TY - JOUR A1 - Paakkari, P. A1 - Paakkari, I. A1 - Landes, P. A1 - Sirén, Anna-Leena A1 - Feuerstein, G. T1 - Respiratory \(\mu\)-Opioid and benzodiazepine interactions in the understrained rat N2 - lnteractions of p-opioid receptors with the benzodiazepine system were studied by examining the modulatory effects of flumazenil (a benzodiazepine antagonist) and alprazolam (a benzodiazepine agonist) on the respiratory effects ofthe opioid peptide dermorphin. Dermorphin, 1-30 nmol administered i.c.v., to conscious, unrestrained rats decreased ventilation rate (VR) and minute volume (MV) dose-dependently. The ventilatory depression was antagonized by naloxone and by the benzodiazepine antagonist flumazenil. The benzodiazepine alprazolam potentiateri the respiratory inhibition of a small (I nmol) dose of dermorphin but antagonized that of a higher dos:~ (3 nmol). The results suggest that the benzodiazepine/GABA receptor complex modulates respiratory depression induced by centrat p-receptor Stimulation in the rat. KW - Neurobiologie KW - dermorphin KW - opioid receptors KW - opioid-benzodiazepine interactions KW - respiration KW - flumazenil KW - benzodiazepine antagonist. Y1 - 1993 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62974 ER - TY - JOUR A1 - Oehler, Beatrice A1 - Mohammadi, Milad A1 - Perpina Viciano, Cristina A1 - Hackel, Dagmar A1 - Hoffmann, Carsten A1 - Brack, Alexander A1 - Rittner, Heike L. T1 - Peripheral interaction of Resolvin D1 and E1 with opioid receptor antagonists for antinociception in inflammatory pain in rats JF - Frontiers in Molecular Neuroscience N2 - Antinociceptive pathways are activated in the periphery in inflammatory pain, for instance resolvins and opioid peptides. Resolvins are biosynthesized from omega-3 polyunsaturated fatty acids such as eicosapentaenoic acid and docosahexaenoic acid. Resolvin D1 (RvD1) and resolvin E1 (RvE1) initiate the resolution of inflammation and control of hypersensitivity via induction of anti-inflammatory signaling cascades. RvD1 binds to lipoxin A4/annexin-A1 receptor/formyl-peptide receptor 2 (ALX/FPR2), RvE1 to chemerin receptor 23 (ChemR23). Antinociception of RvD1 is mediated by interaction with transient receptor potential channels ankyrin 1 (TRPA1). Endogenous opioid peptides are synthesized and released from leukocytes in the tissue and bind to opioid receptors on nociceptor terminals. Here, we further explored peripheral mechanisms of RvD1 and chemerin (Chem), the ligand of ChemR23, in complete Freund’s adjuvant (CFA)-induced hindpaw inflammation in male Wistar rats. RvD1 and Chem ameliorated CFA-induced hypersensitivity in early and late inflammatory phases. This was prevented by peripheral blockade of the μ-opioid peptide receptor (MOR) using low dose local naloxone or by local injection of anti-β-endorphin and anti-met-enkephalin (anti-ENK) antibodies. Naloxone also hindered antinociception by the TRPA1 inhibitor HC-030031. RvD1 did not stimulate the release of β-endorphin from macrophages and neutrophils, nor did RvD1 itself activate G-proteins coupled MOR or initiate β-arrestin recruitment to the membrane. TRPA1 blockade by HC-030031 in inflammation in vivo as well as inhibition of the TRPA1-mediated calcium influx in dorsal root ganglia neurons in vitro was hampered by naloxone. Peripheral application of naloxone alone in vivo already lowered mechanical nociceptive thresholds. Therefore, either a perturbation of the balance of endogenous pro- and antinociceptive mechanisms in early and late inflammation, or an interaction of TRPA1 and opioid receptors weaken the antinociceptive potency of RvD1 and TRPA1 blockers. KW - transient receptor potential channels KW - pain behavior KW - resolvin KW - opioid receptors KW - opioid peptides KW - inflammation KW - animals Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-158642 VL - 10 IS - 242 ER - TY - THES A1 - Mambretti, Egle Maria T1 - Opioid receptors as therapeutic targets for nociceptor specific regional analgesia T1 - Opioidrezeptoren als therapeutisches Target einer nozizeptionsspezifischen Regionalanalgesie N2 - Opioids have been, since centuries, the gold standard for pain treatment and relief. They exert their effects after binding to opioid receptors (OP) that are expressed and functional in the central (CNS) and peripheral nervous system (PNS). As their systemic application has many side effects, including sedation and respiratory depression, a peripheral application of opioids and selective targeting of µ-OP (MOP) in nociceptive axons would be extremely beneficial. MOP presence and function has been conclusively demonstrated at nerve terminals; however it is still controversial whether functional MOPs are available on the membrane of peripheral nociceptive axons to mediate opioid-induced antinociception. While under pathologic conditions (i.e. nerve injury) exogenous as well as endogenous MOP agonists applied at the damaged nerve can elicit potent antinociception or anti-allodynia, under physiological conditions no antinociception was seen in rats. This could be caused by either a lack of functional opioid receptors in the axonal membranes or by the inability of injected opioids to cross the intact perineurial barrier and to reach nociceptors. Previous behavioral test results showed an antinociceptive effect (up to 5h) following perisciatic application of the hydrophilic DAMGO (MOP agonist) if coinjected with hypertonic saline solution (HTS; 10% NaCl), a treatment suited to open the perineural barrier. The effect was inhibited by naloxone, a MOP antagonist, documenting its specific action via MOP. Fentanyl, a lipophilic opioid, elicited an effect, which was enhanced by HTS treatment, indicating that HTS may act not only on the barrier but also directly on axonal MOP presence and/or functionality. To provide a basis for testing this hypothesis, the present work was designed to study the axonal localization of MOP in experimental animals under different conditions using molecular and morphological methods. Initially four different commercial antibodies were tested for MOP detection. Immunoreactions with these antibodies specifically detected MOP in the hippocampus and in amygdala, while in the peripheral nervous system the reactions showed varying labeling patterns pointing towards less specificity with low signal-to-noise ratio. Double labelling with calcitonin gene related peptide (CGRP), a neuropeptide expressed in sensory fibers, with the non-compacted myelin marker S100 or with the neuronal marker PGP9.5 documented significant immunoreaction signals outside sensory nerve fibers. Therefore, none of these antibodies appeared suitable. Taking advantage of a new commercial monoclonal rabbit antibody (RabMAb) and of genetically modified mice in which the fluorescent protein mcherry was inserted in the C-tail of MOP (MOP-mcherry knock-in mice), MOP fusion protein expression in rat and mouse CGRP+ sciatic nerve fibers and fiber bundles was confirmed by immunofluorescence labeling. Immunoelectron microscopic analysis indicated MOP/MOP-mcherry-localization in the cytoplasm and the membranes of unmyelinated axons organized in Remak bundles. Both antibodies detected bands of appropriate size in Western Blot in the CNS and additional larger bands in the PNS. Quantitative analyses 60 min after HTS-treatment revealed no change in MOP mRNA in the sciatic nerve and DRG as well as no change in MOP immunoreactivity in the sciatic nerve. Thus, the opioid-induced long lasting antinociception enhanced by perisciatic injection of HTS were not due to a sustained increased MOP expression or content in sensory, putative nociceptive axons. In summary, the current study succeeded to unequivocally document the presence of MOP protein in intact sensory axons of rat and mouse sciatic nerve. Thus, axonal MOPs may indeed mediate antinociceptive opioid effects observed in behavioral studies in naive animals possibly via activation of potassium or calcium channels. As HTS treatment does not lead to a sustained increase in axonal MOP protein or MOP mRNA expression, other mechanisms might enhance MOP function, including inhibition of MOP recycling or changes in functional coupling. Future studies should further explore the axonal mechanisms of antinociception by opioids and enhancing treatments. N2 - Opioide sind seit Jahrhunderten der Goldstandard für die Schmerzbehandlung. Sie entfalten ihre Wirkung nach der Bindung mit Opioidrezeptoren (OP), die im zentralen (ZNS) und peripheren (PNS) Nervensystem exprimiert und funktionell sind. Da die systemische Anwendung viele Nebenwirkungen hat, wie die Beruhigung und Atemdepression, wäre eine Anwendung von Opioiden und die gezielte Targeting von µ-OP (MOP) in nozizeptiven Axone in Rahmen einer Regionalanalgesie besser. Die Anwesenheit und die Funktionalität der MOP wurden zwar schon in Nervenendungen gezeigt, aber es ist noch strittig, ob funktionelle MOP in der Membran von peripheren nozizeptiven Axonen sind, um opioid-induzierte Antinozizeption zu vermitteln. Während bei Erkrankungen der Nerven (z.B. traumatische Nervenbeschädigung) exogene und endogene MOP-Agonisten Antinozizeption und Antiallodynie bewirken, konnte in gesunden Ratten kein Effekt bei perineuraler Injektion am Nerven beobachtet werden. Dies könnte entweder durch einen Mangel an funktionellen OP in axonalen Membranen verursacht sein. Alternativ könnte die mangelde Penetration der injizierten Opioide durch die Barriere des Perineuriums verantwortlich sein, die es verhindert, dass die Opioide die Nozizeptoren erreichen. Vorherige Ergebnisse aus Schmerzverhaltenstests zeigten eine Anhebung von mechanischen nozizeptiven Schwellen (bis 5 h) nach perineuraler Anwendung des hydrophilen MOP-Agonisten DAMGO, wenn dieser mit einer hypertonen Lösung (HTS; 10% NaCl) ko-injiziert war. Denn dies ist eine geeignete Behandlung, die die Barriere des Perineuriums öffnet. Der Effekt wurde von Naloxon, einem MOP-Antagonist, gehemmt, was eine spezifische Wirkung via MOP unterstützt. Die Wirkung von Fentanyl, einem lipophilen Opioid, wurde ebenfalls durch die HTS-Behandlung verbessert. Das führt zu unserer Hypothese, dass HTS nicht nur die Schranke öffnet, sondern auch direkt Expression und/oder Funktionalität von axonalen MOP verbessert. Um eine Grundlage für die Untersuchung dieser Hypothese zu schaffen, war das Ziel dieser Arbeit, die axonale MOP bei Versuchstieren unter verschiedenen Bedingungen mit molekularen und morphologischen Methoden zu charaktiersieren. Am Anfang wurden vier verschiedene kommerzielle Antikörper für die Erkennung der MOP getestet. Immunreaktionen mit diesen Antikörpern wiesen spezifisch MOP in dem Hippocampus und in der Amygdala nach, während im peripheren Nervensystem die Immunreaktion veränderliche Markierungsmuster und weniger Spezifität mit einem ungünstigeren Signal-zu-Hintergund Verhältnis zeigte. Die Doppelmarkierung mit calcitonin gene-related peptide (CGRP), einem Neuropeptid, das in sensorischen Fasern exprimiert ist, mit dem Marker für non-compacted Myelin S100 oder mit dem neuronalen Marker PGP9.5, bestätigte ein reproduzierbares Färbemuster außerhalb sensorischer Nervenfasern. Deshalb war keiner dieser Antikörper geeignet. Mit der Anwendung eines neuen kommerziell erhältlichen monoklonalen Kaninchen Antikörpers (RabMAb) gegen MOP sowie gentechnisch veränderten Mäusen, bei denen das fluoreszierende Protein mCherry in das C-terminale Ende von MOP eingefügt wurde (MOP-mcherry knock-in Mäusen), wurden MOP und das MOP-Fusionprotein im CGRP+ im Ischiasnerv und Fasernbündeln durch Immunfluoreszenzmarkierung von Ratten und Mäuse bestätigt. Die immunelectron-mikroscopische Analyse zeigte MOP/MOP-mcherry im Zytoplasma und der Membran unmyelinizierter Axone, die in Remak Bündlen organisiert sind. Beide Antikörper erkannten Banden in richtige Größe in Western Blot in ZNS und mehrere größere Banden in PNS. Quantitative Analysen 60 min nach HTS-Behandlung zeigten keine Veränderung in MOP mRNA in dem Ischiasnerv und Hinterwurzelganglion sowie keine Veränderung in der MOP-Immunreaktivität in dem Ischiasnerv. Daher müssen noch weitere Ursachen für die verbesserte Wirkung von Opioiden am Nerven nach HTS in Betracht gezogen werden. Zusammenfassend konnte diese Studie die MOP-Proteins in intakten sensorischen Axonen des N. ischiadicus der Ratte und Maus eindeutig nachweisen. Axonale MOPs könnten über Kaliumkanäle oder Calciumkanäle in den Verhaltenstests bei naiven Tiere antinozizeptiv wirken. Da die HTS Behandlung zu keiner deutlichen Steigerung von axonalem MOP-Protein führen kann, sollten anderen Mechanismen wie MOP-Recycling oder Veränderung der intrazellulären Singaltransduktion untersucht werden, die die Funktionalität von MOP erhöhen. Zukünftige Studien ferner den genauen Mechanismus klären, wie axonal Opioide antinozizeptiv wirken, um so die Behandlung von Schmerzen mit Regionalanalgesie weiter zu verbessert. KW - opioid receptors KW - nociceptors KW - regional analgesia KW - Opioide KW - Rezeptor KW - Nozizeptor KW - Lokalanästhesie Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-128866 ER - TY - JOUR A1 - Drakopoulos, Antonios A1 - Decker, Michael T1 - Development and Biological Applications of Fluorescent Opioid Ligands JF - ChemPlusChem N2 - Opioid receptors (ORs) are classified among the oldest and best investigated drug targets due to their fundamental role in the treatment of pain and related disorders. ORs are divided in three conventional subtypes (μ, κ, δ) and the non‐classical nocicepetin receptor. All ORs are family A G protein‐coupled receptors (GPCRs), and are located on the cell surface. Modern biophysical methods use light to investigate physiological processes at organismal, cellular and subcellular level. Many of these methods rely on fluorescent ligands, thus highlighting their importance. This review addresses the advancements in the development of opioid fluorescent ligands and their use in biological, pharmacological and imaging applications. KW - biophysics KW - fluorescent ligands KW - imaging KW - microscopy KW - opioid receptors Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-216068 VL - 85 IS - 6 SP - 1354 EP - 1364 ER -