Supraspinal μ2-opioid receptors mediate spinal/supraspinal morphine synergy

TRIMU-5 (Tyr-D-Ala-Gly-NHC₂H₄CH(CH₃)₂) is a potent μ₂-opioid agonist/μ₁-opioid antagonist. A supraspinal dose (0.5 μg i.c.v.) of TRIMU-5 which is not analgesic when given alone antagonizes the analgesia produced by intracerebroventricular (i.c.v.) morphine, a μ₁ action. In contrast, in a synergy model consisting of the simultaneous administration of intrathecal morphine (0.1 μg) with multiple doses of i.c.v. morphine, the same supraspinal TRIMU-5 dose (0.5 μg i.c.v.) enhances analgesia. Supraspinal TRIMU-5 also potentiates spinal morphine directly, shifting its dose-response to the left. These results imply that within the brainstem μ₁ receptors mediate supraspinal analgesia while μ₂ receptors mediate the synergy with spinal μ systems.     

Involvement of mu1-opioid receptor on oxycodone-induced antinociception in diabetic mice

The effect of naloxonazine, a selective μ₁-opioid receptor antagonist, on oxycodone-induced antinociception was examined in streptozotocin-induced diabetic mice. Oxycodone (5 mg/kg, s.c.) induced significant antinociception in both non-diabetic and diabetic mice. This antinociceptive effect of oxycodone was completely antagonized by pretreatment with naloxonazine (35 mg/kg, s.c.) in both non-diabetic and diabetic mice. The selective κ-opioid receptor antagonist nor-binaltorphimine (20 mg/kg, s.c.) also antagonized oxycodone-induced antinociception in diabetic mice, but only had a partial effect in non-diabetic mice. These results suggest that although primarily interacts with μ₁-opioid receptor, κ-opioid receptors are also strongly involved in oxycodone-induced antinociception.     

Involvement of spinal mu1-opioid receptors on the Tyr-d-Arg-Phe-sarcosine-induced antinociception

The involvement of spinal mu-opioid receptor subtypes on the antinociception induced by i.t.-administered Tyr-D-Arg-Phe-sarcosine (TAPS), a N-terminal tetrapeptide analog of dermorphin, was determined in mice tail-flick test. Intrathecal administration of TAPS produced the marked inhibition of the tail-flick response in a dose-dependent manner. The antinociception induced by TAPS was completely eliminated by i.t.-co-administration of Tyr-D-Pro-Phe-Phe-NH2 (D-Pro2-endomorphin-2), the mu1-opioid receptor antagonist, whereas i.t. co-treatment with Tyr-D-Pro-Trp-Phe-NH2 (D-Pro2-endomorphin-1) or Tyr-D-Pro-Trp-Gly-NH2 (D-Pro2-Tyr-W-MIF-1), the mu2-opioid receptor antagonists, did not affect the TAPS-induced antinociception. In contrast, the antinociception induced by i.t.-administered [D-Ala2,N-MePhe4,Gly-ol5]enkephalin was significantly attenuated by i.t.-co-administration of D-Pro2-endomorphin-1 or D-Pro2-Tyr-W-MIF-1, but not D-Pro2-endomorphin-2. These results suggest that TAPS may stimulate spinal mu1-opioid receptors to produce the antinociception.     

Possible involvement of the locus coeruleus in inhibition by prostanoid EP(3) receptor-selective agonists of morphine withdrawal syndrome in rats

We examined the mechanism of the inhibitory effect of prostanoid EP₃ receptor agonists on naloxone-precipitated withdrawal syndrome in morphine-dependent rats. Rats were rendered morphine dependent by subcutaneous (s.c.) implantation of two pellets containing 75 mg morphine for 5 days. Morphine withdrawal syndrome was precipitated by i.p. injection of naloxone (3 mg/kg). Intracerebroventricular (i.c.v.) administration of (±)-15-hydroxy-9-oxo-16-phenoxy-17,18,19,20-tetranorprost-13-trans-enoic acid (M&B28,767: prostanoid EP₃ receptor agonist) or sulprostone (prostanoid EP₁/EP₃ receptor agonist) significantly suppressed many withdrawal signs. Northern blotting and in situ hybridization studies revealed that i.c.v. administration of M&B28,767 (1 pg/rat) attenuated the elevation of c-fos mRNA during naloxone-precipitated withdrawal in many brain regions, including the cerebral cortex, thalamus, hypothalamus and locus coeruleus. Double in situ hybridization analysis revealed that in the locus coeruleus most of the tyrosine hydroxylase mRNA-positive neurons expressed μ-opioid receptor mRNA and more than half of these neurons were positive for prostanoid EP₃ receptor mRNA. These results indicate that the suppression by prostanoid EP₃ receptor agonists of naloxone-precipitated morphine withdrawal syndrome can be attributed to the inhibition of neuronal activity in several brain regions, including the locus coeruleus, the largest source of central noradrenergic neurons.     

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.     

Opioid receptor-mediated inhibition of evoked catecholamine release from cultured neurons of rat ventral mesencephalon and locus coeruleus

The effects of selective opioid agonists on the evoked release of [³H]dopamine and [³H]noradrenaline were studied in cultured dopaminergic neurons of the ventral mesencephalon (containing the substantia nigra and ventral tegmental area) and in cultured neurons of the noradrenergic locus coeruleus, respectively. The cultures were prepared from embroyonic day 15 rat brains. After 9 days in culture, the calcium-dependent release of [³H]dopamine from dopaminergic substantia nigra/ventral tegmental aera neurons induced by 23 mM k⁺ appeared to be inhibited exclusively by activation of κ-opioid receptors, as [³H]dopamine release was inhibited selectively by the κ- agonists U69,593 and dynorphin-(1–13) (EC₅₀ 8 and 5 nM, respectively), and this inhibitory effect was antagonized by the κ-selective antagonist nor-binaltorphine (K_i 0.07 nM). In contrast, cultured noradrenergic locus coeruleus neurons appeared to contain release-inhibitory μ-opioid receptors only, as evoked [³H]noradrenaline release was inhibited selectively by the μ agonist [D-Ala², MePhe⁴, Gly-ol⁵]enkephalin (EC₅₀ 45 nM), a response that was antagonized by the preferential μ antagonist naloxone (K_i = 0.7 nM). The δ-opioid receptor agonist [D-Ser²(O-butyl), Leu⁵]enkephaly-Thr⁶ did not affect catecholamine release. Dopamine release from cultured ventral mesencephalic neurons, induced by 100 μM N-Methyl-D-Aspartate (NMDA), also appeared to be subject to κ receptor-mediated inhibition, whereas NMDA-induced noradrenaline release from cultured locus coeruleus neurons was under the inhibitor control of μ receptors. It is therefore concluded that in rat brain neurotransmitter release from dopaminergic and noradrenergic neurons, originating from the substantia nigra/vental tegmental area and the locus coeruleus, is liable to inhibition by homogenous populations of κ- and μ-opioid receptors, respectively, independent of the input of non-opioid neurons from distict nuclei.     

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.     

Chronic intrathecal morphine treatment does not cause down-regulation of spinal adenosine A1 receptors in rats

We have shown previously that systemic chronic morphine treatment causes down-regulation of spinal adenosine A₁ receptors in rats. Recently, we have found that chronic supraspinal morphine treatment also causes this effect. In the present study, we investigated whether chronic spinal morphine treatment has the same effect of down-regulation of spinal adenosine A₁ receptors. Adult male Sprague-Dawley rats were rendered tolerant to morphine either by multiple intrathecal (i.t.) injections or continuous Lt. infusion by osmotic pump administration for 2 or 4 days. Spinal A₁-adenosine receptor binding activity was measured by using the selective A₁ adenosine agonist [³H]cyclohexyladenosine. No significant decrease in [³H]cyclohexyladenosine binding was found in the spinal cord after 2 or 4 days of multiple Lt. injections of morphine. There was also no significant change in the amount of spinal [³H]cyclohexyladenosine bound after 4 days of continuous Lt. infusion of morphine by osmotic pump. From these and our previous results, it is concluded that only supraspinal chronic morphine treatment down regulates the spinal A₁ adenosine receptor and this may play a role in the mechanism of supraspinal morphine tolerance but not spinal morphine tolerance.     

Development and validation of a radioreceptor assay for the determination of morphine and its active metabolites in serum

This article describes the development and validation of a radioreceptor assay for the determination of morphine and morphine-6-β-glucuronide (M6G) in serum. The assay is based on competitive inhibition of the μ-opioid-selective radiolabeled ligand [³H]-DAMGO by opioid ligands (e.g. M6G) for binding to the striatal opioid receptor. The assay has been validated according to the Washington Conference Report on Analytical Method Validation. The radioreceptor assay can be performed in serum without prior pre-treatment of the sample. Direct addition of the sample results in no significant loss in maximal binding sites, and therefore, no loss in sensitivity. The assay proves to be selective for a multitude of opioid agonists and antagonists (e.g. morphine IC₅₀ = 4.1 nM and M6G IC₅₀ = 12.8 nM). Moreover, morphine-3-glucuronide (M3G) displays a low affinity (IC₅₀ = 1100 nM) for the μ-opioid receptor and according to the literature demonstrates no analgesic activity. This makes discrimination, in relation to the analgesic effect, of the two metabolites of morphine possible. The assay is fast (assay time <4 h, analysis 5 min/sample), easy and the sensitivity (limit of detection (LOD) = 1.6 nM M6G-equivalents) is such that very potent agonists, like morphine and M6G, can be measured at the desired serum levels. The assay is accurate (<18%), but precision is limited if measured over several days (>35%). The assay is most accurate and precise if measured over a range from 3.5 to 40 nM M6G-equivalents. Based on the limited inter-assay precision, we propose to use this receptor assay mainly as a screening tool for neonates treated with morphine.     

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.     

Peripheral κ1-opioid receptor-mediated analgesia in mice

When injected directly into the tail, U50,488H is a potent analgesic in the tailflick assay (ED₅₀ 3.1 μg). The analgesic activity is lost if the radiant heat is focused 1 cm away from the site of injection. The κ₁-opioid receptor antagonist nor-binaltorphimine given systemically reverses the local analgesic response of U50,488H, but the antagonist is 100-fold more potent when injected directly into the tail. Intrathecal antisense treatment with a probe targeting the mRNA encoding the κ₁-opioid receptor blocks the local analgesic actions of U50,488H in the tail, suggesting that U50,488H is acting on dorsal ganglia neurons.     

Role of spinal and supraspinal muscarinic receptors in the expression of morphine withdrawal symptoms in the rat

Previous studies in this laboratory have demonstrated that prior intracerebroventricular (i.c.v.) administration of the muscarinic antagonist, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) in morphine dependent rats significantly attenuates the development of cardiovascular and certain behavioral responses precipitated by the opiate antagonist, naloxone. The purpose of this study was to determine whether both supraspinal and spinal cholinergic neurons are involved in the expression of withdrawal symptoms. Employing localized (i.c.v. or intrathecal, i.t.) infusions of muscarinic antagonists, it was determined that a significant antiwithdrawal action could be produced through both an inhibition of supraspinal and spinal cholinergic neurons. Pharmacological differences emerged regarding the antiwithdrawal potential of 4-DAMP and the partially M₁ selective antagonist, pirenzepine. While our previous studies had revealed that pirenzepine had essentially no antiwithdrawal activity when administered by the i.c.v. route, in the present study, pirenzepine evoked a marked antiwithdrawal action by the i.t. route, significantly inhibiting both cardiovascular and behavioral signs of withdrawal. In contrast, 4-DAMP which was effective by the i.c.v. route (especially for the cardiovascular symptoms), elicited no antiwithdrawal action by the i.t. route. As a muscarinic antagonist (ability to block the presser response to central injection of carbachol) 4-DAMP was equally active by i.c.v. or i.t. injection. However, pirenzepine was clearly more effective in this regard by the i.t. route. These results are consistent with ability of muscarinic antagonists to offer significant anti-morphine withdrawal activity at both supraspinal and spinal locations. They also suggest that different muscarinic systems, possibly different receptor subtypes, mediate the expression of morphine withdrawal symptoms within the two regions of the CNS.     

Binding of [H](+)-BW373U86 to δ-opioid receptors in rat brain membranes

A tritiated form of the non-peptidic δ-opioid receptor agonist (+)-BW373U86 ((+)-4-((-R)--((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-hydroxybenzyl)-N,N-diethylbenzamide) was synthesized and its binding characteristics studied. [³H](+)-BW373U86 bound with subnanomolar affinity to rat brain membranes and was displaced most effectively by ligands selective for δ-opioid receptors. Naltrindole, naltriben, and 7-benzylidenenaltrexone exhibited apparent inhibition constants of 0.06, 1.54, and 4.49 nM, respectively, while μ- or κ-selective ligands showed little affinity for this site. [³H](+)-BW373U86 binding was sensitive to the presence of guanine nucleotides; GDP caused a 3-fold decrease and 5′-guanylyl-imidodiphosphate (Gpp[NH]p) caused a 25% increase in binding affinity.     

Regional reward differences within the ventral pallidum are revealed by microinjections of a mu opiate receptor agonist

The ventral pallidum receives a major projection from the nucleus accumbens, a heavily studied terminus of the mesolimbic dopamine system that is known to be involved in a variety of reward and behavioral functions. Recently, ventral pallidum microinjections of the μ opiate receptor agonist Tyr- -Ala-Gly-NMe-Phe-Gly-ol-enkephalin (DAMGO) have been shown to increase motor activity while ventral pallidum lesions have been shown to reduce opiate and cocaine self-administration behaviors. These results suggest a possible continuation of the mesolimbic reward/motor circuit from the nucleus accumbens into the ventral pallidum. This study investigated the effects of ventral pallidum DAMGO microinjections on reward and motor/performance through the use of the intracranial self-stimulation rate-frequency curve-shift paradigm. Microinjections of DAMGO (vehicle, 0.03 nmol, and 0.33 nmol) were administered bilaterally in a random dose order with a minimum of 3 days between injections. Rats were tested over three consecutive rate-frequency curves immediately following the opiate microinjections to investigate the time course of drug effects. DAMGO microinjections in the rostral ventral pallidum produced decreases in reward and motor/performance when compared to normal baseline activity or vehicle microinjections. In contrast, DAMGO microinjections into the caudal ventral pallidum produced increases in reward and motor/performance. These data confirm a role for the ventral pallidum in limbic function and extend it to intracranial self-stimulation reward. They also suggest reward modulation in the ventral pallidum is a regionally heterogeneous function and that the rostral ventral pallidum may be a transition area between the nucleus accumbens and the ventral pallidum.     

Endomorphin-1 and endomorphin-2 activate µ-opioid receptors in myenteric neurons of the guinea-pig small intestine

The novel opioid tetrapeptides, endomorphin-1 and endomorphin-2, recently isolated from bovine and human brain bind with high affinity and selectivity to central μ-opioid receptors. In the digestive tract, a comprehensive pharmacological analysis of the receptors involved in endomorphin action has not been reported. In this study, we analyzed the effects of endomorphin-1 and endomorphin-2 on longitudinal muscle-myenteric plexus preparations (LMMPs) from the guinea-pig ileum. Both peptides (30 pM–1 μM) inhibited (–log EC₅₀ values: 8.61 and 8.59, respectively) the amplitude of electrically-induced twitch contractions in a concentration-dependent fashion, up to its abolition. Conversely, in unstimulated LMMPs, they failed to affect contractions to applied acetylcholine (100 nM). In stimulated LMMPs, the highly selective μ-opioid receptor antagonist, d-Phe-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr-NH₂ (CTOP), caused a concentration-dependent (30 nM–1 μM), parallel rightward shift of endomorphin-1 and endomorphin-2 inhibitory curves, without depression of their maximum. Following Schild analysis, calculated pA ₂ values were 7.81 and 7.85, respectively, with slopes not different from unity. Concentration-response curves to both peptides were not affected by 30 nM naltrindole (a selective δ-receptor antagonist) or 30 nM nor-binaltorphimine (a selective κ-receptor antagonist). These results demonstrate that endomorphins selectively activate μ-opioid receptors located on excitatory myenteric plexus neurons, and that they act as full agonists. Received: 6 August 1998 / Accepted: 5 October 1998     

Blockade of morphine reward through the activation of κ-opioid receptors in mice

The effects of systemic (s.c.) treatment with the κ-agonists U-50,488H and E-2078 (a stable dynorphin analog) on the morphine-induced place preference were examined in mice. Morphine (s.c.) caused a dose-related preference for the drug-associated place; the effects at doses of 3 and 5 mg/kg were significant. On the other hand, U-50,488H or E-2078 produced a dose-related conditioned place aversion. Both U-50,488H (1 mg/kg, s.c.) and E-2078 (0.1 mg/kg, s.c.) induced a slight, nonsignificant place aversion. Pretreatment with U-50,488H (1 mg/kg) abolished the morphine (3 mg/kg)-induced place preference. The morphine-induced place preference was also significantly decreased by pretreatment with E-2078 (0.1 mg/kg). The inhibitory effects of the κ-agonists were antagonized by the κ-antagonist nor-binaltorphimine (nor-BNI; 3 mg/kg, s.c.). In contrast, pretreatment with U-50,488H did not affect the place preference induced by the dopamine (DA) receptor agonist apomorphine (1 mg/kg, s.c.). In addition, morphine (3 mg/kg, s.c.) significantly increased the levels of the DA metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the limbic forebrain (nucleus accumbens and olfactory tubercle) but not in the striatum, implying that activation of the mesolimbic DA system may play an important role in the morphine-induced place preference in mice. Pretreatment with U-50,488H significantly reduced the morphine-induced elevation of DA metabolites in the limbic forebrain. These results suggest that κ-agonists suppress the morphine-induced place preference, and that activation of κ-opioid receptors could suppress the reinforcing effects of morphine which may be induced by enhanced DA transmission in the mesolimbic DA system.     

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.     

Phorbol ester blocks the increase of a high affinity GTPase activity induced by δ2-opioid receptor agonist in the mouse spinal cord

The high affinity GTPase activity in the mouse spinal cord was increased in a concentration-dependent manner by a selective δ₂-opioid receptor agonist, [ -Ala²]deltorphin II (0.1-1 μM). This increase of GTPase activity induced by [ -Ala²]deltorphin II was completely blocked by co-incubation with a selective δ₂-opioid receptor antagonist, naltriben (0.1 μM). A protein kinase C activator, phorbol 12,13-dibutyrate (PDB; 0.1-10 μM), which given alone had no effect on basal GTPase activity, blocked dose-dependently the increase of GTPase activity induced by [ -Ala²]deltorphin II (1 μM). Our results indicate the possibility that activation of protein kinase C by phorbol ester uncouples the δ₂-opioid receptor from G-proteins in the spinal cord.     

Binding site of loperamide: automated docking of loperamide in human mu- and delta-opioid receptors

Loperamide is a piperidine analogue, acting as agonist on peripheral opioid receptors, exhibiting affinity and selectivity for the cloned mu human opioid receptor compared with the delta human opioid receptor. Automatic docking studies of loperamide, using AutoDock, on human mu- and delta-opioid receptors is described. Whilst no meaningful difference was detected concerning the docking of the arylpiperidine moiety, mu/delta selectivity could be explained as a different accommodation of the two phenyl groups in two lipophylic pockets of receptors.     

Possible involvement of the locus coeruleus in inhibition by prostanoid EP3 receptor-selective agonists of morphine withdrawal syndrome in rats

We examined the mechanism of the inhibitory effect of prostanoid EP₃ receptor agonists on naloxone-precipitated withdrawal syndrome in morphine-dependent rats. Rats were rendered morphine dependent by subcutaneous (s.c.) implantation of two pellets containing 75 mg morphine for 5 days. Morphine withdrawal syndrome was precipitated by i.p. injection of naloxone (3 mg/kg). Intracerebroventricular (i.c.v.) administration of (±)-15α-hydroxy-9-oxo-16-phenoxy-17,18,19,20-tetranorprost-13-trans-enoic acid (M&B28,767: prostanoid EP₃ receptor agonist) or sulprostone (prostanoid EP₁/EP₃ receptor agonist) significantly suppressed many withdrawal signs. Northern blotting and in situ hybridization studies revealed that i.c.v. administration of M&B28,767 (1 pg/rat) attenuated the elevation of c-fos mRNA during naloxone-precipitated withdrawal in many brain regions, including the cerebral cortex, thalamus, hypothalamus and locus coeruleus. Double in situ hybridization analysis revealed that in the locus coeruleus most of the tyrosine hydroxylase mRNA-positive neurons expressed μ-opioid receptor mRNA and more than half of these neurons were positive for prostanoid EP₃ receptor mRNA. These results indicate that the suppression by prostanoid EP₃ receptor agonists of naloxone-precipitated morphine withdrawal syndrome can be attributed to the inhibition of neuronal activity in several brain regions, including the locus coeruleus, the largest source of central noradrenergic neurons.