ABSTRACT
Opioids in skin function during stress response, regeneration, ageing and, particularly in regulating sensation. In chronic pruritus, topical treatment with Naltrexone changes µ-opioid receptor (µ-OR) localization to relieve itch. The molecular mechanisms behind the effects of Naltrexone on µ-OR function in reduction of itching behavior has not been studied. There is an immediate need to understand the endogenous complexity of µ-OR dynamics in normal and pathological skin conditions. Here we evaluate real-time behavior of µ-OR-Endomorphine complexes in the presence of agonist and antagonists. The µ-OR ligand Endomorphine-1 (EM) was conjugated to the fluorescent dye Tetramethylrhodamine (TAMRA) to investigate the effects of agonist and antagonists in N/TERT-1 keratinocytes. The cellular localization of the EM-TAMRA was followed through time resolved confocal microscopy and population analysis was performed by flow cytometry. The in vitro analyses demonstrate fast internalization and trafficking of the endogenous EM-TAMRA-µ-OR interactions in a qualitative manner. Competition with Endomorphine-1, Naltrexone and CTOP show both canonical and non-canonical effects in basal and differentiated keratinocytes. Acute and chronic treatment with Naltrexone and Endomorphine-1 increases EM-TAMRA binding to skin cells. Although Naltrexone is clinically effective in relieving itch, the mechanisms behind re-distribution of µ-ORs during clinical treatments are not known. Our study has given insight into cellular mechanisms of µ-OR ligand-receptor interactions after opioid agonist and antagonist treatments in vitro. These findings potentially offer opportunities in using novel treatment strategies for skin and peripheral sensory disorders.
