Year: 2009
Type: dissertation
Abstract: Chronic agonist treatment of opioid receptors causes sensitization of adenylyl cyclase (AC), which is characterized by an increased basal level of AC activity, and can be observed as a cAMP overshoot upon re.While the mechanisms behind sensitization are still unknown, recent studies have shown that G protein-coupled receptors and associated signaling elements are localized in cholesterol-enriched membrane microdomains called lipid rafts.In order to investigate the role of cholesterol and lipid rafts in AC sensitization, we used C6 cells stably expressing the mu-opioid receptor (C6μ).Chronic treatment of C6μ cells using selective muopioid agonist DAMGO ([D-Ala 2 , N-MePhe 4 , Gly-ol]-enkephalin) and morphine was used to sensitize AC.Depletion of cellular cholesterol using the cholesterol-sequestering agent methyl-βcyclodextrin (MβCD) prior to sensitization of AC caused a significant decrease in the forskolinstimulated cAMP production, which could be recovered by the inclusion of cholesterol with MβCD.Cholesterol depletion after sensitization, on the other hand, had no effect on overshoot, suggesting that the development and expression of overshoot utilize different biochemical pathways.Further investigation using isolated lipid raft and non-raft membranes from C6μ cells demonstrated that mu-receptors are localized in lipid rafts and associate with cholesterol throughout the plasma membrane.AC sensitization experiments using these membranes also supported our conclusions from the whole cells studies.Together, our findings suggest that lipid rafts and membrane cholesterol have important roles in mu-opioid receptor signaling and cellular responses to chronic opioid exposure.
Author: Jason Chen
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W570115209