Specific Localization of β-Arrestin2 in Myenteric Plexus of Mouse Gastrointestinal Tract

Abstract

β-arrestin2 is a key molecule involved in signaling and internalization of activated G protein-coupled receptors including µ-opioid receptors (MOR). Previously we have shown that decreased expression of β-arrestin2 upon chronic morphine is associated with the development of opioid tolerance in the gastrointestinal tract. However, the localization of β-arrestin2 within the gastrointestinal wall is not known. In this study we found that β-arrestin2 is localized in the soma of a select group of neurons in the myenteric ganglia but not in smooth muscle. The density of β-arestin2 was significantly higher in the ileum than the colon. We identified four variants of β-arrestin2 in the ileum, with ARRB-005 and ARRB-013 being the most abundant. Further, the current study utilized multiple-labeling immunofluorescence to characterize the chemical coding of neurons expressing β-arrestin2 in the murine myenteric plexus and the co-localization of MOR1 and β-arrestin2. β-arrestin2 co-localized with choline acetyltransferase and calretinin. In contrast, β-arrestin2 neither co-localized with substance P, nitric oxide synthase nor calbindin. Genetic deletion of β-arrestin2 did not affect cholinergic neuron activation by nicotine in the isolated ileum (-log M EC50: wild type = 5.8 vs. β-arrestin2 knockout = 5.9). Our findings suggest specificity in the localization of β-arrestin2 in the myenteric plexus within MOR1-expressing neurons and provide a relation for direct intracellular crosstalk between MOR1 receptor activation and β-arrestin2 signaling in the myenteric neurons. β-arrestin2 deletion does not directly alter basal enteric cholinergic neuronal function.

Toward Directing Opioid Receptor Signaling to Refine Opioid Therapeutics

“…The barr2-KO mice displayed less delay in colonic bead expulsion and less overall fecal accumulation in response to morphine, but ileum transport of a charcoal gavage was the same between the genotypes (33). In additional studies, both barr2 and MOR were shown to be colocalized in neurons dissociated from the myenteric plexus of ileum and colon (34), and barr2 may play differential roles in morphine-sensitive neurons from the ileum versus from the colon (35). In contrast, the phosphorylation site mutant mice displayed no protection from morphine-induced constipation (21), while mice lacking the exon4 (but not the exon7) C-terminus of MOR were less responsive to morphine as well (22).…”

Connexin‐purinergic signaling in enteric glia mediates the prolonged effect of morphine on constipation

“…Whole‐mount LMMP preparations of colonic myenteric plexus were prepared by removing the mucosal layers and circular muscle layers according to a published method in Maguma et al . (21). Colon LMMP preparations were fixed in 4% formaldehyde overnight.…”

Site and mechanism of morphine tolerance in the gastrointestinal tract

“…Recent studies have demonstrated that β ‐arrestin2 is localized to choline acetyltransferase and μ ‐opioid receptor positive neurons in the myenteric plexus . These findings suggest that unlike tolerance development in the spinal and supraspinal neurons that mediate antinociception, the regulation of μ ‐opioid receptors in the enteric neurons are differentially regulated by β ‐arrestin2.…”