Mo1638 Agonist- and Peptidase-Dependent Regulation of Somatostatin Receptor 2A Trafficking in Myenteric Neurons of the Mouse

Abstract

Background and Aims. Substance P (SP) and the neurokinin 1 receptor (NK1R) control gastrointestinal motility and secretion, and mediate neurogenic inflammation and pain. SPstimulated trafficking regulates NK1R signaling. β-arrestin-mediated NK1R desensitization and endocytosis attenuate plasma membrane signaling, endosomal NK1R signals via βarrestin signalosomes, and NK1R recycling is required for subsequent responses to SP. Since SP and the NK1R mediate intestinal inflammation, we identified neuronal subtypes expressing NK1R and examined the impact of colitis on SP-induced trafficking of the NK1R in enteric neurons. Methods and Results. The neurochemical coding of NK1R-positive neurons of the submucosal and myenteric plexuses of mouse colon was characterized by immunofluorescence. NK1R was expressed by 88% and 22% of submucosal and myenteric neurons, respectively, identified using PGP9.5. NK1R expression was restricted to non-cholinergic secretomotor neurons (NK1R/NPY: 88%; NK1R/VIP: 99%) of the submucosal plexus. In the myenteric plexus, NK1R was localized to intrinsic primary afferent neurons expressing CGRP (76%) and to Dogiel type I neurons containing NOS (9%). Under unstimulated conditions, the NK1R was localized to the plasma membrane of the soma and neurites of myenteric and submucosal neurons. Incubation of colonic explants with SP induced NK1R endocytosis in myenteric and submucosal neurons. The impact of inflammation on NK1R trafficking was examined in IL10 mice treated with piroxicam to induce chronic colitis. In mice with colitis, 55% of NK1R in myenteric neurons was detected in endosomes, compared to 27% in neurons from control mice without colitis (p<0.05). Inflammation-induced NK1R internalization was not detected in submucosal neurons. When explants of myenteric plexus from mice with colitis were incubated in SP-free medium, internalized NK1R recycled to the plasma membrane within 60 min. Subsequent treatment with SP induced NK1R endocytosis. There was no significant difference in the kinetics of SP-induced NK1R endocytosis in myenteric neurons from mice with colitis compared to uninflamed controls. In contrast, NK1R recycling after removal of SP was delayed in myenteric neurons of mice with colitis, with intracellular retention of receptor in inflamed neurons (% NK1R in endosomes at 60 min recovery in SP-free medium: inflamed 32%, control 20%, p<0.05). Conclusions. The NK1R redistributes from the plasma membrane to endosomes of myenteric but not submucosal neurons during colitis. Although SP can evoke NK1R endocytosis in inflamed and uninflamed neurons with similar kinetics, NK1R recycles more slowly in inflamed neurons. During colitis, chronic release of SP and defective recycling machinery, results in intracellular retention of the NK1R, which may transmit inflammatory signals from endosomes. Supported by NIH and NHMRC.