Whiteman, Sara

Abstract Enteroendocrine cells (EECs) are specialized sensors of luminal forces and chemicals in the gastrointestinal (GI) epithelium that respond to stimulation with a release of signalling molecules such as serotonin (5‐HT). For mechanosensitive EECs, force activates Piezo2 channels, which generate a very rapidly activating and inactivating (∼10 ms) cationic (Na + , K + , Ca 2+ ) receptor current. Piezo2 receptor currents lead to a large and persistent increase in intracellular calcium (Ca 2+ ) that lasts many seconds to sometimes minutes, suggesting signal amplification. However, intracellular calcium dynamics in EEC mechanotransduction remain poorly understood. The aim of this study was to determine the role of Ca 2+ stores in EEC mechanotransduction. Mechanical stimulation of a human EEC cell model (QGP‐1) resulted in a rapid increase in cytoplasmic Ca 2+ and a slower decrease in ER stores Ca 2+ , suggesting the involvement of intracellular Ca 2+ stores. Comparing murine primary colonic EECs with colonocytes showed expression of intercellular Ca 2+ store receptors, a similar expression of IP 3 receptors, but a >30‐fold enriched expression of Ryr3 in EECs. In mechanically stimulated primary EECs, Ca 2+ responses decreased dramatically by emptying stores and pharmacologically blocking IP 3 and RyR1/3 receptors. RyR3 genetic knockdown by siRNA led to a significant decrease in mechanosensitive Ca 2+ responses and 5‐HT release. In tissue, pressure‐induced increase in the Ussing short circuit current was significantly decreased by ryanodine receptor blockade. Our data show that mechanosensitive EECs use intracellular Ca 2+ stores to amplify mechanically induced Ca 2+ entry, with RyR3 receptors selectively expressed in EECs and involved in Ca 2+ signalling, 5‐HT release and epithelial secretion. image Key points A population of enteroendocrine cells (EECs) are specialized mechanosensors of the gastrointestinal (GI) epithelium that respond to mechanical stimulation with the release of important signalling molecules such as serotonin. Mechanical activation of these EECs leads to an increase in intracellular calcium (Ca 2+ ) with a longer duration than the stimulus, suggesting intracellular Ca 2+ signal amplification. In this study, we profiled the expression of intracellular Ca 2+ store receptors and found an enriched expression of the intracellular Ca 2+ receptor Ryr3, which contributed to the mechanically evoked increases in intracellular calcium, 5‐HT release and epithelial secretion. Our data suggest that mechanosensitive EECs rely on intracellular Ca 2+ stores and are selective in their use of Ryr3 for amplification of intracellular Ca 2+ . This work advances our understanding of EEC mechanotransduction and may provide novel diagnostic and therapeutic targets for GI motility disorders.

AbstractBackgroundThe gut is the only organ system with intrinsic neural reflexes. Intrinsic primary afferent neurons (IPANs) of the enteric nervous system initiate intrinsic reflexes, form gut‐brain connections, and undergo considerable neuroplasticity to cause digestive diseases. They remain inaccessible to study in mice in the absence of a selective marker. Advillin is used as a marker for primary afferent neurons in dorsal root ganglia. The aim of this study was to test the hypothesis that advillin is expressed in IPANs of the mouse jejunum.MethodsAdvillin expression was assessed with immunohistochemistry and using transgenic mice expressing an inducible Cre recombinase under the advillin promoter were used to drive tdTomato and the genetically encoded calcium indicator GCaMP5. These mice were used to characterize the morphology and physiology of advillin‐expressing enteric neurons using confocal microscopy, calcium imaging, and whole‐cell patch‐clamp electrophysiology.Key ResultsAdvillin is expressed in about 25% of myenteric neurons of the mouse jejunum, and these neurons demonstrate the requisite properties of IPANs. Functionally, they demonstrate calcium responses following mechanical stimuli of the mucosa and during antidromic action potentials. They have Dogiel type II morphology with neural processes that mostly remain within the myenteric plexus, but also project to the mucosa and express NeuN and calcitonin gene‐related peptide (CGRP), but not nNOS.Conclusions and InferencesAdvillin marks jejunal IPANs providing accessibility to this important neuronal population to study and model digestive disease.

BACKGROUND Mechanical stimulation of the gastrointestinal (GI) epithelium causes changes in epithelial ion transport by an unclear mechanism. We recently discovered the mechanosensitive ion channel Piezo2 is specifically expressed by the enteroendocrine cells (EECs) and is necessary for EEC mechanosensitivity. Further, in mouse small bowel, the pharmacologic inhibition or genetic knockout of the epithelial Piezo2 led to an inhibition of the pressure‐induced short‐circuit current (I sc ) in the Ussing chamber. These results suggested that EEC Piezo2 plays a role in pressure‐induced epithelial ion transport, but the mechanism and whether these responses are region‐specific remains uknown. AIM Determine whether epithelial Piezo2‐mediated pressure‐induced I sc is due to Clsecretion in the mouse colon. METHODS Full‐thickness mid‐colon preparations of Vil Cre/+ ::Piezo2 f/f ( Piezo2 CKO ) or Vil +/+ ::Piezo2 f/f ( Piezo2 WT ) mice at 0.3 cm 2 were mounted in 4mL Ussing chambers with normal Krebs solution. The epithelial side was intermittently pressure‐clamped at a range of pressures (5, 10, 20, 30, 40, 60 mmHg) for 10s each. Forskolin (FSK, 10μM) was applied at the end of the experiments, and FSK response was used to normalize the pressure‐induced responses (ΔI sc /ΔI sc,FSK ). RESULTS Pressure applied to the mucosa of Piezo2 CKO and Piezo2 WT colons caused a pressure‐dependent increase in I sc . Responses in Piezo2 CKO colons were smaller than Piezo2 WT at 40mmHg (0.41±0.1 vs. 0.76±0.1) and 60mmHg (0.51±0.1 vs. 0.87±0.1) (n=9–11, P <0.05, two‐way ANOVA). To determine the nature of the pressure‐induced I sc we removed Cl − from the Krebs solutions. Cl‐removal resulted in XX. We found a nearloss in the mechanically induced ΔI sc in both Piezo2 WT tissues and Piezo2 CKO : 5mmHg (− 88.85±0.1% and −87.44±0.1% Piezo2 WT ), 10mmHg (−86.46±0.1% Piezo2 CKO and − 90.30±0.1% Piezo2 WT ), 20mmHg (−88.50±0.01% Piezo2 CKO and −89.86±0.1% Piezo2 WT ), 40mmHg (−91.78±0.1% Piezo2 CKO and −91.35±0.04% Piezo2 WT ), and 60mmHg (−93.93±0.04% Piezo2 CKO and −92.08±0.04% Piezo2 WT (n= 5–10, P <0.05, unpaired t ‐test). Replacement of Cl − led to full recovery of the mechanically induced ΔI sc in both Piezo2 WT and Piezo2 CKO tissues when tested at 30 mmHg ( Piezo2 WT : 2.51±1.4 vs. 73.52±33.8, n=6 and 3, P <0.05, unpaired t ‐test), ( Piezo2 CKO : 1.80±1.0 vs. 45.14±12.4, n=5 and 4, P <0.05, unpaired t ‐test). CONCLUSIONS Hydrostatic pressure applied to the colon epithelium leads to an increase in epithelial secretion that depends chloride and Piezo2 contributes to this mechanosensitive chloride secretion. Support or Funding Information Supported by AGA RSA, NIH DK106456, NIH DK119683, NIH DK123549