Pharmacological characterization of Protease-Activated Receptor signaling in the human enteric nervous system

[Speaker] Carla Cirillo:1,2
[Co-author] Laurent Alric:3, Nathalie Vergnolle:1
1:Pathophysiology of intestinal epithelium, Digestive Health Research Institute (IRSD), INSERM UMR 1220, INRA UMR 1416, ENVT, University of Toulouse Paul Sabatier, Toulouse, France, 2:Laboratory for Enteric Neuroscience, TARGID, University of Leuven, Leuven, Belgium, 3:Department of Internal Medicine, Toulouse Hospital, Toulouse, France

Background: Proteases that are particularly up-regulated in pathologies such as inflammatory or functional bowel disorders can signal to the enteric nervous system (ENS) through protease-activated receptors (PARs) activation. Three of those receptors, PAR1, PAR2 and PAR4, have been detected in the ENS. In humans, electrophysiological recordings highlighted PAR1, rather than PAR2 or PAR4 agonist responses, in submucosal and myenteric neurons. We have recently described a PAR2-dependent calcium signal, driven by the endogenous agonist trypsin-3, in human submucosal neurons (Rolland et al. 2017). We aimed at better deciphering PAR-induced calcium signaling in the human ENS.
Methods: Tissues were obtained from macroscopically healthy portions of colon from 5 patients with colon cancer (ethic approval DC-2015-2443). Submucosal ganglia were isolated and cultured as previously described (Cirillo et al. 2013). PAR1, PAR2 and PAR4 agonists were used at a concentration of 1-100 microM and calcium transients were monitored. Post-hoc immunohistochemistry was performed to identify PARs in human submucosal plexus, in combination with neuronal (HuCD and neurofilament200) and glial (S100) markers.
Results: Calcium imaging showed that PAR2 agonist, at a concentration of 1 and 10microM, induces a significant increase in calcium transients (1microM: 1.85+/-0.33; 10microM: 1.74+/-0.05, all p<0.01), compared to basal, but also to PAR1 (1microM: 1.05+/-0.01; 10microM: 1.2+/-0.06) and PAR4 (1microM: 1.12+/-0.03; 10microM: 1.47+/-0.06) agonists (see figure). PAR2 agonist, at a concentration of 1 and 10microM, was also able to activate a higher number of submucosal neurons (1microM: 63+/-5.5%; 10microM: 95.2+/-3.9%; 100microM: 84.1+/-9.6%), compared to PAR1 (1microM: 26.4+/-13.4%; 10microM: 27.3+/-7%; 100microM: 37.5+/-14.6%) and PAR4 agonists (1microM: 58.1+/-29.5%; 10microM: 43.4+/-6.6%; 100microM: 26.6+/-12.3%). Immunohistochemistry showed that PAR1 is not uniformly expressed in all submucosal neurons, while strong PAR2 expression is observed in all neurons, as well as in glia. PAR4 is only slightly expressed in neurons.
Conclusions: In human submucosal neurons, PAR2 signaling prevails compared to PAR1 and PAR4. This is supported by the strong expression of PAR2 on submucosal neurons, compared to PAR1 and PAR4. Further investigations in human samples are warrant to better unravel protease signaling and to design drugs capable to regulate this signaling in the human ENS.

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