LP1 and LP2: Dual-Target MOPr/DOPr Ligands as Drug Candidates for Persistent Pain Relief.

Although persistent pain is estimated to affect about 20% of the adult population, current treatments have poor results. Polypharmacology, which is the administration of more than one drug targeting on two or more different sites of action, represents a prominent therapeutic approach for the clinical management of persistent pain. Thus, in the drug discovery process the "one-molecule-multiple targets" strategy nowadays is highly recognized. Indeed, multitarget ligands displaying a better antinociceptive activity with fewer side effects, combined with favorable pharmacokinetic and pharmacodynamic characteristics, have already been shown. Multitarget ligands possessing non-opioid/opioid and opioid/opioid mechanisms of action are considered as potential drug candidates for the management of various pain conditions. In particular, dual-target MOPr (mu opioid peptide receptor)/DOPr (delta opioid peptide receptor) ligands exhibit an improved antinociceptive profile associated with a reduced tolerance-inducing capability. The benzomorphan-based compounds LP1 and LP2 belong to this class of dual-target MOPr/DOPr ligands. In the present manuscript, the structure-activity relationships and the pharmacological fingerprint of LP1 and LP2 compounds as suitable drug candidates for persistent pain relief is described.

Emerging Treatment Targets for Migraine and Other Headaches.

Migraine is a complex disorder that is characterized by an assortment of neurological and systemic effects. While headache is the most prominent feature of migraine, a host of symptoms affecting many physiological functions are also observed before, during, and after an attack. Furthermore, migraineurs are heterogeneous and have a wide range of responses to migraine therapies. The recent approval of calcitonin gene-related-peptide based therapies has opened up the treatment of migraine and generated a renewed interest in migraine research and discovery. Ongoing advances in migraine research have identified a number of other promising therapeutic targets for this disorder. In this review, we highlight emergent treatments within the following biological systems: pituitary adenylate cyclase activating peptdie, 2 non-mu opioid receptors that have low abuse liability - the delta and kappa opioid receptors, orexin, and nitric oxide-based therapies. Multiple mechanisms have been identified in the induction and maintenance of migraine symptoms; and this divergent set of targets have highly distinct biological effects. Increasing the mechanistic diversity of the migraine tool box will lead to more treatment options and better patient care.

Met-enkephalin improves metabolic syndrome in high fat diet challenged mice through promotion of adipose tissue browning.

Obesity and its related metabolic disorders including insulin resistance and fatty liver become major public health concerns in both developed and developing countries. Brown adipose tissue (BAT), a critical organ of energy expenditure due to thermogenesis, has been considered as an attractive target for prevention or treatment of obesity and obesity related diseases. Previous studies indicate Met-enkephalin (MetEnk) has the potential on adipocyte browning, however, whether MetEnk displays the impact on adipocyte browning in vivo to improve obesity associated morbidities is still unclear. In the present study, we showed that MetEnk effectively prevented high fat diet (HFD) induced C57BL/6J mice weight gain, clearly enhanced glucose tolerance and insulin sensitivity, and dramatically reduced hepatic steatosis in HFD fed mice. Mechanically, MetEnk restored protein kinase A (PKA) signaling pathway in HFD challenged mice and promoted subcutaneous white adipose tissue (WAT) browning. Our study suggests that MetEnk can be considered as a potential therapeutic peptide for diet-induced obesity and metabolic disorders.

Contribution of Delta-Opioid Receptors to Pathophysiological Events Explored by Endogenous Enkephalins.

Very few discoveries in the neurosciences have triggered clinical speculation and experimentation regarding the etiology of psychiatric illness to the same extent as that following identification of the opiate receptor(s) and subsequent isolation of endogenous morphine-like peptides. There is overwhelming evidence in animals and in human that opioids are involved in behaviorally relevant issues such as the modulation of pain, the response to stress, motivation, addiction, sexuality, food intake, etc., but our knowledge on the possible relation between opioids and mental illness is still very limited.These responses could be explored eitheir by using higlhy selective delta agonist or by emphasizing the effects of phasically secreted endogenous opioid peptides, enkephalin. Both approaches were investigated in particular through protection of enkephalin degradation by dual enkephalinase ihibitors DENKIs such as RB101, PL37 or PL265.

Pharmacologic Evidence for a Putative Conserved Allosteric Site on Opioid Receptors.

Allosteric modulators of G protein-coupled receptors, including opioid receptors, have been proposed as possible therapeutic agents with enhanced selectivity. BMS-986122 is a positive allosteric modulator (PAM) of the µ-opioid receptor (µ-OR). BMS-986187 is a structurally distinct PAM for the δ-opioid receptor (δ-OR) that has been reported to exhibit 100-fold selectivity in promoting δ-OR over µ-OR agonism. We used ligand binding and second-messenger assays to show that BMS-986187 is an effective PAM at the µ-OR and at the κ-opioid receptor (κ-OR), but it is ineffective at the nociceptin receptor. The affinity of BMS-986187 for δ-ORs and κ-ORs is approximately 20- to 30-fold higher than for µ-ORs, determined using an allosteric ternary complex model. Moreover, we provide evidence, using a silent allosteric modulator as an allosteric antagonist, that BMS-986187 and BMS-986122 bind to a similar region on all three traditional opioid receptor types (µ-OR, δ-OR, and κ-OR). In contrast to the dogma surrounding allosteric modulators, the results indicate a possible conserved allosteric binding site across the opioid receptor family that can accommodate structurally diverse molecules. These findings have implications for the development of selective allosteric modulators.

T-Cell Mediation of Pregnancy Analgesia Affecting Chronic Pain in Mice.

It has been reported consistently that many female chronic pain sufferers have an attenuation of symptoms during pregnancy. Rats display increased pain tolerance during pregnancy due to an increase in opioid receptors in the spinal cord. Past studies did not consider the role of non-neuronal cells, which are now known to play an important role in chronic pain processing. Using an inflammatory (complete Freund's adjuvant) or neuropathic (spared nerve injury) model of persistent pain, we observed that young adult female mice in early pregnancy switch from a microglia-independent to a microglia-dependent pain hypersensitivity mechanism. During late pregnancy, female mice show no evidence of chronic pain whatsoever. This pregnancy-related analgesia is reversible by intrathecal administration of naloxone, suggesting an opioid-mediated mechanism; pharmacological and genetic data suggest the importance of δ-opioid receptors. We also observe that T-cell-deficient (nude and Rag1-null mutant) pregnant mice do not exhibit pregnancy analgesia, which can be rescued with the adoptive transfer of CD4+ or CD8+ T cells from late-pregnant wild-type mice. These results suggest that T cells are a mediator of the opioid analgesia exhibited during pregnancy.SIGNIFICANCE STATEMENT Chronic pain symptoms often subside during pregnancy. This pregnancy-related analgesia has been demonstrated for acute pain in rats. Here, we show that pregnancy analgesia can produce a complete cessation of chronic pain behaviors in mice. We show that the phenomenon is dependent on pregnancy hormones (estrogen and progesterone), δ-opioid receptors, and T cells of the adaptive immune system. These findings add to the recent but growing evidence of sex-specific T-cell involvement in chronic pain processing.

Regulation of δ Opioid Receptor-Mediated Signaling and Antinociception in Peripheral Sensory Neurons by Arachidonic Acid-Dependent 12/15-Lipoxygenase Metabolites.

The function of δ opioid receptors (DOR) expressed by peripheral pain-sensing neurons (nociceptors) is regulated by both cyclooxygenase- and lipoxygenase (LOX)-dependent arachidonic acid (AA) metabolites. Whereas cyclooxygenase metabolites enhance responsiveness, LOX metabolites elicit a refractory, nonsignaling state of the DOR receptor system for antinociceptive signaling. In this study, using high-performance liquid chromatography-tandem mass spectrometry analyses, we have found that the 12-/15-LOX metabolites, 12-hydroxyeicosatetraenoic acid (HETE) and 15-HETE, were elevated after treatment of adult rat primary sensory neuron cultures with AA. Exogenously applied 12-HETE and 15-HETE, but not 5-HETE, completely prevented DOR and κ opioid receptor (KOR) agonist-mediated inhibition of prostaglandin E2 (PGE2)-stimulated cAMP accumulation, but not inhibition, by the 5-HT1 receptor agonist 5-carboxamidotryptamine in cultured peripheral sensory neurons and in Chinese hamster ovary (CHO) cells heterologously expressing DOR or KOR. Similarly, intraplantar injection of 12- or 15-HETE, either alone or in combination, prevented DOR agonist-mediated inhibition of PGE2-evoked thermal allodynia. Further, both AA- and carrageenan-mediated induction of the nonresponsive state of the DOR system was blocked by an intraplantar coinjection of the 12-/15-LOX inhibitors baicalein and luteolin. In contrast to the regulation of cAMP signaling, pretreatment with 12- and 15-HETE had no effect on either DOR or KOR agonist- mediated activation of extracellular signal-regulated kinase in peripheral sensory neurons or CHO cells. These results suggest that the analgesic efficacy of peripherally restricted opioids for treatment of inflammatory pain may be enhanced by adjunct inhibition of LOX activity.

Intrathecal morphine-3-glucuronide-induced nociceptive behavior via Delta-2 opioid receptors in the spinal cord.

Intrathecal (i.t.) injection of morphine-3-glucuronide (M3G), a major metabolite of morphine without analgesic actions, produces severe hindlimb scratching followed by biting and licking in mice. The M3G-induced behavioral response was inhibited dose-dependently by pretreatment with an antisera against dynorphin. However, the selective κ-opioid receptor antagonist, nor-BNI did not prevent the M3G-induced behavioral response. Dynorphin is rapidly degraded by a dynorphin-converting enzyme (cystein protease), to leucine-enkephalin (Leu-ENK). The M3G-induced behavioral response was inhibited dose-dependently by pretreatment with the antisera against Leu-ENK. We also showed that M3G co-administered with Leu-ENK-converting enzyme inhibitors, phosphoramidon and bestatin produced much stronger behavioral responses than M3G alone. Furthermore, the M3G-induced behavioral responses were inhibited dose-dependently by i.t. co-administration of the non-selective δ-opioid receptor antagonist, naltrindole or the selective δ2-opioid receptor antagonist, naltriben, whereas the selective δ1-opioid receptor antagonist, BNTX had no effect. An i.t. injection of M3G also produced a definite activation of ERK in the lumbar dorsal spinal cord. Western blotting analysis revealed that antisera against dynorphin, antisera against Leu-ENK, naltrindole or naltriben resulted in a significant blockade of ERK activation induced by M3G in the spinal cord. Taken together, these results suggest that M3G-induced nociceptive responses and ERK activation may be triggered via δ2-opioid receptors activated by Leu-ENK, which is formed from dynorphin in the spinal cord.

Structure-Activity Relationships of the Peptide Kappa Opioid Receptor Antagonist Zyklophin.

The dynorphin (Dyn) A analogue zyklophin ([N-benzyl-Tyr(1)-cyclo(d-Asp(5),Dap(8))]dynorphin A(1-11)NH2) is a kappa opioid receptor (KOR)-selective antagonist in vitro, is active in vivo, and antagonizes KOR in the CNS after systemic administration. Hence, we synthesized zyklophin analogues to explore the structure-activity relationships of this peptide. The synthesis of selected analogues required modification to introduce the N-terminal amino acid due to poor solubility and/or to avoid epimerization of this residue. Among the N-terminal modifications, the N-phenethyl and N-cyclopropylmethyl substitutions resulted in analogues with the highest KOR affinities. Pharmacological results for the alanine-substituted analogues indicated that Phe(4) and Arg(6), but interestingly not the Tyr(1) phenol, are important for zyklophin's KOR affinity and that Arg(7) was important for KOR antagonist activity. In the GTPγS assay, while all of the cyclic analogues exhibited negligible KOR efficacy, the N-cyclopropylmethyl-Tyr(1) and N-benzyl-Phe(1) analogues were 28- and 11-fold more potent KOR antagonists, respectively, than zyklophin.

δ-opioid receptor activation leads to neurite outgrowth and neuronal differentiation via a STAT5B-Gαi/o pathway.

It remains unclear how opioid receptors (δ, µ, κ) are implicated in mechanisms controlling differentiation, cell proliferation, and survival. Opioid receptors are coupled to Gi/Go proteins and recent findings have shown that opioid receptors can form a multicomponent signaling complex, consisting of members of G protein and the signal transducer and activator of transcription (STAT)5B. We thus wondered whether activation of the opioid receptors could direct differentiation and neurite outgrowth through a molecular pathway involving STAT5B and other signaling intermediates. We demonstrate that prolonged δ-opioid receptor (δ-OR) activation with opioid agonists induces STAT5B phosphorylation in Neuro-2A cells. Moreover, [D-Ser2, Leu5, Thr6]-enkephalin-activation of δ-OR triggers neurite outgrowth and neuronal survival; these effects are blocked by the selective antagonist naltrindole, by treatment with pertussis toxin, and after expression of a dominant negative mutant of STAT5B (DN-STAT5B), suggesting that the signaling pathway participating in this mechanism involves Gi/o proteins and p-STAT5B. Additional studies have shown that while [D-Ser(2) , Leu(5) , Thr(6) ]-enkephalin exposure of neuroblastoma cells induces a marked increase in the differentiation marker proteins, ßIII-tubulin (Tuj-1), synaptophysin, and neural cell adhesion molecule, over-expression of the DN-STAT5B attenuated significantly their expression levels. Taken together, our findings demonstrate that δ-OR activation leads to a number of neurotropic events via a Gαi/o-linked and STAT5B-dependent manner. We propose a novel signalling pathway for δ-opioid receptor (δ-ΟR)-mediated neurotropic events. STAT5B interacts with the δ-ΟR and upon prolonged receptor activation phosphorylates STAT5B in a Gi/Go dependent manner leading to increased neuronal survival, neurite outgrowth and differentiation. These findings contribute to a better understanding of the molecular and cellular events following δ-OR activation and suggest a possible neuroprotective role opioids could exert.

PKA and ERK1/2 are involved in dopamine D1 receptor-induced heterologous desensitization of the δ opioid receptor.

AIMS: Chronic administration of cocaine attenuates delta opioid receptor (DOPR) signaling in the striatum and the desensitization is mediated by the indirect actions of cocaine on dopamine D1 receptors (D1R). In addition, DOPR and D1R co-exist in some rat striatal neurons. In the present study, we examined the underlying mechanism of DOPR desensitization by D1R activation. MAIN METHODS: NG 108-15 cells stably expressing HA-rat D1 receptor (HA-D1R) and Chinese hamster ovary (CHO) cells stably expressing both FLAG-mouse DOPR (FLAG-DOPR) and HA-D1R were used as the cell models. Receptor binding, [(35)S]GTPγS binding, receptor phosphorylation and western blot were conducted to examine DOPR affinity, expression, internalization, downregulation, desensitization, phosphorylation and phosphorylated ERK1/2. KEY FINDINGS: Pretreatment with either the DOPR agonist DPDPE or the D1R agonist SKF-82958 for 30min attenuated DPDPE-stimulated [(35)S]GTPγS binding to G proteins, demonstrating homologous and heterologous desensitization of the DOPR, respectively. SKF-82958 pretreatment did not affect the level of DOPR or affinity of DOPR antagonist or agonists, nor did it induce phosphorylation, internalization or down-regulation of the DOPR in the CHO-FLAG-DOPR/HA-D1R cells. Pretreatment of cells with inhibitors of PKA, MEK1 and PI3K, but not PKC, attenuated SKF-82958-induced desensitization of the DOPR. The D1R agonist SKF-82958 enhanced phosphorylation of ERK1/2, and pretreatment with inhibitors of MEK1 and PI3K, but not PKA and PKC, reduced the effect. These results indicate that activation of ERK1/2 and/or PKA, but not PKC, is involved in D1 receptor-induced heterologous desensitization of the DOPR. SIGNIFICANCE: This study provides possible mechanisms underlying D1R activation-induced DOPR desensitization.

Introduction of target cliffs as a concept to identify and describe complex molecular selectivity patterns.

The study of target specificity or selectivity of small molecules is an important task in drug design. In an ideal situation, a compound would exclusively interact with an individual target and hence be target specific. However, such exclusive binding events are likely to be rare, as increasing evidence suggests. Because many compounds are active against more than one target, apparent selectivity often results from potency differences, i.e., a compound that is highly potent against a given target and weakly potent against one or more others displays target selectivity. In a simple case, a compound might have known activity against a pair of targets and be selective for one over the other. Then, selectivity is straightforward to rationalize. However, there are many more complex selectivity relationships associated with multi-target activities of compounds that are difficult to analyze and compare in a consistent manner. For this purpose, we introduce herein target cliffs as a concept to describe complex selectivity patterns. A target cliff is defined as a pair of targets against which at least one compound displays a large difference in potency. As such, target cliffs are distinct from activity cliffs. However, qualifying target pairs (target cliffs) and compound pairs (activity cliffs) can be systematically extracted from the same data structure termed target-compound matrices. Furthermore, these two types of cliffs can be compared to identify and prioritize compounds that are selective and reveal structure-activity relationship (SAR) information.

Characterization of ligand binding to the σ(1) receptor in a human tumor cell line (RPMI 8226) and establishment of a competitive receptor binding assay.

The standard assay for the determination of σ(1) receptor affinities of novel compounds is a competitive binding assay using [(3)H]-(+)-pentazocine as radioligand and membrane preparations from guinea pig brain. Herein, a novel competitive binding assay was developed employing the hematopoietic cell line of human multiple myeloma (RPMI 8226), which expresses a large amount of the human σ(1) receptor. Membrane fragments of RPMI 8226 cells were prepared and characterized. A Western blot analysis confirmed the high density of σ(1) receptors in this cell line. Assay conditions were carefully optimized leading to an incubation period of 120 min, an incubation temperature of 37°C, and receptor material for each well was prepared from 300,000 cells. It was shown that a large excess (10 µM) of (+)-pentazocine, haloperidol, and di-o-tolylguanidine provided the same results during determination of the nonspecific binding. Saturation experiments with the radioligand [(3)H]-(+)-pentazocine led to a K(d)-value of 36±0.3 nM and a B(max)-value of 477±7 fmol/mg protein. These data resulted in approximately 122,000 σ(1) binding sites per cell. The assay was validated by using six known σ(1) ligands and eight σ(1) ligands prepared in our lab. The K(i)-values determined with RPMI 8226-derived receptor material are in good accordance with the K(i)-values obtained with guinea pig brain membrane preparations. Compared with guinea pig brain preparations, the RPMI 8226-derived receptor material represents a better standardized receptor material with a high density of human σ(1) receptors.

Delta-opioid receptor activation promotes mesenchymal stem cell survival via PKC/STAT3 signaling pathway.

BACKGROUND: The survival of stem cells upon transplantation into ischemic myocardium is a major concern in cell-based therapy. In this study, we tested the hypothesis that activation of opioid receptors would enhance the survival of mesenchymal stem cells (MSCs) upon exposure to an injury stimulus. METHODS AND RESULTS: MSCs were obtained from rat bone marrow and cultured in basal DMEM cell culture medium. Delta-opioid receptor (DOR) was present in MSCs as examined by reverse transcription-polymerase chain reaction and immunochemistry. Activation of DOR with 5µmol/L SNC80 (DOR agonist) for 24h significantly enhanced MSC viability upon exposure to 5µg/ml actinomycin D as determined by TUNEL and MTT assays. The cytoprotection was abolished with 20µmol/L naltrindole hydrochloride (a DOR antagonist). Treatment of the cells with 1.5µmol/L chelerythrine (protein kinase C inhibitor) and 1.25µmol/L WP1066 (signal transducer and activator of transcription 3 (STAT3) inhibitor) blocked SNC80-induced cytoprotection. Furthermore, treatment of the cells with chelerythrine also blocked STAT3-phosphorylation and Mcl-1 gene expression. CONCLUSIONS: Taken together, the results indicate that DOR plays a critical role in MSC survival upon exposure to actinomycin D through activation of protein kinase C and its downstream signaling molecules STAT3 and Mcl-1. DOR may be a novel therapeutic target for stem cell survival during cell-based therapy.

Novel potent and selective σ ligands: evaluation of their agonist and antagonist properties.

Novel enantiomers and diastereoisomers structurally related to σ ligand (+)-MR200 were synthesized to improve σ(1)/σ(2) subtype selectivity. The selective σ(1) ligand (-)-8 showed an antagonist profile determined by phenytoin differential modulation of binding affinity in vitro, confirmed in vivo by an increase of κ opioid analgesia. The σ(2) ligand (-)-9 displayed agonist properties in an in vitro isolated organ bath assay and antiproliferative effects on LNCaP and PC3 prostate cancer cell lines.

Gi/o-coupled receptors compete for signaling to adenylyl cyclase in SH-SY5Y cells and reduce opioid-mediated cAMP overshoot.

Organization of G protein-coupled receptors and cognate signaling partners at the plasma membrane has been proposed to occur via multiple mechanisms, including membrane microdomains, receptor oligomerization, and protein scaffolding. Here, we investigate the organization of six types of Gi/o-coupled receptors endogenously expressed in SH-SY5Y cells. The most abundant receptor in these cells was the µ-opioid receptor (MOR), the activation of which occluded acute inhibition of adenylyl cyclase (AC) by agonists to δ-opioid (DOR), nociceptin/orphanin FQ peptide (NOPr), α2-adrenergic (α2AR), cannabinoid 1, and serotonin 1A receptors. We further demonstrate that all receptor pairs share a common pool of AC. The MOR agonist [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) also occluded the ability of DOR agonist to stimulate G proteins. However, at lower agonist concentrations and at shorter incubation times when G proteins were not limiting, the relationship between MOR and DOR agonists was additive. The additive relationship was confirmed by isobolographic analysis. Long-term coadministration of MOR and DOR agonists caused cAMP overshoot that was not additive, suggesting that sensitization of AC mediated by these two receptors occurs by a common pathway. Furthermore, heterologous inhibition of AC by agonists to DOR, NOPr, and α2AR reduced the expression of cAMP overshoot in DAMGO-dependent cells. However, this cross-talk did not lead to heterologous tolerance. These results indicate that multiple receptors could be tethered into complexes with cognate signaling proteins and that access to shared AC by multiple receptor types may provide a means to prevent opioid withdrawal.

Adenosine A(1) receptor agonist N(6)-cyclohexyl-adenosine induced phosphorylation of delta opioid receptor and desensitization of its signaling.

AIM: To define the effect of adenosine A(1) receptor (A(1)R) on delta opioid receptor (DOR)-mediated signal transduction. METHODS: CHO cells stably expressing HA-tagged A(1)R and DOR-CFP fusion protein were used. The localization of receptors was observed using confocal microscope. DOR-mediated inhibition of adenylyl cyclase was measured using cyclic AMP assay. Western blots were employed to detect the phosphorylation of Akt and the DOR. The effect of A(1)R agonist N(6)-cyclohexyladenosine (CHA) on DOR down-regulation was assessed using radioligand binding assay. RESULTS: CHA 1 micromol/L time-dependently attenuated DOR agonist [D-Pen(2,5)]enkephalin (DPDPE)-induced inhibition of intracellular cAMP accumulation with a t(1/2)=2.56 (2.09-3.31) h. Pretreatment with 1 micromol/L CHA for 24 h caused a right shift of the dose-response curve of DPDPE-mediated inhibition of cAMP accumulation, with a significant increase in EC(50) but no change in E(max). Pretreatment with 1 micromol/L CHA for 1 h also induced a significant attenuation of DPDPE-stimulated phosphorylation of Akt. Moreover, CHA time-dependently phosphorylated DOR (Ser363), and this effect was inhibited by A(1)R antagonist 1,3-Dipropyl-8-cyclopentylxanthine (DPCPX) but not by DOR antagonist naloxone. However, CHA failed to produce the down-regulation of DOR, as neither receptor affinity (K(d)) nor receptor density (B(max)) of DOR showed significant change after chronic CHA exposure. CONCLUSION: Activation of A(1)R by its agonist caused heterologous desensitization of DOR-mediated inhibition of intracellular cAMP accumulation and phosphorylation of Akt. Activation of A(1)R by its agonist also induced heterologous phosphorylation but not down-regulation of DOR.

Hypertensive state, independent of hypertrophy, exhibits an attenuated decrease in systolic function on cardiac kappa-opioid receptor stimulation.

Opioids/opiates are commonly administered to alleviate pain, unload the heart, or decrease breathlessness in patients with advanced heart failure. As such, it is important to evaluate whether the myocardial opioidergic system is altered in cardiac disease. A hamster model of spontaneous hypertension was investigated before the development of hypertension (1 mo of age) and in the hypertensive state (10 mo of age) to evaluate the effect of prolonged hypertension on myocardial opioidergic activity. Plasma beta-endorphin was decreased before the development of hypertension and in the hypertensive state (P < 0.05). There was no change in cardiac beta-endorphin content at either time point. No differences were detected in cardiac or plasma dynorphin A, Met-enkephalin, or Leu-enkephalin, or in cardiac peptide expression of kappa- or delta-opioid receptors. mu-Opioid receptor was not detected in either model. To determine how hypertension affects myocardial opioid signaling, the ex vivo work-performing heart was used to assess the cardiac response to opioid administration in healthy hearts and those subjected to chronic hypertension. Agonists selective for the kappa- and delta-opioid receptors, but not mu-opioid receptors, induced a concentration-dependent decrease in cardiac function. The decrease in left ventricular systolic pressure on administration of the kappa-opioid receptor-selective agonist, U50488H, was attenuated in hearts from hamsters subjected to chronic, untreated hypertension (P < 0.05) compared with control. These results show that peripheral and myocardial opioid expression and signaling are altered in hypertension.

Pharmacological characterization of ATPM [(-)-3-aminothiazolo[5,4-b]-N-cyclopropylmethylmorphinan hydrochloride], a novel mixed kappa-agonist and mu-agonist/-antagonist that attenuates morphine antinociceptive tolerance and heroin self-administration behavior.

ATPM [(-)-3-amino-thiazolo[5,4-b]-N-cyclopropylmethylmorphinan hydrochloride] was found to have mixed kappa- and mu-opioid activity and identified to act as a full kappa-agonist and a partial mu-agonist by in vitro binding assays. The present study was undertaken to characterize its in vivo effects on morphine antinociceptive tolerance in mice and heroin self-administration in rats. ATPM was demonstrated to yield more potent antinociceptive effects than (-)U50,488H (trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide). It was further found that the antinociceptive effects of ATPM were mediated by kappa- and mu-, but not delta-opioid, receptors. In addition to its agonist profile on the mu-receptor, ATPM also acted as a mu-antagonist, as measured by its inhibition of morphine-induced antinociception. It is more important that ATPM had a greater ratio of the ED(50) value of sedation to that of antinociception than (-)U50,488 (11.8 versus 3.7), indicative of a less sedative effect than (-)U50,488H. In addition, ATPM showed less potential to develop antinociceptive tolerance relative to (-)U50,488H and morphine. Moreover, it dose-dependently inhibited morphine-induced antinociceptive tolerance. Furthermore, it was found that chronic treatment of rats for 8 consecutive days with ATPM (0.5 mg/kg s.c.) produced sustained decreases in heroin self-administration. (-)U50,488H (2 mg/kg s.c.) also produced similar inhibitory effect. Taken together, our findings demonstrated that ATPM, a novel mixed kappa-agonist and mu-agonist/-antagonist, could inhibit morphine-induced antinociceptive tolerance, with less potential to develop tolerance and reduce heroin self-administration with less sedative effect. kappa-Agonists with some mu-activity appear to offer some advantages over selective kappa-agonists for the treatment of heroin abuse.

delta-Opioid receptor antagonism induces NMDA receptor-dependent excitotoxicity in anoxic turtle cortex.

delta-Opioid receptor (DOR) activation is neuroprotective against short-term anoxic insults in the mammalian brain. This protection may be conferred by inhibition of N-methyl-d-aspartate receptors (NMDARs), whose over-activation during anoxia otherwise leads to a deleterious accumulation of cytosolic calcium ([Ca(2+)](c)), severe membrane potential (E(m)) depolarization and excitotoxic cell death (ECD). Conversely, NMDAR activity is decreased by approximately 50% with anoxia in the cortex of the painted turtle, and large elevations in [Ca(2+)](c), severe E(m) depolarization and ECD are avoided. DORs are expressed in high quantity throughout the turtle brain relative to the mammalian brain; however, the role of DORs in anoxic NMDAR regulation has not been investigated in turtles. We examined the effect of DOR blockade with naltrindole (1-10 micromol l(-1)) on E(m), NMDAR activity and [Ca(2+)](c) homeostasis in turtle cortical neurons during normoxia and the transition to anoxia. Naltrindole potentiated normoxic NMDAR currents by 78+/-5% and increased [Ca(2+)](c) by 13+/-4%. Anoxic neurons treated with naltrindole were strongly depolarized, NMDAR currents were potentiated by 70+/-15%, and [Ca(2+)](c) increased 5-fold compared with anoxic controls. Following naltrindole washout, E(m) remained depolarized and [Ca(2+)](c) became further elevated in all neurons. The naltrindole-mediated depolarization and increased [Ca(2+)](c) were prevented by NMDAR antagonism or by perfusion of the G(i) protein agonist mastoparan-7, which also reversed the naltrindole-mediated potentiation of NMDAR currents. Together, these data suggest that DORs mediate NMDAR activity in a G(i)-dependent manner and prevent deleterious NMDAR-mediated [Ca(2+)](c) influx during anoxic insults in the turtle cortex.