@phdthesis{Mambretti2015,
  author    = {Mambretti, Egle Maria},
  title     = {Opioid receptors as therapeutic targets for nociceptor specific regional analgesia},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-128866},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {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.},
  subject      = {Opioide},
  language  = {en}
}
@phdthesis{Schmid2020,
  author    = {Schmid, Benedikt},
  title     = {Molecular Signaling Mechanisms at the µ-Opioid Receptor},
  doi       = {10.25972/OPUS-17685},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-176850},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {To this day, opioids represent the most effective class of drugs for the treatment of severe pain. On a molecular level, all opioids in use today are agonists at the μ-opioid receptor (μ receptor). The μ receptor is a class A G protein-coupled receptor (GPCR). GPCRs are among the biological structures most frequently targeted by pharmaceuticals. They are membrane bound receptors, which confer their signals into the cell primarily by activating a variety of GTPases called G proteins. In the course of the signaling process, the μ receptor will be phosphorylated by GRKs, increasing its affinity for another entity of signaling proteins called β-arrestins (β-arrs). The binding of a β-arr to the activated μ receptor will end the G protein signal and cause the receptor to be internalized into the cell. Past research showed that the μ receptor's G protein signal puts into effect the desired pain relieving properties of opioid drugs, whereas β-arr recruitment is more often linked to adverse effects like obstipation, tolerance, and respiratory depression. Recent work in academic and industrial research picked up on these findings and looked into the possibility of enhancing G protein signaling while suppressing β-arr recruitment. The conceptual groundwork of such approaches is the phenomenon of biased agonism. It appreciates the fact that different ligands can change the relative contribution of any given pathway to the overall downstream signaling, thus enabling not only receptor-specific but even pathway-specific signaling. This work examined the ability of a variety of common opioid drugs to specifically activate the different signaling pathways and quantify it by means of resonance energy transfer and protein complementation experiments in living cells. Phosphorylation of the activated receptor is a central step in the canonical GPCR signaling process. Therefore, in a second step, expression levels of the phosphorylating GRKs were enhanced in search for possible effects on receptor signaling and ligand bias. In short, detailed pharmacological profiles of 17 opioid ligands were recorded. Comparison with known clinical properties of the compounds showed robust correlation of G protein activation efficacy and analgesic potency. Ligand bias (i.e. significant preference of any path- way over another by a given agonist) was found for a number of opioids in native HEK293 cells overexpressing μ receptor and β-arrs. Furthermore, overexpression of GRK2 was shown to fundamentally change β-arr pharmacodynamics of nearly all opioids. As a consequence, any ligand bias as detected earlier was abolished with GRK2 overexpression, with the exception of buprenorhin. In summary, the following key findings stand out: (1) Common opioid drugs exert biased agonism at the μ receptor to a small extent. (2) Ligand bias is influenced by expression levels of GRK2, which may vary between individuals, target tissues or even over time. (3) One of the opioids, buprenorhin, did not change its signaling properties with the overexpression of GRK2. This might serve as a starting point for the development of new opioids which could lack the ability of β-arr recruitment altogether and thus might help reduce adverse side effects in the treatment of severe pain.},
  subject      = {Opiatrezeptor},
  language  = {en}
}
@phdthesis{Mayer2021,
  author    = {Mayer, Stefanie},
  title     = {Differenzierte β-Arrestin2 Rekrutierung am μ-Opioid Rezeptor durch klinisch eingesetzte Opioide},
  doi       = {10.25972/OPUS-24094},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-240949},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Opioide geh{\"o}ren zu den potentesten Analgetika f{\"u}r die Behandlung akuter und chronischer Schmerzen, werden jedoch in ihrer Anwendung durch analgetische Toleranz aber auch Nebenwirkungen wie Abh{\"a}ngigkeit, Atemdepression und Obstipation limitiert. Opioid-Analgetika vermitteln dabei nahezu alle klinisch relevanten Wirkungen durch Stimulation des μ-Opioidrezeptors, einem G- Protein-gekoppelten Rezeptor. Die „klassische" Signaltransduktion durch Aktivierung inhibitorischer Gi/0-Proteine kann durch G-Protein gekoppelte Rezeptorkinasen (GRKs) und β-Arrestine negativ reguliert werden. Zus{\"a}tzlich k{\"o}nnen durch β-Arrestin-Bindung an den Rezeptor G-Protein-unabh{\"a}ngige Signalwege aktiviert werden. Die genauen Mechanismen wie β-Arrestin- assoziierte Rezeptordesensibilisierung, -internalisierung und G-Protein- unabh{\"a}ngige Signalwege an der physiologischen Antwort und insbesondere an Toleranzentwicklung und Abh{\"a}ngigkeit von Opioid-Analgetika beteiligt sind, k{\"o}nnen bislang nicht ausreichend erkl{\"a}rt werden. In dieser Arbeit konnte in HEK293-Zellen mit Lebendzell-Konfokalmikroskopie und Luciferase-Komplementierung f{\"u}r 17 Opioide eine differenzierte β-Arrestin2- Rekrutierung zum μ-Opioidrezeptor gezeigt werden. Von den untersuchten Opioiden sind 13 h{\"a}ufig eingesetzte Opioid-Analgetika. Durch die Erstellung detaillierter pharmakologischer Profile ließen sich die Opioide bez{\"u}glich ihres β- Arrestin2-Rekrutierungsverm{\"o}gens in Voll-, Partial und Antagonisten eingruppieren. Bemerkenswert war die fehlende β-Arrestin2-Rekrutierung f{\"u}r Buprenorphin, Tramadol und Tilidin, sodass diese interessante Substanzen f{\"u}r weitere Untersuchungen in physiologischerem Kontext sind. Durch {\"U}berexpression von GRK2 konnte die β-Arrestin2-Rekrutierung insbesondere f{\"u}r Partialagonisten gesteigert werden, was die Abh{\"a}ngigkeit der β-Arrestin- Rekrutierung vom GRK-Expressionslevel, das in verschiedenen Assays und Gewebetypen variieren kann, zeigt. Außerdem konnte ein heterogenes Bild der Rezeptorregulierung demonstriert werden, welches indirekt durch Endozytosehemmung unter Verwendung von Dynamin-Inhibitoren erfasst wurde. Die erhobenen Daten dienen als Ankn{\"u}pfungspunkt f{\"u}r weiteren Arbeiten auf dem Gebiet der μ-Opioidrezeptorregulation. Ein besseres Verst{\"a}ndnis der molekularen Mechanismen ist n{\"o}tig, um sichere und nebenwirkungs{\"a}rmere Opioid-Analgetika entwickeln zu k{\"o}nnen.},
  subject      = {Opiatrezeptor},
  language  = {de}
}