American journal of physiology. Gastrointestinal and liver physiology最新文献

Fibroblast-like cells (FLCs) exist in the smooth muscle layers of visceral organs, yet in many instances their functional role(s) have not been identified. FLCs express platelet-derived growth factor receptor (PDGFR) α and are a novel class of excitable cells recently described in visceral organs. Crenolanib is a benzamidine quinolone derivative originally developed as an inhibitor of PDGFR to treat certain solid tumors with PDGFRα overexpression mutations. In the present study, we used crenolanib to disrupt PDGFRα expression and signaling in the gastrointestinal (GI) tracts of BALB/c mice. Intraperitoneal injections of crenolanib (100 µg/g body wt) or DMSO control vehicle were given to littermates from postpartum P1 through P15. Crenolanib-injected mice were smaller in size and weight. The gastrointestinal tracts were also shorter and appeared partially distended. qPCR revealed downregulation of key gene transcripts involved in PDGFRα cell signaling including Pdgfra, Kcnn3, and P2ry1. Confocal immunofluorescence demonstrated significant decreases in PDGFRα and SK3 protein expression. c-Kit expression was slightly inhibited, but gastric, intestinal, and colonic pacemaker activity was not affected by crenolanib. Purinergic inhibitory postjunctional motor responses were greatly attenuated in the GI tracts of crenolanib-treated animals compared with vehicle-treated controls in response to electric field-evoked nerve stimulation. These data provide evidence for a functional role of PDGFRα⁺ cells in inhibitory neuroeffector motor responses throughout the gastrointestinal tract.NEW & NOTEWORTHY The physiological roles of newly described PDGFRα⁺ interstitial cells in neurotransmission within the gastrointestinal (GI) tract have predominantly come from studies on isolated cells. Here we used an inhibitor of PDGFRα, crenolanib, to examine the effects of PDGFRα⁺ cells in enteric inhibitory neurotransmission. Crenolanib caused loss of PDGFRα⁺ cells and neurally evoked fast inhibitory junction potentials associated with purine neurotransmission, providing evidence for the function of PDGFRα⁺ cells within intact tissues of the GI tract.

Transient receptor potential vanilloid 6 (TRPV6) is a highly Ca²⁺-permeable cation channel predominantly expressed in the intestinal epithelium. It plays a crucial role in maintaining systemic calcium homeostasis by regulating Ca²⁺ absorption in the intestine. However, its local physiological and pathophysiological roles in the intestine remain unexplored. The exact cause of inflammatory bowel disease is not fully understood; however, disruption of the intestinal epithelial barrier is a key pathogenic mechanism. In this study, we aimed to elucidate the role of TRPV6 in the pathogenesis of colitis. Experimental colitis was induced in TRPV6-deficient [knockout (KO)] and wild-type (WT) mice by administering 2% dextran sulfate sodium (DSS) solution in drinking water for 7 days. DSS treatment resulted in weight loss, diarrhea/bloody stool, histological colonic injury, and colon shortening. The systemic symptoms and colonic injury were significantly worse in TRPV6KO mice than in WT mice. DSS treatment increased tumor necrosis factor-α, interleukin-1β, interleukin-6 mRNA expressions, and myeloperoxidase activity, and these responses were significantly enhanced in TRPV6KO mice compared with WT mice. Under normal (no DSS-treated) conditions, TRPV6KO mice exhibited increased intestinal permeability compared with WT mice. No difference was observed in the number of goblet cells between WT and TRPV6KO mice; however, the expression of intercellular junction proteins, including E-cadherin, claudin-3, and occludin, was significantly suppressed in TRPV6KO mice compared with WT mice. These findings suggest that TRPV6 protects against DSS-induced colitis, potentially by regulating epithelial barrier function through intracellular junction protein expressions.NEW & NOTEWORTHY This study is the first to reveal the local role of TRPV6 in the intestine. TRPV6KO exacerbated dextran sulfate sodium-induced colitis and increased intestinal permeability compared with wild-type mice. Furthermore, intercellular junction protein expression was lower in TRPV6KO mice. TRPV6 protects against colitis by maintaining epithelial barrier function by regulating intercellular junction protein expression. Thus, TRPV6 may be a novel therapeutic candidate for treating inflammatory bowel disease.

Biological sex has been shown to influence aging outcomes, contributing to distinct trajectories in disease susceptibility and lifespan. DNA methylation patterns provide a quantitative measure of biological aging. This study investigated whether aged male and female mice display distinct liver DNA methylation patterns and differences in epigenetic aging. Liver samples were collected from 17 aged c57BL/6 mice (6 males, 11 females). Genomic DNA was extracted and bisulfite-converted before targeted enrichment of 2,045 murine age-associated CpG loci. Biological age (DNAge) was estimated using a previously developed DNA methylation-based predictor generated through elastic net regression. The difference (ΔDNAge) between DNAge and chronological age was computed. Sex-specific differences were assessed by comparing site-specific methylation ratios, ΔDNAge values, and through principal component analysis (PCA) and multiple linear regression. Twelve CpG sites across six genes (Fam84b, Zswim6, Hsf4, Mn1, Qprt, and Rapgefl1) showed significant sex-associated differences in methylation. Fam84b demonstrated the largest and most consistent sex-associated effect, with all three associated CpG sites showing higher methylation in males (regression coefficients: -0.204, -0.281, and -0.294). Zswim6 exhibited consistent lower methylation ratios in females, whereas the other genes showed higher methylation in females. There were no sex differences in biological age or ΔDNAge (P = 0.596). Although the epigenetic clock did not reveal differences between sexes in aging, aged mice did exhibit sex-specific liver methylation patterns different from those reported in younger mice, suggesting that sex-dependent epigenetic changes may emerge later in life and may reflect sexual dimorphism in liver function with age.NEW & NOTEWORTHY Males and females are known to age differently and develop certain diseases at different rates. Here, we examined the livers of aged male and female mice to see if they show different DNA methylation patterns. We found that aged male and female mice had distinct DNA methylation patterns at specific genes. Interestingly, most of these methylation differences were not present in younger mice, suggesting that sex differences in the genome may change with age.

Anticipation of daily recurring changes in the environment is critical for survival. When food access is limited to a few hours during the daytime, nocturnal rodents exhibit food-anticipatory activity, which appears a few hours before scheduled mealtime. The rodents are also known to exhibit anticipatory activity for time-restricted palatable meals under ad libitum access to chow. When 1 h of chocolate chip access was given during the day, mice exhibited robust anticipatory activity. In contrast, despite the peanut butter-fed mice eating two times the calories of peanut butter than the chocolate-fed mice did of chocolate chips, we observed only negligible anticipatory activity for daily 1-h peanut butter administration. In ex vivo explants, the phase of the liver in mice subjected to timed-chocolate chip access was significantly advanced, similarly to that in mice subjected to 4-h restricted feeding during the day. Similar to anticipatory activity, negligible phase changes in the liver were observed in the mice given 1 h of peanut butter access during the day. Therefore, robustness of palatable meal-anticipatory activity and phase advance in the liver are unlikely to be in direct response to increased calorie intake during the day. We measured food-seeking nose-poking behavior during food deprivation following daily 1-h chocolate chip access. Mice expressed anticipatory food seeking around the time that they had previously been given daily chocolate chips. This suggests that the time of chocolate chip access is encoded to the same circadian pacemaker that controls food-anticipatory activity.NEW & NOTEWORTHY Anticipatory activity for daily chocolate chip access is stronger than that for daily peanut butter access. Mice given daily chocolate chip access, but not peanut butter access, exhibited altered circadian organization among peripheral clocks. During food deprivation, mice exhibited anticipatory food-seeking behavior at the time they had previously been given chocolate chip access, suggesting that the time of palatable meals is encoded in the circadian pacemaker controlling food-anticipatory activity.

Gastric slow waves fail to propagate through the pyloric sphincter (PS), thus isolating the specialized motility patterns of the stomach and small intestine. We investigated the role of interstitial cells of Cajal (ICC) in PS of mice. Ca²⁺ waves in ICC, events responsible for electrical slow waves, propagated along the gastric wall but failed to propagate into the PS. ICC in PS fired localized Ca²⁺ transients and displayed low expression of voltage-dependent Ca²⁺ conductances. These are properties of intramuscular ICC (ICC-IM) that cannot regenerate and propagate slow waves. A T-type Ca²⁺ channel antagonist had no effect on Ca²⁺ transients, but these events were blocked by thapsigargin and cyclopiazonic acid, suggesting that they result from Ca²⁺ release. PS ICC expressed ANO1, a Ca²⁺-activated Cl^- conductance. Ca²⁺ released from stores activates ANO1 channels, thus exerting a depolarizing influence on PS. Ani9, a selective antagonist of ANO1 channels, hyperpolarized cells and reduced contractile tone. Electrical field stimulation (EFS) of intrinsic neurons yielded inhibitory junction potentials (IJPs), and cessation of EFS resulted in poststimulus depolarization and contraction. N^ω-nitro-l-arginine (L-NNA) abolished relaxation responses to EFS and switched responses to contractions. Application of atropine or Ani9 (in the presence of L-NNA) abolished contraction during EFS. Our results describe new and fundamental functions of ICC-IM in the PS. The inability of these cells to propagate slow waves provides the insulator function of PS muscles and localized Ca²⁺ transients, and activation of ANO1 regulates PS tone and mediates inputs from enteric neurons.NEW & NOTEWORTHY The pyloric sphincter is an electrical insulator between the stomach and small intestine, isolating and maintaining the unique motility patterns of these organs. Using Ca²⁺ imaging techniques, this study demonstrates why slow waves do not propagate between organs and how interstitial cells of Cajal serve to regulate pyloric tone and responses to enteric motor neurons.

Colonic motility is controlled by the enteric nervous system that is modulated not only by autonomic neurotransmitters but also by substances released from enteroendocrine cells. Glucagon-like peptide-1 (GLP-1) secreted from L cells accelerates peristalsis of the proximal colon, whereas somatostatin released from D cells inhibits GLP-1 secretion via the activation of somatostatin receptor subtype 5 (SST₅). Here, effects of somatostatin on GLP-1-mediated acceleration of colonic peristalsis were investigated. Cannulated segments of rat proximal colon were serosally perfused with oxygenated physiological salt solution and luminally perfused with degassed solution. Colonic wall motion was imaged and converted into spatiotemporal maps. Distributions of somatostatin and SST₅ receptors were assessed via immunohistochemistry. Intraluminal administration of somatostatin alone did not affect oro-aboral propagating peristaltic contractions, but prevented luminal-applied GLP-1 (30 nM)-induced acceleration of peristaltic waves. Somatostatin also prevented the prokinetic action of endogenous GLP-1 that was released upon the stimulation by luminal-applied short-chain fatty acids (SCFA, 3 mM) or lipopolysaccharide. In colonic segments that had been pretreated with selective SST₅ receptor antagonist 1, a lower concentration of GLP-1 (10 nM) or SCFA (300 µM) became capable of accelerating peristaltic waves. GLP-1-positive epithelial cells coexpressed SST₅ receptors, whereas GLP-1 receptor-positive epithelial cells and afferent neurons contained somatostatin. Thus, L cell-derived GLP-1 that accelerates colonic peristalsis may simultaneously stimulate D cells and afferents to release somatostatin that serves as a break on prokinetic actions of GLP-1 by activating SST₅ receptors on L cells. This interplay between GLP-1 and somatostatin would prevent excessive colonic motility.NEW & NOTEWORTHY Somatostatin inhibited the prokinetic action of glucagon-like peptide-1 (GLP-1), short-chain fatty acids, and lipopolysaccharide. The inhibitory effect of somatostatin was mediated by the activation of somatostatin receptor subtype 5 that was expressed on L cells. Sources of somatostatin were D cells and intrinsic primary afferent neurons, both of which expressed GLP-1 receptors. Thus, colonic peristalsis may well be regulated by the functional interaction between excitatory GLP-1 and inhibitory somatostatin.

Current clinical decision-making is hindered by the absence of predictive preclinical models that faithfully bridge molecular diversity to patient outcomes. Here, we apply the principle of abstraction-deriving essential features from human tissues to build next-generation new approach methodologies (NAMs) that transform patient-derived organoids (PDOs) into predictive vehicles for Crohn's disease (CD). From our living biobank of adult stem cell-derived colonic PDOs, we previously defined two molecular CD subtypes: immune-deficient infectious CD (IDICD) and stress and senescence-induced fibrostenotic CD (S2FCD), each defined by unique genomic, transcriptomic, and functional profiles with matched therapeutic vulnerabilities. In this study, we prospectively anchored PDO-derived molecular phenotypes to real-world clinical outcomes, revealing that S2FCD maps to baseline and progressive colonic disease activity, whereas IDICD tracks with prior ileocecal surgery, penetrating disease behavior, as well as baseline and progressive ileal disease activity. By abstracting NAMs from human tissues and cycling insights between small-"n" organoids and Phase 3-sized datasets, this framework recasts PDOs as dynamic, predictive platforms that capture the past, present, and future of disease behavior. Beyond oncology, this work establishes PDOs as vehicles for prospective clinical trial-like studies in inflammatory diseases and highlights colonic immune dysfunction as a potential driver of ileal CD.NEW & NOTEWORTHY In this prospective study, Penrose et al. evaluate a living biobank of genotyped and phenotyped patient-derived organoids (PDOs) as predictive tools in Crohn's disease, demonstrating their ability to faithfully capture past, present, and future disease behavior. By positioning PDOs as new approach methodologies (NAMs), this work extends PDO-informed precision medicine beyond oncology and into complex inflammatory disorders, translating molecular diversity into actionable clinical insights.

Injury and aging of the external anal sphincter (EAS) muscle lead to fibrosis and muscle dysfunction, major contributors to fecal incontinence. Activation of the WNT/β-catenin signaling pathway has been linked to fibrosis in various tissues, including skeletal muscle. This study examined whether the WNT agonist Wnt3a induces fibrosis and dysfunction in the EAS muscle. Adult female New Zealand White rabbits received four local injections of Wnt3a or saline into the EAS muscle. Anal canal pressure was measured by manometry every 2 wk for 8 wk, followed by histological, immunofluorescent, immunohistochemistry (IHC), Western blot, and proteomic analyses of the EAS muscle. Rabbits treated with Wnt3a exhibited a significant reduction in anal canal pressure 8 wk postinjection (P ≤ 0.05) compared with controls. Histologic evaluation revealed increased connective tissue (P = 0.06), significant collagen deposition, and decreased muscle area and fiber thickness (P ≤ 0.05). Western blot analysis showed elevated levels of β-catenin, nuclear active β-catenin (PY489), Smad1/2/3, signal transducer and activator of transcription 3 (Stat3), transforming growth factor-β (TGF-β), and vimentin (P ≤ 0.05), with p-Stat3, p-Smad3, and collagen-4 trending upward. Immunofluorescence and IHC confirmed increased β-catenin, collagen-4, and TGF-β levels, and proteomic data indicated altered pathways related to muscle contraction, fibrosis, and atrophy. These findings demonstrate that direct administration of a WNT agonist promotes EAS fibrosis and dysfunction, mirroring changes associated with aging and injury. Local application of WNT antagonists may represent a therapeutic strategy to prevent anal sphincter dysfunction following injury.NEW & NOTEWORTHY In the current study, for the first time we investigated the effects of a local injection of Wnt3a-an agonist of the WNT signaling pathway-on EAS function, muscle replacement by fibrosis, and the activation of downstream WNT signaling pathways. Wnt3a injection resulted in impaired EAS function and replacement of muscle with fibrosis. Notably, the downstream signaling remained active even 8 wk after the Wnt3a injection.

Dysphagia is common in patients with preserved contraction phase of esophageal peristalsis, such as those diagnosed with functional dysphagia (FD) or esophagogastric junction outflow obstruction (EGJOO); it amounts to ≥50% patients undergoing high-resolution manometry impedance (HRMZ) study. We aimed to characterize the distension phase of esophageal peristalsis (bolus domain) and identify abnormalities that may contribute to dysphagia in these patients. HRMZ recordings from 35 healthy controls, 35 patients with FD, and 35 patients with EGJOO were analyzed. Distension-contraction plots were used to assess the luminal cross-sectional area, bolus pressure, and regions without bolus ("no-bolus areas") within the bolus domain of peristalsis. The proportion of no-bolus area was compared among groups, and receiver operating characteristic (ROC) analysis evaluated diagnostic performance. Esophageal wall compliance during the distension phase was also determined. Both FD and EGJOO groups exhibited a significantly greater proportion of no-bolus area within the bolus domain compared with controls, particularly in the distal esophagus (P < 0.001). Pressure peaks frequently occurred in the absence of bolus, indicating pseudo-bolus pressures. Ultrasound imaging revealed transient luminal collapse against the manometry catheter in the "pseudo-bolus" zone. Esophageal wall compliance was reduced in both patient groups. ROC analysis demonstrated that the percentage of no-bolus area discriminated patients from controls with high accuracy (area under the curve 0.83 for FD, 0.88 for EGJOO). We propose that impaired esophageal distensibility and transient luminal collapse within the bolus domain cause functional obstruction and possibly dysphagia.NEW & NOTEWORTHY This study introduces a novel impedance-based method to analyze the bolus domain of esophageal peristalsis, emphasizing bolus pressure and bolus mismatch. Patients with functional dysphagia and EGJOO demonstrated impaired esophageal distension, reduced wall compliance, and frequent pseudo-bolus pressures from transient luminal collapse during peristalsis. These findings suggest that dysphagia may result from dynamic obstruction due to esophageal collapse during peristalsis rather than fixed outflow obstruction, highlighting bolus domain mechanics as a complementary diagnostic tool.