Delta and kappa opioid receptors are differentially regulated by dynamin-dependent endocytosis when activated by the same alkaloid agonist

Many alkaloid drugs used as analgesics activate multiple opioid receptors. Mechanisms that distinguish the actions of these drugs on the regulation of individual micro, delta, and kappa receptors are not understood. We have observed that individual cloned opioid receptors differ significantly in their regulation by rapid endocytosis in the presence of alkaloid drug etorphine, a potent agonist of mu, delta, and kappa opioid receptors. Internalization of epitope-tagged delta opioid receptors from the plasma membrane is detectable within 10 min in the presence of etorphine. In contrast, kappa receptors expressed in the same cells remain in the plasma membrane and are not internalized for >/=60 min, even when cells are exposed to saturating concentrations of etorphine. The rapid internalization of delta receptors is specifically inhibited in cells expressing K44E mutant dynamin I, suggesting that type-specific internalization of opioid receptors is mediated by clathrin-coated pits. Examination of a series of chimeric mutant kappa/delta receptors indicates that at least two receptor domains, including the highly divergent carboxyl-terminal cytoplasmic tail, determine the type specificity of this endocytic mechanism. We conclude that structurally homologous opioid receptors are differentially sorted by clathrin-mediated endocytosis following activation by the same agonist ligand. These studies identify a fundamental mechanism of receptor regulation mediating type-specific effects of analgesic drugs that activate more than one type of opioid receptor.     

Expression of the human antigen SPAG2 in the testis and localization to the outer dense fibers in spermatozoa

There is increasing evidence to suggest that opioid peptides may have widespread effects as regulators of growth. To evaluate the hypothesis that endogenous opioids control cellular proliferation during neural development, we have used in situ hybridization to examine opioid peptide and receptor mRNA expression in neuroepithelial zones of fetal rat brain and spinal cord. Our data show that proenkephalin mRNA is widely expressed in forebrain germinal zones and choroid plexus during the second half of gestation. In contrast, prodynorphin mRNA expression is restricted to the periventricular region of the ventral spinal cord. Little mu or delta receptor mRNA expression was detected in any regions of neuronal proliferation prior to birth. However, kappa receptor mRNA is widely expressed in hindbrain germinal zones during the 3rd week of gestation. Our present findings support the hypothesis that endogenous opioids may regulate proliferation of both neuronal and non-neuronal cells during central nervous system development. Given the segregated expression of proenkephalin mRNA in forebrain neuroepithelium and kappa receptor mRNA within hindbrain, different opioid mechanisms may regulate cell division in rostral and caudal brain regions.     

Deleterious brain cell membrane effects after NMDA receptor antagonist administration to newborn piglets

Heroin administered i.c.v. acts on supraspinal mu opioid receptors in ICR mice but on delta receptors in Swiss Webster mice. The purpose of this study was to determine the degree to which genotype plays a role in the opioid receptor selectivity of heroin across a range of fully inbred strains of mice. Six inbred strains were given heroin i.c.v. 10 min before the tail-flick test. Differences in the descending neurotransmitter systems involved in supraspinal opioid-induced analgesia were evaluated as the first step. Antagonism by bicuculline given intrathecally indicated the involvement of supraspinal delta receptors in activating spinal gamma-aminobutyric acid (GABA) receptors; antagonism by intrathecal methysergide indicated either mu or kappa receptor involvement. Antagonism by intrathecal yohimbine implicated mu and eliminated kappa receptor involvement. Intracerbroventricular opioid antagonists (beta-funaltrexamine, 7-benzylidenenaltrexone, naltriben, or nor-binaltorphimine) provided further differentiation. Based on these initial results, receptor selectivity was determined by more extensive ED50 experiments with i.c.v. administration of heroin with opioid antagonists, beta-funaltrexamine (for mu), naltrindole (for delta), and nor-binaltorphimine (for kappa). The combined results indicated that heroin analgesia was predominantly mediated in C57BL/6J by delta, in DBA/2J and CBA/J by mu, and in BALB/cByJ and AKR/J by kappa receptors. The response in C3H/HeJ appeared to involve mu receptors. The results indicate that the opioid receptor selectivity of heroin is genotype-dependent. Because these genotypes are fully inbred, the genetically determined molecular and neurochemical substrate mediating the different opioid receptor selectivities of heroin can be studied further.     

The anti-gonadotropic effects of cytokines: the role of neuropeptides

The inhibitory effect of inflammation and endotoxins on the secretion of reproductive hormones from the hypothalamo-pituitary axis is well documented. A comparison of the luteinizing hormone (LH) suppressing effects of several pro-inflammatory cytokines revealed that centrally administered IL-1 beta was the most potent inhibitor of pituitary LH secretion; interleukin (IL)-1 alpha and tumor necrosis factor (TNF) alpha were relatively less effective, whereas IL-6 was ineffective. This order of potency suggested that the anti-gonadotropic effects of an immune challenge are most likely attributable to the action of centrally released IL-1 beta, and this was supported by the demonstration that IL-1 beta suppressed hypothalamic luteinizing hormone releasing hormone (LHRH) release. We used a multifaceted approach to identify the afferent signals in the brain that convey immune messages to hypothalamic LHRH neurons. Pharmacological studies with specific antagonists of opioid receptor subtypes demonstrated that activation of the mu 1 receptor subtype was required to transmit the cytokine signal. Furthermore, icv IL-1 beta upregulated hypothalamic POMC mRNA and increased the concentration and release of beta-endorphin, the primary ligand of mu 1 receptors. We have obtained evidence that IL-1 beta also enhanced the gene expression and concentration of tachykinins, a family of nociceptive neuropeptides in the hypothalamus. Blockade of tachykinergic NK2 receptors attenuated IL-1 beta induced inhibition of LH secretion. Collectively, these results demonstrate that IL-1 beta, generated centrally in response to inflammation, upregulates the opioid and tachykinin peptides in the hypothalamus. These two groups of neuropeptides are critically involved in relaying the cytokine signal to neuroendocrine neurons and causing the suppression of hypothalamic LHRH and pituitary LH release.     

Differential regulation of the cloned kappa and mu opioid receptors

To directly compare the regulation of the cloned kappa and mu opioid receptor, we expressed them in the same cells, the mouse anterior pituitary cell line AtT-20. The coupling of an endogenous somatostatin receptor to adenylyl cyclase and an inward rectifier K+ current has been well characterized in these cells, enabling us to do parallel studies comparing the regulation of both the kappa and the mu receptor to this somatostatin receptor. We show that the kappa receptor readily uncoupled from the K+ current and from adenylyl cyclase after a 1 h pretreatment with agonist, as indicated by the loss in the ability of the agonist to induce a functional response. The desensitization of the kappa receptor was homologous, as the ability of somatostatin to mediate inhibition of adenylyl cyclase or potentiation of the K+ current was not altered by kappa receptor desensitization. The mu receptor uncoupled from the K+ current but not adenylyl cyclase after a 1 h pretreatment with agonist. Somatostatin was no longer able to potentiate the K+ current after mu receptor desensitization, thus this desensitization was heterologous. Interestingly, pretreatment with a somatostatin agonist caused uncoupling of the mu receptor but not the kappa receptor from the K+ current. These results show that in the same cell line, after a 1 h pretreatment with agonist, the kappa receptor displays homologous regulation, whereas the mu receptor undergoes only a heterologous form of desensitization. mu receptor desensitization may lead to the alterations of diverse downstream events, whereas kappa receptor regulation apparently occurs at the level of the receptor itself. Broad alterations of non-opioid systems by the mu receptor could be relevant to the addictive properties of mu agonists. Comparison of kappa and mu receptor regulation may help define the properties of the mu receptor which are important in the development of addiction, tolerance, and withdrawal to opioid drugs. These are the first studies to directly compare the coupling of the kappa and mu receptors to two different effectors in the same mammalian expression system.     

Synthesis and characterization of 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY)-labeled fluorescent ligands for the mu opioid receptor

A series of opioid ligands utilizing the 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) fluorophores 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene++ +-3-propionic acid or 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza- s-indacene-3-propionic acid were synthesized and characterized for their ability to act as a suitable fluorescent label for the mu opioid receptor. All compounds displaced the mu opioid receptor binding of [3H]Tyr-D-Ala-Gly-(Me)Phe-Gly-ol in monkey brain membranes with high affinity. The binding of fluorescent ligands to delta and kappa receptors was highly variable. 5,7-Dimethyl-BODIPY naltrexamine, "6-BNX," displayed subnanomolar affinities for the mu and kappa opioid receptors (Ki 0.07 and 0.43 nM, respectively) and nanomolar affinity at the delta (Ki 1.4 nM) receptor. Using fluorescence spectroscopy, the binding of 6-BNX in membranes from C6 glioma cells transfected with the cloned mu opioid receptor was investigated. In these membranes containing a high receptor density (10-80 pmol/mg protein), 6-BNX labeling was saturable, mu opioid specific, stereoselective (as determined with the isomers dextrorphan and levorphanol), and more than 90% specific. The results describe a series of newly developed fluorescent ligands for the mu opioid receptor and the use of one of these ligands as a label for the cloned mu receptor. These ligands provide a new approach for studying the structural and biophysical nature of opioid receptors.     

Mu and kappa opioid receptors in periaqueductal gray and rostral ventromedial medulla

The periaqueductal gray (PAG) and rostral ventromedial medulla (RVM) are important brain stem pain modulating regions. Recent evidence suggests that kappa opioids antagonize the effects of mu opioids in the RVM. However, the anatomical relationship between mu and kappa opioid receptors in PAG and RVM is not well characterized. This study examined relationships between mu and kappa opioid receptor immunoreactivity (IR) and mRNA in PAG and RVM. Brain slices were processed for either immunocytochemistry or in situ hybridization. We found considerable anatomical overlap of mu and kappa opioid IR and mRNA in the RVM and PAG. These results provide an anatomical basis for recent behavioral and electrophysiological findings in RVM, and suggest modulatory interactions between mu and kappa opioids in PAG.     

The Stimulated Raman Spectrum of Symmetric 13C Cyanogen, 13C2N2

BACKGROUND: The authors examined the interaction of ketamine with recombinant mu, kappa, and delta opioid receptors and recombinant orphan opioid receptors expressed in Chinese hamster ovary cells (CHO-mu, CHO-kappa, CHO-delta, and CHO(ORL1), respectively). METHODS: CHO-mu, CHO-kappa, and CHO-delta membranes were incubated with the opioid receptor radioligand [3H]diprenorphine at room temperature. Ketamine (racemic, R(-) and S(+)) was included at concentrations covering the clinical range. CHO(ORL1) membranes were incubated with [125I]Tyr(14)nociceptin and racemic ketamine at room temperature. The effects of racemic ketamine and selective opioid receptor agonists (mu: [D-Ala2, MePhe4, Gly(ol)5] enkephalin (DAMGO); kappa: spiradoline or delta: [D-pen2, D-pen5] enkephalin (DPDPE)) on forskolin-stimulated cyclic adenosine monophosphate formation also were examined. Data are mean +/- SEM. RESULTS: Racemic ketamine increased the radioligand equilibrium dissociation constant for [3H]diprenorphine from 85+/-5 to 273+/-11, 91+/-6 to 154+/-16, and 372+/-15 to 855+/-42 pM in CHO-mu, CHO-kappa, and CHO-delta, respectively. The concentration of radioligand bound at saturation was unaffected. In CHO-mu and CHO-kappa cells, racemic ketamine did not slow the rate of naloxone-induced [3H]diprenorphine dissociation. Ketamine and its isomers also displaced [3H]diprenorphine binding to mu, kappa, and delta receptors in a dose-dependent manner, with pKi values for racemic ketamine of 4.38+/-0.02, 4.55+/-0.04, and 3.57+/-0.02, respectively. S(+)-ketamine was two to three times more potent than R(-)-ketamine at mu and kappa receptors. Racemic ketamine displaced [125I]Tyr(14)nociceptin with an estimated affinity constant of 0.5 mM. Racemic ketamine inhibited the formation of cyclic adenosine monophosphate (naloxone insensitive) in a dose-dependent manner (concentration producing 50% inhibition approximately 2 mM) in all cell lines, including untransfected CHO cells. Ketamine (100 microM) reversed DAMGO (mu) and spiradoline (kappa) inhibition of formation of cyclic adenosine monophosphate. CONCLUSIONS: Ketamine interacts stereoselectively with recombinant mu and kappa opioid receptors.     

Alterations in hypothalamic mu-opiate receptor-mediated responses but not methionine enkephalin or proenkephalin messenger RNA levels in rats with acute cholestasis

Endogenous opioids, including methionine enkephalin, have been implicated in the control of adrenocorticotrophic hormone release by acting through mu-opiate receptors in the hypothalamus. Recently, alterations in the central opioid system have been postulated to occur in cholestasis. In addition, alterations in hypothalamic corticotropin-releasing hormone content and messenger RNA levels, as well as basal release, have been described in bile duct-resected rats, and hypothalamic methionine enkephalin is colocalized with corticotropin-releasing hormone in hypothalamic neurons. Therefore hypothalamic and pituitary methionine enkephalin content and hypothalamic proenkephalin messenger RNA levels, as well as hypothalamic mu-opiate receptor-mediated responses in vitro and in vivo, were studied in rats with acute cholestasis caused by bile duct resection and in respective controls. Hypothalamic and pituitary methionine enkephalin levels were similar in bile duct-resected, sham-resected and unoperated control rats. In addition, hypothalamic proenkephalin steady state messenger RNA levels were similar in the three groups of animals. mu-Opiate receptor stimulation of hypothalamic explants in vitro with the specific mu-opiate receptor agonist ligand [D-Ala2,N-Me-Phe4,Gly-ol]-Enkephalin resulted in 8.2% and 16.9% inhibition of corticotropin-releasing hormone release in sham-resected and unoperated control rats, respectively. In contrast, treatment of hypothalamic explants from bile duct-resected rats with [D-Ala2,N-Me-Phe4,Gly-ol]-Enkephalin resulted in a significant 22.5% increase in corticotropin-releasing hormone release. Systemic administration of the mu-opiate receptor agonist morphine to rats in vivo resulted in significantly higher incremental rises in plasma adrenocorticotropic hormone levels in sham-resected and unoperated control animals than in bile duct-resected rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Beta-endorphin response to exercise. An update

Opioid receptor subtype antagonists differentially alter food intake under deprivation (24 h), glucoprivic (2-deoxy-D-glucose, 500 mg/kg, i.p.) or palatable (10% sucrose) conditions with mu (beta-funaltrexamine) and kappa (nor-binaltorphamine), but not delta1 ([D-Ala2,Leu5,Cys6]enkephalin) opioid antagonists reducing each form of intake following ventricular microinjection. Both mu and kappa opioid antagonists microinjected into either the hypothalamic paraventricular nucleus or the nucleus accumbens reduce intake under deprivation and glucoprivic conditions. Palatable intake is reduced by both antagonists in the paraventricular nucleus, but only mu antagonists are active in the accumbens. Food intake is stimulated by mu and delta, but not kappa, opioid agonists microinjected into the ventral tegmental area. The present study examined whether food intake under either deprivation, glucoprivic or palatable conditions was altered by bilateral administration of general (naltrexone), mu, kappa, delta1 or delta2 (naltrindole isothiocyanate) opioid antagonists into the ventral tegmental area. Deprivation (24 h)-induced feeding was significantly reduced by high (50 microg), but not lower (10-20 microg) doses of naltrexone (21%), and by delta2 (4 microg, 19%) antagonism in the ventral tegmental area. 2-Deoxy-D-glucose (500 mg/kg, i.p.)-induced hyperphagia was significantly reduced by high (50 microg), but not lower (20 microg) doses of naltrexone (64%), and by delta2 (4 microg, 27%) antagonism in the ventral tegmental area. Sucrose (10%) intake was significantly reduced by naltrexone (20-50 microg, 25-39%) and delta2 (4 microg, 25%) antagonism in the ventral tegmental area. Neither mu, kappa nor delta1 antagonists were effective in reducing any form of intake following microinjection into the ventral tegmental area. These data indicate that the ventral tegmental area plays a relatively minor role in the elicitation of these forms of food intake, and that delta2, rather than mu, kappa or delta1 opioid receptors appear responsible for mediation of these forms of intake by this nucleus.     

Differential expression of thyroid hormone receptor isoforms in neurons and astroglial cells

The brain has abundant nuclear T3-binding sites and contains messenger RNAs (mRNAs) encoding multiple thyroid hormone receptor (TR) isoforms; the cellular distribution of these different TR isoforms is unknown. To determine whether the TR isoforms are differentially expressed in neuronal and astroglial cells, we examined the relative abundance of the mRNAs encoding TR alpha 1, c-erbA alpha 2, and TR beta 1 in primary cultures of fetal rat brain and in several cell lines of neural and glial origin. Additionally, the TR isoform polypeptides were identified by immunocytochemistry using isoform-specific antibodies. Northern blot analysis showed that fetal brain cell cultures contain mRNAs encoding the T3-binding isoforms TR alpha 1 and TR beta 1 as well as the mRNA encoding the non-T3-binding c-erbA alpha 2. c-erbA alpha 2 mRNA was most abundant, comprising more than 85% of the TR mRNAs in the primary cultures. Neuronal enrichment by antimitotic selection increased TR beta 1 mRNA approximately 3-fold, decreased c-erbA alpha 2 mRNA 70%, and had little or no effect on TR alpha 1 mRNA. Neuronal depletion resulted in the complete loss of TR beta 1 mRNA without changing c-erb alpha 2 or TR alpha 1 mRNA levels. Primary cultures of rat astrocytes, the astrocytoma cell line C6, and the pheochromocytoma cell line PC12 contained only the c-erbA alpha 2 mRNA. Immunocytochemistry using isoform-specific anti-sera revealed that TR beta 1 was exclusively localized to neuronal nuclei, and c-erbA alpha 2 was only found in the nuclei of astrocytes. These results show that TR beta 1 is localized to the nuclei of neuronal cells, and that c-erbA alpha 2 is restricted to the nuclei of astrocytes.     

Recent advances in molecular recognition and signal transduction of active peptides: receptors for opioid peptides

1. Opioid peptides are a family of structurally related neuromodulators which play a major role in the control of nociceptive pathways. These peptides act through membrane receptors of the nervous system, defined as mu, delta and kappa and endowed with overlapping but distinct pharmacological, anatomical and functional properties. 2. Recent cloning of an opioid receptor gene family has opened the way to the use of recombinant DNA technology at the receptor level. 3. This review focuses on the molecular cloning and functional characterization of opioid receptors and provides first insights into molecular aspects of opioid peptide recognition and signal transduction mechanisms, using the cloned receptors as investigation tools.     

Postnatal development of multiple opioid receptors in the spinal cord and development of spinal morphine analgesia

The postnatal ontogeny of mu, delta and kappa opioid receptor binding sites in the spinal cord of rat pups at various postnatal days was determined using in vitro autoradiographical methods. The functional effect of spinal morphine was also assessed using in vivo electrophysiological methods in rats at P14, P21 and adults (P56). Both mu and kappa opioid receptor binding-sites are present from P0 and spread relatively diffusely throughout the spinal cord. Overall binding peaks at P7 and subsequently decreases to adult levels with the mu opioid receptor binding sites regressing to become denser in the superficial dorsal horn. delta Opioid receptor binding was first seen at P7, and no distinction between superficial and deeper laminae was seen. In the adult, the relative proportions of the opiate receptors in the superficial dorsal horn are 63%, 22% and 15%, for mu, delta and kappa receptor binding sites, respectively. C-fibre evoked dorsal horn neuronal responses recorded from anaesthetized rat pups were highly sensitive to spinal morphine at P21, (EC50 0.005 microgram), compared to the adult (EC50 0.9 microgram). However, the EC50 (0.2 microgram) at P14 was greater than at P21 despite the fact that mu receptor binding was greater at P14. Opioid receptor binding is developmentally regulated and undergoes substantial postnatal reorganization. However, the number of mu receptor binding sites appears not to be the only determinant of functional sensitivity to spinal morphine. Other factors, such as coupling of the receptors are likely to be important.     

Proxy use of the Canadian SF-36 in rating health status of the disabled elderly

A kappa opioid receptor binding inhibitor was isolated from the fermentation broth of a basidiomycete, Hericium ramosum CL24240 and identified as erinacine E (1). Three analogs of 1 were produced by fermentation in other media and by microbial biotransformation. Of these compounds, 1 was shown to be the most potent binding inhibitor. Preliminary SAR studies of these compounds indicated that all functional groups and side chains were required for the activity. Compound 1 was a highly-selective binding inhibitor for the kappa opioid receptor: 0.8 microM (IC50) for kappa, >200 microM for mu, and >200 microM for delta opioid receptor. Compound 1 suppressed electrically-stimulated twitch responses of rabbit vas deferens with an ED50 of 14 microM. The suppression was recovered by adding a selective kappa opioid receptor antagonist nor-binaltorphimine, indicating that 1 is a kappa opioid receptor agonist.     

Opioid receptors altered binding nature in guinea-pig brain following the development of morphine dependence

Morphine is well known to produce tolerance and dependence. The mechanisms for these phenomena are not clearly understood and there are a number of conflicting reports that chronic morphine administration decreases, increases, or leaves unchanged the number of opioid binding sites. We examined the potency of MScontin (oral controlled-release preparation of morphine) to induce morphine dependence and also determined the change of mu, delta and kappa opioid receptor types in brain homogenates obtained from morphine-dependent guinea-pigs. 1. Guinea-pigs were implanted subcutaneously with MScontin (300 mg.kg-1) and naloxone was employed to precipitate jumping behavior of withdrawal symptoms at various times. The highest degree of physical dependence was observed on the 2nd day after implantation. Therefore, this period was chosen to investigate opioid receptor binding assay. 2. Two days after implantation, the binding of 3H-DAGO (mu agonist), 3H-DPDPE (delta agonist) and 3H-U69593 (kappa agonist) to brain membranes prepared from morphine dependent and control guinea-pigs was determined. Scatchard plot of the saturation binding data revealed an increase in Bmax values (maximum specific binding) and no change in the Kd values (equilibrium dissociation constants) of 3H-opioid ligand bindings obtained from morphine-dependent animals as compared to controls. These results indicate that brain mu, delta and kappa opioid receptors are up-regulated in morphine dependent guinea-pigs.     

Extended life of human corneal endothelial cells transfected with the SV40 large T antigen

PURPOSE: To transfect human corneal endothelial cells with a plasmid vector coding for the SV40 large T antigen to extend the life of the cells in culture. METHODS: Human corneal endothelial cells were transfected with the SV40 large T antigen-coding plasmid pSV3neo using the electroporation method. Transfected and control cells were propagated in culture until senescence. Polymerase chain reaction and immunofluorescence were used to demonstrate messenger RNA and protein, respectively, for the Simian virus 40 large T antigen in the transfected cells. Polymerase chain reaction and hot blotting were used to demonstrate messenger RNA coding for several growth factors and receptors in transfected and control cells. RESULTS: The transfected cells continued to proliferate to 38 passages (more than 120 population doublings) in culture (control cells, 8 population doublings). Transfected cells, but not control cells, expressed messenger RNA coding for the Simian virus 40 large T antigen. Similarly, immunofluorescent staining with monoclonal antibodies demonstrated that the Simian virus 40 large T antigen protein was present in the nucleus of the transfected cells. Transfected cells were shown to produce messenger RNA coding for epidermal growth factor, epidermal growth factor receptor, basic fibroblast growth factor, fibroblast growth factor receptor-1, interleukin-1 alpha, the interleukin-1 receptor, transforming growth factor beta-1, and the glucocorticoid receptor. Qualitative expression of the messenger RNA coding for each of these modulators was similar in proliferating primary corneal endothelial cells and proliferating or confluent transfected corneal endothelial cells. CONCLUSIONS: In culture, the life of human corneal endothelial cells transfected with a plasmid vector coding for the Simian virus 40 large T antigen is extended. This study suggests that human corneal endothelial cells have the capacity for extensive proliferation, but the proliferation of untransfected cells is regulated through mechanisms that have not yet been characterized.     

Dual label in situ hybridization studies provide evidence that luteinizing hormone-releasing hormone neurons do not synthesize messenger ribonucleic acid for mu, kappa, or delta opiate receptors

Abundant evidence suggests that opiatergic neurons play an important intermediary role in the regulation of LHRH release by ovarian steroids; however, it is unclear whether opiates communicate directly or indirectly with LHRH neurons. To investigate this issue, we used dual label in situ hybridization histochemistry to determine whether LHRH neurons synthesize messenger RNA (mRNA) for mu, kappa, and/or delta opiate receptors. For these studies, we examined both intact (n = 3) and ovariectomized, steroid-treated rats. Ten of the ovariectomized rats were implanted 1 week later (day 0) with SILASTIC brand (Dow Corning) capsules of estradiol. On the morning of day 2, half of the estradiol-treated rats were injected with 5 mg progesterone. All animals were killed at approximately 1530 h on day 2. We found that cells expressing mu, kappa, and delta opiate receptor mRNAs were in all sections that also contained LHRH neurons. In every case, LHRH neurons were seen to be surrounded by or in close proximity to cells containing mu, kappa, or delta mRNAs. However, regardless of steroid treatment, we found no neurons containing both LHRH mRNA and mRNAs encoding any of the three receptor subtypes. These results support the hypothesis that LHRH neurons are regulated indirectly by opiatergic neurons.     

Identification of an opioid kappa receptor subtype-selective N-substituent for (+)-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine

A three-component library of compounds was prepared in parallel using multiple simultaneous solution-phase synthetic methodology. The compounds were biased toward opioid receptor antagonist activity by incorporating (+)-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (a potent, nonselective opioid pure antagonist) as one of the monomers. The other two monomers, which included N-substituted or unsubstituted Boc-protected amino acids and a range of substituted aryl carboxylic acids, were selected to add chemical diversity. Screening of these compounds in competitive binding experiments with the kappa opioid receptor selective ligand [3H]U69,593 led to the discovery of a novel kappa opioid receptor selective ligand, N-?(2'S)-[3-(4-hydroxyphenyl)propanamido]-3'-methylbutyl?-(3R, 4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (8, RTI-5989-29). Additional structure-activity relationship studies suggested that 8 possesses lipophilic and hydrogen-bonding sites that are important to its opioid receptor potency and selectivity. These sites appear to exist predominantly within the kappa receptor since the selectivity arises from a 530-fold loss of affinity of 8 for the mu receptor and an 18-fold increase in affinity for the kappa receptor relative to the mu-selective ligand, (+)-N-[trans-4-phenyl-2-butenyl]-(3R, 4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (5a). The degree of selectivity observed in the radioligand binding experiments was not observed in the functional assay. According to its ability to inhibit agonist stimulated binding of [35S]GTPgammaS at all three opioid receptors, compound 8 behaves as a mu/kappa opioid receptor pure antagonist with negligible affinity for the delta receptor.     

Pharmacological characterization of endomorphin-1 and endomorphin-2 in mouse brain

The recently isolated peptides endomorphin-1 and endomorphin-2 have been suggested to be the endogenous ligands for the mu receptor. In traditional opioid receptor binding assays in mouse brain homogenates, both endomorphin-1 and endomorphin-2 competed both mu1 and mu2 receptor sites quite potently. Neither compound had appreciable affinity for either delta or kappa1 receptors, confirming an earlier report. However, the two endomorphins displayed reasonable affinities for kappa3 binding sites, with Ki values between 20 and 30 nM. Both endomorphins competed 3H-[D-Ala2, MePhe4,Gly(ol)5] enkephalin binding to MOR-1 receptors expressed in CHO cells with high affinity. In mouse brain homogenates 125I-endomorphin-1 and 125I-endomorphin-2 binding was selectively competed by mu ligands. 125I-Endomorphin-1 and 125I-endomorphin-2 also labeled MOR-1 receptors expressed in CHO cells with high affinity. Autoradiography of the two 125I-labeled endomorphins demonstrated regional patterns in the brain similar to those previously observed for mu drugs. Pharmacologically, the endomorphins were potent analgesics. Although they were equipotent supraspinally, endomorphin-1 was more potent spinally. Endomorphin analgesia was effectively blocked by naloxone, as well as the mu-selective antagonists beta-funaltrexamine and naloxonazine. In CXBK mice, which are insensitive to supraspinal morphine, neither endomorphin was active, consistent with a mu mechanism of action. Finally, the endomorphins inhibited gastrointestinal transit. In conclusion, these results support the mu selectivity of these agents.