Opioid Receptor Subtypes Research Articles

Commentary

Recent research has documented the involvement of the endogenous opioid system in neural development, including neurogenesis and neuronal differentiation. However, the expression of opioid receptors (ORs) in different cell types of the human ventricular and subventricular zones (VZ and SVZ) has not been studied during early gestation. In the present study, we have used immunohistochemistry and quantified the results to demonstrate that the levels of δ- and μ-OR subtypes were high in the VZ and SVZ between 11 and 16 gestation weeks (GW) and decreased by 20 GW. These results have also been confirmed by studying OR mRNA expression in the VZ and SVZ. Both δ- and μ-OR subtypes were expressed by multipotential stem cells, newly differentiated neurons and developing glial cells to different extents. However, migrating neurons expressed negligible levels of both OR subtypes. Our results suggest that the opioid system may affect cellular proliferation and/or differentiation of stem cells into neurons and glia during the first and second trimesters of gestation in humans. Since layers II and III of the cerebral cortex are being formed during the second trimester, their development is most likely affected by the opioid system mediated through δ- and μ-ORs.

Studies on the Determination of the Pharmacophoric Elements of the Naturally Occurring Opioid Receptor Agonist, Mitragynine and Related Alkaloids from Mitragyna speciosa

Leaves from the Southeast Asian tree Mitragyna speciosa, have been traditionally utilized for their ability to attenuate opium withdrawal and addiction. Mitragynine is the major alkaloid in this species and has been reported to be a partial opioid agonist. Moreover, the oxidative derivative, 7-hydroxymitragynine, a minor component, has been reported to be a full opioid agonist. Over 20 other alkaloids have been isolated from the tree yet all have not been characterized for opioid receptor affinity. We have isolated several known compounds from the dried leaves and produced simplified synthetic analogs of mitragynine in order to determine the active pharmacophore at opioid receptors. In a similar fashion to morphine-related synthetic ligands, we have systematically removed certain portions of the structure to determine the necessary structural requirements for opioid receptor affinity and activity. Known alkaloids and synthetic derivatives were screened on MOP, KOP, and DOP for opioid receptor affinities and some were screened in functional assays of GTPgS. Some of these compounds have shown interesting affinity at the opioid receptor subtypes as well as agonist and antagonist activities. Together, these data start to decipher the structural requirements of opioid receptor activity of mitragynine and related analogs. Additional synthetic modifications as well as in vivo studies may provide insights to novel analgesics and potential treatments for opioid withdrawal/addiction.

Methionine-enkephalin modulation of hydrogen peroxide (H2O2) release by rat peritoneal macrophages involves different types of opioid receptors

We investigated the involvement of specific types of opioid receptors in methionine-enkephalin (MET)-induced modulation of hydrogen peroxide (H2O2) release by rat macrophages primed with sub-optimal concentrations of phorbol myristate acetate (PMA). Peritoneal macrophages in vitro treated with different concentrations of MET were tested for H2O2 release in phenol red assay. In the antagonistic study macrophages were treated with MET and one opioid receptor antagonist, or combination of MET and two or three opioid receptor antagonists. MET decreased H2O2 release in eight individual macrophage samples, and increased it in 10 samples. The increase of H2O2 release induced by MET in macrophages was blocked with combination of opioid receptor antagonists specific delta1,2 and mu receptors, as well as with combination of antagonists specific for delta1,2 and kappa opioid receptors. MET-induced decrease of the H2O2 release in macrophages was prevented by opioid receptor antagonists specific for delta1,2 or mu receptors, and also with combination of two or three opioid receptor antagonists. MET-induced enhancement of H2O2 release was mediated via delta1 or delta2 opioid receptor subtypes, or by mu-kappa opioid receptor functional interactions, while MET-induced suppression involved functional interactions between delta1 and mu, delta2 and mu, or delta1 and kappa opioid receptors. It is possible that individual differences in basal or induced macrophage capacity to produce H2O2 might shape the repertoire of opioid receptors expression and in that way pre-determine the direction of MET-induced changes after the in vitro treatment.

The Role of Amygdalar Mu-Opioid Receptors in Anxiety-Related Responses in Two Rat Models

Amygdala opioids such as enkephalin appear to play some role in the control of anxiety and the anxiolytic effects of benzodiazepines, although the opioid receptor subtypes mediating such effects are unclear. This study compared the influences of mu-opioid receptor (MOR) activation in the central nucleus of the amygdala (CEA) on unconditioned fear or anxiety-like responses in two models, the elevated plus maze, and the defensive burying test. The role of MORs in the anxiolytic actions of the benzodiazepine agonist diazepam was also examined using both models. Either the MOR agonist [D-Ala(2), NMe-Phe(4), Gly-ol(5)]-enkephalin (DAMGO), or the MOR antagonists Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH(2) (CTAP) or beta-funaltrexamine (FNA) were bilaterally infused into the CEA of rats before testing. The results show that microinjection of DAMGO in the CEA decreased open-arm time in the plus maze, whereas CTAP increased open-arm behaviors. In contrast, DAMGO injections in the CEA reduced burying behaviors and increased rearing following exposure to a predator odor, suggesting a shift in the behavioral response in this context. Amygdala injections of the MOR agonist DAMGO or the MOR antagonist CTAP failed to change the anxiolytic effects of diazepam in either test. Our results demonstrate that MOR activation in the central amygdala exerts distinctive effects in two different models of unconditioned fear or anxiety-like responses, and suggest that opioids may exert context-specific regulation of amygdalar output circuits and behavioral responses during exposure to potential threats (open arms of the maze) vs discrete threats (predator odor).

Synthetic studies of neoclerodane diterpenes from Salvia divinorum: Exploration of the 1-position

Modification of the C-1 ketone of salvinorin A ( 2a) produces analogues with opioid antagonist properties. Of particular significance is the finding that 1-deoxo-1,10-dehydrosalvinorin A ( 11a) is a moderately potent antagonist at all three opioid receptor subtypes, and that herkinorin ( 2b), a μ agonist, is converted to a weak antagonist by removal of the C-1 ketone ( 3b and 11b). These observations suggest that the ketone of 2b is a key structural feature responsible for μ agonist activity.

Comparison of opioid receptor binding in horse, guinea pig, and rat cerebral cortex and cerebellum

ObjectiveTo compare the density and binding characteristics of opioid receptor subtypes in horse, rat, and guinea pig cerebral cortex and cerebellum. Study designProspective receptor binding study. AnimalsWhole brains were obtained from four neurologically normal adult horses during necropsy. Rat and guinea pig brains were obtained commercially. MethodsThe cerebellum and cerebral cortex were dissected from each brain, and tissue homogenates prepared. A radioligand binding technique with the highly selective ligands [3H]-DAMGO, [3H]-U69593, and [3H]-DPDPE was used to identify the mu- (μ), kappa- (κ) and delta- (δ) opioid receptors, respectively. Competitive binding assays were performed with these ligands and varying concentrations of one of multiple unlabeled ligands. ResultsWhile there were marked species differences in relative densities of opioid receptors, all radioligands interacted with their binding sites with high, nanomolar affinity in both the cerebral cortex and cerebellum. In the horse cerebral cortex, the percentages of total opioid binding sites for the μ-, κ- and δ-receptors were 71%, 14% and 15%, respectively. In the rat and guinea pig cerebral cortex, the corresponding values were 56%μ-, 4%κ- and 40%δ-receptors, and 25%μ-, 37%κ- and 38%δ-receptors, respectively. In horse and guinea pig cerebellum, the binding was 37%μ-, 59%κ- and 4%δ-receptors, and 15%μ-, 76%κ- and 10%δ-receptors, respectively. For competitive analysis, all competitors of the μ-, κ- and δ-receptors completely displaced [3H]-DAMGO, [3H]-U69593, and [3H]-DPDPE and had inhibitory constants in the nanomolar range. Conclusion and clinical relevanceHorses used in this study had a greater density of μ-receptors in the cerebral cortex compared with rats and guinea pigs but without further characterization of the functional role of these receptors it is impossible to determine the clinical significance of these data.

Morphine Postconditioning Protects Against Reperfusion Injury in the Isolated Rat Hearts

Postconditioning is a novel strategy of attaining cardioprotection. Previous studies have suggested morphine mimics the effects of ischemic preconditioning. Whether it is also capable of producing postconditioning or not is still unclear. The purpose of this study was to determine (1) whether morphine postconditioning (MPostcond) would protect the heart against reperfusion injury and the subtype(s) of opioid receptors (OR) involved, (2) whether combining MPostcond with morphine preconditioning (MPC) would afford additive cardioprotection, and (3) to evaluate the role mitochondrial adenosine triphosphate-sensitive potassium (mito-K atp) channel played in MPostcond. Isolated perfused rat hearts were subjected to 45 min of ischemia followed by 1 h of reperfusion. First, three morphine concentrations (0.3, 3.0 and 30 microM) were used to study the protective effect of MPostcond. Second, the effect of blockade of OR subtypes by three antagonists (nonselective OR antagonist naloxone, kappa-OR antagonist nor-binaltorphimine, and delta-OR antagonist naltrindole) on MPostcond was investigated. Third, the protective effects of MPC, MPostcond and the combining MPC with MPostcond on reperfusion injury were compared. Last, the effect of blockade of mito-K atp by 5-hydroxydecanoate on MPostcond was studied. MPostcond was induced by a 10-min perfusion of morphine in Krebs-Ringer's solution performed at the onset of reperfusion, and MPC was produced by a 20-min perfusion of morphine 10 min before ischemia. Infarct size (IS/AAR, as a percentage of the area at risk) was determined by 2,3,5-triphenyltetrazolium staining. IS/AAR was significantly reduced after MPostcond from 58% +/- 8% (control) to 37% +/- 6% (morphine 3.0 microM, P < 0.01). This effect was abolished by coadministering naloxone (58% +/- 7%), nor-binaltorphimine (52% +/- 5%), but not naltrindole (34% +/- 5%). MPC and MPostcond had similar extent of protective effect on IS/AAR, and combining MPC with MPostcond did not afford further cardiprotection. 5-Hydroxydecanoate also abolished the cardioprotection of MPostcond. Unexpectedly, all three OR antagonists and 5-hydroxydecanoate themselves also afforded some extent of cardioprotection. MPost confers cardioprotection via activating kappa-OR but not delta-OR and opening mito-K atp channels. MPost and MPC have no additive protection. kappa-OR and mito-K atp channel may play a dual role in protecting ischemia-reperfusion injury.

Structure–activity relationship studies of carboxamido-biaryl ethers as opioid receptor antagonists (OpRAs). Part 1

A structurally unique and new class of opioid receptor antagonists (OpRAs) that bear no structural resemblance with morphine or endogenous opioid peptides has been discovered. A series of carboxamido-biaryl ethers were identified as potent receptor antagonists against mu, kappa and delta opioid receptors. The structure–activity relationship indicated para-substituted aryloxyaryl primary carboxamide bearing an amine tether on the distal phenyl ring was optimal for potent in vitro functional antagonism against three opioid receptor subtypes.

Synthesis of a novel 4,6′-epoxymorphinan derivative and a highly strained novel conjugated ketone

A modification of the 4,5-epoxymorphinan skeleton of naltrexone was carried out to improve the potency and the selectivity of the ligand for an opioid receptor subtype. As one of the modified structures, we newly designed and synthesized a novel 7-membered ring ether derivative, which had an inserted OCH 2 group between the 4- and 6-positions of the morphinan skeleton. The derivative with a 7-membered ring ether, 4,6′-epoxymorphinan, has a more fixed chair form than the 4,5-epoxymorphinan. In addition, we found a new cleavage reaction of the 4,5-epoxy ring in naltrexone, and also obtained a highly strained novel conjugated ketone.

Isolation and Chemical Modification of Clerodane Diterpenoids from Salvia Species as Potential Agonists at the κ‐Opioid Receptor

The clerodane diterpenoid salvinorin A (1), the main active component of the psychotropic herb Salvia divinorum, has been reported to be a potent agonist at the kappa-opioid receptor. Computer modeling suggested that splendidin (2) from S. splendens, as well as related compounds, might possess similar activities. In the present study, this hypothesis was tested by determination of the binding properties of a series of structural congeners, compounds 2-8, at the mu-, delta-, and kappa-opioid receptors. However, none of these compounds showed significant binding to any of the opioid-receptor subtypes, thus disproving the above hypothesis. The novel compounds 7 and 8 were obtained semi-synthetically by selective modification of salvifarin (5), isolated from Salvia farinacea, upon epoxide-ring opening with AcOH in the presence of indium(III) triflate. Also, the X-ray crystal structure of salvifaricin (6; Fig.), obtained from S. farinacea, was determined for the first time and used, in combination with in-depth NMR experiments, to elucidate the absolute configurations of the new products. Our experiments demonstrate that the relatively well-accessible diterpenoid 6 could be used as starting material for future studies into the structure-activity relationship at the kappa-opioid receptor.

Micro-opioid receptor alkylation in the ventral pallidum and ventral tegmental area, but not in the nucleus accumbens, attenuates the effects of heroin on cocaine self-administration in rats.

The concurrent use of cocaine and heroin, often referred to as speedball, is a powerful reinforcer that has been reported in humans to sometimes result in heightened euphoria compared with either drug alone. Data from animal research indicate that the reinforcing efficacy of low doses of cocaine is potentiated by the addition of small amounts of heroin and that this potentiation is accompanied by synergistic increases in nucleus accumbens (NAc) extracellular fluid levels of dopamine. Although micro- and/or delta-opioid receptors may underlie this potentiation, the opioid receptor subtype or the loci responsible for this enhancement is not known. This experiment used intracranial administration of a selective micro-opioid receptor alkylating agent (beta-funaltrexamine (beta-FNA)) to assess the role of mu-opioid receptors in the NAc, ventral pallidum (VP), and ventral tegmental area (VTA) on the ability of heroin to alter cocaine self-administration. Rats were trained to self-administer cocaine, heroin, or their combination and were administered either vehicle or beta-FNA into one of each brain region and the effects upon drug intake assessed. beta-FNA administered into the VP or VTA shifted the dose-effect curve for the cocaine/heroin combination towards that maintained by cocaine alone. beta-FNA had no effect on self-administration of the combination of cocaine and heroin when injected into the NAc. These data suggest that heroin may attenuate feedback inhibition from the NAc to the VP and VTA when co-self-administered with cocaine, resulting in a positive modulation of the effects of cocaine.

Central κ-opioid receptor-mediated antidepressant-like effects of nor-Binaltorphimine: Behavioral and BDNF mRNA expression studies

κ-opioid receptor antagonists such as nor-Binaltorphimine (nor-BNI) have been shown to produce antidepressant-like behavioral effects in animal models of depression. The aim of this study was to investigate further the duration of centrally administered nor-BNI-induced antidepressant-like actions measured by both behavior and brain-derived neurotrophic factor (BDNF) gene expression. In addition, antagonist studies were conducted to determine the role of opioid receptor subtypes and the time course of nor-BNI's pharmacological actions. Antidepressant-like behavioral effects were measured by decreased immobility in the rat forced swim test and BDNF mRNA expression was determined by in situ hybridization. Centrally administered nor-BNI (20 μg, i.c.v.) decreased immobility and increased BDNF mRNA expression in the hippocampus on day 1, not on days 3–14, post-administration. Systemic administration of selective μ-, δ- and κ-opioid receptor antagonists did not block nor-BNI-induced antidepressant-like effects. In contrast, i.c.v. administration of nor-BNI 7 or 14 days earlier significantly blocked subsequent nor-BNI-induced decreased immobility and upregulation of BDNF mRNA expression. Although the duration of nor-BNI's antidepressant-like effects did not synchronize with that of its κ-opioid receptor antagonist effects, this study is the first to show that centrally administered nor-BNI, like most clinically used antidepressants, can upregulate BDNF mRNA expression in the rat hippocampus. These findings further demonstrate that central κ-opioid receptor mediates antidepressant-like effects of nor-BNI measured by both behavior and BDNF gene expression.

Central inhibition of opioid receptor subtypes and its effect on haemorrhagic hypotension in conscious sheep

To investigate the contribution of cerebral mu-, kappa- and delta-opioid receptors in causing the hypotension, bradycardia and renal hypoperfusion evoked by haemorrhage. Adult conscious ewes were bled continuously from a jugular vein until mean arterial blood pressure (MAP) was reduced to below 50 mmHg. Starting 30 min before and continuing until 60 min after haemorrhage either artificial cerebrospinal fluid (control), d-Phe-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr-NH(2) (CTOP micro-receptor antagonist), ICI 174,864 (delta-receptor antagonist) or nor-binaltorphimine dihydrochloride (nor-BNI, kappa-receptor antagonist) were infused intracerebroventricularly. In a randomized crossover fashion the effect of antagonizing one central opioid receptor subtype was compared to control experiments in the same animal (n = 6 in all groups). Compared to corresponding controls, nor-BNI and ICI 174,864 significantly increased the haemorrhage volume needed to reduce MAP to below 50 mmHg (+4.7 mL kg(-1), SD 1.8 and +3.1 mL kg(-1), SD 3.0 respectively). In the nor-BNI group this was accompanied by a significantly augmented tachycardia before MAP fell. Both nor-BNI and ICI 174,864 also postponed haemorrhagic bradycardia and prolonged adequate blood flow to the kidney. The infusions did not affect the circulation per se or the recovery after haemorrhage. The micro-opioid receptor blockade had no effect on baseline circulation or the response to haemorrhage. Activation of kappa- and delta-opioid receptors adjacent to the ventricular compartment contributes to initiating haemorrhagic hypotension and bradycardia in conscious sheep. However, other parts of the brain and different receptors are likely to play a role as well.

Effects of Selective Opioid Receptor Antagonists on Alcohol‐Induced and Nicotine‐Induced Antinociception

Despite synergistic increases in risks of various cancers, the incidence of concomitant smoking and drinking remains high. An additive or synergistic analgesic effect of combined alcohol and nicotine may contribute to their coabuse. Recently, we provided evidence that doses of alcohol and nicotine that are ineffective in inducing an antinociceptive effect alone, when combined, can induce such an effect. Moreover, this antinociceptive effect could be attenuated by pretreatment with the nonselective opioid antagonist naloxone. The purpose of this study was to determine the role of selective opioid receptor subtypes (micro, delta, and kappa) in mediating the antinociceptive effects of alcohol, nicotine, and their combination. Adult male Wistar rats were administered selective opioid antagonists, D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH(2) (CTAP, for micro receptor, 1.0 mg/kg), naltrindole (for delta receptors, 10 mg/kg), and norbinaltorphimine (nor-BNI, for kappa receptor, 10 mg/kg) before injection of alcohol, nicotine or their combination. The animals were tested in hot-plate and tail-flick assays, representing nociception mediated predominantly via brain or spinal pathways, respectively. All the injections were administered acutely and the nociceptive tests were carried out 20 minutes after alcohol and 10 minutes after nicotine administration. In general, the antagonists were more effective in blocking the effects of alcohol, nicotine, or their combination in the tail-flick versus the hot-plate assay. CTAP was most effective in blocking the effects of alcohol alone and nicotine alone in the tail-flick test, whereas in the hot-plate test both CTAP and naltrindole were more effective than nor-BNI. All 3 antagonists had a very similar profile in attenuating the combination of alcohol and nicotine effect in the hot-plate assay. None of the antagonists had a significant effect against the highest alcohol-nicotine dose in this test. In the tail-flick test, however, CTAP and naltrindole were more effective than nor-BNI in attenuating the highest alcohol-nicotine dose. The data suggest the utility of all 3 opioid antagonists in blocking the effects of alcohol, nicotine, or their combination in spinally mediated antinociception. Although the supraspinally mediated antinociception was also attenuated by the opioid antagonists, further investigation of combination doses of these antagonists in fully blocking the supraspinal effects or attenuating voluntary alcohol and nicotine intake is warranted.

Pharmacological Evidence for a Motivational Role of κ-Opioid Systems in Ethanol Dependence

The purpose of this study was to test the hypothesis that activation of the dynorphin/kappa (kappa)-opioid system has a role in the increased consumption of ethanol in dependent animals. The effects of three opioid receptor antagonists with different effects on opioid receptors, naltrexone, nalmefene, and nor-binaltorphimine (nor-BNI), were compared in their ability to decrease ethanol self-administration in nondependent and ethanol-dependent male Wistar rats. Nalmefene and naltrexone are both opioid receptor ligands with comparable molecular weights and pharmacokinetic profiles, but differing specificity for the three opioid receptor subtypes at low doses, while nor-BNI is a selective kappa-opioid receptor antagonist. Dependence was induced in half the animals by subjecting them to a 4-week intermittent vapor exposure period in which animals were exposed to ethanol vapor for 14 h per day. Subsequent to dependence induction, nalmefene, naltrexone, and nor-BNI were tested for their ability to modulate self-administration of ethanol in vapor-exposed and control rats. The results indicated that both nalmefene and naltrexone induced a significant dose-dependent decrease in the number of lever presses for ethanol in both groups of animals. However, in ethanol-dependent animals, nalmefene was significantly more effective in suppressing ethanol intake than naltrexone. Nor-BNI selectively attenuated ethanol-dependent self-administration while leaving nondependent ethanol self-administration intact. Because naltrexone is primarily selective for the mu-opioid receptor, and nalmefene is primarily selective for the mu- and kappa-opioid receptor subtypes, the fact that nalmefene demonstrates more suppression in dependent animals suggests that opioid systems distinct from the mu-regulated portion may be involved in the increased drinking seen during withdrawal in dependent animals. The results with nor-BNI confirm that kappa-opioid receptor antagonism selectively decreases dependence-induced ethanol self-administration, which supports the hypothesis that dynorphin/kappa-opioid systems are dysregulated in dependence and contribute to the increased drinking seen during acute withdrawal in dependent rats.

Pharmacological studies with a nonpeptidic, delta-opioid (−)-(1 R,5 R,9 R)-5,9-dimethyl-2′-hydroxy-2-(6-hydroxyhexyl)-6,7-benzomorphan hydrochloride ((−)-NIH 11082)

In the search for a selective delta-opioid receptor agonist, (−)-(1 R,5 R,9 R)-5,9-dimethyl-2′-hydroxy-2-(6-hydroxyhexyl)-6,7-benzomorphan hydrochloride ((−)-NIH 11082) and the (+)-enantiomer were synthesized and tested. (−)-NIH 11082 displayed antinociceptive activity in the paraphenylquinone test (PPQ test) in male ICR mice [ED 50 = 1.9 (0.7–5.3) mg/kg, s.c.] and showed little, if any, activity in the tail-flick and hot-plate assays. The (+)-enantiomer was essentially inactive indicating stereoselectivity. Opioid receptor subtype characterization studies indicated that naltrindole, a delta-opioid receptor antagonist, was potent versus the ED 80 of (−)-NIH 11082 in the PPQ test [AD 50 = 0.75 (0.26–2.20) mg/kg, s.c]. beta-Funaltrexamine and nor-binaltorphimine, selective mu- and kappa-receptor antagonists, respectively, were inactive versus the ED 80 of (−)-NIH 11082. In rats with inflammation-induced pain, (−)-NIH 11082 produced antihyperalgesic effects that were attenuated by naltrindole. In morphine-dependent rhesus monkeys of both sexes, (−)-NIH 11082 neither substituted for morphine nor exacerbated withdrawal signs in the dose range of 4.0 to 32.0 mg/kg, s.c. Neither convulsions nor other overt behavioral signs were observed in any of the species tested. The results indicate that (−)-NIH 11082 has delta-opioid receptor properties.

The in vitro pharmacology of the peripherally restricted opioid receptor antagonists, alvimopan, ADL 08-0011 and methylnaltrexone

This study characterized the pharmacology of the peripherally restricted opioid receptor antagonists, alvimopan, its metabolite, ADL 08-0011, and methylnaltrexone. The activities of the compounds were investigated with respect to human or guinea pig opioid receptor binding and function in recombinant cell lines and mechanical responsiveness of the guinea pig ileum. Alvimopan and ADL 08-0011 had higher binding affinity than methylnaltrexone at human mu opioid receptors (pK (i) values of 9.6, 9.6, and 8.0, respectively). The compounds had different selectivities for the mu receptor over human delta and guinea pig kappa opioid receptors. ADL 08-0011 had the highest mu receptor selectivity. With respect to their mu opioid receptor functional activity ([(35)S]GTPgammaS incorporation), methylnaltrexone had a positive intrinsic activity, consistent with partial agonism, unlike alvimopan and ADL 08-0011, which had negative intrinsic activities. Alvimopan, ADL 08-0011, and methylnaltrexone antagonized inhibitory responses mediated by the mu opioid agonist, endomorphin-1 (pA (2) values of 9.6, 9.4, and 7.6, respectively) and by U69593, a kappa opioid agonist (pA (2) values of 8.4, 7.2, and 6.7, respectively). In morphine-naive guinea pig ileum, methylnaltrexone reduced, while alvimopan and ADL 08-0011 increased, the amplitude of electrically evoked contractions and spontaneous mechanical activity. In tissue from morphine-dependent animals, alvimopan and ADL 08-0011 increased spontaneous activity to a greater degree than methylnaltrexone. The data suggested that alvimopan-induced contractions resulted predominantly from an interaction with kappa opioid receptors. It is concluded that alvimopan, ADL 08-0011, and methylnaltrexone differ in their in vitro pharmacological properties, particularly with respect to opioid receptor subtype selectivity and intrinsic activity. The clinical significance of the data from this study remains to be determined.

Effects of opioid subtypes on detrusor overactivity in rats with cerebral infarction

In order to determine the influence of different opioid receptor subtypes on detrusor overactivity after left middle cerebral artery (MCA) occlusion, cystometric recordings were obtained in conscious rats. Female Sprague-Dawley rats were used in this study. Control cystometrography was followed by left MCA occlusion. The sham-operated (SO) rats underwent the same procedures except for MCA occlusion. [D-Ala(2), Phe(4), Gly(5)]-enkephalin (DAGO; mu-opioid agonist), [D-Pen(2,5)]-enkephalin (DPDPE; delta1-opioid agonist), deltorpin II (delta2-opioid agonist), and U-50488 (kappa-opioid agonist) were administered intracerebroventricularly at graded doses. The bladder capacity, residual volume, micturition threshold pressure, and bladder contraction pressure were determined. Finally, the volume of the infarction was measured. The intracerebroventricular administration of DAGO and DPDPE significantly increased the bladder capacity in the cerebrally infarcted (CI) and SO rats, but differences in the changes in bladder capacity between the CI and SO rats were not significant. Deltorpin II did not produce any changes in the bladder capacity in the CI or SO rats at any dose examined. However, the intracerebroventricular administration of U-50488 significantly increased the bladder capacity in the CI rats but not in the SO rats. None of the drugs affected the residual volume, micturition threshold pressure or bladder contraction pressure at any dosage examined. The mean infarcted volumes were not significantly different from those in the vehicle-treated rats. These results suggest that the opioid receptor subtypes, mu and delta1 in the brain, are related to the micturition reflex. Furthermore, the kappa opioid agonist might be useful for the suppression of detrusor overactivity caused by cerebral infarction.

Antagonism of phosphoramidon-induced antinociception in mice by μ- but not κ-opioid receptor blockers

Intracerebroventricular (i.c.v.) administration of the neutral endopeptidase 24.11-inhibitor phosphoramidon evoked a dose-dependent antinociceptive effect in the mouse acetic acid abdominal constriction test. The present study was conducted to identify the opioid receptor subtype(s) that mediate phosphoramidon antinociception in this paradigm. Mice were pretreated with different opioid antagonists prior to being challenged with phosphoramidon, i.c.v., the μ-opioid agonist sufentanil, s.c., or the κ-opioid agonist U-50,488H, s.c. Naltrexone significantly attenuated phosphoramidon-induced antinociception at an i.c.v. dose that also blocked both sufentanil and U-50,488H. The μ-opioid antagonist β-funaltrexamine (β-FNA) blocked phosphoramidon and sufentanil at an i.c.v. dose that did not block U-50,488H. The κ-opioid antagonist nor-binaltorphimine (nor-BNI) produced dose-related effects. A low dose (10 μg) of nor-BNI had no effect on either phosphoramidon or sufentanil but did reduce U-50,488H antinociception. A higher dose (30 μg) of nor-BNI blocked phosphoramidon, sufentanil, and U-50,488H, suggesting a loss of κ-opioid receptor selectivity at this dose. These findings suggest that μ- but not κ-opioid receptors mediate phosphoramidon-induced antinociception in the abdominal constriction test.

Sex differences in opioid-mediated pain inhibitory mechanisms during the interphase in the formalin test

Many chronic pain conditions are more prevalent in women than men and both fundamental and clinical research supports the implication of endogenous pain inhibitory mechanisms. The goal of this study was to verify if sex differences on endogenous pain inhibitory mechanisms during the formalin test are opioidergic and modulated by sex hormones. Formalin tests were performed with naloxone hydrochloride, a non-selective opioid antagonist in intact and gonadectomized Sprague–Dawley rats of both sexes. Considering the sexual dimorphisms we found, where naloxone preferentially blocked the interphase in female rats, injections of all the possible combinations of μ- (naltrexone hydrochloride), δ- (naltrindole hydrochloride) and κ-selective antagonists (norbinaltorphimine dihydrochloride) were given to evaluate the contribution of these opioid-receptor subtypes to the inhibitory mechanism during the interphase in intact females. Finally, the systemic administration of naloxone methiodide and intrathecal administration of naloxone hydrochloride in intact females allowed us to verify if the action of endogenous opioids that are liberated during the interphase takes place at the periphery or spinally, respectively. The results show that the interphase was almost completely inhibited by naloxone in females while it produced only a slight blockade in males. These results permitted us to conclude that opioids play a major role in the pain inhibitory mechanism of the interphase in females while a non-opioid mechanism seems to be responsible for this inhibitory pathway in males. Using gonadectomized animals of both sexes, we demonstrated the modulation of the opioidergic system of the interphase by sex hormones. The administration of different combinations of selective antagonists for μ-, κ- and δ-opioid receptors in intact females permitted us to conclude that only the combination of κ- and δ-selective antagonists significantly blocked the interphase. The same result was obtained with the combination of the three antagonists, confirming the results with systemic naloxone hydrochloride. Finally, intrathecal administration permitted us to support that the action of naloxone is primarily at the spinal level, even if a supraspinal action cannot be ruled out. These results are of particular interest in showing sexual dimorphisms in endogenous pain modulation mechanisms during the interphase of the formalin test. A clearer understanding of the difference between male and female endogenous pain inhibitory pathways should lead to a better understanding of the role of endogenous pain modulation deficits in certain chronic pain conditions.