Knutson, Kaitlyn R.

Age-related gastrointestinal dysfunction is common, but the mechanisms of aging-associated smooth muscle failure remain unclear. We show that aging in mice slows whole gut and colonic transit, increases regional stiffness, and reduces smooth muscle contractility. Inducible smooth muscle cell (SMC)– specific deletion of Piezo1 preserved youthful transit and force generation, whereas Piezo1 activation in young mice phenocopied aging-associated transit delay. Single-cell transcriptomics, RNA velocity, stiffness-controlled cell and tissue cultures, and pharmacologic studies revealed that Piezo1 couples increased stiffness to Ca 2+ –calcineurin–NFAT signaling, loss of contractile gene programs, leading to age- related contractile loss and contractile-to-synthetic SMC remodeling and gut wall stiffening. Human intestinal SMCs supported conservation of this pathway, and PIEZO1 gain-of-function carriers showed a trend toward delayed colonic transit. Thus, maladaptive SMC Piezo1 mechanotransduction is a targetable mechanism of aging-associated gut dysmotility. Aging alters organ structure, yet how these changes drive functional decline remains poorly defined. We show that progressive stiffening of the gut with age activates the mechanosensitive ion channel Piezo1 in smooth muscle cells, initiating a maladaptive response that impairs contractility and slows intestinal transit. On the other hand, deletion of smooth muscle Piezo1 prevents age-related gut dysmotility. These findings reveal that age-related physical shifts actively drive functional decline through mechanotransduction pathways. By identifying Piezo1 as a critical link between tissue mechanics and organ aging, this work provides a framework for understanding how mechanical cues contribute to age-related dysfunction in the gut and possibly other smooth muscle organs.

Background: Gastrointestinal (GI) disorders like chronic constipation are more prevalent with aging. Aging GI tract becomes stiffer and exhibits delayed GI transit. Smooth muscle cells (SMCs) are involved in controlling GI transit and show reduced contractility with age. Piezo1 is a mechanically activated ion channel that responds to membrane tension by opening its pore to allow flow of ions such as calcium (Ca2+) and regulates various cellular processes through Ca2+-calcineurin signaling pathway. We wondered GI stiffness changes might control SMC contractile to non-contractile phenotype through Piezo1. Objective: To determine how substrate stiffness regulates GI SMC contractile phenotype through Piezo1. Hypothesis: SMC Piezo1 senses GI tissue stiffness changes and alters SMC phenotype. Methods: Pathway analysis on human colon single cell sequencing data was performed using Gene Set Enrichment Analysis (GSEA) pre-ranked module with publicly available gene-sets from Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Immortalized human jejunal SMCs (HuSMCs) and freshly dissociated SMCs from control (CTRL, Myh11-creERT2) and SMC specific Piezo1 knockout [Myh11-creERT2xPiezo1 Flox (Piezo1-KO)] mouse colons (primary SMCs) were cultured on soft (1kPa) and stiff (50kPa) substrates to model age-dependent shifts in intestinal stiffness for 48 hours either in the presence/absence of Piezo1 siRNA, Piezo1 agonist (Yoda1, 1μM), and calcineurin blocker (Cyclosporin A, CsA, 1μM) and used for Ca2+ imaging, RT-qPCR and immunostaining. Results: GSEA showed Piezo1+ve SMCs were significantly enriched in pathways involved in the regulation of Ca2+ signaling compared to Piezo1−ve SMCs (p<0.05). HuSMCs and primary SMCs showed increased Piezo1 expression (HuSMCs:1.1±0.2-fold, n=8, p<0.05; primary SMCs:2.1±0.1-fold, n=3, p<0.05) and cytoplasmic Ca2+ (3.6-fold, n=10, p<0.05) in response to Yoda1 on stiff substrates compared to soft. HuSMCs on stiff substrate showed decreased expression of contractile SMC markers smoothelin ( SMTN:0.7±0.2-fold, n=7, p<0.05) and transgelin ( TAGLN:0.6±0.1-fold, n=7, p<0.05) which was rescued by Piezo1 siRNA treatment ( SMTN:1.2±0.1-fold, n=2-7, p<0.05; TAGLN:1.15±0.02-fold, n=2-7, p<0.05) (Piezo1 is necessary). Stimulation by Yoda1 of HuSMCs on the soft substrate resulted in decreased expression of SMTN (0.7±0.3-fold, n=2-7, p<0.05) and TAGLN (0.7±0.1-fold, n=2-7, p<0.05) (Piezo1 is suffcient). CTRL primary SMCs on stiff substrate showed decreased expression of Smtn (0.4±0.1-fold, n=3, p<0.05) and Tagln (0.3±0.2-fold, n=3, p<0.05) compared to SMCs on soft substrate which was reversed when SMCs from Piezo1-KO mice were cultured on these substrates ( Smtn:1.8±0.5-fold, n=3, p<0.05; Tagln:2.4±0.07-fold, n=3, p<0.05). Treatment of CsA in HuSMCs cultured on stiff substrate also rescued from loss of SMTN (1.3±0.1-fold, n=3, p<0.05) and TAGLN (1.7±0.3-fold, n=3, p<0.05) expression and prevented nuclear factor of activated T cells (NFAT) nuclear translocation. Summary and Conclusion: GI SMC Piezo1 senses stiffness changes and regulates SMC phenotype through Ca2+-calcineurin signaling pathway. Targeting Piezo1 might be protective from age induced reduced GI SMC contractility. NIH DK052766, DK123549. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

AbstractBackgroundIn preclinical studies whole gut transit (WGT) in mice is a gold‐standard “leading‐edge” approach that measures the time between orogastric gavage of carmine red and defecation of the first carmine red pellet. Transit studies in humans are performed during the active day because GI motility and transit are suppressed during the night. Since mice are nocturnal, WGT studies traditionally done during the day occur during their rest phase. How circadian rhythm affects WGT in mice is not known.MethodsWe used an automated approach for high temporal resolution uninterrupted testing of mouse WGT and activity. We housed wild‐type Bl6/C57 mice under the standard 12 h light–dark cycles. At 8 weeks, we performed carmine red orogastric gavage and assessed WGT during Light (rest) conditions. Then, we exposed mice to a reverse 12 h light–dark cycle for 2 weeks and tested them in the Dark (active) under red light conditions. Timelapse videos were analyzed to quantify activity and to timestamp all pellets, and multiple parameters were analyzed.Key ResultWhen complementary light cycle reversal experiments were performed, we found a significant increase in mouse activity when mice were tested during their Dark (active) phase, compared to their Light (rest) phase. In mice tested in the Active phase compared to the Rest phase, we found a significant acceleration in WGT, increased rate and total number of pellets produced, and more pellet clustering. These data show that the mice tested in the Active phase have important differences in activity that correlate with multiple alterations in gastrointestinal transit.Conclusion & InferencesDuring the Active phase mice have faster WGT, produce more pellets, and cluster their output compared to testing in the Rest phase. Like in humans, circadian rhythm is an important consideration for transit studies in mice, and a simple reverse light cycle approach facilitates further studies on the role of circadian rhythm in GI motility.

BACKGROUND In the gastrointestinal (GI) epithelium, enterochromaffin (EC) cells are enteroendocrine cells that use tryptophan hydroxylase 1 (TPH‐1) to produce serotonin (5‐hydroxytryptamine, 5‐HT). EC cells release 5‐HT in response chemical and mechanical stimuli. EC cell 5‐HT affects GI secretion, motility and sensation. Other neuroendocrine cells are electrically excitable, and several types use SCN3A ‐encoded voltage‐gated sodium channel Na V 1.3 for electrical excitability. It is unknown whether EC cells are electrically excitable. AIM To determine if the EC cells are electrically excitable. METHODS Murine TPH1‐CFP small bowel was dissociated, FAC sorted for TPH1‐CFP cells, and ion channel expression was analyzed by RNA‐seq. Murine TPH1‐CFP and human small bowel and colon were analyzed by immunohistochemistry (IHC). After dissociation, primary cultures from TPH1‐CFP mouse jejunum and colon were maintained for 72 hours. Scn3a expression in CFP+, CFP− cultured cells and media was analyzed by single cell polymerase chain reaction (PCR). Voltage‐ and current‐clamp experiments were performed on TPH1‐CFP cells at 24–48 hours. RESULTS RNA‐seq of FACS TPH1‐CFP EC cells from murine small bowel showed that Scn3a was the 3 rd most densely expressed ion channel gene, and no other ScnXa genes showed significant expression. Scn3a encodes a voltage‐gated sodium channel Na V 1.3. By IHC, Na V 1.3 is expressed in ~89–93% of the human and mouse small bowel and colon EC cells, which were defined as TPH1‐CFP+ for mouse and 5‐HT+ for human. Single cell PCR of CFP+ cells from primary cultures confirmed Scn3a expression. In voltage‐clamp, we found voltage‐gated fast activating and inactivating voltage‐gated currents in TPH1‐CFP EC cells from murine small bowel (78% of cells, −86±16 pA/pF, n=28), and colon (65% of cells, −66±12 pA/pF, n=14). These currents were abolished by Na + substitution by NMDG + and blocked by the Na V 1.3 inhibitor ICA‐121431 (IC 50 = 131±53 nM). Current‐clamped EC cells showed both elicited and spontaneous action potentials, also abolished by Na + substitution and Na V 1.3 block by ICA‐121431. Spontaneous current‐clamp activity showed action potential bursts of variable amplitude and frequency. CONCLUSIONS SCN3A ‐encoded voltage‐gated sodium channel Na V 1.3 is densely and specifically expressed by the EC cells of the human and mouse small and large bowel. In mouse EC cells, Na V 1.3 generates voltage‐gated sodium current which is critical for EC cell electrical excitability. Support or Funding Information NIH DK106456, DK84567, DK052766

BACKGROUND Enterochromaffin (EC) cells are enteroendocrine cells in the gastrointestinal (GI) epithelium that release serotonin (5‐HT) in response to a variety of stimuli including mechanical force. In turn, EC cell 5‐HT affects GI secretion, motility and sensation. The molecular mechanism of EC cell mechanotransduction is unknown. The recently discovered mechanosensitive ion channel Piezo2 is important in several mechanosensory cell types. AIM to determine if the mechanosensitive ion channel Piezo2 is expressed and functional in EC cells. METHODS Murine small bowel was collected, fixed in 4% paraformaldehyde, and frozen. Human and mouse RNA from human jejunum and mouse duodenum, jejunum and ileum was analyzed by quantitative RT‐PCR for Piezo2 mRNA. Piezo2 and 5‐HT were also identified by fluorescence immunolabeling. QGP‐1, a human enteroendocrine cell model, was analyzed by RT‐PCR for the presence of Piezo2 and immunolabeling was done to detect Piezo2 and 5‐HT. Piezo2 mechanically activated currents were recorded from QGP‐1 cells in whole‐cell configuration. Voltage‐clamped QGP‐1 cells were subjected to a series of mechanical perturbations using a piezo‐electrically driven glass probe at steps of 0.3 μm for stimulation. RESULTS Human jejunum and all segments of mouse small bowel mucosa (duodenum, jejunum, ileum) expressed Piezo2 mRNA. In human jejunum and all segments of mouse small bowel Piezo2 co‐localized with 5‐HT in >90% of 5‐HT containing cells. No Piezo2 immunoreactivity was seen outside of 5‐HT positive enterochromaffin cells. The enteroendocrine cell model QGP‐1 expressed both Piezo2 and 5‐HT by immunohistochemistry and RT‐PCR showed message for Piezo2. Application of force to the surface of voltage‐clamped QGP‐1 cells elicited whole cell fast inward non‐selective cation currents of ~200 pA (at −60 mV) that were blocked by Gd 3+ with kinetic properties compatible with Piezo2 channels. CONCLUSIONS Piezo2 is specifically expressed in human and murine small bowel EC cells and in the enteroendocrine cell model QGP‐1. QGP‐1 cells have a mechanosensitive inward cation current with kinetic and pharmacological properties similar to Piezo2. Our data suggest that Piezo2 is involved in EC cell mechanotransduction. Support or Funding Information Supported by NIH DK106456 , DK84567, DK052766

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.

BACKGROUND Enteroendocrine cells (EECs) in the gastrointestinal (GI) epithelium are specialized sensors of luminal chemicals and forces. Upon stimulation, EECs release a variety of neurotransmitters and hormones, but cellular mechanisms of excitation‐secretion coupling are poorly understood. We recently discovered that a population of EECs expresses the mechanosensitive ion channel Piezo2, which is necessary for force‐dependent intracellular Ca2+ increase and serotonin release. However, the molecular mechanism that links Piezo2 and serotonin release is unknown. In other sensory systems, the Cacna1h ‐encoded t‐type voltage‐gated calcium channel Cav3.2 is important in mechanotransduction for linking mechanical activation with neurotransmitter release. AIM to determine if Cav3.2 is expressed and functionally relevant in EECs. METHODS Quantitative RT‐PCR for Cacna1h mRNA expression was evaluated in colon epithelium of control and NeuroD1 Cre x RiboTag mice, in which actively transcribed mRNA specifically from EECs is tagged with hemagglutinin (HA). NeuroD1 Cre x tdTomato colon cross‐sections were fixed and immunolabeled with anti‐Cav3.2 antibody. Single primary NeuroD1 cre x GCaMP5 EECs were mechanically stimulated and Cav3.2 role in mechanotransduction was tested by pharmacology (Ca2+ free, nickel, mibefradil, ω‐agatoxin IVA) and Cacna1h siRNA knockdown. RESULTS Murine colon epithelium expressed Cav3.2 mRNA. In NeuroD1 Cre x RiboTag mouse, HA co‐localized with EEC marker Chromogranin A (Chga). NeuroD1+ EEC ribosomal trapped mRNA was enriched for markers of epithelium (Vil1), EEC (Chga), and enriched 7.0‐fold for Cav3.2 (n=3, p<0.05 compared to whole epithelium). Immunohistochemistry in NeuroD1 Cre x tdTomato showed that Cav3.2 co‐localized specifically with tdTomato (NeuroD1+) EECs. In primary NeuroD1+ EECs, Cav3.2 inhibition by all pharmacologic agents and siRNA knockdown inhibited mechanically induced Ca2+ response (96±1% for Ca2+ free, 71±9% nickel, 61±6% mibefradil, 80±4% ω‐agatoxin IVA and 96±1% Cav3.2 siRNA, n=3–18, p<0.05 compared to control untreated cells or cells treated with non‐targeted siRNA). CONCLUSIONS Cav3.2 is densely and specifically expressed in mouse colon EECs and it is important for regulating Ca2+ in EEC mechanotransduction. Future studies will determine how Cav3.2 channels contribute to EEC electrical excitability, excitation‐secretion coupling, and ultimately EEC Cav3.2 roles in GI physiology. Support or Funding Information Supported by NIH DK106456, NIH DK084567, and AGA RSA This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

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 .

AbstractBackgroundGastrointestinal (GI) motility is a complex physiological process that is critical for normal GI function. Disruption of GI motility frequently occurs in GI diseases or as side effects of therapeutics. Whole gut transit measurements, like carmine red leading‐edge transit, in mice form the cornerstone of in vivo preclinical GI motility studies.MethodWe have developed an easily achievable, labor‐saving method to measure whole gut transit time in mice. This approach uses inexpensive, commercially available materials to monitor pellet production over time via high definition cameras capturing time‐lapse video for offline analysis.Key ResultWe describe the assembly of our automated gut transit setup and validate this approach by comparing the results with loperamide to delay transit and conventional transit measurements. We demonstrate that compared to the control group, the loperamide group had slowed transit, evidenced by a decrease in total pellet production and prolonged whole gut transit time. The control group had an extended transit time compared with the results reported in the literature. Whole gut transit rates accelerated to times comparable to the literature by disrupting cages every 10‐15 min to imitate the conventional approach, suggesting that disruption affects the assay and supports the use of an automated approach.Conclusion & InferencesA novel automated, inexpensive, and easily assembled whole gut transit setup is labor‐saving and allows minimal disruption to animal behavior compared with the conventional approach.

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