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.
Highlights
Pharmacological compounds targeting opioid receptors (ORs) have been used as effective anti-nociceptive drugs
The conjugated product was characterized using Electrospray Ionization (ESI) mass spectroscopy to determine the molecular mass of EM-TAMRA
We reported a potential role for Naltrexone in relieving the itch sensation by increasing the functional presence of μ-opioid receptor (μ-OR) in skin keratinocytes within two weeks of topical treatment [20]
Summary
Pharmacological compounds targeting opioid receptors (ORs) have been used as effective anti-nociceptive drugs. Monitoring μ-opioid receptor-ligand dynamics in skin cells. ORs are G protein-coupled receptors (GPCR), which upon binding of a ligand undergo conformational changes and activate an associated inhibitory Gi/o protein. This can result in multiple responses including inhibition of adenylyl cyclase, attenuation of the influx of calcium, increase in potassium channel conductance and activation of mitogen-activated protein kinases[1]. Neuropeptides, the opioidergic system, are one of the components coordinating this stress response to modulate nociceptive and inflammatory pathways in skin homeostasis and differentiation [4]
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