrations. Moreover, effects of the opioid agonists were not consistent with the reported involvement of spinal 5-HT neurotransmission in the mediation of their antinociceptive action. Thus, the ability of opioids to modulate spinal 5-HT release appears to be of minimal physiological significance.

1. Male Sprague-Dawley rats were chronically tested with intrathecal (i.t.) receptor selective opioid antagonists to determine if antinociceptive supersensitivity developed to selective i.t. opioid receptor agonists. 2. A subcutaneously implanted osmotic minipump was used to deliver the mu-opioid receptor antagonist CTOP (0.3 nmol) or the delta-opioid receptor antagonist naltrindole (5.5 nmol) for 7 days. 3. Following a 24 hr washout period, rats received a single i.t. dose (ED50) of either DAMPGO (for CTOP-treated animals) or DPDPE (for naltrindole-treated animals) and the antinociceptive effects of the agents were tested on the tail-flick test. 4. Our findings revealed that chronic spinal treatment with selective opioid receptor antagonists did not induce an antinociceptive supersensitivity to selective opioid receptor agonists. 5. Perhaps this lack of supersensitivity is reflective of difficulties inherent to opioid receptor antagonists that do not possess negative intrinsic activity.

The brain sites which may be involved in delta-opioid receptor agonist-mediated actions in vivo were examined using quantitative [1-14C]deoxyglucose autoradiography. For this purpose [D-Pen2,D-Pen5]enkephalin (DPDPE)--one of the most selective delta-opioid receptor agonists available--was employed. DPDPE was most effective at a dose of 25 micrograms i.c.v. All of the motor regions measured displayed significant increases in glucose utilization. Further, increases were widespread in limbic forebrain regions and were also detected in components of the limbic midbrain. The ventroposterolateral thalamic nucleus, a region relaying somatosensory information to the cerebral cortex, displayed the strongest enhancement of glucose utilization. This regional pattern of changes is assumed to underlie the modulatory role in the processing of somatosensory and nociceptive information of DPDPE, its rewarding properties and the behavioral arousal produced by the delta-opioid agonist. A selective involvement of delta-opioid receptors in these effects was indicated by their antagonism by the delta-opioid receptor antagonist ICI 174,865 (N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH).

This study examined the influence of acute and repeated administration of the kappa-opioid receptor antagonist, nor-binaltorphimine, upon opioid-induced antinociception as measured by the tail-pressure test. A single intracerebroventricular (i.c.v.) injection of nor-binaltorphimine (30 micrograms) administered 1, 10 or 30 days prior to algesiometric testing prevented the analgesic effect of the kappa-opioid receptor agonist, (5 alpha, 7 alpha, 8 beta)-(-)-N- methyl-N-(7-(1-pyrrolidinyl)-1-oxaspiro(4,5)dec-8-yl)benzenacet amide (U69593). The analgesic effect of the mu-opioid receptor agonist, [D-Ala2,N-methyl-Phe4,Gly5-ol]enkephalin (DAMGO), and the delta-opioid receptor agonist, [D-Pen2,D-Pen5]enkephalin (DPDPE), was not modified. In contrast, when nor-binaltorphimine was administered repeatedly (twice daily i.c.v. administration of 30 micrograms nor-binaltorphimine for 10 days), the analgesic effect of DAMGO, DPDPE as well as U69593 was abolished. In the case of mu- and delta-opioid receptor agonists, this abolition was apparent when testing occurred 1 or 2, but not 5 days after termination of nor-binaltorphimine treatment. This treatment regimen also resulted in a long-lasting antagonism (e.g. 20 days) of U69593-induced analgesia. These data show that, depending on the treatment regimen employed, nor-binaltorphimine can function as a selective kappa-opioid receptor antagonist, or as an antagonist at multiple opioid receptor subtypes. Further, they demonstrate that nor-binaltorphimine functions as a long-lasting kappa-opioid receptor antagonist in vivo.

Various doses of MK-801 ((+/-)-5-methyl-10,11-dihydro-5H-dibenzo(a,d) cyclohepten-5, 10-imine maleate), a non-competitive N-methyl-D-aspartic acid (NMDA) receptor antagonist (0.001-1 microgram) injected intracerebroventricularly (i.c.v.) alone did not show any antinociceptive effect. MK-801 (0.001-1 microgram i.c.v.) dose dependently attenuated the inhibition of the tail-flick and hot plate responses induced by i.c.v. administered morphine (1 microgram), [D-Pen2, D-Pen5]enkephalin (DPDPE; 10 micrograms), and U50,488H (trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeoce tamide ) 60 micrograms). However, the inhibition of the tail-flick and hot plate responses induced by i.c.v. administered beta-endorphin (1 microgram) was not changed by i.c.v. administered MK-801. Our results indicate that, at the supraspinal level, NMDA receptors are involved in the production of antinociception induced by supraspinally administered morphine, DPDPE, and U50,488H but not beta-endorphin.

(+)-cis-Dioxolane (0.5-2 micrograms), a muscarinic receptor agonist, given intracerebroventricularly (i.c.v.) produced a dose-dependent inhibition of the tail-flick response in male ICR mice. (+)-cis-Dioxolane given i.c.v. at a subanalgesic dose (0.25 micrograms), selectively potentiated the antinociceptive response induced by i.c.v. administered beta-endorphin, an epsilon-opioid receptor agonist, but not morphine or [D-Ala2,NMePhe4,Gly5-ol]enkephalin (DAMGO), mu-opioid receptor agonists, [D-Pen2,D-Pen5]enkephalin (DPDPE), a delta receptor agonist, or trans(+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]- benzeneacetamide methane sulfonate (U50,488H), a kappa-opioid receptor agonist. The antinociceptive response induced by (+)-cis-dioxolane given i.c.v. was attenuated by i.c.v. treatment with N omega-nitro-L-arginine (1 microgram), hemoglobin (120 micrograms) or methylene blue (10 micrograms). The antinociception induced by carbachol given i.c.v. was also antagonized by the i.c.v. treatment with N omega-nitro-L-arginine (1 microgram). However, the same treatment with N omega-nitro-L-arginine, hemoglobin or methylene blue did not affect the beta-endorphin-induced antinociception. The potentiation of beta-endorphin-induced antinociception by (+)-cis-dioxolane was reversed by i.c.v. treatment with N omega-nitro-L-arginine (1 microgram), hemoglobin (120 micrograms) or methylene blue (10 micrograms). On the other hand, the antinociceptive response induced by (+)-cis-dioxolane (1 microgram) given i.c.v. was potentiated by i.c.v. administered L-arginine (20 micrograms) but not D-arginine (20 micrograms). Dibutyryl cyclic GMP at 0.5-2.0 micrograms given i.c.v. produced an antinociceptive response and at subanalgesic dose (0.1 microgram) potentiated i.c.v. beta-endorphin-induced antinociception.(ABSTRACT TRUNCATED AT 250 WORDS)

The present study was undertaken to investigate the possible role of delta-opioid receptors in the neuroregulation of human colonic motility by using a superfusion model. Spontaneous mechanical activity and responses to electrical transmural nerve stimulation of both longitudinal and circular muscle strips from the human sigmoid colon were studied. Exogenously added delta-opioid receptor agonists did not modify spontaneous contractile activities of either type of strip. Nerve stimulation induced a triphasic response composed of an initial contraction followed by a relaxation and an off-contraction. This response was mediated by cholinergic excitatory nerves and non-adrenergic, non-cholinergic excitatory and inhibitory nerves. [Met5]Enkephalin and the synthetic delta-opioid receptor agonist [D-Pen2,D-Pen5]enkephalin (DPDPE) significantly decreased the amplitude of the initial contraction and of the off-contraction. The effects of both delta-opioid receptor agonists were reduced in the presence of either the delta-opioid receptor antagonist, ICI 174864, or another delta-opioid receptor antagonist, naltrindole. ICI 174864 prevented neither the effects of a natural kappa-opioid receptor agonist, dynorphin-(1-13) nor those of the mu-opioid receptor agonist, PL017. Therefore, these data suggest that delta-opioid receptors might be involved in the neuroregulation of smooth muscle of human colon and may mediate inhibition of cholinergic and non-cholinergic excitatory transmission within the myenteric plexus.

1. In previous studies, we have demonstrated that delta-opioid receptors are involved both in the acute control of hypothalamus-pituitary-adrenocortical (HPA) axis activity and in the development of neuroendocrine opioid tolerance. In the present work we studied whether central delta-opioid receptors play a role in the development of neuroendocrine physical dependence to opioids in the rat. 2. Intracerebroventricular (i.c.v.) administration of the delta-selective agonist DPDPE ([D-Pen2,D-Pen2]enkephalin) produced stimulation of HPA activity, as shown by an increase in corticosterone release. This effect was antagonized by i.c.v. co-administration of ICI 174,864, a selective delta-receptor antagonist, which provide direct evidence that the activation of the HPA axis produced by DPDPE is mediated by central delta-opioid receptor. 3. Chronic pretreatment with i.c.v. DPDPE resulted in tolerance to its neuroendocrine effect. Intracerebroventricular injection of ICI 174,864 to DPDPE-tolerant rats produced neither alteration in corticosterone release nor behaviour signs of dependence. 4. It was concluded that delta-opioid receptors do not play a role in the development of opioid neuroendocrine physical dependence at the HPA axis.

Intracerebroventricular (i.c.v.) administration of the venom peptide, mastoparan, to mice decreased to a limited extent opioid-induced supraspinal analgesia in a non-competitive fashion. The mu-opioid receptor agonists, [D-Ala2,N-MePhe4,Gly-ol5]-enkephalin (DAMGO) and morphine, the mu/delta-opioid receptor ligands, human beta-endorphin-(1-31) and [D-Ala2,D-Leu5]-enkephalin (DADLE), and the selective ligands of delta-opioid receptors, [D-Pen2,5]enkephalin (DPDPE) and [D-Ala2]deltorphin II, showed an impaired analgesic effect in mice given mastoparan. Mastoparan diminished the analgesic activity of DPDPE and [D-Ala2]deltorphin II to the same extent as observed after giving the delta-opioid receptor-selective antagonist, ICI 174864. The mu-opioid receptor-mediated analgesia that remained after mastoparan was abolished in the presence of the opioid antagonist, naloxone. Mastoparan after binding to Gi alpha/Go alpha subunits could block opioid antinociception. The existence of a class of G protein functionally coupled to mu-opioid receptors, but resistant to the effect of mastoparan is suggested.

An antisense oligodeoxynucleotide (A-oligo) to delta-opioid receptor mRNA was utilized to block the expression of mouse delta-opioid receptor in the spinal cord of male ICR mice. Intrathecal treatment with A-oligo (1.6-163 pmol) dose-dependently attenuated the antinociception induced by i.t. administered DPDPE ([D-Pen2,5]enkephalin) or [D-Ala2]deltorphin II, delta-opioid receptor agonist, without affecting the antinociception induced by DAMGO ([D-Ala2-MePhe4,Gly(ol)5]enkephalin) or U50,488H, respective mu- or kappa-opioid receptor agonists. Scrambled sense oligodeoxynucleotide (163 pmol) was ineffective against the tail-flick inhibition induced by DPDPE,[D-Ala2]deltorphin, DAMGO or U50,488H. The studies confirm previous pharmacological studies at the molecular level indicating a distinct delta-opioid receptor for antinociception in the spinal cord.

This study with the rat evaluated the contribution of omega-conotoxin GVIA-(omega-CgTx) and verapamil-sensitive Ca2+ channels in behavioural, antinociceptive and thermoregulatory responses to intracerebroventricular (i.c.v.) injection of [D-Ala2,NMePhe4,Gly-ol5]enkephalin (DAMGO), [D-Pen2,D-Pen5]enkephalin (DPDPE) and dynorphin A-(1-17), which are selective agonists for putative mu, delta and kappa-opioid receptors, respectively. The rats treated with omega-CgTx (8-32 pmol i.c.v.) showed transient, dose-dependent shaking behaviour, hyperalgesia and hypothermia which gradually disappeared within 4 h. The behaviour of the rats was normal by 24 h. Histological examination of brain sections showed morphological alterations of neurons in the hippocampus, medial-basal hypothalamus and pyriform cortex. antinociception, catalepsy and thermoregulatory responses elicited by DAMGO (0.4 and 2.0 nmol) were significantly prolonged and potentiated by verapamil (20 pmol i.c.v. 15 min before) or omega-CgTx (8 pmol 24 h before). Antinociception and hypothermia induced by DPDPE were antagonized by verapamil and omega-CgTx, whereas only omega-CgTx prevented the behavioural arousal observed after DPDPE. Similarly, hypothermia induced by dynorphin A-(1-17) (5.0 nmol) and by the kappa-opioid receptor agonist U50,488H (215 nmol) was antagonized by the two Ca2+ channel blockers but only omega-CgTx prevented the barrel rolling and bizarre postures caused by the opioid peptide.

In anesthetized rats, the intrastriatal infusion of the delta-opioid receptor agonist, [D-Pen2,D-Pen5]enkephalin, increased the extracellular concentration of dopamine. This effect was abolished by the NMDA receptor antagonist, 3-[(+/-)-2-carboxypiperazine-4-yl]propyl-1-phosphonate, but was unchanged by the AMPA (D,L-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate) and kainate receptor antagonist, 6,7-dinitroquinoxaline-2,3-dione. This suggests that the dopamine release induced by the delta-opioid agonist depends critically on the involvement of glutamatergic transmission via NMDA receptors.

The recently cloned kappa-opioid receptor has binding characteristics consistent with those of a kappa 1-opioid receptor. Repeated intrathecal administration of an antisense oligodeoxynucleotide against the kappa 1-opioid receptor selectively lowers U50,488H (trans-3,4-dichloro-N-methyl-N-[2-(1- pyrrolidinyl)cyclohexyl]benzeneacetemide) analgesia (P < 0.02) without affecting mu or delta analgesia. A mismatched antisense oligodeoxynucleotide in which 4 bases had been switched is inactive against U50,488H analgesia. These studies confirm at the molecular level traditional pharmacological studies implying a distinct receptor mechanisms for kappa 1 analgesia and demonstrate the utility of antisense approaches in studies of opioid pharmacology.