5-aminosalicylic acid (5-ASA) is a classic anti-inflammatory drug for the treatment of ulcerative colitis. N-acetyltransferase (NAT) enzymes convert 5-ASA to its metabolite N-acetyl-5-ASA, and it is ...unresolved whether 5-ASA or N-acetyl-5-ASA is the effective therapeutic molecule. We previously demonstrated that colonic production of N-acetyl-5-ASA (NAT activity) is decreased in dextran sulfate sodium-induced colitis. Our hypothesis is that 5-ASA is the therapeutic molecule to improve colitis, with the corollary that altered NAT activity affects drug efficacy. Since varying clinical effectiveness of 5-ASA has been reported, we also ask if NAT activity varies with inflammation in pediatric or adult patients.
Acute colonic inflammation was induced in C57BL/6 NAT wild-type (WT) or knockout mice, using 3.5% dextran sulfate sodium (w/v) concurrent with 5-ASA treatment. Adult and pediatric rectosigmoid biopsies were collected from control or patients with ulcerative colitis. Tissue was analyzed for NAT and myeloperoxidase activity.
Dextran sulfate sodium-induced colitis was of similar severity in both NAT WT and knockout mice, and NAT activity was significantly decreased in NAT WT mice. In the setting of colitis, 5-ASA significantly restored colon length and decreased myeloperoxidase activity in NAT knockout but not in WT mice. Myeloperoxidase activity negatively correlated with NAT activity in pediatric patients, but correlation was not observed in adult patients.
Inflammation decreases NAT activity in the colon of mice and human pediatric patients. Decreased NAT activity enhances the therapeutic effect of 5-ASA in mice. A NAT activity assay could be useful to help predict the efficacy of 5-ASA therapy.
Calcium (Ca2+) is a known accelerator for gastric wound repair. We have demonstrated in vivo and in vitro that intracellular Ca2+ increases in the gastric epithelial cells directly adjacent to a ...damaged cell, and that this Ca2+ rise is essential for the cellular migration that rapidly repairs the epithelium (restitution). While intracellular Ca2+ has been shown to be an important signaling factor during epithelial restitution, the source from which this intracellular Ca2+ originates remains unclear. Using gastric organoids derived from mice transgenic for a genetically encoded Ca2+ indicator, we sought to investigate the potential sources of intracellular Ca2+ mobilization. During confocal imaging, photodamage (PD) was induced to 1–2 gastric organoid epithelial cells and epithelial restitution measured simultaneously with changes in intracellular Ca2+ (measured as FRET/CFP ratio in migrating cells adjacent to the damaged area). Inhibition of voltage‐gated Ca2+ channels (verapamil, 10 µM) or store‐operated calcium entry (YM58483, 20 µM) resulted in delayed repair and dampened intracellular Ca2+ response. Furthermore, inhibition of phospholipase C (U73122, 10 µM) or inositol trisphosphate receptor (2‐APB, 50 µM) likewise resulted in delayed repair and dampened Ca2+ response. Results suggest both extracellular and intracellular Ca2+ sources are essential for supplying the Ca2+ mobilization that stimulates repair.
Intracellular calcium mobilization is known to be essential for proper gastric epithelial repair, however the source from which calcium mobilization originates remains unclear. Our work examines intracellular calcium signaling during epithelial repair in response to photodamage of individual cells. Our work demonstrates that gastric organoids exhibit essential parallels with native tissue and provides insight into the complexity of extracellular and intracellular calcium sources needed for intracellular calcium mobilization to occur in specific migrating (restituting) cells juxtaposed to the damaged cell.
Background & Aims: Confocal endomicroscopy is an emerging technology that poses the endoscopist with challenges for identifying epithelial structures in the human intestine. We have shown previously ...that the murine intestinal epithelium is punctuated by gaps caused by cell shedding. The goals of this study were to determine if confocal endomicroscopy could resolve the presence of human epithelial gaps and whether a proinflammatory cytokine could increase cell shedding. Methods: Intestinal mucosa was imaged after staining with acriflavine. Confocal endomicroscopy of 17 patients yielded 6277 images from the human terminal ileum and rectum. Results were validated by parallel studies of anesthetized mice (wild-type and Math1ΔIntestine ) using rigid confocal probe microscopy, 2-photon/confocal microscopy, and scanning electron microscopy. Results: Human terminal ileal and rectal epithelium revealed unstained areas with the diameter of an individual epithelial cell, with 2 distinct morphologies. One had a “target” appearance, shown by mouse studies to be goblet cells. The other morphology had no nucleus and was observed by rigid confocal probe microscopy and scanning electron microscopy in the villi of Math1ΔIntestine mice, which lack goblet cells. In the mouse, tumor necrosis factor α (0.33 μg/g intraperitoneally) increases cell shedding by 27-fold and caused loss of barrier function across 20% of resultant gaps. Conclusions: Confocal endomicroscopy can distinguish between epithelial discontinuities (gaps) and goblet cells in human intestine. Results suggest that the sealing of epithelial gaps must be considered as a component of the intestinal barrier and has potential implications for intestinal barrier dysfunction in human disease.
CFTR and pHi regulation Akiba, Yasutada; Kaunitz, Jonathan D; Montrose, Marshall H
American journal of physiology. Gastrointestinal and liver physiology/American journal of physiology: Gastrointestinal and liver physiology,
06/2016, Letnik:
310, Številka:
11
Journal Article
Background & Aims The peptic ulcer heals through a complex process, although the ulcer relapse often occurs several years later after healing. Our hypothesis is that even after visual evidence of ...healing of gastric ulceration, the regenerated epithelium is aberrant for an extended interval, increasing susceptibility of the regenerated epithelium to damage and further diseases. Methods Gastric ulcers were induced in mice by serosal topical application of acetic acid. Results Gastric ulcers induced by acetic acid visually healed within 30 days. However, regenerated epithelial architecture was poor. The gene profile of regenerated tissue was abnormal, indicating increased stem/progenitor cells, deficient differentiated gastric cell types, and deranged cell homeostasis. Despite up-regulation of PDX1 in the regenerated epithelium, no mature antral cell type was observed. Four months after healing, the regenerated epithelium lacks parietal cells, trefoil factor 2 (TFF2) and (sex-determining region Y)-box 9 (SOX9) remain up-regulated deep in the gastric gland, and the Na/H exchanger 2 (a TFF2 effector in gastric healing) remains down-regulated. Gastric ulcer healing was strongly delayed in TFF2 knockout mice, and re-epithelialization was accompanied with mucous metaplasia. After Helicobacter pylori inoculum 30 days after ulceration, we observed that the gastric ulcer selectively relapses at the same site where it originally was induced. Follow-up evaluation at 8 months showed that the relapsed ulcer was not healed in H pylori –infected tissues. Conclusions These findings show that this macroscopically regenerated epithelium has prolonged abnormal cell distribution and is differentially susceptible to subsequent damage by H pylori.
Disruption of circadian rhythms is a risk factor for several human gastrointestinal (GI) diseases, ranging from diarrhea to ulcers to cancer. Four-dimensional tissue culture models that faithfully ...mimic the circadian clock of the GI epithelium would provide an invaluable tool to understand circadian regulation of GI health and disease. We hypothesized that rhythmicity of a key circadian component, PERIOD2 (PER2), would diminish along a continuum from ex vivo intestinal organoids (epithelial 'miniguts'), nontransformed mouse small intestinal epithelial (MSIE) cells and transformed human colorectal adenocarcinoma (Caco-2) cells. Here, we show that bioluminescent jejunal explants from PERIOD2::LUCIFERASE (PER2::LUC) mice displayed robust circadian rhythms for >72 hours post-excision. Circadian rhythms in primary or passaged PER2::LUC jejunal organoids were similarly robust; they also synchronized upon serum shock and persisted beyond 2 weeks in culture. Remarkably, unshocked organoids autonomously synchronized rhythms within 12 hours of recording. The onset of this autonomous synchronization was slowed by >2 hours in the presence of the glucocorticoid receptor antagonist RU486 (20 μM). Doubling standard concentrations of the organoid growth factors EGF, Noggin and R-spondin enhanced PER2 oscillations, whereas subtraction of these factors individually at 24 hours following serum shock produced no detectable effects on PER2 oscillations. Growth factor pulses induced modest phase delays in unshocked, but not serum-shocked, organoids. Circadian oscillations of PER2::LUC bioluminescence aligned with Per2 mRNA expression upon analysis using quantitative PCR. Concordant findings of robust circadian rhythms in bioluminescent jejunal explants and organoids provide further evidence for a peripheral clock that is intrinsic to the intestinal epithelium. The rhythmic and organotypic features of organoids should offer unprecedented advantages as a resource for elucidating the role of circadian rhythms in GI stem cell dynamics, epithelial homeostasis and disease.
Background & Aims:
Epithelial cells of the small intestine migrate to the tip of the villus at which they are shed. It is not understood how the intestinal barrier is maintained during this high cell ...turnover. The aim of this study was to use high-resolution in vivo light microscopy to investigate the mechanism of epithelial shedding and the site of the permeability barrier during cell shedding.
Methods:
A laparotomy was performed on anesthetized mice, and a segment of small intestine was opened. The exposed epithelial surface of the intestine was imaged by multiphoton microscopy. Nuclei, cytosol, and cell membranes were imaged using the dyes Hoescht 33258, BCECF, a transgenically expressed fluorescent protein, and the membrane dye DiI. The fluorescent caspase substrate PhiPhiLux was used to detect apoptosis.
Results:
In the epithelial monolayer, gaps were observed that lacked nuclei or cytosol but appeared to be filled with an impermeable substance. Studies with membrane impermeant fluorophores (Lucifer Yellow and Alexa-dextran) showed that the impermeable substance completely fills the void left by the absent cell. Only a fraction of gaps have either ZO-1 staining or cytoplasmic extensions from neighboring cells at the basal pole. Time-lapse studies reveal that cell shedding results in genesis of a gap and that shedding usually occurs prior to detectable cellular activation of caspase 3 or nuclear condensation.
Conclusions:
Results suggest that epithelial barrier function is sustained at the apical pole of the epithelial layer, despite discontinuities in the cellular layer.
Novel dual emission, pH-sensitive variants of the green fluorescent protein (GFP) have been constructed and are suitable for ratiometric emission measurements in vivo. This new class of GFPs, termed ...deGPFs, results from substitution of wild-type residue 65 with threonine and residues 148 and/or 203 with cysteine. deGFPs display pK a values ranging from 6.8 to 8.0 and emission that switches from a green form (λmax ∼515 nm) to a blue form (λmax ∼460 nm) with acidifying pH. In this report we analyze in most detail the deGFP1 variant (S65T/H148G/T203C, pK a ∼8.0) and the deGFP4 variant (S65T/C48S/H148C/T203C, pK a ∼7.3). In the following paper McAnaney, T. B., Park, E. S., Hanson, G. T., Remington, S. J., and Boxer, S. G. (2002) Biochemistry 41, 15489−15494, data obtained by ultrafast fluorescence upconversion spectroscopy can be described by a kinetic model that includes an excited-state proton-transfer pathway at high pH but not at low pH. Crystal structure analyses of deGFP1 at high-pH and low-pH conformations were performed to elucidate the basis for the dual emission characteristics. At low pH the structure does not contain a hydrogen bond network that would support rapid transfer of a proton from the excited state of the neutral chromophore to a suitable acceptor; hence blue emission is observed. At high pH, backbone rearrangements induced by changes in the associated hydrogen bond network permit excited-state proton transfer from the excited state of the neutral chromophore to the bulk solvent via Ser147 and bound water molecules, resulting in green emission from the anionic chromophore. Comparative analysis suggests that the basis for dual emission is elimination of the wild-type proton-transfer network by the S65T substitution, a general reduction in hydrogen-bonding opportunities, and a concomitant increase in the hydrophobic nature of the chromophore environment resulting from the cysteine substitutions. We evaluated the suitability of the deGFP4 variant for intracellular pH measurements in mammalian cells by transient expression in PS120 fibroblasts. The responses of deGFP4 and a commercially available pH-sensitive dye, SNARF-1, to changes in pH were compared in the same cells. Results show that the dynamic range of the emission ratio change is comparable between the two pH sensors over the range examined. Two-photon excitation was found to elicit a better deGFP4 fluorescent signal above cellular autofluorescence when compared to conventional confocal microscopy. Given their favorable optical characteristics, suitable pK a's for the physiological pH range, and suitability for ratiometric measurements, dual emission GFPs should make excellent probes for studying pH in vivo.
We report that a localized intracellular and extracellular Ca2+ mobilization occurs at the site of microscopic epithelial damage in vivo and is required to mediate tissue repair. Intravital ...confocal/two-photon microscopy continuously imaged the surgically exposed stomach mucosa of anesthetized mice while photodamage of gastric epithelial surface cells created a microscopic lesion that healed within 15 min. Transgenic mice with an intracellular Ca2+-sensitive protein (yellow cameleon 3.0) report that intracellular Ca2+ selectively increases in restituting gastric epithelial cells adjacent to the damaged cells. Pretreatment with U-73122, indomethacin, 2-aminoethoxydiphenylborane, or verapamil inhibits repair of the damage and also inhibits the intracellular Ca2+ increase. Confocal imaging of Fura-Red dye in luminal superfusate shows a localized extracellular Ca2+ increase at the gastric surface adjacent to the damage that temporally follows intracellular Ca2+ mobilization. Indomethacin and verapamil also inhibit the luminal Ca2+ increase. Intracellular Ca2+ chelation (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid/acetoxymethyl ester, BAPTA/AM) fully inhibits intracellular and luminal Ca2+ increases, whereas luminal calcium chelation (N-(2-hydroxyetheyl)-ethylendiamin-N,N,N′-triacetic acid trisodium, HEDTA) blocks the increase of luminal Ca2+ and unevenly inhibits late-phase intracellular Ca2+ mobilization. Both modes of Ca2+ chelation slow gastric repair. In plasma membrane Ca-ATPase 1+/− mice, but not plasma membrane Ca-ATPase 4−/− mice, there is slowed epithelial repair and a diminished gastric surface Ca2+ increase. We conclude that endogenous Ca2+, mobilized by signaling pathways and transmembrane Ca2+ transport, causes increased Ca2+ levels at the epithelial damage site that are essential to gastric epithelial cell restitution in vivo.
Background: Calcium promotes gastric wound repair, but the in vivo mechanisms are unknown.
Results: Signaling pathways sequentially mobilize intracellular and extracellular calcium as a requirement to repair gastric lesions. PMCA1 mediates extracellular calcium increases.
Conclusion: Endogenous calcium is an essential second and third messenger driving gastric repair.
Significance: Signaling and ion flux pathways are newly identified targets for enhancing gastric repair.
The authors' goal was to measure pH at the gastric surface (pH₀) to understand how acid secretion affects the repair of microscopic injury to the gastric epithelium.
Microscopic gastric damage was ...induced by laser light, during confocal/two-photon imaging of pH-sensitive dyes (Cl-NERF, BCECF) that were superfused over the mucosal surface of the exposed gastric corpus of anaesthetised mice. The progression of repair was measured in parallel with pH₀. Experimental conditions included varying pH of luminal superfusates, and using omeprazole (60 mg/kg ip) or famotidine (30 mg/kg ip) to inhibit acid secretion.
Similar rates of epithelial repair and resting pH₀ values (∼pH 4) were reported in the presence of luminal pH 3 or pH 5. Epithelial repair was unreliable at luminal pH 2 and pH₀ was lower (2.5±0.2, P <0.05 vs pH 3). Epithelial repair was slower at luminal pH 7 and pH₀ was higher (6.4±0.1, P<0.001). In all conditions, pH₀ increased adjacent to damage. At luminal pH 3 or pH 7, omeprazole reduced maximal damage size and accelerated epithelial repair, although only at pH 3 did omeprazole further increase surface pH above the level caused by imposed damage. At luminal pH 7, famotidine also reduced maximal damage size and accelerated epithelial repair. Neither famotidine nor omeprazole raised plasma gastrin levels during the time course of the experiments.
Epithelial repair in vivo is affected by luminal pH variation, but the beneficial effects of acutely blocking acid secretion extend beyond simply raising luminal and/or surface pH.
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