Internalization and Ser375 phosphorylation induced by MA-13 - a novel analogue of the μ-opioid agonist oxymorphone
Independent thesis Advanced level (professional degree), 20 credits / 30 HE creditsStudent thesis
Opioids are the most common and efficient medical treatment of acute and chronic pain. Although very efficient in regard to attenuating pain, opioids are accompanied by severe adverse effects, such as opioid tolerance, thus limiting their degree of usage. One focus in the research of opioid tolerance is internalization of the µ-opioid receptors (MORs); the G-protein coupled receptors that opioids primarily bind to. Phosphorylation of MOR precedes internalization in receptor regulation, where serine 375 (Ser375) is assumed to be the site of most importance. A new lead in the research of the role of internalization concerns the dissociation rate of opioid agonists, where a slower dissociation rate seems to result in greater internalization. The -opioid agonist oxymorphone has a fast dissociation rate and induces little internalization, supporting this theory. An analogue to oxymorphone, MA-13, with a hydrophobic side chain has been synthesized, showing a slow dissociation rate resulting in binding to the receptor at greater time intervals.
The aim of this project is to determine the degree of internalization and phosphorylation of MA-13 compared to its parent compound, oxymorphone. By using mouse pituitary cells stably transfected with FLAG-tagged µ-opioid receptors (AtT20-MOR cells) and treated with varying concentrations of MA-13, immunohistochemistry experiments and confocal microscopy was performed in order to determine the internalization as well as the Ser375 phosphorylation. Throughout the project, MA-13 will be compared to the well documented µ-agonists oxymorphone, morphine and DAMGO, the latter a highly internalization-inducing opioid agonist.
MA-13 induced significantly more internalization than its parent compound oxymorphone; 53 % of internalization when normalized to DAMGO. MA-13 also produced significantly more Ser375 phosphorylation than oxymorphone, although this experiment is in need of repetition since the data was normalized to previous results done at the lab.
The findings that MA-13 induced more internalization than oxymorphone are in line with earlier results showing that a slower dissociation rate leads to greater induced internalization. Since phosphorylation precedes internalization, the results showing a higher degree of Ser375 phosphorylation occurs after MA-13 treatment when compared to oxymorphone, also supports earlier studies.
Place, publisher, year, edition, pages
2015. , 33 p.
opioid, tolerance, internalization
IdentifiersURN: urn:nbn:se:uu:diva-242760OAI: oai:DiVA.org:uu-242760DiVA: diva2:784920
Subject / course
Master of Science Programme in Pharmacy
Christie, MacDonald, Associate Dean
Nylander, Ingrid, Professor