Investigating endogenous µ-opioid receptors in human keratinocytes as pharmacological targets using novel fluorescent ligand

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.

Definition of pain revised after four decades

“…The release of inflammatory factors, notably IL-1β, induces the expression and release of opioids by these cells, which, once attached to the receptors of peripheral nerve fibers, inhibit neuronal triggering and transmitter release [12][13][14][15][16] . The present study demonstrated that supernatant from keratinocyte and fibroblast culture promotes analgesia during inflammatory pain induced by carrageenan in a model of mechanical hyperalgesia, corroborating previous studies [16][17][18][32][33][34] . The data obtained suggest that the opioid receptor seems to be involved in the analgesic effect of keratinocyte culture.…”

Considerations about the new concept of pain

“…The release of inflammatory factors, notably IL-1β, induces the expression and release of opioids by these cells, which, once attached to the receptors of peripheral nerve fibers, inhibit neuronal triggering and transmitter release [12][13][14][15][16] . The present study demonstrated that supernatant from keratinocyte and fibroblast culture promotes analgesia during inflammatory pain induced by carrageenan in a model of mechanical hyperalgesia, corroborating previous studies [16][17][18][32][33][34] . The data obtained suggest that the opioid receptor seems to be involved in the analgesic effect of keratinocyte culture.…”

FBNTI, a DOR-Selective Antagonist That Allosterically Activates MOR within a MOR–DOR Heteromer

“…34 The finding that both FBNTI and MDAN-21 display 100 nM range antinociception via topical administration is of particular significance in light of evidence for the presence of multiple opioid receptors (MOR, DOR, and KOR) in the skin. 35,36,27,28,37,38 Given our recent finding 3 that loperamide and oxymorphindole synergize in the central terminals of nociceptive afferents, it is likely that FBNTI and MDAN-21 are acting at the peripheral terminals of these afferents after topical application. Notably, a recent report 39 has described the presence of a subepidermal meshlike network of two sensorreceptor cell types that influence the sensation of pain.…”