Ethylketocyclazocine Research Articles

P‐91: The Causes and Improvement of Circular Mura in Micro OLED Displays

There is a huge impact on the image quality of Micro‐OLED display in the production, which looks like a white ring or white dot under the microscope. This phenomenon is called Circular Mura in this paper. The root caucus locks in the common effect of residual sulfur hexafluoride (SF6) and the solution of ethyl ketocy clazocine (EKC) on the anode surface. Circular Mura was finally successfully solved through equipment modification and process optimization.

Effects of kappa opioid agonists alone and in combination with cocaine on heart rate and blood pressure in conscious squirrel monkeys

As kappa agonists have been proposed as treatments for cocaine abuse, the cardiovascular effects of the kappa opioid receptor agonists ethylketocyclazocine (EKC) and enadoline were investigated in conscious squirrel monkeys. Both EKC and enadoline increased heart rate with little effect on blood pressure. This effect appeared to be specific for kappa receptors as the mu opioid agonist morphine did not mimic the effects of the kappa agonists. The opioid antagonist naltrexone, at a dose of 1.0 mg/kg, blocked the effect of EKC. An action at both central and peripheral receptors may be responsible for the heart rate increase following kappa agonist treatment. The ganglionic blocker chlorisondamine partially antagonized the effect of EKC on heart rate, suggesting central involvement, while the peripherally-acting agonist ICI 204,448 ((±)-1-[2,3- (Dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol hydrochloride) also increased heart rate, supporting a peripheral site of action. When given in combination with cocaine, EKC produced effects that were sub-additive, suggesting that the kappa agonists may be used safely as cocaine abuse treatments.

The inhibitory effect of opioids on HepG2 cells is mediated via interaction with somatostatin receptors

Opioids, acting via G-protein coupled membrane receptors, induce analgesia. However their role is not limited to their anti-nociceptive action. They are found in several peripheral tissues acting as negative regulators of cellular processes. Even though that is not fully elucidated, it becomes obvious that opioids exert their effects in close relation to other neuropeptides such as somatostatin. Hepatocellular carcinoma is one tumor, among others, which secrete bioactive peptides while somatostatin analogs exert an inhibitory effect. We have used the human hepatocyte-derived cancer cell line HepG2, in order to examine the effect of opioids on cell growth and their possible mode of action. Our results show that the opioid ethylketocyclazocine (EKC) inhibits cell proliferation and induces apoptosis. This inhibitory effect is not exerted via opioids receptors since it was not reversed by the opioid antagonist diprenorphine and functional opioid receptors were not found on HepG2 cells. On the contrary, we show that EKC binds to somatostatin receptors, and activates a PTP signalling cascade. In this respect, the interaction of opioids with somatostatin receptors on hepatocellular carcinoma cells, and the fact that they are widely used for pain control, may provide some additional clues for the discrepancies during treatment with somatostatin analogues.

The opioid agonist ethylketocyclazocine reverts the rapid, non-genomic effects of membrane testosterone receptors in the human prostate LNCaP cell line

Neuropeptides influence cancer cell replication and growth. Opioid peptides, and opiergic neurons are found in the prostate gland, and they are proposed to exert a role in tumor regulation, influencing cancer cell growth, as opioid agonists inhibit cell growth in several systems, including the human prostate cancer cell line LNCaP. In the same cell line, the existence of membrane testosterone receptors was recently reported, which increase, in a non-genomic manner, the secretion of PSA, and modify actin cytoskeleton dynamics, through the signaling cascade FAK→PI-3 kinase→Cdc42/Rac1. In the present work, we present data supporting that the general opioid agonist Ethylketocyclazocine (EKC) decreases testosterone-BSA (a non-internalizable testosterone analog) induced PSA secretion. Furthermore, we report that this opioid affects this non-genomic testosterone action, by modifying the distribution of the actin cytoskeleton in the cells, disrupting the above signaling cascade. In addition, after long (>24 h) incubation, opioids decrease the number of membrane testosterone receptors, and reverse their effect on the signaling molecules. In conclusion, our results provide some new insights of a possible action of opioids in prostate cancer control by interfering with the action and the expression of membrane testosterone receptors and signaling.

Effects of structural modifications of N-CPM-normorphine derivatives on agonist and antagonist activities in isolated organs.

The agonistic and antagonistic properties of N-cyclopropylmethyl (N-CPM) morphine derivatives were observed in mouse vas deferens (MVD), longitudinal muscle of guinea pig ileum (GPI) and rabbit vas deferens (LVD). In MVD the K(e) values of the titled compounds (N-CPM-morphine, N-CPM-isomorphine, N-CPM-dihydromorphine, N-CPM-dihydroisomorpPhine, N-CPM-dihydromorphone and naltrexone) were measured for mu-, kappa- and delta-receptors using normorphine, ethylketocyclazocine (EKC) and D-Pen2-D-Pen5-enkephaline (DPDPE) as selective agonists on the receptors, respectively. For mu-receptors of MVD the tested compounds showed similar affinity. For kappa-receptors the non-iso-6-OH derivatives possessed much less affinity than the iso-derivatives. Similar difference could be observed for delta-receptors. The agonistic activities of these compounds in MVD were observed to be between 0-20% of the inhibition of muscle contractions. In GPI the compounds except naltrexone possessed strong agonistic activities effectively antagonized by nor-binaltorphimine (nor-BNI) (K(e) of nor-BNI was 0.23 nM) suggesting that they were strong kappa-receptor agonists. We investigated these agents in LVD too, which contains kappa-receptors, but they did not produce any agonist potencies. It raises the possibility that the kappa-receptor subtypes of LVD and MVD are different from the kappa-receptor subtype of GPI or the vasa deferentia contain much fewer kappa-receptors than GPI and the intrinsic activities of these compounds are too small to reach the 50% inhibition of the contractions.

A subset of kappa opioid ligands bind to the membrane glucocorticoid receptor in an amphibian brain.

Previous studies demonstrated that a membrane receptor for glucocorticoids (mGR) exists in neuronal membranes from the roughskin newt (Taricha granulosa) and that this receptor appears to be a G protein-coupled receptor (GPCR). The present study investigated the question of whether this mGR recognizes nonsteroid ligands that bind to cognate receptors in the GPCR superfamily. To address this question, ligand-binding competition studies evaluated the potencies of various ligands to displace [3H]corticosterone (CORT) binding to neuronal membranes. Initial screening studies tested 21 different competitors and found that [3H]CORT binding was displaced only by dynorphin 1-13 amide (an endogenous kappa-selective opioid peptide), U50,488 (a synthetic kappa-specific agonist) and naloxone (a nonselective opioid antagonist). Follow-up studies revealed that the kappa agonists bremazocine (BRE) and ethylketocyclazocine (EKC) also displaced [3H]CORT binding to neuronal membranes, but that U69,593 (a kappa specific agonist) and nor-BNI (a kappa specific antagonist) were ineffective. The Ki values measured for the opioid competitors were in the subnanomolar to low micromolar range and had the following rank-order: dynorphin > U50,488 > naloxone > BRE > EKC. Because these ligands displaced, at most, only 70% of [3H]CORT specific binding, it appears that some [3H]CORT binding sites are opioid insensitive. Kinetic analysis of [3H]CORT off-rates in the presence of U50,488 and/or CORT revealed no differences in dissociation rate constants, suggesting that there is a direct, rather than allosteric, interaction with the [3H]CORT binding site. In summary, these results are consistent with the hypothesis that the high-affinity membrane binding site for [3H] CORT is located on a kappa opioid-like receptor.

Receptor selectivity of Met-enkephalin-Arg 6-Phe 7, an endogenous opioid peptide, in cerebral cortex of human and rat

This study was undertaken to examine the receptor selectivity of Met-enkephalin-Arg 6-Phe 7 (MERF) employing radioreceptor binding assays in human cerebral cortex membranes, and to elucidate the responsible receptors that mediate the regulatory action of MERF on high (20 mM) K +-stimulated release of [ 3H]norepinephrine ([ 3H]-NE) in rat cortex slices. Specific binding of [ 3H]MERF was inhibited by DAMGO, Tyr-D-Arg-Phe-Sar(TAPS), bremazocine and ethylketocyclazocine (EKC), but not by U69,593 (U69) and DPDPE. MERF showed high affinity for specific binding sites of [ 3H]DAMGO. However, MERF had little influence on the specific binding of [ 3H]DPDPE, [ 3H]U69 and [ 3H]diprenorphine ([ 3H]DIP) in the presence of 1 μM each of DAMGO, DPDPE and U69. In [ 3H]NE release experiments using rat cortex slices, DAMGO, MERF and EKC, in order of their potency, inhibited K +-stimulated release of [ 3H]NE. The inhibitory effects of MERF and DAMGO were more sensitive than that of EKC to antagonism by CTAP, nor-binaltorphimine (nor-BNI) and naloxone. These results suggested that MERF possesses high affinity for μ-receptors, but not for δ-, κ 1-, and very low affinity for κ 2-receptors in human cerebral cortex membranes. Also, the inhibitory effect of MERF on the K +-stimulated release of [ 3H]NE appears to be mediated by μ-receptors in rat cerebral cortex slices.

Systemic effects of E-2078, a stabilized dynorphin A(1-8) analog, in rhesus monkeys.

E-2078 ([N-methyl-Tyr1, N-methyl-Arg7, D-Leu8] dynorphin A(1-8) ethylamide) is a dynorphin A(1-8) analog with a reduced tendency to be biotransformed, when compared to the unmodified opioid peptide. E-2078 has been found to produce kappa-opioid agonist effects in vivo in rodents. In the present studies, we investigated whether systemically administered E-2078 could produce kappa-agonist effects in rhesus monkeys, in tests of antinociception, diuresis and ethyl-ketocyclazocine (EKC) discrimination. E-2078 (0.32-18 mg/kg, SC, IM or IV) was tested in the warm water (50 degrees, 55 degrees C) tail withdrawal assay of thermal antinociception. The diuretic effects of E-2078 (0.056-1.8 mg/kg, SC) were also compared to those of the kappa-agonist, U69,593 (0.01-0.32 mg/kg, SC). Lastly, the effects of E-2078 (0.1-3.2 mg/kg, SC or IV) were studied in rhesus monkeys trained to discriminate EKC (0.0056 mg/kg SC) from vehicle, in a food-reinforced operant procedure. E-2078 did not produce thermal antinociception in rhesus monkeys following SC or IM administration, up to the largest doses presently studied (i.e., 18 and 10 mg/kg, respectively). E-2078 caused thermal antinociception by the IV route, but this effect was not apparently mediated by kappa- or mu-opioid receptors, as shown by its insensitivity to quadazocine (1 mg/kg) pretreatment. However, SC E-2078 caused diuresis, and this effect was blocked by quadazocine pretreatment, consistent with mediation by kappa-opioid receptors. E-2078 generalized in EKC-discriminating monkeys, but only after the largest dose (3.2 mg/kg), and only following IV administration. The present studies suggest that systemically administered E-2078 can produce some kappa-receptor mediated effects in rhesus monkeys, but its profile of action is not identical to non-peptidic kappa-agonists following all routes of administration, or across all experimental situations.

Effects of kappa opioid agonists on the discriminative stimulus effects of cocaine in rhesus monkeys.

This study examined the effects of the kappa opioid agonists U50,488 and ethylketocyclazocine (EKC) on cocaine discrimination in rhesus monkeys trained to discriminate cocaine (0.4 mg/kg) from saline. Administration of U50,488 and EKC alone produced primarily saline-appropriate responding. Kappa agonist pretreatments produced variable effects on cocaine discrimination across monkeys, attenuating the discriminative stimulus effects of cocaine in some monkeys, but either having no effect on cocaine discrimination or enhancing the discriminative stimulus effects of cocaine in other monkeys. The effects of kappa agonists on cocaine discrimination were reversed by pretreatment with the opioid antagonist naloxone (1.0 mg/kg). These results indicate that kappa agonists do not consistently block the discriminative stimulus effects of cocaine in rhesus monkeys.

Modulation of the estrogen-regulated proteins cathepsin D and pS2 by opioid agonists in hormone-sensitive breast cancer cell lines (MCF7 and T47D): evidence for an interaction between the two systems.

In many cancer cell lines, including breast, prostate, lung, brain, head and neck, retina, and the gastrointestinal tract, opioids decrease cell proliferation in a dose-dependent and reversible manner. Opioid and/or other neuropeptide receptors mediate this decrease. We report that only the steroid-hormone-sensitive cell lines MCF7 and T47D respond to opioid growth inhibition in a dose-dependent manner. Therefore, an interaction of the opioid and steroid receptor system might exist, as is the case with insulin. To investigate this interaction, we have assayed two estrogen-inducible proteins (pS2 and the lysosomal enzyme cathepsin D) in MCF7 and T47D cells. When cells were grown in the presence of FBS (in which case a minimal quantity of estrogens and/or opioids is provided by the serum), we observed either no effect of etorphine or ethylketocyclazocine (EKC) or an increase of secretion and/or production of pS2 and cathepsin D. However, when cells were cultured in charcoal-stripped serum and in the absence of phenol red, the effect of the two opioids is different: EKC decreased the production and/or secretion of pS2 and cathepsin D, whereas etorphine increased their synthesis and/or secretion. The differential effect of the two general opioids was attributed to their different receptor selectivity. Furthermore, the variations of the ratio of secreted/produced protein and the use of cycloheximide indicate that opioids selectively modify the regulatory pathway of each protein discretely. In conclusion, through the interaction with opioid and perhaps other membrane-receptor sites, opioid agonists modify in a dose-dependent manner the production and the secretion of two estrogen-regulated proteins. Opioids may therefore disturb hormonal signals mediated by the estrogen receptors. Hence, these chemicals may have potential endocrine disrupting activities.

NTI4F: a non-peptide fluorescent probe selective for functional delta opioid receptors

A non-peptide fluorescent probe, NTI4F, has been developed for the delta ( δ) opioid receptor. The probe is a potent δ-antagonist in the mouse vas deferens (MVD) smooth muscle assay and it binds to the δ opioid receptor with high affinity ( K i=1 nM) and selectivity. Confocal microscopy indicates that the probe binds to Madin–Darby canine kidney (MDCK) cells transfected with the δ opioid receptor. This binding can be blocked by the δ opioid receptor antagonist, naltrindole (NTI), but not by morphine or ethylketazocine (EK) which are mu ( μ)- and kappa ( κ)-selective ligands. This fluorescent probe should prove useful in the study of the distribution of the δ opioid receptor.

Receptor-Mediated Activation of G-Proteins by κ Opioid Agonists in Frog ( Rana esculenta) Brain Membranes

This study delineates the heterotrimeric guanine nucleotide binding regulatory protein (G-protein) types in frog ( Rana esculenta) brain membranes and their activation by κ opioid agonists. Ethylketocyclazocine (EKC), trans-(±)-3,4-dichloro- N-methy- N-(2-[1-pyrrolidinyl]cyclohexyl)benzene-acetamide (U-50,488) and bremazocine displayed dose-dependent, norbinaltorphimine-reversible stimulation of guanosine-5′-O-(3-[ 35S]thio)triphosphate ([ 35S]GTPγS) binding in crude membrane preparations. G-proteins were identified by Western-blotting using previously characterized specific antisera that were generated against mammalian G-protein α-subunits and β-subunits. A photoreactive guanosine 5′-triphosphate (GTP) analog, [α- 32P]GTP azidoanilide ([α- 32P]AA-GTP) irreversibly labeled four proteins in the molecular weight range of 39–43 kDa. Ethylketocyclazocine and U-50,488 stimulated photolabelling of these proteins among which the 39 kDa band comigrated with the protein specifically labelled with the α i2 antibody and the 40 kDa band was identified as α o1. The other two bands were also stained with the α common antibody, but were not further identified. These results suggest that the endogenously expressed κ opioid receptors that are present in frog brain interact with multiple G-proteins in situ. Furthermore, the structure of most G-proteins seems to be well preserved during phylogenesis.

Autoradiographic determination of changes in opioid receptor binding in the limbic system of the Columbian ground squirrel at different hibernation states

To localize and quantify the state-dependent changes in various opioid receptor subtypes in the limbic system of non-hibernating and hibernating Columbian ground squirrels, quantitative receptor-binding autoradiography was used. Compared to the non-hibernating animals, the binding density of [ 3H]-[ d-Pen 2,5]-enkephalin (DPDPE) to the δ receptor in the lateral septum, CA3, and the hippocampal fissure of the hippocampal formation was significantly decreased in the hibernating ground squirrels. A significant reduction in the binding density of [ 3H]-[ d-Ala 2,N-Me-Phe 4,Gly-ol 5]-enkephalin (DAGO) to μ receptor was also observed in the medial septum and the CA3 region of the hippocampus of the hibernating animals. In contrast, a decrease in [ 3H]ethylketocyclazocine (EKC) binding to the κ receptor was only observed in the claustrum and CA3 of the hippocampus during hibernation. The differential changes in binding to various opioid receptors suggest that different opioid subtypes may exert different physiological roles in regulating the specific states (entrance, maintenance and arousal) of a hibernation bout.

Perinatal treatment of rats with opiates affects the development of the blood-brain barrier transport system PTS-1.

Previous results have shown that treatment of rats with morphine during the neonatal period can influence development of peptide transport system-1 (PTS-1), the blood-brain barrier transport system for Tyr-MIF-1 and methionine enkephalin. Previous work has suggested that the activity level of PTS-1 correlates with the concentration of methionine enkephalin in the brain. We show here that rats treated peripherally with morphine sulfate (MS) in both the prenatal and neonatal periods have enhanced activity of PTS-1. The degree of enhancement increases with age to reach a 66% increase in comparison with controls at age 9 weeks. The mu agonist MS was more powerful than the kappa agonist ethylketocyclazocine (EKC) or the delta agonist [D-Pen2.5,pCl-Phe4]enkephalin (pCl-DPDPE) in producing this effect. Opiate antagonists had complex effects with methylnaltrexone blocking the action of MS on PTS-1. These results show that the level of PTS-1 activity in adult rats can be modified by perinatal events that affect opiate tone during development.

Ligand binding profiles of U-69,593-sensitive and -insensitive sites in human cerebral cortex membranes: Evidence of kappa opioid receptors heterogeneity

Receptor binding studies were performed to characterize the properties of subtypes of k opioid receptors in membrane preparations of human cerebral cortex. [ 3H]U69,593 ([ 3H]U69), a selective κ 1-agonist, and [ 3H]diprenorphine ([ 3H]DIP), a non-selective opioid antagonist, in the presence of 1 μM each of DAMGO, DPDPE and U-69 to block μ-, δ-, and κ 1-sites, labeled single population of binding sites, respectively. [ 3H]U-69 binding sites (Kd = 3.8 ± 0.2 nM, B max = 6.3 ± 0.2 fmol/mg protein) had a binding profile that correspond to κ 1-receptor. That is, dynorphin A (1–13) (Dyn A), bremazocine (BZC), U50,488H (U50), (−)ethylketocyclazocine (EKC) and nor-binaltorphimine (nor-BNI) bound to this site with high affinities. [ 3H]DIP labeled binding sites (Kd = 7.3 ± 0.2 nM, B max = 102 ± 9 fmol/mg protein) that were not sensitive to U-50, but to BZC, EKC and nor-BNI. These results indicate that κ 1, and κ 2 opioid receptors exist in human cerebral cortex with different ligand binding profiles.

κ-Opioid receptor recognition sites are not modulated by local anaesthetics

Local anaesthetics and opioid drugs function synergistically to provide analgesia. In the present study, the nature of this synergy has been investigated using in vitro radioligand binding to determine whether the local anaesthetics bupivacaine and tetracaine modulate the binding of two κ-opioid receptor ligands, [ 3H]U-69593 (5- α-,7- α,8- β-(−)- N-methyl- N-[7-(1-pyrrolodinyl)-1-oxaspiro(4,5)dec-8- yl]-benzene acetamide) and [ 3H](−)-EKC (ethylketocyclazocine). [ 3H]U-69593 bound with a K D of 0.88 nM and a B max of 2.39 ± 0.22 fmol/mg wet weight in guinea pig cerebellar membranes. The binding was inhibited by bremazocine and morphine with Hill slopes near unity and pI 50 values of 9.96 and 6.84–6.86, respectively. [ 3H]U-69593 binding was inhibited by Gpp(NH)p (5′-guanylyl imidodiphosphate) and NaCl, consistent with an agonist action of the compound. The binding characteristics of the ligand were not changed by bupivacaine or tetracaine. [ 3H](−)-EKC bound with K D values of 0.55 and 0.97 nM and B max values of 4.22 and 0.99 fmol/mg wet weight in guinea pig cerebellar membranes and rat spinal cords, respectively. In the rat spinal cord, [ 3H](−)-EKC appeared to act as an agonist/antagonist, since the presence of Gpp(NH)p and NaCl only produced a small (21%) reduction in binding, but reduced the pI 50 for the residual binding to inhibition by morphine from 6.33–6.39 to 5.95. As with [ 3H]U-69593, the binding characteristics of [ 3H](−)-EKC were not affected by bupivacaine or tetracaine. These studies demonstrate that effects on κ-opioid receptor recognition site conformation are unlikely to explain the clinically observed synergy between local anaesthetics and opioids.

Unexpected prosecretory action component of loperamide at mu-opioid receptors in the guinea-pig colonic mucosa in vitro.

1. In a voltage clamp setting (Ussing chamber), the antidiarrhoeal drug, loperamide (Lop) slightly augmented prostaglandin E1 (PGE1) plus theophylline-stimulated net chloride secretion above control values at low concentrations (10(-10) and 10(-9) M) but inhibited it at higher concentrations (10(-6) and 10(-5) M). The apparently weak prosecretory action component of Lop was turned into a clear cut antisecretory effect by pretreatment with 2 x 10(-7) M naloxonazine plus 10(-7) M CTOP-NH2 (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2), two selective mu opioid receptor antagonists. This indicates a prosecretory effect of loperamide at mu opioid receptors. The antisecretory effect of low Lop concentrations, uncovered by mu opioid receptor blockade, was prevented by additional blockade of kappa opioid receptors by 5 x 10(-9) M nor-binaltorphimine (nor-BNI). 2. The nonselective opioid antagonist, naloxone, at 10(-6) M did not significantly reduce either PGE1 plus theophylline-stimulated net chloride secretion in Lop-free controls or the antisecretory action of Lop. By contrast, the partial agonist ethylketocyclazocine (EKC), which activates kappa but blocks mu opioid receptors, concentration-dependently inhibited PGE1 plus theophylline-stimulated net chloride secretion without any consistent prosecretory action component. Nor-BNI at 5 x 10(-8) M significantly blocked the antisecretory action of EKC. 3. It is concluded that, in the guinea-pig colonic mucosa under the present conditions, mu opioid receptors mediate enhancement and kappa opioid receptors inhibition of PGE1-stimulated net chloride secretion by low Lop concentrations. The two opposite actions are largely masked by superimposition. An opioid receptor-independent mechanism of action contributes to the antisecretory effect of Lop at high concentrations.

P2-21 - Inhibitory effects of ethylketocyclazocine (EKC) and morphine via δ-opioid receptor on cAMP levels in NG 108-15 cells

P2-21 - Inhibitory effects of ethylketocyclazocine (EKC) and morphine via δ-opioid receptor on cAMP levels in NG 108-15 cells

Patterns of cortical lateralisation in dyslexic children

The effects of several mu and kappa opioid receptor agonists on urinary excretion were examined in the water-loaded rat. Mu agonists induced antidiuresis whereas kappa agonists caused diuresis. Furthermore, high efficacy kappa agonists eg. U50,488H and tifluadom, produced marked diuretic effects, whereas kappa partial agonists eg. nalorphine, produced low maximum diuresis. Compounds having activity at both mu and kappa receptors eg. ethylketocyclazocine(EKC) and MR2034, produced a biphasic effect, an initial anti-diuretic effect followed by a more sustained diuretic effect. The irreversible mu receptor antagonist, β-funaltrexamine (β-FNA) antagonized the anti-diuretic effects elicited by fentanyl, pentazocine and buprenorphine and the initial anti-diuretic effect induced by EKC but did not inhibit the diuretic effects induced by U50,488H and EKC. Thus, the use of β-FNA in the water-loaded rat provides us with a valuable in vivo test which differentiates both mixed and selective opioid receptor agonists.

Differential effects of systemically administered nor-binaltorphimine (nor-BNI) on kappa-opioid agonists in the mouse writhing assay.

The opioid antagonist effects of systemically administered nor-binaltorphimine (nor-BNI) were evaluated against the kappa agonists CI-977, U69,593, U50,488, ethylketocyclazocine (EKC), Mr2034 and bremazocine, the mu agonist morphine and the alkaloid delta agonist BW-373U86 in the acetic acid-induced writhing assay in mice. All eight agonists completely and dose-dependently inhibited writhing. Antagonism of CI-977 was apparent 1 h after administration of 32 mg/kg nor-BNI, peaking after 4 h and was maintained for at least 4 weeks; no antagonist effects of nor-BNI were apparent after 8 weeks. Nor-BNI (32 mg/kg) caused little or no antagonism of morphine or BW-373U86 at 1 h and none at 24 h after nor-BNI administration. Subsequently, dose-effect curves for CI-977, U50,488, U69,593, EKC, Mr2034 and bremazocine were determined 24 h after pretreatment with 3.2, 10 and 32 mg/kg nor-BNI. Pretreatment with 3.2 mg/kg nor-BNI produced significant antagonism of all six kappa agonists, suggesting that their antinociceptive effects were mediated at least in part by nor-BNI-sensitive kappa receptors. At higher doses, nor-BNI dose-dependently shifted the agonist dose-effect curves of CI-977, U50,488, U69,593 and bremazocine, but not those of EKC and Mr2034, suggesting that the latter compounds may be producing effects via nor-BNI-insensitive receptors. Mu receptor involvement was demonstrated following a 24 h pretreatment with 32 mg/kg beta-FNA in combination with nor-BNI, which significantly increased the degree of antagonism of Mr2034 and EKC from that seen with nor-BNI alone.2+ off