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Opioid and N-methyl-D-aspartate (NMDA) receptors mediate different effects of fluoxetine. We investigated whether opioid and NMDA receptors are involved in the protective effect of fluoxetine against the behavioral despair induced by acute physical stress in male mice. We used the forced swimming test (FST), tail suspension test (TST), and open-field test (OFT) for behavioral evaluation. We used fluoxetine, naltrexone (opioid receptor antagonist), MK-801 (NMDA receptor antagonist), morphine (opioid receptor agonist), and NMDA (NMDA receptor agonist). Acute foot-shock stress (FSS) significantly induced behavioral despair (depressive-like) and anxiety-like behaviors in tests. Fluoxetine (5 mg/kg) reversed the depressant-like effect of FSS, but it did not alter the locomotion and anxiety-like behavior in animals. Acute administration of subeffective doses of naltrexone (0.3 mg/kg) or MK-801 (0.01 mg/kg) potentiated the antidepressant-like effect of fluoxetine, while subeffective doses of morphine (1 mg/kg) and NMDA (75 mg/kg) abolished this effect of fluoxetine. Also, co-administration of subeffective doses of naltrexone (0.05 mg/kg) and MK-801 (0.003 mg/kg) with fluoxetine (1 mg/kg) induced a significant decrease in the immobility time in FST and TST. Our results showed that opioid and NMDA receptors (alone or in combination) are involved in the antidepressant- like effect of fluoxetine against physical stress. Key words: fluoxetine, opioid receptor, NMDA receptor, acute foot shock stress, depression. Les recepteurs d'opioides et du N-methyl-D-aspartate (NMDA) medient differents effets de la fluoxetine. Les auteurs ont examine si les recepteurs d'opioides et du NMDA sont impliques dans les effets protecteurs de la fluoxetine contre le<> comportemental induit par un stress physique aigu chez la souris male. Ils ont utilise un test de la nage forcee, un test de suspension par la queue et le test du champ ouvert aux fins devaluation comportementale. Ils ont utilise de la fluoxetine, de la naltrexone (antagoniste du recepteur d'opioides), du MK-801 (antagoniste du recepteur du NMDA), de la morphine (agoniste du recepteur d'opioides) et du NMDA (agoniste du recepteur du NMDA). Le stress cause par un choc electrique aigu aux pattes induisait un <> comportemental (de type depressif) de maniere significative et des comportements de types anxieux. La fluoxetine (5 mg/kg) renversait l'effet depresseur du choc electrique aigu aux pattes mais elle n'affectait pas la locomotion et le comportement de type anxieux des animaux. L'administration aigue de doses sous-efficaces de naltrexone (0,3 mg/kg) ou de MK-801 (0,01 mg/kg) potentialisait l'effet antidepresseur de la fluoxetine. Or, des doses sous-efficaces de morphine (1 mg/kg) et de NMDA (75 mg/kg) abolissaient cet effet de la fluoxetine. Aussi, la co-administration de doses sous-efficaces de naltrexone (0,05 mg/kg) et de MK-801 (0,003 mg/kg) avec la fluoxetine (1 mg/kg) induisait une diminution significative du temps d'immobilite dans le test de la nage forcee et le test de suspension par la queue. Les resultats des auteurs ont montre que les recepteurs d'opioides et du NMDA (seuls ou en combinaison) sont impliques dans l'effet antidepresseur de la fluoxetine contre le stress physique. [Traduit par la Redaction] Mots-cles: fluoxetine, recepteur d'opioides, recepteur du NMDA, stress cause par un choc electrique aigu aux pattes, depression., Introduction Exposure to stress produces a variety of physiological and pathophysiological alterations, including neurophysiological responses in the brain (Kirby et al. 2013) that may consequently lead to development of behavioral [...]

μ-Opioid receptors (pORs) are G-protein-coupled receptors that are activated by a structurally diverse spectrum of natural and synthetic agonists including endogenous endorphin peptides, morphine and methadone. The recent structures of [...]

Purpose Opioid-induced spastic paraplegia after transient spinal cord ischemia during aortic surgery has been reported. Opioids modulate neurotransmission through mu (l) opioid receptors (MORs) in the spinal ventral horn. However, their effects during ischemic insult are not understood. Methods The effects of the selective μ agonist [D-[Ala.sup.2], N-Me-[Phe.sup.4], [Gly.sup.5]-ol]enkephalin (DAMGO) on ischemia-induced agonal currents were examined in the spinal lamina IX neurons of neonatal rats by using the whole-cell patch-clamp technique. Ischemia was simulated in vitro by oxygen/glucose deprivation. Results DAMGO (1 µM) produced outward currents in ~60% of spinal lamina IX neurons at a holding potential of -70 mV. Superfusion with ischemia-simulating medium elicited an agonal current. The latency was 457 [+ or -] 18 s. Despite its neuromodulatory effects, DAMGO did not significantly change the latencies of the agonal currents with (440 ± 23 s) or without (454 ± 33 s) DAMGO-induced currents. Conclusion Activation of MORs does not influence ongoing ischemia-induced neuronal death. Our findings indicate that MOR agonist administration should be suitable as an anesthetic during aortic surgery. Keywords μ Opioid * Oxygen and glucose deprivation * Spinal cord ischemia * Spinal motoneuron, Introduction One of the most worrisome complications of thoracoabdominal aneurysm repair is paraplegia caused by spinal cord ischemia because it results in serious physical disability [1]. Because the extent of [...]

Opioids represent widely prescribed and abused medications, although their signal transduction mechanisms are not well understood. Here we present the 1.8 Å high-resolution crystal structure of the human δ-opioid receptor (δ-OR), revealing the presence and fundamental role of a sodium ion in mediating allosteric control of receptor functional selectivity and constitutive activity. The distinctive δ-OR sodium ion site architecture is centrally located in a polar interaction network in the seven-transmembrane bundle core, with the sodium ion stabilizing a reduced agonist affinity state, and thereby modulating signal transduction. Site-directed mutagenesis and functional studies reveal that changing the allosteric sodium site residue Asn 131 to an alanine or a valine augments constitutive β-arrestin-mediated signalling. Asp95Ala, Asn310Ala and Asn314Ala mutations transform classical δ-opioid antagonists such as naltrindole into potent β-arrestin-biased agonists. The data establish the molecular basis for allosteric sodium ion control in opioid signalling, revealing that sodium-coordinating residues act as 'efficacy switches' at a prototypic G-protein-coupled receptor., The three classical opioid receptors (μ, κ and δ-OR) and the related nociceptin/orphanin FQ peptide receptor (NOP) are G-protein-coupled receptors (GPCRs) essential for regulating nociception, mood and awareness (1). These [...]

Purpose Pruritus associated with intrathecal opioid administration is a common side effect. There is evidence that κ-opioid receptor agonists have antipruritic activity. Butorphanol has agonist actions at both κ-opioid and μ-opioid receptors. This study was designed to evaluate the antipruritic efficacy of butorphanol after intrathecal morphine administration in the setting of a randomized, double-blind study of parturients undergoing cesarean section. Methods Ninety-one women who received combined spinal-epidural anesthesia with 1.2 ml 0.5 % isobaric bupivacaine and 0.1 mg preservative-free morphine were included in this study. After delivery of the baby, the parturients were randomly allocated to two groups: butorphanol group (n = 46) and physiological saline group (n = 45). In the butorphanol group, parturients received an intravenous loading dose of 1 mg butorphanol followed by infusion of 0.2 mg/h butorphanol. The physiological saline group received an infusion of the same volume of physiological saline. The presence of pruritus, visual analog scores for pain, sedation scores, and adverse effects were recorded 1, 2, 4, 6, 8, 10, 12, and 24 h after intrathecal morphine administration. Results The incidence of pruritus at 24 h was significantly more frequent in the physiological saline group than in the butorphanol group (48.9 vs. 13.0 %, P < 0.001). The severity of pruritus was significantly greater in the saline group than in the butorphanol group 2, 4, 6, 8 and 10 h after intrathecal morphine injection (P = 0.004, 0.001, 0.002, and 0.003, respectively). The visual analog scale scores at 24 h were significantly lower in the butorphanol group than in physiological saline group (P < 0.001). The Ramsay sedation score in the butorphanol group was significantly higher than that in the physiological saline group after 1, 2, 4, 6, 8, 10, 12, and 24 h (P < 0.05). There were no significant differences between the two groups in nausea/vomiting and other adverse effects. Conclusion Administration of intravenous butorphanol after delivery of the baby can reduce pruritus that has been induced by intrathecal morphine administration in cesarean delivery with combined spinal-epidural anesthesia. Keywords Pruritus * Intrathecal opioid administration * κ-Opioid receptor, Introduction Intraspinal administration of opioids is one the most effective analgesic methods in many surgical procedures. However, intrathecal opioid injections are often accompanied by many troublesome adverse reactions, e.g., nausea, [...]

The opioid receptor family comprises three members, the μ-, δ- and κ-opioid receptors, which respond to classical opioid alkaloids such as morphine and heroin as well as to endogenous peptide ligands like endorphins. They belong to the G-proteincoupled receptor (GPCR) superfamily, and are excellent therapeutic targets for pain control. The δ-opioid receptor (δ-OR) has a role in analgesia, as well as in other neurological functions that remain poorly understood (1). The structures of the μ-OR and κ-OR have recently been solved (2,3). Here we report the crystal structure of the mouse δ-OR, bound to the subtype-selective antagonist naltrindole. Together with the structures of the μ-OR and κ-OR, the δ-OR structure provides insights into conserved elements of opioid ligand recognition while also revealing structural features associated with ligand-subtype selectivity. The binding pocket of opioid receptors can be divided into two distinct regions. Whereas the lower part of this pocket is highly conserved among opioid receptors, the upper part contains divergent residues that confer subtype selectivity. This provides a structural explanation and validation for the 'message-address' model of opioid receptor pharmacology (4,5), in which distinct 'message' (efficacy) and 'address' (selectivity) determinants are contained within a single ligand. Comparison of the address region of the o-OR with other GPCRs reveals that this structural organization may be a more general phenomenon, extending to other GPCR families as well., Opioid receptors have an important role in the central nervous system, regulating pain perception, hedonic homeostasis, mood and wellbeing (1). Thus, they have long been the focus of physiological and [...]

Members of the opioid receptor family of G-protein-coupled receptors (GPCRs) are found throughout the peripheral and central nervous system, where they have key roles in nociception and analgesia. Unlike the 'classical' opioid receptors, δ, κ and μ (δ-OR, κ-OR and µ-OR), which were delineated by pharmacological criteria in the 1970s and 1980s, the nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP, also known as ORL-1) was discovered relatively recently by molecular cloning and characterization of an orphan GPCR (1). Although it shares high sequence similarity with classical opioid GPCR subtypes (~60%), NOP has a markedly distinct pharmacology, featuring activation by the endogenous peptide N/OFQ, and unique selectivity for exogenous ligands (2,3). Here we report the crystal structure of human NOP, solved in complex with the peptide mimetic antagonist compound-24 (C-24) (ref. 4), revealing atomic details of ligand-receptor recognition and selectivity. Compound-24 mimics the first four amino-terminal residues of the NOP-selective peptide antagonist UFP-101, a close derivative of N/OFQ, and provides important clues to the binding of these peptides. The X-ray structure also shows substantial conformational differences in the pocket regions between NOP and the classical opioid receptors κ (ref. 5) and μ (ref. 6), and these are probably due to a small number of residues that vary between these receptors. The NOP-compound-24 structure explains the divergent selectivity profile of NOP and provides a new structural template for the design of NOP ligands., The pharmacological effects of NOP are complex and distinct from classical opioid receptors. N/OFQ shares sequence similarity with other opioid peptides, notably the κ-OR endogenous ligand dynorphin A, but does [...]

Opioid receptors mediate the actions of endogenous and exogenous opioids on many physiological processes, including the regulation of pain, respiratory drive, mood, and--in the case of κ-opioid receptor (κ-OR)--dysphoria and psychotomimesis. Here we report the crystal structure of the human κ-OR in complex with the selective antagonist JDTic, arranged in parallel dimers, at 2.9 A resolution. The structure reveals important features of the ligand-binding pocket that contribute to the high affinity and subtype selectivity of JDTic for the human κ-OR. Modelling of other important κ-OR-selective ligands, including the morphinan-derived antagonists norbinaltorphimine and 5'-guanidinonaltrindole, and the diterpene agonist salvinorin A analogue RB-64, reveals both common and distinct features for binding these diverse chemotypes. Analysis of site-directed mutagenesis and ligand structure-activity relationships confirms the interactions observed in the crystal structure, thereby providing a molecular explanation for κ-OR subtype selectivity, and essential insights for the design of compounds with new pharmacological properties targeting the human κ-OR., The four opioid receptors, μ, δ, κ and the nociceptin/orphanin FQ peptide receptor, belong to the class A (rhodopsin-like) γ subfamily of G-protein-coupled receptors (GPCRs) (1) with a common seven-transmembrane [...]

Sexually dimorphic nociception and opioid antinociception is very pervasive but poorly understood. We had demonstrated that spinal morphine antinociception in females, but not males, requires the concomitant activation of spinal [mu]- and [kappa]-opioid receptors (MOR and KOR, respectively). This finding suggests an interrelationship between MOR and KOR in females that is not manifest in males. Here, we show that expression of a MOR/KOR heterodimer is vastly more prevalent in the spinal cord of proestrous vs. diestrous females and vs. males. Cross-linking experiments in combination with in vivo pharmacological analyses indicate that heterodimeric MOR/KOR utilizes spinal dynorphin 1-17 as a substrate and is likely to be the molecular transducer for the female-specific KOR component of spinal morphine antinociception. The activation of KOR within the heterodimeric MOR/KOR provides a mechanism for recruiting spinal KOR-mediated antinociception without activating the concomitant pronociceptive functions that monomeric KOR also subserves. Spinal cord MOR/KOR heterodimers represent a unique pharmacological target for female-specific pain control. estrous cycle | sexual dimorphism | sex steroids | signaling complexes | estrogen and progesterone doi/ 10.1073/pnas.1009923107

We previously reported that soymorphins, [mu]-opioid agonist peptides derived from soy [beta]-conglycinin [beta]-subunit, have anxiolytic-like activity. The aim of this study was to investigate the effects of soymorphins on food intake and gut motility, along with their mechanism. We found that soymorphins decreases food intake after oral administration in fasted mice. Orally administered soymorphins suppressed small intestinal transit at lower dose than that of anorexigenic activity. Suppression of food intake and small intestinal transit after oral administration of soymorphins was inhibited by naloxone or naloxonazine, antagonists of [mu]- or [[mu].sub.1]-opioid receptor, respectively, after oral but not intraperitoneal administration. The inhibitory activities of small intestinal transit by soymorphins were also inhibited by WAY100135, raclopride, or saclofen, antagonists for serotonin 5-HTIA, dopamine 02, or GABAB receptor, respectively. We then examined the order of activation of 5-[HT.sub.1A], [D.sub.2], and [GABA.sub.B] receptors, using their agonists and antagonists. The inhibitory effect of 8-hydroxy-2-dipropylaminotetralin hydrobromide, a 5-HT1A agonist, after oral administration on small intestinal transit was blocked by raclopride or saclofen. Bromocriptine, a [D.sub.2] agonistinduced small intestinal transit suppression, was inhibited by saclofen, but not by WAY100135. Baclofen, a [GABA.sub.B] agonist-induced small intestinal transit suppression, was not blocked by WAY100135 or raclopride. These results suggest that 5-[HT.sub.1A] activation elicits [D.sub.2] followed by GABAB activations in small intestinal motility. We conclude that orally administered soymorphins suppress food intake and small intestinal transit via [[mu].sub.1]-opioid receptor coupled to 5-[HT.sub.1A], [D.sub.2], and [GABA.sub.B] systems. anorexigenic activity; small intestinal motility; [[mu].sub.1]-opioid receptor; soy [beta]-conglycinin [beta]-subunit doi: 10.1152/ajpgi.00081.2010.

Morphine-induced analgesia and antinociceptive tolerance are known to be modulated by interaction between [delta]-opioid receptors (DORs) and [mu]-opioid receptors (MORs) in the pain pathway. However, evidence for expression of DORs in nociceptive small-diameter neurons in dorsal root ganglia (DRG) and for coexistence of DORs with MORs and neuropeptides has recently been challenged. We now report, using in situ hybridization, single-cell PCR, and immunostaining, that DORs are widely expressed not only in large DRG neurons but in small ones and coexist with MORs in peptidergic small DRG neurons, with protachykinin-dependent localization in large dense-core vesicles. Importantly, both DOR and MOR agonists reduce depolarization-induced [Ca.sup.2+] currents in single small DRG neurons and inhibit afferent C-fiber synaptic transmission in the dorsal spinal cord. Thus, coexistence of DORs and MORs in small DRG neurons is a basis for direct interaction of opioid receptors in modulation of nociceptive afferent transmission and opioid analgesia. dorsal root ganglion | spinal cord | peptides | pain | tolerance doi/ 10.1073/pnas.1008382107

ABSTRACT The isolation, structures and biological activity of the neoclerodane and other natural products obtained from the Mexican hallucinogenic sage, Salvia divinorum are reviewed. Keywords: Salvia divinorum, salviarins, opioid receptors, [...]

We previously demonstrated that several epoxyeicosatrienoic acids (EETs) produce reductions in myocardial infarct size in rats and dogs. Since a recent study demonstrated the release of opioids in mediating the antinociceptive effect of 14,15-EET, we hypothesized that endogenous opioids may also be involved in mediating the cardioprotective effect of the EETs. To test this hypothesis, we used an in vivo rat model of infarction and a rat Langendorff model. In the infarct model, hearts were subjected to 30 min occlusion of the left coronary artery and 2 h reperfusion. Animals were treated with 11,12-EET or 14,15-EET (2.5 mg/kg) alone 15 min before occlusion or with opioid antagonists [naloxone, naltrindole, nor-binaltorphimine (nor-BNI), and D-Phe-Cys-Tyr-D-Trp-Om-Thr-Pen-Thr-N[H.sub.2] (CTOP), a nonselective, a selective [delta], a selective [kappa], and a selective [mu] receptor antagonist, respectively] 10 min before EET administration. In four separate groups, antiserum to Met- and Leuenkephalin and dynorphin-A-(1-17) was administered 50 min before the 11,12-EET administration. Infarct size expressed as a percent of the area at risk (IS/AAR) was 63.5 [+ or -] 1.2, 45.3 [+ or -] 1.0, and 40.9 [+ or -] 1.2% for control, 11,12-EET, and 14,15-EET, respectively. The protective effects of 11,12-EET were abolished by pretreatment with either naloxone (60.5 [+ or -] 1.8%), naltrindole (60.8 [+ or -] 1.0%), nor-BNI (62.3 [+ or -] 2.8%), or Met-enkephalin antiserum (63.2 [+ or -] 1.7%) but not CTOP (42.0 [+ or -] 3.0%). In isolated heart experiments, 11,12-EET was administered to the perfusate 15 min before 20 min global ischemia followed by 45 min reperfusion in control hearts or in those pretreated with pertussis toxin (48 h). 11,12-EET increased the recovery of left ventricular developed pressure from 33 [+ or -] 1 to 45 [+ or -] 6% (P < 0.05) and reduced ISIAAR from 37 [+ or -] 4 to 20 [+ or -] 3% (P < 0.05). Both pertussis toxin and naloxone abolished these beneficial effects of 11,12-EET. Taken together, these results suggest that the major cardioprotective effects of the EETs depend on activation of a [G.sub.i/o] protein-coupled [delta]- and/or [kappa]-opioid receptor. nor-binaltorphimine; infarct size; area at risk doi: 10.1152/ajpheart.00815.2009.

Epidermal growth factor (EGF), a mitogen, also stimulates neurite extension during development, but the underlying mechanism is elusive. This study reveals a functional role for kappa opioid receptor (KOR) in EGF-stimulated neurite extension, and the underlying mechanism. EGF and activated EGF receptor (EGFR) levels are elevated in embryonic spinal cords during late gestation stages, with concurrent rise in protein levels of KOR and axon extension markers, growth-associated protein 43 (GAP43), and transient axonal glycoprotein-1 (TAG-1). Both GAP43 and TAG-1 levels are significantly lower in KOR-null ([KOR.sup.-/-]) spinal cords, and EGFR inhibitors effectively reduce the levels of KOR, GAP43, and TAG-1 in wildtype embryonic spinal cords. For [KOR.sup.-/-] or KOR-knockdown dorsal root ganglion (DRG) neurons, EGF can no longer effectively stimulate axon extension, which can be rescued by introducing a constitutive KOR expressing vector but not by a regulated KOR vector carrying its 5' untranslated region, which can be bound and repressed by growth factor receptor-bound protein 7 (Grb7). Furthermore, blocking KOR activation by application of anti-dynorphin, KOR antagonist, or EGFR inhibitor effectively reduces axon extension of DRG neurons. Thus, EGF-stimulated axon extension during development is mediated, at least partially, by specific elevation of KOR protein production at posttranscriptional level, as well as activation of KOR signaling. The result also reveals an action of EGF to augment posttranscriptional regulation of certain mRNAs during developmental stages. 5' untranslated region | dorsal root ganglion | epidermal growth factor | growth factor receptor-bound protein 7 | posttranscriptional regulation www.pnas.org/cgi/doi/10.1073/pnas.0912367107

We report the first example of a coordinated dual action of epidermal growth factor (EGF) in stimulating the nuclear--cytoplasmic export and translation of a select messenger RNA (mRNA). The effect of EGF is mediated by the RNA-binding protein Grb7 (growth factor receptor-bound protein 7), which serves as an adaptor for a specific mRNA--protein export complex and a translational regulator. Using the [kappa]--opioid receptor (OR [KOR]) as a model, we demonstrate that EGF activates nuclear SHP-2 (Src homology region 2--containing tyrosine phosphatase), which dephosphorylates Grb7 in the nucleus. Hypophosphorylated Grb7 binds to the KOR mRNA and recruits the Hu antigen R--exportin-1 (CRM1) complex to form a nuclear--cytoplasmic export complex that exports KOR mRNA. EGF also activates focal adhesion kinase in the cytoplasm to rephosphorylate Grb7, releasing KOR mRNA for active translation. In summary, this study uncovers a coordinated, dual activity of EGF in facilitating nuclear export of a specific mRNA--protein complex as well as translational activation of the exported mRNA. doi/10.1083/jcb.200910083

Although stress has profound effects on motivated behavior, the underlying mechanisms responsible are incompletely understood. In this study we elucidate a functional pathway in mouse brain that encodes the aversive effects of stress and mediates stress-induced reinstatement of cocaine place preference (CPP). Activation of the dynorphin/kappa opioid receptor (KOR) system by either repeated stress or agonist produces conditioned place aversion (CPA). Because KOR inhibition of dopamine release in the mesolimbic pathway has been proposed to mediate the dysphoria underlying this response, we tested dopamine-deficient mice in this study and found that KOR agonist in these mice still produced CPA. However, inactivation of serotonergic KORs by injection of the KOR antagonist norBNI into the dorsal raphe nucleus (DRN), blocked aversive responses to the KOR agonist U50,488 and blocked stress-induced reinstatement of CPP. KOR knockout (KO) mice did not develop CPA to U50,488; however, lentiviral re-expression of KOR in the DRN of KOR KO mice restored place aversion. In contrast, lentiviral expression in DRN of a mutated form of KOR that fails to activate p38 MAPK required for KOR-dependent aversion, did not restore place aversion. DRN serotonergic neurons project broadly throughout the brain, but the inactivation of KOR in the nucleus accumbens (NAc) coupled with viral re-expression in the DRN of KOR KO mice demonstrated that aversion was encoded by a DRN to NAc projection. These results suggest that the adverse effects of stress may converge on the serotonergic system and offers an approach to controlling stress-induced dysphoria and relapse. depression | drug addiction | dynorphin | serotonin www.pnas.org/cgi/doi/10.1073/pnas.0910705106

The cyclic peptide zyklophin {[N-benzyl[Tyr.sup.1],cyclo([D-Asp.sup.5], [Dap.sup.8])dynorphin A-(1-11)N[H.sub.2], Patkar KA, et al. (2005) J Med Chem 48: 4500-4503} is a selective peptide kappa opioid receptor (KOR) antagonist that shows activity following systemic administration. Systemic (1-3 mg/kg s.c.) as well as central (0.3-3 nmol intracerebroventricular, i.c.v.) administration of this peptide dose-dependently antagonizes the antinociception induced by the selective KOR agonist U50,488 in C57BL/6J mice tested in the 55[degrees]C warm water tail withdrawal assay. Zyklophin administration had no effect on morphine-or SNC-80-mediated antinociception, suggesting that zyklophin selectively antagonizes KOR in vivo. Additionally, the antagonism of antinociception induced by centrally (i.c.v.) administered U50,488 following peripheral administration of zyklophin strongly suggests that the peptide crosses the blood-brain barrier to antagonize KOR in the CNS. Most importantly, the antagonist activity of zyklophin (3 mg/kg s.c.) lasts less than 12 h, which contrasts sharply with the exceptionally long duration of antagonism reported for the established small-molecule selective KOR antagonists such as norbinaltorphimine (nor-BNI) that last weeks after a single administration. Systemically administered zyklophin (3 mg/kg s.c.) also prevented stress-induced reinstatement of cocaine-seeking behavior in a conditioned place preference assay. In conclusion, the peptide zyklophin is a KOR-selective antagonist that exhibits the desired shorter duration of action, and represents a significant advance in the development of KOR-selective antagonists. cocaine abuse | dynorphin A analog | kappa opioid receptor antagonist | opioid peptide | stress doi/10.1073/pnas.0910180106

The opioid growth factor (OGF) regulates cell proliferation of human cancer cells through the cyclin-dependent kinase inhibitory pathway, with mediation of this action by the OGF receptor (OGFr). The ubiquity of the OGF-OGFr axis in human cancer is unknown. We used 31 human cancer cell lines, representative of more than 90% of neoplasias occurring in humans, and found that OGF and OGFr were detected in the cytoplasm and nucleus by immunohistochemistry. The addition of OGF to cultures depressed cell number up to 41%, whereas naltrexone (NTX) increased cell proliferation by up to 44%, a total of 85% in the modulating capacity for the OGF-OGFr axis. Neutralization of OGF by specific antibodies led to a marked increase in cell number. Knockdown of OGFr by OGFr-siRNA resulted in a significant increase in the number of cells, even in the face of the addition of exogenous OGF. The cultures to which NTX was added and subjected to OGFr-siRNA were similar to those with OGF-siRNA alone. The OGF-OGFr axis, a physiological determinant of cell-proliferative activity, is a ubiquitous feature of human cancer cells. The identification of this native biological system in neoplasia may be important in understanding the pathophysiology of neoplasia, and in designing treatment modalities that utilize the body's own chemistry. siRNA; immunohistochemistry; tissue culture; naltrexone; opioid antagonists doi: 10.1152/ajpregu.00414.2009

Scientific understanding of social pain--the hurt feelings resulting from social rejection, separation, or loss--has been facilitated by the hypothesis that such feelings arise, in part, from some of the same neural and neurochemical systems that generate the unpleasant feelings resulting from physical pain. Accordingly, in animals, the painkiller morphine not only alleviates the distress of physical pain, but also the distress of social separation. Because morphine acts on the [mu]-opioid receptor, we examined whether variation in the [mu]-opioid receptor gene (OPRM1), as measured by the functional A118G polymorphism, was associated with individual differences in rejection sensitivity. Participants (n = 122) completed a self-report inventory of dispositional sensitivity to social rejection and a subsample (n = 31) completed a functional MRI session in which they were rejected from an online ball-tossing game played with two supposed others. The A118G polymorphism was associated with dispositional sensitivity to rejection in the entire sample and in the fMRI subsample. Consistent with these results, G allele carriers showed greater reactivity to social rejection in neural regions previously shown to be involved in processing social pain as well as the unpleasantness of physical pain, particularly the dorsal anterior cingulate cortex (dACC) and anterior insula. Furthermore, dACC activity mediated the relationship between the A118G polymorphism and dispositional sensitivity to rejection, suggesting that this is a critical site for [mu]-opioid-related influence on social pain. Taken together, these data suggest that the A118G polymorphism specifically, and the [mu]-opioid receptor more generally, are involved in social pain in addition to physical pain. anterior cingulate | brain | exclusion | genetic | social pain

[micro]-Opioid receptor (MOR) agonists represent the gold standard for the treatment of severe pain but may paradoxically also enhance pain sensitivity, that is, lead to opioid-induced hyperalgesia (OIH). We show that abrupt withdrawal from MOR agonists induces long-term potentiation (LTP) at the first synapse in pain pathways. Induction of opioid withdrawal LTP requires postsynaptic activation of heterotrimeric guanine nucleotide-binding proteins and N-methyl-D-aspartate receptors and a rise of postsynaptic calcium concentrations. In contrast, the acute depression by opioids is induced presynaptically at these synapses. Withdrawal LTP can be prevented by tapered withdrawal and shares pharmacology and signal transduction pathways with OIH. These findings provide a previously unrecognized target to selectively combat pro-nociceptive effects of opioids without compromising opioid analgesia.

Sighs, a well-known phenomenon in mammals, are substantially augmented by hypoxia and hypercapnia. Because (D-[Ala.sup.2],N-Me-[Phe.sup.4],Gly-ol)enkephalin (DAMGO), a [micro]-receptor agonist, injected intravenously and locally in the caudal medullary raphe region (cMRR) decreased the ventilatory response to hypoxia and hypercapnia, we hypothesized that these treatments could inhibit sigh responses to these chemical stimuli. The number and amplitude of sighs were recorded during three levels of isocapnic hypoxia (15%, 10%, and 5% [O.sub.2] for 1.5 min) or hypercapnia (3%, 7%, and 10% C[O.sub.2] for 4 min) to test the dependence of sigh responses on the intensity of chemical drive in anesthetized and spontaneously breathing rats. The role of [micro]-receptors in modulating sigh responses to 10% [O.sub.2] or 7% C[O.sub.2] was subsequently evaluated by comparing the sighs before and after 1) intravenous administration of DAMGO (100 [micro]g/kg), 2) microinjection of DAMGO (35 ng/100 nl) into the cMRR, and 3) intravenous administration of DAMGO after microinjection of D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-N[H.sub.2] (CTAP, 100 ng/100 nl), a [micro]-receptor antagonist, into the cMRR. Hypoxia and hypercapnia increased the number, but not amplitude, of sighs in a concentration-dependent manner, and the responses to hypoxia were significantly greater than those to hypercapnia. Systemic and local injection of DAMGO into the cMRR predominantly decreased the number of sighs, while microinjection into the rostral and middle MRR had no or limited effects. Microinjecting CTAP into the cMRR significantly diminished the systemic DAMGO-induced reduction of the number of sighs in response to hypoxia, but not to hypercapnia. Thus we conclude that hypoxia and hypercapnia elevate the number of sighs in a concentration-dependent manner in anesthetized rats, and this response is significantly depressed by activating systemic [micro]-receptors, especially those within the cMRR. hypercapnia; hypoxia

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 Leuenkephalin, or in cardiac peptide expression of [kappa]- or [delta]-opioid receptors. [micro]-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 [micro]-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. hypertension; hamsters; cardiac physiology; cardiac function; [beta]-endorphin; cardiac opioid receptors

Heroin remains a major drug of abuse and is preferred by addicts over morphine. Like morphine, heroin has high affinity and selectivity for [mu]-receptors, but its residual analgesia in exon 1 MOR-1 knockout mice that do not respond to morphine suggests a different mechanism of action. MOR-1 splice variants lacking exon 1 have been observed in mice, humans, and rats, raising the possibility that they might be responsible for the residual heroin and morphine-6[beta]-glucuronide (M6G) analgesia in the exon 1 knockout mice. To test this possibility, we disrupted exon 11 of MOR-1, which eliminates all of the variants that do not contain exon 1. Morphine and methadone analgesia in the exon 11 knockout mouse was normal, but the analgesic actions of heroin, M6G, and fentanyl were markedly diminished in the radiant heat tail-flick and hot-plate assays. Similarly, the ability of M6G to inhibit gastrointestinal transit was greatly diminished in these exon 11 knockout mice, whereas the ability of morphine was unchanged. These findings identify receptors selectively involved with heroin and M6G actions and confirm the relevance of the exon 11-associated variants and raise important issues regarding the importance of atypical truncated G-protein-coupled receptors. knockout | analgesia | opiate receptor

Byline: Dickson Carvalho (1), Fabricia Petronilho (1), Francieli Vuolo (1), Roberta Albino Machado (1), Larissa Constantino (1), Remo Guerrini (2), Girolamo Calo (3), Elaine Cristina Gavioli (4), Emilio Luiz Streck (1), Felipe Dal-Pizzol (1) Keywords: Sepsis; Neutrophil migration; NOP receptor; UFP-101; Nociceptin/orphanin FQ Abstract: Objective The aim of this study was investigate the effects of nociceptin/orphanin FQ (N/OFQ) and ([Nphe.sup.1,Arg.sup.14,Lys.sup.15]N/OFQ--NH.sub.2) (UFP-101), the endogenous N/OFQ peptide receptor (NOP) ligand and a selective NOP antagonist, respectively, in the inflammatory response after cecal ligation and puncture (CLP) model of sepsis in rats. Design Prospective, controlled experiment. Setting Animal basic science laboratory. Subjects Male Wistar rats, weighing 300--350 g. Interventions Rats subjected to CLP were treated with N/OFQ (0.001, 0.01 or 0.1 mg/kg) or UFP-101 (0.03, 0.03 or 0.3 mg/kg) subcutaneously administered immediately after surgery. Measurements and main results Twelve hours after surgery, blood was collected by cardiac puncture and bronchoalveolar (BAL) and peritoneal lavage were performed. In a separate set of experiments mortality was evaluated monitoring CLP rats for 10 days. Our findings showed that UFP-101 (0.03 mg/kg, sc, but not 0.003 mg/kg) modified parameters related to the systemic inflammatory response by effectively preventing cells migration, bacterial dissemination, and by modulating the release of pro-inflammatory cytokines and chemokines, and reducing animal mortality in a clinically relevant model of sepsis. By contrast, N/OFQ (0.1 mg/kg, sc) increased mortality in the CLP model. Conclusions Our findings point to a functional relationship between the N/OFQ-NOP receptor system and inflammatory response in the CLP model of sepsis and suggest that NOP receptor antagonists are worthy of testing as innovative drugs for the treatment of sepsis. Author Affiliation: (1) Laboratorio de Fisiopatologia Experimental, Universidade do Extremo Sul Catarinense, Avenida Universitaria, 1105, Criciuma, SC, 88806-000, Brazil (2) Department of Pharmaceutical Science and Biotechnology Center, University of Ferrara, Ferrara, Italy (3) Department of Experimental and Clinical Medicine, Section of Pharmacology and National Institute of Neuroscience, University of Ferrara, Ferrara, Italy (4) Laboratorio de Neurociencias, Universidade do Extremo Sul Catarinense, Criciuma, SC, Brazil Article History: Registration Date: 30/09/2008 Received Date: 04/01/2008 Accepted Date: 28/09/2008 Online Date: 10/10/2008

For ectothermic vertebrates, such as reptiles, the effects of opioid receptor subtype activation on breathing are poorly understood. On the basis of previous studies on mammals and lampreys, we hypothesized that [mu]- and [delta]-opioid receptor (MOR and DOR, respectively) activation would cause respiratory depression, whereas [kappa]-opioid receptor (KOR) activation would have no effect. To address this question, we measured respiration in awake, freely swimming adult red-eared slider turtles (Trachemys scripta) before and after injection with agonists for specific opioid receptors. Injection of the MOR agonist [D-[Ala.sup.2],NMe-[Phe.sup.4], [Gly.sup.5]-ol]-enkephalin acetate salt (DAMGO, 1.5 or 6.5 mg/ kg) decreased ventilation ([V.sub.E]) by 72 [+ or -] 9% and 95 [+ or -] 3%, respectively, 4.0 h after injection as a result of decreased breathing frequency and no change in tidal volume ([V.sub.T]). DOR agonists, such as [D-[Pen.sup.2.5]]-enkephalin hydrate (DPDPE, 5.0 mg/kg) and [D-[Ala.sup.2],D[Leu.sup.5]]-enkephalin acetate salt (DADLE, 6.3 mg/kg), decreased [V.sub.E] by 44 [+ or -] 10% and 89 [+ or -] 4%, respectively, 4.0 h after injection as a result of decreased breathing frequency and no change in [V.sub.T]. DADLE also increased breath duration by a maximum of 25 [+ or -] 9% at 6.0 h after injection. The KOR agonist U-50488 (6.2 mg/kg) increased [V.sub.T] by a maximum of 52 [+ or -] 30% at 5.0 h after injection, with variable nonsignificant changes in [V.sub.E] and breathing frequency. Naloxone injections (0.25-0.5 mg/kg) 1.0 h before opioid agonist injections blocked all DAMGO-dependent effects, DPDPE-dependent frequency depression, and DADLE-dependent breath duration augmentation for 2.0 h after agonist injections. These results show that MOR and DOR activation causes respiratory depression as a result of decreased breathing frequency, whereas [V.sub.T] is increased after KOR activation. reptile; respiration; chelonian

[mu] opioid receptors are G protein--coupled receptors that mediate the pain-relieving effects of clinically used analgesics, such as morphine. Accumulating evidence shows that [mu]-[delta] opioid heterodimers have a pharmacologic profile distinct from those of the [mu] or [delta] homodimers. Because the heterodimers exhibit distinct signaling properties, the protein and mechanism regulating their levels have significant effects on morphine-mediated physiology. We report the characterization of RTP4, a Golgi chaperone, as a regulator of the levels of heterodimers at the cell surface. We show that the association with RTP4 protects [mu]-[delta] receptors from ubiquitination and degradation. This leads to increases in surface heterodimer levels, thereby affecting signaling. Thus, the oligomeric organization of opioid receptors is controlled by RTP4, and this governs their membrane targeting and functional activity. This work is the first report of the identification of a chaperone involved in the regulation of the biogenesis of a family A GPCR heterodimer. The identification of such factors as RTP4 controlling dimerization will provide insight into the regulation of heterodimers in vivo. This has implications in the modulation of pharmacology of their endogenous ligands, and in the development of drugs with specific therapeutic effects. enkephalin | G protein-coupled receptors | heroin | hetero-oligomerization

The basis for agonist-selective signaling was investigated by using the [mu]-opioid receptor (MOR) as a model. In the absence of agonist, MOR located within the lipid raft domains, whereas etorphine, but not morphine, induced the translocation of MOR from lipid raft to nonraft domains, similar to the action of methyl-[beta]-cyclodextrin. The etorphine-induced MOR translocation required the dissociation of the receptor from G[alpha]i2 first and then the binding of [beta]-arrestin. In contrast, the low affinity of the morphine-MOR complex for [beta]-arrestin and the rebinding of G[alpha]i2 after GTP hydrolysis retained the complex within the lipid raft domains. Disruption of the MOR-G[alpha]i2 interaction, either by deleting the [sup.276][RRITR.sup.280] sequence of MOR or knocking down the level of G[alpha]i2, resulted in the translocation of MOR to the nonraft domains. In addition, lipid raft location of MOR was critical for G protein-dependent signaling, such as etorphine- and morphine-mediated inhibition of adenylyl cyclase activity and morphine-induced ERK phosphorylation, whereas [beta]-arrestin-dependent, etorphine-induced ERK phosphorylation required MOR to translocate into the nonraft domains. Thus, agonist-selective signaling is regulated by the location of MOR, which is determined by interactions of MOR with G proteins and [beta]-arrestin. lipid raft | opioid

Abstract: The aim of the study was to investigate the presence of opioid receptor types in human breast adenocarcinoma MCF-7 cells and to characterize the changes in MOR expression induced [...]

Although opioids are highly effective analgesics, they are also known to induce cellular adaptations resulting in tolerance. Experimental studies are often performed in the absence of painful tissue injury, which precludes extrapolation to the clinical situation. Here we show that rats with chronic morphine treatment do not develop signs of tolerance at peripheral [micro]-opioid receptors ([micro]-receptors) in the presence of painful CFA-induced paw inflammation. In sensory neurons of these animals, internalization of [micro]-receptors was significantly increased and G protein coupling of [micro]-receptors as well as inhibition of cAMP accumulation were preserved. Opioid receptor trafficking and signaling were reduced, and tolerance was restored when endogenous opioid peptides in inflamed tissue were removed by antibodies or by depleting opioid-producing granulocytes, monocytes, and lymphocytes with cyclophosphamide (CTX). Our data indicate that the continuous availability of endogenous opioids in inflamed tissue increases recycling and preserves signaling of [micro]-receptors in sensory neurons, thereby counteracting the development of peripheral opioid tolerance. These findings infer that the use of peripherally acting opioids for the prolonged treatment of inflammatory pain associated with diseases such as chronic arthritis, inflammatory neuropathy, or cancer, is not necessarily accompanied by opioid tolerance., Introduction Opioids are the most widely used drugs in acute and chronic pain. Long-term application of opioids can result in pharmacological tolerance in animals, i.e., a decreased effect with prolonged [...]

This study evaluated the participation of [micro]-opioid-receptor activation in body temperature ([T.sub.b]) during normal and febrile conditions (including activation of heat conservation mechanisms) and in different pathways of LPS-induced fever. The intracerebroventricular treatment of male Wistar rats with the selective opioid [micro]-receptor-antagonist cyclic D-Phe-Cys-Try-DTrp-Arg-Thr-Pen-Thr-N[H.sub.2] (CTAP; 0.1-1.0 [micro]g) reduced fever induced by LPS (5.0 [micro]g/kg) but did not change [T.sub.b] at ambient temperatures of either 20[degrees]C or 28[degrees]C. The subcutaneous, intracerebroventricular, and intrahypothalamic injection of morphine (1.0-10.0 mg/kg, 3.0-30.0 [micro]g, and 1-100 ng, respectively) produced a dose-dependent increase in [T.sub.b]. Intracerebroventricular morphine also produced a peripheral vasoconstriction. Both effects were abolished by CTAP. CTAP (1.0 [micro]g icv) reduced the fever induced by intracerebroventricular administration of TNF-[alpha]] (250 ng), IL-6 (300 ng), CRF (2.5 [micro]g), endothelin-1 (1.0 pmol), and macrophage inflammatory protein (500 pg) and the first phase of the fever induced by PG[F.sub.2][alpha] (500.0 ng) but not the fever induced by IL-1[beta] (3.12 ng) or PG[E.sub.2] (125.0 ng) or the second phase of the fever induced by PGF2[alpha]. Morphine-induced fever was not modified by the cyclooxygenase (COX) inhibitor indomethacin (2.0 mg/kg). In addition, morphine injection did not induce the expression of COX-2 in the hypothalamus, and CTAP did not modify PGE2 levels in cerebrospinal fluid or COX-2 expression in the hypothalamus after LPS injection. In conclusion, our results suggest that LPS and endogenous pyrogens (except IL-1[beta] and prostaglandins) recruit the opioid system to cause a [micro]-receptor-mediated fever. prostaglandin independent; body temperature; morphine; CTAP

[delta]-Opioid receptors (DORs) are associated with ischemic preconditioning and vagal transmission in the sinoatrial (SA) node and atria. Although functional studies suggested that DORs are prejunctional on parasympathetic nerve terminals, their precise location remains unconfirmed. DORs were colocalized in tissue slices and synaptosomes from the canine fight atrium and SA node along with cholinergic and adrenergic markers, vesicular acetylcholine transporter (VAChT), and tyrosine hydroxylase (TH). Synapsin I immunofluorescence verified the neural character of tissue structures and isolated synaptosomes. Acetylcholine and norepinephrinc measurements suggested the presence of both cholinergic and adrenergic synaptosomes. Fluorescent analysis of VAChT and TH signals indicated that >80% of the synapsin-positive synaptosomes were of cholinergic origin and opioids; vagal function

Striatal enkephalin and dynorphin opioid systems mediate reward and negative affect, respectively, relevant to addiction disorders. We examined polymorphisms of proenkephalin (PENK) and prodynorphin (PDYN) genes in relation to heroin abuse and gene expression in the human striatum and the relevance of genetic dopaminergic tone, critical for drug reward and striatal function. Heroin abuse was significantly associated with PENK polymorphic 3' UTR dinucleotide (CA) repeats; 79% of subjects homozygous for the 79-bp allele were heroin abusers. Such individuals tended to express higher PENK mRNA than the 81-bp homozygotes, but PENK levels within the nucleus accumbens (NAc) shell were most strongly correlated to catecholamine-O-methyltransferase (COMT) genotype. Control Met/ Met individuals expressed lower PENK mRNA than Val carriers, a pattern reversed in heroin users. Up-regulation of NAc PENK in Met/Met heroin abusers was accompanied by impaired tyrosine hydroxylase (TH) mRNA expression in mesolimbic dopamine neurons. In contrast to PENK, no association was detected between PDYN genotype (68-bp repeat element containing one to four copies of AP-1 binding sites in the promoter region) and heroin abuse, although there was a clear functional association with striatal PDYN mRNA expression: an increased number of inducible repeats (three and four) correlated with higher PDYN levels than adult or fetal subjects with noninducible (one and two) alleles. Moreover, PDYN expression was not related to COMT genotype. Altogether, the data suggest that dysfunction of the opioid reward system is significantly linked to opiate abuse vulnerability and that heroin use alters the apparent influence of heritable dopamine tone on mesolimbic PENK and TH function. catecholarnine-O-methyltransferase | drug abuse | mu opioid receptor | nucleus accurnbens | prodynorphin

Exercise increases serum opioid levels and improves cardiovascular health. Here we tested the hypothesis that opioids contribute to the acute cardioprotective effects of exercise using a rat model of exercise-induced cardioprotection. For the standard protocol, rats were randomized to 4 days of treadmill training and 1 day of vigorous exercise (day 5), or to a sham exercise control group. On day 6, animals were killed, and global myocardial ischemic tolerance was assessed on a modified Langendorff apparatus. Twenty minutes of ischemia followed by 3 h of reperfusion resulted in a mean infarct size of 42 [+ or -] 4% in hearts from sham exercise controls and 21 [+ or -] 3% (P < 0.001) in the exercised group. The cardioprotective effects of exercise were gone by 5 days after the final exercise period. To determine the role of opioid receptors in exercise-induced cardioprotection, rats were exercised according to the standard protocol; however, just before exercise on days 4 and 5, rats were injected subcutaneously with 10 mg/kg of the opioid receptor antagonist naltrexone. Similar injections were performed in the sham exercise control group. Naltrexone had no significant effect on baseline myocardial ischemic tolerance in controls (infarct size 43 [+ or -] 4%). In contrast, naltrexone treatment completely blocked the cardioprotective effect of exercise (infarct size 40 [+ or -] 5%). Exercise was also associated with an early increase in myocardial mRNA levels for several opioid system genes and with sustained changes in a number of genes that regulate inflammation and apoptosis. These findings demonstrate that the acute cardioprotective effects of exercise are mediated, at least in part, through opioid receptor-dependent mechanisms that may include changes in gene expression. exercise; myocardial ischemic tolerance; enkephalins; opioid receptors

Increased signaling by [G.sub.i]-coupled receptors has been implicated in dilated cardiomyopathy. To investigate the mechanisms, we used transgenic mice that develop dilated cardiomyopathy after conditional expression of a cardiac-targeted [G.sub.i]-coupled receptor (Ro1). Activation of [G.sub.i] signaling by the Ro1 agonist spiradoline caused decreased cellular cAMP levels and bradycardia in Langendorff-perfused hearts. However, acute termination of Ro1 signaling with the antagonist norbinaltorphimine did not reverse the Ro1-induced contractile dysfunction, indicating that Ro1 cardiomyopathy was not due to acute effects of receptor signaling. Early after initiation of Ro1 expression, there was a 40% reduction in the abundance of the sarcoplasmic reticulum [Ca.sup.2+]-ATPase (P < 0.05); thereafter, there was progressive impairment of both [Ca.sup.2+] handling and force development assessed with ventricular trabeculae. Six weeks after initiation of Ro1 expression, systolic [Ca.sup.2+] concentration was reduced to 0.61 [+ or -] 0.08 vs. 0.91 [+ or -] 0.07 [micro]M for control (n = 6-8; P < 0.05), diastolic [Ca.sup.2+] concentration was elevated to 0.41 [+ or -] 0.07 vs. 0.23 [+ or -] 0.06 [micro]M for control (n = 6-8; P < 0.01), and the decline phase of the [Ca.sup.2+] transient (time from peak to 50% decline) was slowed to 0.25 [+ or -] 0.02 s vs. 0.13 [+ or -] 0.02 s for control (n = 6-8; P < 0.01). Early after initiation of Ro1 expression, there was a ninefold elevation of matrix metalloproteinase-2 (P < 0.01), which is known to cause myofilament injury. Consistent with this, 6 wk after initiation of Ro1 expression, [Ca.sup.2+]-saturated myofilament force in skinned trabeculae was reduced to 21 [+ or -] 2 vs. 38 [+ or -] 0.1 mN/[mm.sup.2] for controls (n = 3; P < 0.01). Furthermore, electron micrographs revealed extensive myofilament damage. These findings may have implications for some forms of human heart failure in which increased activity of [G.sub.i]-coupled receptors leads to impaired [Ca.sup.2+] handling and myofilament injury, contributing to impaired ventricular pump function and heart failure. matrix metalloproteinase-2; sarco(endo)plasmic reticulum [Ca.sup.2+]-ATPase; contraction; [kappa]-opioid receptor; conditional expression

The specific [delta]-opioid receptor agonist [D-[Ala.sup.2]-D-[Leu.sup.5]]enkephalin (DADLE) protects against infarction in the heart when given before ischemia. In rabbit, this protection leads to phosphorylation of the pro-survival kinases Akt and extracellular signal-regulated kinase (ERK) and is dependent on transactivation of the epidermal growth factor receptor (EGFR). DADLE reportedly protects rat hearts at reperfusion. We therefore tested whether DADLE at reperfusion could protect isolated rabbit hearts subjected to 30 min of regional ischemia and 120 min of reperfusion and whether this protection is dependent on Akt, ERK, and EGFR. DADLE (40 nM) was infused for 1 h starting 5 min before reperfusion and reduced infarct size from 31.0 [+ or -] 2.3% in the control group to 14.6 [+ or -] 1.6% (P = 0.01). This protection was abolished by cotreatment of the metalloproteinase inhibitor (MPI) and the EGFR inhibitor AG1478. In contrast, 20 nM DADLE, although known to be protective before ischemia, failed to protect. Western blotting revealed that DADLE's protection was correlated to increase in phosphorylation of the kinases Akt and ERK1 and -2 in reperfused hearts (2.5 [+ or -] 0.5, 1.6 [+ or -] 0.2, and 2.3 [+ or -] 0.7-fold of baseline levels, P < 0.05 vs. control). The DADLE-dependent increases in Akt and ERK1/2 phosphorylation were abolished by either MPI or AG1478, confirming a signaling through the EGFR pathway. Additionally, DADLE treatment increased phosphorylation of EGFR (1.4 [+ or -] 0.2-fold, P = 0.03 vs. control). Thus the [delta]-opioid agonist DADLE protects rabbit hearts at reperfusion through activation of the pro-survival kinases Akt and ERK and is dependent on the transactivation of the EGFR. akt cardioprotection; [D-[Ala.sup.2]-D-[Leu.sup.5]]enkephalin; epidermal growth factor receptor; extracellular signal-regulated kinase

The aim of this study was to examine the hypothesis that [delta]-opioid receptor activation before ischemia suppresses gap junction (GJ) permeability by PKC-mediated connexin 43 (Cx43) modulation, which contributes to infarct size limitation afforded by the [delta]-opioid receptor activation. A [delta]-opioid receptor agonist, [D-[Ala.sup.2],D-[Leu.sup.5]]-enkephalin acetate (DADLE, 300 nM), was used in place of preconditioning (PC) ischemia to trigger PC mechanisms in rat hearts. GJ permeability during ischemia, which was assessed by Lucifer yellow, was reduced by DADLE to 47% of the control Level, and this effect of DADLE was almost abolished by a PKC-[epsilon] inhibitor [PKC-[epsilon] translocation inhibitory peptide (PKC-[epsilon]TIP)] but was not affected by a PKC-[delta] inhibitor (rottlerin). After DADLE infusion, PKC-[epsilon], but not PKC-[delta], was coimmunoprecipitated with Cx43, and the level of phosphorylation of Cx43 at a PKC-dependent site ([Ser.sup.368]) was significantly elevated during ischemia. DADLE reduced infarct size after 35 min of ischemia followed by 2 h of reperfusion by 69%, and PKC-[epsilon]-TIP and rottlerin eliminated 48% and 63%, respectively, of the infarct size-limiting effect of DADLE. infusion of a GJ blocker, heptanol, before reperfusion reduced infarct size by 36%, and this protection was not enhanced by preischemic infusion of rottlerin + DADLE, which allows PKC-[epsilon] activation by DADLE. These results suggest that phosphorylation of Cx43 by PKC-[epsilon] plays a crucial role in [delta]-opioid-induced suppression of GJ permeability in ischemic myocardium and that this modulation of the GJ is possibly an adjunct mechanism of infarct size limitation afforded by preischemic [delta]-opioid receptor activation. infarct size; preconditioning

Current ligand-receptor binding assays for G-protein coupled receptors cannot directly measure the system's dissociation constant, [K.sub.d], without purification of the receptor protein. Accurately measured [K.sub.d]'s are essential in the development of a molecular level understanding of ligand-receptor interactions critical in rational drug design. Here we report the introduction of two-photon excitation fluorescence cross-correlation spectroscopy (TPE-FCCS) to the direct analysis of ligand-receptor interactions of the human [micro] opioid receptor (hMOR) for both agonists and antagonists. We have developed the use of fluorescently distinct, dye-labeled hMOR-containing cell membrane nanopatches (~100-nm radius) and ligands, respectively, for this assay. We show that the output from TPE-FCCS data sets can be converted to the conventional Hill format, which provides [K.sub.d] and the number of active receptors per nanopatch. When ligands are labeled with quantum dots, this assay can detect binding with ligand concentrations in the subnanomolar regime. Interestingly, conjugation to a bulky quantum dot did not adversely affect the binding propensity of the hMOR pentapeptide ligand, Leu-enkephalin.

mRNA of [kappa] opioid receptor (KOR) can be transported to nerve fibers, including axons of dorsal root ganglia (DRG), and can be locally translated. Yeast three-hybrid screening identifies Copb1 as a kor mRNA-associated protein that form complexes with endogenous kor mRNA, which are colocalized in the soma and axons of DRG neurons. Axonal transport of kor mRNA is demonstrated, directly, by observing mobilization of biotin-labeled kor mRNA in Campenot chambers. Efficient transport of kor mRNA into the side chamber requires Copb1 and can be blocked by a drug that disrupts microtubules. The requirement for Copb1 in mobilizing kor mRNA is confirmed by using the MS2-GFP mRNA-tagging system. Furthermore, Copb1 also facilitates the translation of kor mRNA in the soma and axons. This study provides evidence for a microtubuledependent, active axonal kor mRNA-transport process that involves Copbl and can stimulate localized translation and suggests coupling of transport and translation of mRNAs destined to the remote areas such as axons. dorsal root ganglia neurons

Abstract: The present study tested the hypothesis that κ-opioid s modulate the arterial baroreflex control of heart rate in conscious young sheep. Various parameters governing the arterial baroreflex control of [...]

In adult animals and humans, activation of [kappa]-opioid receptors results in a diuresis. The aim of the present study was to investigate whether [kappa]-opioids are also diuretic early in life and whether this is altered during postnatal maturation. Therefore, the renal effects of the [kappa]-opioid-receptor agonist U-50488H were measured in two separate age groups of conscious lambs at two stages of postnatal maturation (~1 wk and ~6 wk) under physiological conditions. To evaluate whether the renal responses to U-50488H resulted from receptor-dependent effects, responses to U-50488H were also tested in the presence of the specific [kappa]-opioid-receptor antagonist 5'-guanidinonaltrindole (GNTI). Urinary flow rate, free water clearance, and electrolyte excretions and clearances were measured for 30 min before and for 90 min after intravenous injection of U-50488H or vehicle. An increase in urinary flow rate accompanied by an increase in free water clearance occurred in response to administration of U-50488H but not vehicle. There were no effects of U-50488H on electrolyte excretions or clearances at either 1 or 6 wk of postnatal life. Although there were no effects of GNTI on any of the measured or calculated variables, the aforementioned diuretic response to U-50488H was abolished by pretreatment with GNTI in both age groups. We conclude that [kappa]-opioid receptors are diuretic early in life and that this response does not appear to be altered as postnatal maturation proceeds. Therefore, these data provide evidence that activation of [kappa]-opioid receptors early in life may lead to alterations in fluid balance. 5'-guanidinonaltrindole; newborn; renal function; kidney doi:10.1152/ajpregu.00863.2006

Stimulation of cardiac [[beta].sub.2]-adrenergic receptor ([[beta].sub.2]-AR) or [delta]-opioid receptor (DOR) exerts a similar degree of cardioprotection against myocardial ischemia in experimental models. We hypothesized that [delta]-opioid-initiated cardioprotection is mediated by the intrinsic cardiac adrenergic (ICA) cell via enhanced epinephrine release. Using immunohistochemical and in situ hybridization methods, we detected in situ tyrosine hydroxylase (TH) mRNA and TH immunoreactivity that was colocalized with DOR immunoreactivity in ICA cells in human and rat hearts. Western blot analysis detected DOR protein in ICA cells isolated from rat ventricular myocytes. The physiology of DOR expression was examined by determining changes of cytosolic [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) transients in isolated rat ICA cells using fluorescence spectrophotometry. Exposing the selective [delta]-opioid agonist D-[[Pen.sup.2,5]]enkephalin (DPDPE) to ICA cells increased [[[Ca.sup.2+]].sub.i] transients in a concentration-dependent manner. Such an effect was abolished by the [Ca.sup.2+] channel blocker nifedipine. HPLC-electrochemical detection demonstrated a 2.4-fold increase in epinephrine release from ICA cells following DPDPE application. The significance of the ICA cell and its epinephrine release in [delta]-opioid-initiated cardioprotection was demonstrated in the rat myocardial infarction model and ICA cell-ventricular myocyte coculture. DPDPE administered before coronary artery occlusion or simulated ischemia-reperfusion reduced left ventricular infarct size by 54 [+ or -] 15% or myocyte death by 26 [+ or -] 4%, respectively. [[beta].sub.2]-AR blockade markedly attenuated [delta]-opioid-initiated infarct size-limiting effect and abolished [delta]-opioid-initiated myocyte survival protection in rat ICA cell-myocyte coculture. Furthermore, [delta]-opioid agonist exerted no myocyte survival protection in the absence of cocultured ICA cells during ischemia-reperfusion. We conclude that [delta]-opioid-initiated myocardial infarct size reduction is primarily mediated via endogenous epinephrine/[[beta].sub.2]-AR signaling pathway as a result of ICA cell activation. [delta]-opioid receptor; epinephrine; myocardial ischemia doi:10.1152/ajpheart.01195.2006

These experiments were conducted to determine if 1) syndyphalin-33 (SD33), a [micro]-opioid receptor ligand, affects feed intake; 2) SD33 effects on feed intake are mediated by actions on opioid receptors; and 3) its activity can counteract the reduction in feed intake associated with administration of bacterial endotoxin. In Exp. 1, 5 mixed-breed, castrate male sheep were housed indoors in individual pens. Animals had ad libitum access to water and concentrate feed. Saline (SAL; 0.9% NaC1) or SD33 (0.05 or 0.1 [micro]mol/kg of BW) was injected i.v., and feed intake was determined at 2, 4, 6, 8, 24, and 48 h after the i.v. injections. Both doses of SD33 increased (at least P < 0.01) feed intake at 48 h relative to saline. In Exp. 2, SAL + SAL, SAL + SD33 (0.1 [micro]mol/kg of BW), naloxone (NAL; 1 mg/kg of BW) + SAL, and NAL + SD33 were injected i.v. Food intake was determined as in Exp. 1. The SAL + SD33 treatment increased (P = 0.022) feed intake at 48 h relative to SAL + SAL. The NAL + SAL treatment reduced (at least P < 0.01) feed intake at 4, 6, 8, 24, and 48 h, whereas the combination of NAL and SD33 did not reduce feed intake at 24 (P = 0.969) or 48 h (P = 0.076) relative to the saline-treated sheep. In Exp. 3, sheep received 1 of 4 treatments: SAL + SAL, SAL + 0.1 [micro]mol of SD33/kg of BW, 0.1 [micro]g of lipopolysaccharide (LPS)/ kg of BW + SAL, or LPS + SD33, and feed intake was monitored as in Exp. 1. Lipopolysaccharide suppressed cumulative feed intake for 48 h (P < 0.01) relative to saline control, but SD33 failed to reverse the reduction in feed intake during this period. These data indicate that SD33 increases feed intake in sheep after i.v. injection, and its effects are mediated via opioid receptors. However, the LPS-induced suppression in feed intake cannot be overcome by the opioid receptor ligand, SD33. Key words: appetite, endotoxin, feed intake, opioid receptor, sheep, syndyphalin-33

A study investigates the interactions of the two peptide dynorphin A and dynorphin B with model membrane using model membranes. The outcomes demonstrate that Dyn A and Dyn B interact with phospholipid membranes and that only weak secondary structures are induced in the peptides in spite of the strong interaction with the bicelles.

[kappa]-opioid receptor (KOR) is detected pre- and postsynaptically, but the subcellular localization, translation, and regulation of kor mRNA in presynaptic compartments of sensory neurons remain elusive. In situ hybridization detected axonal distribution of kor mRNA in primary neurons of dorsal root ganglia (DRG). The MS2-fused GFP tracked kor mRNA transport from DRG neuronal soma to axons, requiring its 5' and 3' UTRs. In Campenot chambers, axonal translation of kor mRNA was demonstrated for DRG neurons, which depended on its 5' UTR and was stimulated by KCI depolarization. KCI depolarization of DRG neurons rendered redistribution of kor mRNA from the postpolysomal fraction to the translationally active polysomal fraction. This study provided evidence for mRNA transport and regulation of presynaptic protein synthesis of nonstructural proteins like KOR in primary sensory neurons and demonstrated a mechanism of KCI depolarization-stimulated axonal mRNA redistribution for localized translational regulation. axonal translation