Li, Joyce H.

INTRODUCTION Intestinal enterochromaffin (EC) cells are the specialized epithelial mechanosensors of the gastrointestinal (GI) epithelium and are functionally and developmentally like Merkel cells in the skin. EC cells release serotonin (5‐HT) in response to mechanical stimuli. Recently, we discovered that like Merkel cells, human and mouse EC cells express the mechanosensitive (MS) ion channel Piezo2. However, little is known about the functional properties of this channel in primary EC cells. AIM To determine the role of Piezo2 in primary EC cells. METHODS The enteroendocrine cell transcription factor NeuroD1 was used to mark the late stage of EC cell development and generate NeuroD1‐cre::GCaMP5/tdTomato mouse to identify EC cells and examine their Ca 2+ dynamics. We examined colocalization of tdTomato with 5‐HT and Piezo2 by immunohistochemistry. We established and used primary colon epithelium cultures for whole‐cell patch clamp and Ca 2+ imaging experiments and mechanically stimulated by membrane displacement with a piezoelectrically driven glass probe or shear flow. RESULTS Immunostaining showed no tdTomato labeling outside of the GI epithelium, and epithelial tdTomato cells were 5‐HT+ and Piezo2+. Mechanical stimulation of primary EC cells by membrane displacement produced inward MS currents (20.8±4.9 pA/pF, C m =3.5 pF, n=6) with fast activation and inactivation kinetics (τ inact 11.1±2.8 ms), steep mechanosensitivity (Boltzmann function fit with a mid‐point of 3.46±0.80 μm and slope 0.79±0.27 μm) and a linear current‐voltage relationship (slope 0.61±0.17 pA/mV, x‐intercept −4.26±1.34 mV, n=5). Shear flow (20 s) transiently increased intracellular calcium ((Ca 2+ ) i ) (2.15±0.5 ΔF/F0, upstroke 3–5 s, return to baseline in 60 s, n=12). MS currents were inhibited by the MS ion channel blocker Gd 3+ (20.8±4.9 pA/pF versus 5.3±1.9 pA/pF), Piezo channel blocker GsMTx‐4 (20.8±4.9 pA/pF versus 2.5±0.9 pA/pF) and Piezo2 siRNA (20.8±4.9 pA/pF versus 1.1±0.3 pA/pF), but not non‐targeted (NT) siRNA (20.8±4.9 pA/pF versus 16.0±5.2 pA/pF and) (n=4–6 for each condition, p <0.05 for all except NT siRNA). Similarly, the (Ca 2+ ) i responses to force were inhibited by Gd 3+ (2.28±0.7 ΔF/F0 to 0.09±0.05 ΔF/F0), GsMTx‐4 (2.0±0.6 ΔF/F0 to 0.3±0.1 ΔF/F0), Piezo2 siRNA (2.15±0.5 ΔF/F0 to 0.08±0.05 ΔF/F0), but not NT siRNA (2.15±0.5 ΔF/F0 to 2.7±0.3 ΔF/F0) (n=4–6, p <0.05 for all except NT siRNA). CONCLUSIONS In the mouse epithelium, Piezo2+ EC cells were specifically marked by NeuroD1. Primary colonic NeuroD1+ cells had inward and non‐rectifying MS currents that could be blocked by Gd 3+ , GsMTx‐4 and Piezo2 siRNA. Similarly, shear flow evoked changes in (Ca 2+ ) i that were also blocked by Piezo2 inhibitors and dependent on extracellular Ca 2+ . These results suggest that the MS ion channel Piezo2 acts as the primary EC cell mechanosensor, whose activation by force leads to MS ionic current and (Ca 2+ ) i increase that may mediate mechanically‐induced 5‐HT release. Support or Funding Information This work was supported by NIH K08 DK106456, AGA Research Scholar Award, Mayo Clinic Center for Biomedical Discovery, Mayo Clinic Center for Cell Signaling in Gastroenterology (P30DK084567) and DK52766. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Significance Mechanical forces are important for normal gastrointestinal tract function. The enterochromaffin cells in the gastrointestinal epithelium have been proposed, but not previously shown, to be specialized sensors that convert forces into serotonin release, and serotonin released from these cells is important for normal gastrointestinal secretion and motility. The findings in this study show that some enterochromaffin cells are indeed mechanosensitive, and that they use mechanosensitive Piezo2 channels to generate an ionic current that is critical for the intracellular Ca 2+ increase, serotonin release, and epithelial fluid secretion.