Beyder, Arthur

ABSTRACT Background The circadian rhythm regulates gastrointestinal motility. In humans and preclinical models, such as rodents, whole gut transit (WGT) is slower during the rest phase compared to the active phase. Investigators typically study GI transit in rodents during the day, which is their rest phase, rather than during the night, which is their active phase. A circadian rhythm reversal in which mice are in a dark room during the working day (reverse light) allows studies on nocturnal animals during their active phase and has been previously shown to reduce WGT time. GI motility is often disrupted in individuals with disorders of gut–brain interaction (DGBI), which are female predominant. However, the effect of circadian rhythm on regional transit and sex dependence of the differences is not known, as most motility studies looking at circadian rhythm reversal are done in male mice. Methods We tested C57BL/6 wild‐type male and female mice in rest (12 h of light during the day) and active (reverse cycle for 2 weeks: 12 h of dark during the day) phases. We noted female estrous cycle by visual inspection. We performed carmine WGT by monitoring time‐lapse videos of pellet production. We performed fluorescence imaging of excised intestines 30 min after gavage to assess percent fluorescence for each GI region and then examined small intestinal transit (SIT) by measuring geometric center and leading edge. For colonic transit, we monitored bead expulsion time from distal colon to anus. Key Results Compared to rest phase, in the active phase, like male mice, female mice had (1) faster WGT, (2) increased frequency of pellet expulsion in the first 3 h of transit, (3) and greater total pellet production. Both male and female mice in their active phase exhibited (4) more contrast emptied from the stomach and they had (5) further leading edge of fluorescence and (6) geometric center, in SIT, and (7) faster colonic bead expulsion times. There were no significant sex differences in the active phase of WGT. In SIT, male mice had further leading edge in the rest phase than female mice, but this difference was not seen in the active phase, and in colonic transit, male mice in both the active and rest phases had faster bead expulsion than female mice. Conclusions Mice in the active phase have faster regional transit in small and large bowel than mice in the rest phase that collectively contributes to faster WGT times in the active phase of both male and female mice. These findings highlight the importance of circadian biology in sex‐dependent rodent GI transit.

The gastrointestinal (GI) tract plays a crucial role in nutrient absorption, motility, and metabolism, while serving as a barrier against pathogens. The intestinal epithelial layer, particularly enteroendocrine cells (EEC), is pivotal in sensing luminal stimuli due to its strategic position. Although comprising only 1% of the GI epithelium, in total EEC comprises perhaps the largest endocrine organ that couples GI luminal stimuli with release of hormones and neurotransmitters such as serotonin, glucagon-like peptide-1 (GLP-1), and peptide–tyrosine–tyrosine (PYY), which regulate local and systemic physiology. In this review, we use EECs as a model to explore critical intracellular signaling pathways—centering around calcium (Ca²⁺) and cyclic adenosine monophosphate (cAMP). EECs utilize ionotropic and metabotropic receptors to detect luminal stimuli, leading to increased intracellular cytoplasmic Ca²⁺ and altered intracellular cAMP dynamics. Ca²⁺ influx is critical for the exocytosis of signaling molecules. The interplay between Ca²⁺ and cAMP signaling pathways enhances the release of these molecules, affecting both local and systemic responses. We make a point to discuss the conventional and emerging methodologies for measuring Ca²⁺ and cAMP dynamics in EECs, which is a rapidly expanding toolbox, containing genetically encoded indicators and resonance energy transfer techniques. Understanding these signaling cascades is essential for deciphering the complex roles of EECs in GI physiology and their impact on systemic health.

Key Points PIEZO1 activation reduces cystogenesis: Yoda1 activates PIEZO1, raising calcium and lowering cAMP, reducing cyst growth in autosomal dominant polycystic kidney disease models. Context-dependent role of PIEZO1: PIEZO1 knockout in mice with or without polycystic kidneys does not affect cyst formation, suggesting redundancy in mechanosensitive pathways. Therapeutic potential: Findings support PIEZO1 activation as a part of combination therapy to slow cyst growth in autosomal dominant polycystic kidney disease, needing more in vivo studies. Background The disruption of calcium signaling associated with polycystin deficiency is a key factor in abnormal epithelial growth in autosomal dominant polycystic kidney disease. Calcium homeostasis can be influenced by mechanotransduction. The mechanosensitive cation channel PIEZO1 has been implicated in sensing intrarenal pressure and regulating urinary osmoregulation, but its role in kidney cystogenesis is unclear. Methods We hypothesized that altered mechanotransduction contributes to cystogenesis in autosomal dominant polycystic kidney disease and that activation of mechanosensitive cation channels could be a therapeutic strategy. Results We demonstrate that Yoda1, a PIEZO1 activator, increases intracellular calcium and reduces forskolin-induced cAMP levels in mouse inner medullary collecting duct (mIMCD3) cells. Notably, knockout of polycystin-2 attenuated the efficacy of Yoda1 in reducing cAMP levels in mIMCD3 cells. Yoda1 also reduced forskolin-induced mIMCD3 cyst surface area in vitro and cystic index in mouse metanephros ex vivo in a dose-dependent manner. However, collecting duct–specific PIEZO1 knockout neither induced cystogenesis in wild-type mice nor altered cystogenesis in the Pkd1 RC/RC mouse model. Conclusions These findings support the potential role of PIEZO1 agonists in mitigating cystogenesis by increasing intracellular calcium and reducing cAMP levels, but the unaltered in vivo cystic phenotype after PIEZO1 knockout in the collecting duct suggests possible redundancy in mechanotransductive pathways.

Piezo2 is a mechanically gated ion channel most commonly expressed by specialized mechanoreceptors, such as the enteroendocrine cells (EECs) of the gastrointestinal epithelium. A subpopulation of EECs expresses Piezo2 and functionally resembles the skin’s touch sensors, called Merkel cells. Low-magnitude mechanical stimuli delivered to the mucosal layer are primarily sensed by mechanosensitive EECs in a process we term “gut touch.” Piezo2 transduces cellular forces into ionic currents, a process that is sensitive to bilayer tension and cytoskeletal depolymerization. E-cadherin is a widely expressed protein that mediates cell–cell adhesion in epithelia and interacts with scaffold proteins that anchor it to actin fibers. E-cadherin was shown to interact with Piezo2 in immortalized cell models. We hypothesized that the Piezo2–E-cadherin interaction is important for EEC mechanosensitivity. To test this, we used super-resolution imaging, co-immunoprecipitation, and functional assays in primary tissues from mice and gut organoids. In tissue EECs and intestinal organoids, we observed multiple Piezo2 cellular pools, including one that overlaps with actin and E-cadherin at the cells’ lateral walls. Further, E-cadherin co-immunoprecipitated with Piezo2 in the primary colonic epithelium. We found that E-cadherin knockdown decreases mechanosensitive calcium responses in mechanically stimulated primary EECs. In all, our results demonstrate that Piezo2 localizes to the lateral wall of EECs, where it physically interacts with E-cadherin and actin. They suggest that the Piezo2–E-cadherin–actin interaction is important for mechanosensitivity in the gut epithelium and possibly in tissues where E-cadherin and Piezo2 are co-expressed in epithelial mechanoreceptors, such as skin, lung, and bladder.

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.

TMEM63A has biophysical features distinctive for mechano-gated cation channels, but some properties are less clear, including no functional information in whole cells. We report that pressure-dependent yet voltage-independent TMEM63A currents in cell membrane patches correlated with cell size. In addition, radial stretch of whole cells on flexible membranes increased the [Ca2+]i responses more in TMEM63A-transfected cells. Inward TMEM63A currents in response to high stretch can depolarize plasma membranes and contribute to a sustained [Ca2+]i increase.

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

Background SCN5A ‐encoded Na V 1.5 is a mechanosensitive voltage‐gated sodium channel expressed in human gastrointestinal smooth muscle cells. In the gastrointestinal tract, alterations in Na V 1.5 current density by naturally occurring mutations or by pharmaceutical block of channel function are associated with constipation. In the heart loss of Na V 1.5 current is associated with cardiac contractility disorders such as dilated cardiomyopathy. Ion channel expression level is regulated by miRNAs, and Na V 1.5 is known to be regulated by miRNA in heart. However , it is not known if miRNAs regulate Na V 1.5 expression in GI smooth muscle. Aim To identify miRNAs that regulate Na V 1.5 expression in gastrointestinal smooth muscle in health and disease. Methods A qPCR array was used to screen for differential expression of 372 of the most common miRNAs in the miRBase database to test the differential expression of miRNA between control tissue and tissues from patients with slow transit constipation. qRT‐PCR was done to determine expression levels of SCN5A mRNA. A cell line (HuJej) was derived from human jejunum smooth muscle cells by immortalization using the SV40 large T antigen. Whole cell voltage clamp experiments were done by standard patch clamp techniques. Results SCN5A mRNA expression was reduced by 66±0.08% in patients with slow transit constipation (57.5±1.8 years) compared to age matched controls (53±1.7 years). qPCR array screening showed that 12 miRNAs were significantly upregulated in colonic muscularis externa from patients with STC compared to controls. Let‐7f miRNA was increased 6.46‐fold in slow transit constipation compared to controls and was the only miRNA with strong negative correlation to SCN5A mRNA expression in the same tissues. HuJej cells express Na V 1.5; therefore, we investigated the effect of let‐7f on Na V 1.5 currents by transfection with a stable miRNA mimic. 48 hours after transfection with let‐7f mimic, Na V 1.5 currents were significantly decreased compared to untransfected cells or to cells transfected with non‐targeting miRNA (let‐7f, −0.6±0.1 pA/pF versus untransfected, −3.9±0.6 pA/pF or non‐targeting, −2.3±0.6 pA/pF). Summary and Conclusions SCN5A mRNA is decreased in slow transit constipation. Expression levels of let‐7f and Na V 1.5 RNA are negatively correlated in human colonic smooth muscle. miRNA let‐7f regulates Na V 1.5 current density. Regulation of Na V 1.5 current density may have relevance in health and disease. Support or Funding Information This work was supported by grant DK52766.