scholarly journals Ligand-induced μ opioid receptor internalization in enteric neurons following chronic treatment with the opiate fentanyl

2013 ◽  
Vol 91 (6) ◽  
pp. 854-860 ◽  
Author(s):  
Laura Anselmi ◽  
Ingrid Jaramillo ◽  
Michelle Palacios ◽  
Jennifer Huynh ◽  
Catia Sternini
Keyword(s):  
Opioid Receptor ◽  
Chronic Treatment ◽  
Receptor Internalization ◽  
Enteric Neurons ◽  
Μ Opioid Receptor

The effect of chronic treatment with naltrindole, a selective δ-opioid antagonist, on μ-opioid receptor-mediated antinociception in diabetic mice

1993 ◽  
Vol 113 (2) ◽  
pp. 167-171 ◽  
Author(s):  
Junzo Kamei ◽  
Naoya Kawashima ◽  
Yuriko Iwamoto ◽  
Tsutomu Suzuki ◽  
Hiroshi Nagase ◽  
...  
Keyword(s):  
Opioid Receptor ◽  
Chronic Treatment ◽  
Opioid Antagonist ◽  
Diabetic Mice ◽  
Μ Opioid Receptor

scholarly journals Clathrin and GRK2/3 inhibitors block δ-opioid receptor internalization in myenteric neurons and inhibit neuromuscular transmission in the mouse colon

2019 ◽  
Vol 317 (2) ◽  
pp. G79-G89 ◽  
Author(s):  
Jesse J. DiCello ◽  
Pradeep Rajasekhar ◽  
Emily M. Eriksson ◽  
Ayame Saito ◽  
Arisbel B. Gondin ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
G Protein ◽  
Opioid Receptor ◽  
Neuromuscular Transmission ◽  
G Protein Coupled Receptors ◽  
Receptor Internalization ◽  
Enteric Neurons ◽  
Myenteric Neurons ◽  
Gut Function ◽  
G Protein Coupled

Endocytosis is a major mechanism through which cellular signaling by G protein-coupled receptors (GPCRs) is terminated. However, recent studies demonstrate that GPCRs are internalized in an active state and continue to signal from within endosomes, resulting in effects on cellular function that are distinct to those arising at the cell surface. Endocytosis inhibitors are commonly used to define the importance of GPCR internalization for physiological and pathophysiological processes. Here, we provide the first detailed examination of the effects of these inhibitors on neurogenic contractions of gastrointestinal smooth muscle, a key preliminary step to evaluate the importance of GPCR endocytosis for gut function. Inhibitors of clathrin-mediated endocytosis (Pitstop2, PS2) or G protein-coupled receptor kinase-2/3-dependent phosphorylation (Takeda compound 101, Cmpd101), significantly reduced GPCR internalization. However, they also attenuated cholinergic contractions through different mechanisms. PS2 abolished contractile responses by colonic muscle to SNC80 and morphine, which strongly and weakly internalize δ-opioid and μ-opioid receptors, respectively. PS2 did not affect the increased myogenic contractile activity following removal of an inhibitory neural influence (tetrodotoxin) but suppressed electrically evoked neurogenic contractions. Ca2+ signaling by myenteric neurons in response to exogenous ATP was unaffected by PS2, suggesting inhibitory actions on neurotransmitter release rather than neurotransmission. In contrast, Cmpd101 attenuated contractions to the cholinergic agonist carbachol, indicating direct effects on smooth muscle. We conclude that, although PS2 and Cmpd101 are effective blockers of GPCR endocytosis in enteric neurons, these inhibitors are unsuitable for the study of neurally mediated gut function due to their inhibitory effects on neuromuscular transmission and smooth muscle contractility. NEW & NOTEWORTHY Internalization of activated G protein-coupled receptors is a major determinant of the type and duration of subsequent downstream signaling events. Inhibitors of endocytosis effectively block opioid receptor internalization in enteric neurons. The clathrin-dependent endocytosis inhibitor Pitstop2 blocks effects of opioids on neurogenic contractions of the colon in an internalization-independent manner. These inhibitors also significantly impact cholinergic neuromuscular transmission. We conclude that these tools are unsuitable for examination of the contribution of neuronal G protein-coupled receptor endocytosis to gastrointestinal motility.

Ligand-induced μ opioid receptor endocytosis and recycling in enteric neurons

Neuroscience ◽  
2003 ◽  
Vol 119 (1) ◽  
pp. 33-42 ◽  
Author(s):  
J.G. Minnis ◽  
S. Patierno ◽  
S.E. Kohlmeier ◽  
N.C. Brecha ◽  
M. Tonini ◽  
...  
Keyword(s):  
Opioid Receptor ◽  
Enteric Neurons ◽  
Receptor Endocytosis ◽  
Μ Opioid Receptor

The effect of chronic treatment with naltrindole on μ-opioid receptor-mediated antinociception in diabetic mice

1994 ◽  
Vol 53 ◽  
pp. S85-S86
Author(s):  
J Kamei ◽  
N Kawashima ◽  
Y Iwamoto ◽  
T Suzuki ◽  
H Nagase ◽  
...  
Keyword(s):  
Opioid Receptor ◽  
Chronic Treatment ◽  
Diabetic Mice ◽  
Μ Opioid Receptor

scholarly journals Dissecting the roles of GRK2 and GRK3 in μ-opioid receptor internalization and β-arrestin2 recruitment using CRISPR/Cas9-edited HEK293 cells

2020 ◽  
Author(s):  
Thor C. Møller ◽  
Mie F. Pedersen ◽  
Jeffrey R. van Senten ◽  
Sofie D. Seiersen ◽  
Jesper M. Mathiesen ◽  
...  
Keyword(s):  
Opioid Receptor ◽  
G Protein Coupled Receptors ◽  
Receptor Internalization ◽  
Hek293 Cells ◽  
Molecule Inhibitor ◽  
Wide Range ◽  
Individual Contributions ◽  
Μ Opioid Receptor ◽  
G Protein Coupled ◽  
Target Effects

AbstractMost G protein-coupled receptors (GPCRs) recruit β-arrestins and internalize upon agonist stimulation. For the μ-opioid receptor (μ-OR), this process has been linked to development of opioid tolerance. GPCR kinases (GRKs), particularly GRK2 and GRK3, have been shown to be important for μ-OR recruitment of β-arrestin and internalization. However, the contribution of GRK2 and GRK3 to β-arrestin recruitment and receptor internalization, remain to be determined in their complete absence. Using CRISPR/Cas9-mediated genome editing we established HEK293 cells with knockout of GRK2, GRK3 or both to dissect their individual contributions in β-arrestin2 recruitment and μ-OR internalization upon stimulation with four different agonists. We showed that GRK2/3 removal reduced agonist-induced μ-OR internalization and β-arrestin2 recruitment substantially and we found GRK2 to be more important for these processes than GRK3. Furthermore, we observed a sustained and GRK2/3 independent component of β-arrestin2 recruitment to the plasma membrane upon μ-OR activation. Rescue expression experiments restored GRK2/3 functions. Inhibition of GRK2/3 using the small molecule inhibitor CMPD101 showed a high similarity between the genetic and pharmacological approaches, cross-validating the specificity of both. However, off-target effects were observed at high CMPD101 concentrations. These GRK2/3 KO cell lines should prove useful for a wide range of studies on GPCR function.

scholarly journals Dissecting the roles of GRK2 and GRK3 in μ-opioid receptor internalization and β-arrestin2 recruitment using CRISPR/Cas9-edited HEK293 cells

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Thor C. Møller ◽  
Mie F. Pedersen ◽  
Jeffrey R. van Senten ◽  
Sofie D. Seiersen ◽  
Jesper M. Mathiesen ◽  
...  
Keyword(s):  
Opioid Receptor ◽  
G Protein Coupled Receptors ◽  
Receptor Internalization ◽  
Hek293 Cells ◽  
Molecule Inhibitor ◽  
Wide Range ◽  
Individual Contributions ◽  
Μ Opioid Receptor ◽  
G Protein Coupled ◽  
Target Effects

Abstract Most G protein-coupled receptors (GPCRs) recruit β-arrestins and internalize upon agonist stimulation. For the μ-opioid receptor (μ-OR), this process has been linked to development of opioid tolerance. GPCR kinases (GRKs), particularly GRK2 and GRK3, have been shown to be important for μ-OR recruitment of β-arrestin and internalization. However, the contribution of GRK2 and GRK3 to β-arrestin recruitment and receptor internalization, remain to be determined in their complete absence. Using CRISPR/Cas9-mediated genome editing we established HEK293 cells with knockout of GRK2, GRK3 or both to dissect their individual contributions in β-arrestin2 recruitment and μ-OR internalization upon stimulation with four different agonists. We showed that GRK2/3 removal reduced agonist-induced μ-OR internalization and β-arrestin2 recruitment substantially and we found GRK2 to be more important for these processes than GRK3. Furthermore, we observed a sustained and GRK2/3 independent component of β-arrestin2 recruitment to the plasma membrane upon μ-OR activation. Rescue expression experiments restored GRK2/3 functions. Inhibition of GRK2/3 using the small molecule inhibitor CMPD101 showed a high similarity between the genetic and pharmacological approaches, cross-validating the specificity of both. However, off-target effects were observed at high CMPD101 concentrations. These GRK2/3 KO cell lines should prove useful for a wide range of studies on GPCR function.

μ-Opioid Receptor Internalization: Opiate Drugs Have Differential Effects on a Conserved Endocytic Mechanism In Vitro and in the Mammalian Brain

1998 ◽  
Vol 53 (3) ◽  
pp. 377-384 ◽  
Author(s):  
Duane E. Keith ◽  
Benito Anton ◽  
Stephen R. Murray ◽  
Paulette A. Zaki ◽  
Peter C. Chu ◽  
...  
Keyword(s):  
Opioid Receptor ◽  
Receptor Internalization ◽  
Mammalian Brain ◽  
Differential Effects ◽  
Opiate Drugs ◽  
Μ Opioid Receptor

scholarly journals Distribution and trafficking of the μ-opioid receptor in enteric neurons of the guinea pig

2016 ◽  
Vol 311 (2) ◽  
pp. G252-G266 ◽  
Author(s):  
Joslyn Lay ◽  
Simona E. Carbone ◽  
Jesse J. DiCello ◽  
Nigel W. Bunnett ◽  
Meritxell Canals ◽  
...  
Keyword(s):  
Guinea Pig ◽  
Opioid Receptor ◽  
Cholinergic Neurons ◽  
Distal Colon ◽  
Bowel Dysfunction ◽  
Enteric Neurons ◽  
Neurochemical Coding ◽  
Opioid Receptor Agonist ◽  
Μ Opioid Receptor ◽  
Δ Opioid Receptor

The μ-opioid receptor (MOR) is a major regulator of gastrointestinal motility and secretion and mediates opiate-induced bowel dysfunction. Although MOR is of physiological and therapeutic importance to gut function, the cellular and subcellular distribution and regulation of MOR within the enteric nervous system are largely undefined. Herein, we defined the neurochemical coding of MOR-expressing neurons in the guinea pig gut and examined the effects of opioids on MOR trafficking and regulation. MOR expression was restricted to subsets of enteric neurons. In the stomach MOR was mainly localized to nitrergic neurons (∼88%), with some overlap with neuropeptide Y (NPY) and no expression by cholinergic neurons. These neurons are likely to have inhibitory motor and secretomotor functions. MOR was restricted to noncholinergic secretomotor neurons (VIP-positive) of the ileum and distal colon submucosal plexus. MOR was mainly detected in nitrergic neurons of the colon (nitric oxide synthase positive, 87%), with some overlap with choline acetyltransferase (ChAT). No expression of MOR by intrinsic sensory neurons was detected. [d-Ala2, MePhe4, Gly(ol)5]enkephalin (DAMGO), morphiceptin, and loperamide induced MOR endocytosis in myenteric neurons. After stimulation with DAMGO and morphiceptin, MOR recycled, whereas MOR was retained within endosomes following loperamide treatment. Herkinorin or the δ-opioid receptor agonist [d-Ala2, d-Leu5]enkephalin (DADLE) did not evoke MOR endocytosis. In summary, we have identified the neurochemical coding of MOR-positive enteric neurons and have demonstrated differential trafficking of MOR in these neurons in response to established and putative MOR agonists.

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