Chronic ethanol imbibition interferes with δ-, but not with μ-opiate receptors

Treatment of rats with ethanol for 3 weeks resulted in a significant increase in δ-opiate receptor binding whereas no change in μ-opiate receptor binding was observed.

Scatchard analysis showed an increase in δ-receptor affinity without a change in the receptor density. Acute treatment with ethanol did not alter receptor characteristics. The data provide evidence that δ- and μ-opiate receptor sites can be differentially modulated in vivo.     

Role of spinal κ opioid receptors in the blockade of the development of antinociceptive tolerance to morphine

The site of action of the k opioid receptor agonist, U-50,488H in suppressing the development of tolerance to morphine antinociception was examined by local application, either intrathecal (i.t., spinal) or intracerebroventricular (i.c.v., supraspinal) in mice. Mice given morphine s.c., i.c.v. or i.t. daily developed toleunce regardless of the route. Co-administration of U-50.488H i.p. at a subanalgcsic dose suppressed the development of tolerance to s.c and i.t. administered morphine without affecting the antinociceptive effect of morphine. U-50.488K did not influence the development of tolerance to i.c.v. administered morphine. The antinociceptive effect of s.c. administered morphine was not affected by co-administration of U-50,488H given i.t. or i.c.v.: however, the development of tolerance to morphine was suppressed by i.t. but not i.c.v. administered U-50.488H. The suppressive effect of U-50.488H on the development of tolerance to morphine was abolished by pretreatment with nor-binaltorphiminc (nor-BNI) given i.p. or i.t. Intraccrebroventricularly administered nor-BNI failed to abolish the effect of U-50.488H. We suggest that U-50.488H suppresses the development of tolerance to morphine at the spinal level by interacting with κ opioid receptors in this area.     

Response of rat globus pallidus neurons to microiontophoretically applied μ and κ opioid receptor agonists

The effects of the microiontophoretic application of dynorphin A-(1–13) (DYN 13) and the benzomorphans ethylketocyclazocine (EKC), bremazocine and MRZ 2549, (κ) opioid agonists, and of morphine and morphiceptin, (μ) opioid agonists, were compared on spontaneous or glutamate-evoked discharge of globus pallidus (GP) neurons in rat. Our results demonstrate that μ and κ opioid agonists are able to depress the excitability of pallidal neurons, possibly by interacting with μ and κ opioid receptor subtypes, respectively. In addition, the μ agonists and dynorphin A-(1–13), but not the benzomorphans, enhanced the excitability of a number of pallidal neurons. We have proposed a presynaptic site as the basis for this opioid-induced excitation, possibly also mediated by a μ opioid receptor. The selectivity of dynorphin A-(1–13) for benzomorphan κ opioid receptors in the rat GP appears to be low and dynorphin A-(1–13) may elicit effects that are different from those produced by the benzomorphan κ agonists by virtue of its ability to interact with other opioid receptor subtypes, for example μ opioid receptors.     

κ-Agonist analogesics: Evidence for μ2 and δ opioid receptor antagonism

Despite a unique in vivo pharmacology, K agonists possess moderate to high affinities for μ, δ, and κ binding sites in vitro. By monitoring the antagonist activity of κ agents on morphine- and DADLE-dependent changes in dopamine and acetylcholine metabolism we were able to demonstrate a specific μ₂ and δ receptor antagonism for κ analgesics. Furthermore, this antagonism was probably directly at the receptor level and not physiological.     

FR143166 attenuates spinal pain transmission through activation of the serotonergic system

We investigated the antinociceptive effect of 1-(4-fluorophenyl)-3-methyl-5-[4-(methylsulfinyl)phenyl]pyrazole (FR143166) in the tail-pinch test in mice. The p.o. and i.t. injection of FR143166 exerted dose-dependent antinociceptive actions with ED(50) values of 24 mg/kg and 15 micro g/mouse, respectively. However, i.c.v. injection of FR143166 at a maximum dose of 128 micro g/mouse did not show any antinociceptive effect. The antinociceptive effect of FR143166 injected i.t. was abolished by co-administration of the nonselective serotonin (5-hydroxytryptamine, 5-HT) receptor antagonist, methysergide, but not by the adrenoceptor antagonists, phentolamine and propranolol. Moreover, the effect of FR143166 was also reversed by the 5-HT(2A) receptor antagonist, ketanserin, and the 5-HT(3) receptor antagonist, MDL-72222 (3-tropanyl-3,5-dichlorobenzoate). The effect of FR143166 was attenuated by p-chlorophenylalanine, but not by 6-hydroxydopamine plus nomifensine pretreatment. These results suggest that the descending serotonergic system, especially spinal 5-HT(2A) and 5-HT(3) receptors, is involved in the antinociceptive activity of spinally administered FR143166 on noxious mechanical stimuli.     

Multiple opiate receptors: Differential binding of μ, κ and δ agonists

Much data has accumulated suggesting that μ and δ opiate receptors represent distinct topographical units within the CNS. In addition, in vitro binding studies by the present authors also suggest that a unique κ receptor may be present in the brain. In this regard, [³H]ethylketocyclazocine binding to membrane preparations of rat brain was found to be potently displaced by κ partial μ and agonist/antagonist analgesics. However, μ, δ, σ and ε receptor agonists were much less active at this binding site, suggesting that the κ site possesses a different pharmacology from μ and δ sites. These data therefore support previous in vivo experiments which demonstrated a unique behavioral pharmacology and CNS distribution for κ-mediated analgesia.     

Effects of intracerebroventricular injection of opioid peptides selective for μ, δ and κ receptors on discriminative stimulus properties of pentazocine in the rat

The present study was designed to investigate the effects of centrally administered morphine and opioid peptides on the discriminative stimulus properties of pentazocine in the rat. Rats were trained to discriminate 3 mg/kg (s.c.) of pentazocine from vehicle in a shock avoidance paradigm. A 3 mg/kg (s.c.) dose of pentazocine produced stimulus effects in common with those induced by a training dose (3 mg/kg, s.c.) of pentazocine. Morphine (0·1–3 μg, i.c.v.) produced a dose-dependent increase in responding appropriate for pentazocine lever. The μ-selective opioid receptor agonist [D-Ala²,NMePhe⁴,Gly-ol] enkephalin (DAMGO) (0·0003–0·03 μg, i.c.v.) generalized to pentazocine cue. d -Pen², l -Pen⁵] enkephalin (DPLPE) (3 and 10 μg, i.c.v.), a δ-selective opioid receptor agonist, produced partial generalization to pentazocine cue. However, the κ-selective opioid receptor agonist dynorphin A-(1–13) (3 and 10 μg, i.c.v.) did not generalize to pentazocine cue. The pentazocine-like discriminative stimulus effects of morphine (3 μg, i.c.v.) and DAMGO (0·03 μg, i.c.v.) were fully reversed by intracerebroventricular injection of the μ-selective opioid receptor antagonist β-funaltrexamine (5 μg, i.c.v.). These results suggest that μ-opioid receptors play a major role in the discriminative stimulus effects of pentazocine, while δ-opioid receptors only partially contribute to them. © 1998 John Wiley & Sons, Ltd.     

Antinociceptive activity of N-(4-hydroxyphenacetyl)-4-aminoclonidine, a novel analog of clonidine: role of opioid receptors and alpha-adrenoceptors

N-(4-hydroxyphenacetyl)-4-aminoclonidine, a derivative of the alpha-adrenoceptor agonist p-aminoclonidine, was found to exhibit dose-dependent antinociceptive activity in the mouse writhing assay. In this measure of antinociceptive activity it was less potent than clonidine or xylazine. Naloxone, an opioid receptor antagonist, at a dose sufficient to abolish the antinociceptive activity of morphine, did not affect the antinociceptive activity of N-(4-hydroxyphenacetyl)-4-aminoclonidine, clonidine or xylazine. In contrast, yohimbine, a alpha-adrenoceptor antagonist, reduced the antinociceptive activity of N-(4-hydroxyphenacetyl)-4-aminoclonidine, clonidine and xylazine, but not morphine. The affinity of N-(4-hydroxyphenacetyl)-4-aminoclonidine, clonidine and xylazine for alpha-adrenoceptors in rat aorta was correlated highly with the relative potency for writhing inhibition. These results suggest that the antinociceptive activity of N-(4-hydroxyphenacetyl)-4-aminoclonidine is mediated by alpha-adrenoceptors.     

Down-regulation of brain and spinal cord μ-opiate receptors in morphine tolerant-dependent rats

The effect of chronic administration of morphine and its withdrawal on the characteristics of μ-opiate receptors was determined in male Sprague-Dawley rats. The ligand used for characterizing the receptors was [³H][D-Ala², MePhe⁴,Gly⁵-ol]enkephalin ([³H]DAMGO). Rats were implanted s.c. under light ether anesthesia with six morphine pellets (each containing 75 mg of morphine free base). Rats serving as controls were implanted similarly with placebo pellets. Two sets of animals were used. In one group of rats, the pellets were left intact (tolerant-dependent) at the time of killing and in the other the pellets had been removed 18 h earlier (abstinent). The spinal cord and brain regions (amygdala, hippocampus, hypothalamus, corpus striatum, midbrain, pons and medulla, and cortex) were dissected. In morphine-abstinent rats, the binding of ligands of μ-opiate receptors to membranes of spinal cord and brain regions did not change. In non-abstinent morphine-tolerant-dependent rats, the binding of [³H]DAMGO to membranes of spinal cord, pons and medulla, and cerebral cortex was found to be decreased. These changes were due to decreases in the B_max values rather than K_d values for the binding of [³H]DAMGO. The results clearly indicate that morphine-induced tolerance-dependence in the rat is associated with changes in the selected brain regions and spinal cord with μ-opiate receptors being down-regulated in spinal cord, pons and medulla, and cerebral cortex. It is concluded that tolerance to morphine in the rats may be due to down-regulation of central μ-opiate receptors. However, μ-opiate receptors are unaffected in morphine abstinence.     

A chimeric opioid peptide with mixed μ agonist/δ antagonist properties

Abstract: There is evidence to indicate that opioid compounds with mixed μ agonist/δ antagonist properties are analgesics with low propensity to produce tolerance and physical dependence. A chimeric peptide containing the potent and selective μ agonist H‐Dmt‐D‐Arg‐Phe‐Lys‐NH₂ ([Dmt¹]DALDA) (Dmt = 2′,6′‐dimethyltyrosine) and the potent and selective δ antagonist H‐Tyr‐TicΨ[CH₂‐NH]Cha‐Phe‐OH (TICP[Ψ]) (Cha = cyclohexylalanine), connected ‘tail‐to‐tail’ via a short linker, was synthesized using a combination of solid‐phase and solution techniques. The resulting peptide, H‐Dmt→D‐Arg→Phe→Lys‐NH‐CH₂‐CH₂‐NH‐Phe←Cha[NH‐CH₂]ΨTic←Tyr‐H, showed the expected μ agonist/δ antagonist profile in the guinea‐pig ileum and mouse vas deferens assays. Its μ and δ receptor binding affinities were in the low nanomolar range, as determined in rat brain membrane binding assays.     

Synthesis and binding properties of specific photoaffinity ligands for μ and δ opioid receptor subtypes

We report the synthesis and binding properties of specific photoaffinity ligands for μ and δ opioid receptor subtypes. These ligands are derived from DAGO: Tyr-D-Ala-Gly-NMePhe-Gly-ol, a μ selective probe and DTLET: Tyr-D-Thr-Gly-Phe-Leu-Thr, a δ selective probe by modifying the Phe 4 residue. These modifications are: i) a nitro group on the para position of Phe ring as Phe(4 NO₂) or Nip, ii) an azido group as Phe(4 N₃) or AZ. Pharmacological responses on mouse vas deferens (δ sites) and guinea pig ileum (μ sites), as well as competition experiments with [³H] DAGO and [³H] DTLET on crude rat brain membranes have been performed. The nitro group on the phenyl ring of the Phe residue preserves the affinity and selectivity of each probe: NipDAGO for the μ sites, NipDTLET for the δ ones. However the nitro probes do not appear to be photo-activable by u.v. irradiation. Likewise, azidation of the phenyl ring of the Phe residue does not change the receptor selectivity of each probe, but AZDAGO has less affinity than its parent molecule DAGO, while AZDTLET has more affinity than DTLET. These compounds are photoactivable and provide an efficient tool to characterize and isolate the different receptor subtypes, especially the δ site.     

Analogues of β-LPH61–64 posessing selective agonist activity at μ-opiate receptors

Peptides based on the stabilised tetrapeptide HTyr-D-Ala-Gly-MePheOH have been synthesised and shown to have substantial opioid activity both in vitro and in vitro. The selectivity of these compounds for different receptor populations has been investigated using both isolated tissue assays and binding studies. Results suggest that the compounds are potent agonists at μ-receptors with little or no affinity for the δ-receptor population. One of the compounds, RX783006 (HTyr-D-Ala-Gly-MePhe-NH(CH₂)₂OH), has been tritiated to high specific radioactivity and may prove to be a useful probe in the elucidation of the function of the heterogenous opiate receptor population.     

Opioid receptor types and antinociceptive activity in chronic inflammation: bothe κ- and μ-opiate agonistic effects are enhanced in arthritic rats

The antinociceptive effects obtained in arthritic rats with morphine, the opioid μ-agonist DAGO 9D-Al²,MePhe⁴,Gly-ol⁵ol⁵]enkephalin, the δ-selective agonist DTLET [D-Thr²,Leu⁵]enkephalyl-Thr, and the κ-agonist U-50,488H were compared to their corresponding effects in normal animals and morphine-pretreated arthritic rats, respectively, using a paw pressure test. The effects of the μ- and κ-agonists were increased in arthritic rats. While morphine-treated rats were cross-tolerant to the μ- and κ-agonists, no tolerance to the δ-selective agonist was found. The possibility that the potent action of morphine on this model for chronic inflammatory pain is mediated partly through κ-mechanisms is discussed.     

Spectrum of the μ-, δ- and κ-binding sites in homogenates of rat brain

1 In homogenates of rat brain, the binding characteristics of tritiated opiates and opioid peptides were examined and the relative capacities of μ-, δ- and κ-binding sites of the opiate receptor determined by saturation analysis.

2 In competition experiments, binding of the selective μ-ligand [³H]-[D-Ala²,MePhe⁴,Gly-ol⁵]enkephalin at the μ-site was displaced by [D-Ala²,D-Leu⁵]enkephalin with rather low affinity (K_I = 12.6 nM) and more readily by the ketazocine-like compounds (-)-ethylketazocine (K_I = 3.1 nM) and (-)-bremazocine (K_I = 0.32 nM), which also displaced the binding of [³H]-[D-Ala²,D-Leu⁵]enkephalin from the δ-site. In contrast, the binding to the κ-site was easily displaced by ethylketazocine (1.0 nM) and bremazocine (0.37 nM) but not by the μ-ligand [D-Ala²,MePhe⁴,Gly-ol⁵]enkephalin (K_I = 2000-3000 nM) or the δ-ligand [D-Ala²,D-Leu⁵]enkephalin (K_I > 20,000 nM).

3 The dissociation equilibrium constant (K_D) and the binding capacity (pmol/g) of the μ-binding site were determined with the selective μ-ligand [³H]-[D-Ala²,MePhe⁴,Gly-ol⁵]enkephalin. For the δ-site, [³H]-[D-Ala²,D-Leu⁵]enkephalin was used in the presence of unlabelled [D-Ala²,MePhe⁴,Gly-ol⁵]enkephalin in order to suppress cross-reactivity to the μ-binding site. For the estimation of κ-binding, [³H]-(±)-ethylketazocine or [³H]-(-)-bremazocine were used in the presence of unlabelled μ- and δ-ligands for the suppression of cross-reactivities to the μ- and δ-binding sites.

4 In rat brain the capacity of the μ-binding site was 7.3 pmol/g brain, that of the δ-binding site 6.7 pmol/g brain and that of the κ-binding site 2.0 pmol/g brain. Thus, the κ-binding site had the lowest value whereas in the guinea-pig brain the capacity of the μ-binding site was lower than that of the δ- or κ-binding site.

     

Syntheses of Novel Pyridazinomorphinans by Inverse Electron Demand Cycloaddition and their Binding to μ and κ Receptors

A number of novel pyridazinomorphinans have been synthesized by the inverse electron demand Diels-Alder reaction of various 3,6-disubstituted 1,2,4,5-tetrazines with enamines derived from dihydrocodeinone and with codeinone. Reduction of some of the pyridazinomorphinans did not furnish the expected pyrroloepoxymorphinans; in all cases investigated reductive cleavage of the epoxybridge was observed to yield dihydropyridazino- or pyrrolomorphinans. The structures of all new compounds were assigned by the spectral data, that of the cycloadduct of codeinone was additionally verified by X-ray crystallography. Compounds 5a, 8, 11a , and 16 have been evaluated for their affinity at μ and κ opioid receptors in radioligand binding assays. Their ability to inhibit [³H]DAMGO binding at μ and [³H]U 69.593 binding at κ receptors, respectively as compared to codeine has been found to be lower.     

Separation of δ-, γ- and α-tocopherols by CEC

In this study capillary electrochromatography (CEC) was used for the separation of three tocopherols (TOHs), namely delta-, gamma- and alpha-TOH and the antioxidant compound, butylated hydroxytoluene (BHT). The CEC experiments were carried out using an octadecylsilica (ODS) stationary phase packed, in our laboratory, in a fused-silica capillary (100 microm I.D., 365 microm O.D. x 33 cm of total length and 24.6 or 8.4 cm effective length). The mobile phase was composed by a mixture of methanol (MeOH) and acetonitrile (ACN), at different concentrations and 0.01% (w/v) of ammonium acetate. Retention time (t(R)), retention factor (k), resolution (R(s)) of the three TOHs were strongly influenced by the organic solvent composition of the run buffer and by the effective length of the capillary. Optimum experimental conditions were found even employing the short effective length of the capillary achieving the baseline separation of the studied analytes in a relatively short time (less than 5 min). The optimized method was applied to the qualitative analysis of vitamin E (alpha-TOH) present in a human serum extract.     

Antinociceptive evaluation of 14β-(bromoacetamido)- 7,8-dihydro-N(cyclopropylmethyl)-normorphinone in mice

This study evaluated the supraspinal opioid effects of 14β-(bromoacetamido)-7,8-dihydro-N(cyclopropylmethyl)-normorphinone (N-CPM-H₂BAMO) in the mouse acetic acid-induced writhing and tail-flick assays. In the writhing test, N-CPM-H₂BAMO produced a time- and dose-dependent antinociception after i.c.v. administration, with a 50% antinociceptive response being obtained with 0.28 (0.19–0.39) nmol when given 10 min before testing. The antinociceptive effect of N-CPM-H₂BAMO was antagonized in a dose-dependent manner by the κ-selective opioid receptor antagonist, nor-binaltorphimine. In the mouse tail-flick assay, N-CPM-H₂BAMO failed to produce any antinociception after i.c.v. administration. N-CPM-H₂BAMO produced a dose-dependent antagonism of morphine-induced antinociception but not antinociception induced by the δ-opioid receptor agonist [D-Pen²,D-Pen⁵]enkephalin. Nor-binaltorphimine (0.3 nmol) at dose that completely antagonized N-CPM-H₂BAMO-induced antinociception in the writhing assay did not prevent the antagonistic effect of N-CPM-H₂BAMO on morphine-induced antinociception. Therefore, these data indicate that N-CPM-H₂BAMO produces antinociception by acting at supraspinal κ-opioid receptors in the writhing assay, and also acts as a μ-opioid receptor antagonist.     

14β-Chlorocinnamoylamino derivatives of metopon: long-term μ-opioid receptor antagonists

The affinity, selectivity and antinociceptive properties of 5β-methyl-14β-(p-chlorocinnamoylamino)-7,8-dihydromorphinone (MET-Cl-CAMO) and N-cyclopropyl-methyl-5β-methyl-14β-(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][

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-Ala²,(Me)-Phe⁴,Gly(ol)⁵]enkephalin (DAMGO) with IC₅₀ values of less than 2 nM. Preincubation of membranes with MET-Cl-CAMO and N-CPM-MET-Cl-CAMO produced a concentration-dependent, wash-resistant inhibition of μ-opioid receptor binding. Saturation binding experiments with N-CPM-MET-Cl-CAMO showed a reduction in the number of μ-opioid binding sites without a change in affinity. In the mouse 55°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 δ- or κ-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, μ-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 μ-opioid receptor.     

Molecular modeling of salsolinol,a full Gi protein agonist of the μ‐opioid receptor,within the receptor binding site

(R/S)‐Salsolinol is a full agonist of the μ‐opioid receptor (μOR) G_i protein pathway via its (S)‐enantiomer and is functionally selective as it does not promote β‐arrestin recruitment. Compared to (S)‐salsolinol, the (R)‐enantiomer is a less potent agonist of the G_i protein pathway. We have now studied the interactions of the salsolinol enantiomers docked in the binding pocket of the μOR to determine the molecular interactions that promote enantiomeric specificity and functional selectivity of (R/S)‐salsolinol. Molecular dynamics simulations showed that (S)‐salsolinol interacted with 8 of the 11 residues of the μOR binding site, enough to stabilize the molecule. (R)‐Salsolinol showed higher mobility with fewer prevalent bonds. Hence, the methyl group bound to the (S)‐stereogenic center promoted more favorable interactions in the μOR binding site than in the (R)‐orientation. Because (S)‐salsolinol is a small molecule (179.2 Da), it did not interact with residues implicated in the binding of larger morphinan agonists that are located toward the extracellular portion of the binding pocket: W318^7.35, I322^7.39, and Y326^7.43. Our results suggest that contact with residues which (S)‐salsolinol interacts with are enough to elicit G_i protein activation, and possibly define a minimum set required by μOR ligands to promote activation of the G_i protein pathway.     

Lack of significant changes in μ, δ opioid binding sites and neutral endopeptidase EC 3.4.24.11 in the brain and spinal cord of arthritic rats

The possible changes in neutral endopeptidase EC 3.4.24.11 (“enkephalinase”, NEP), μ and δ opioid binding sites, were investigated using in vitro quantitative radioautography in various regions of the central nervous system of the Freund's adjuvant-induced arthritic rat, a model of chronic pain. Enkephalinase was labelled by a specific tritiated inhibitor, [³H]N-[(2RS)-3-hydroxyaminocarbonyl-2-benzyl-l-oxopropyl]glycine ([³H]HACBO-Gly), while μ and δ opioid binding sites were selectively labelled with [³H]Tyr- -Ala-Gly-(Me)Phe-Gly-ol ([³H]DAGO) and [³H]Tyr- -Thr-Gly-Phe-Leu-Thr ([³H]DTLET), respectively. As compared to controls, no significant modifications were found in NEP, μ or δ binding sites at both supraspinal and spinal levels of arthritic rats. These results suggest that the enhanced efficiency of exogenous opioids or endogenous enkephalins, reported to occur in this model of chronic inflammatory pain, are not directly related to changes in μ and δ opioid binding sites or steady state levels of NEP.