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BackgroundPeptic ulcers recur, suggesting that ulcer healing may leave tissue predisposed to subsequent damage. In mice, we have identified that the regenerated epithelium found after ulcer healing will remain abnormal for months after healing.AimTo determine whether healed gastric mucosa has altered epithelial function, as measured by electrophysiologic parameters.MethodUlcers were induced in mouse gastric corpus by serosal local application of acetic acid. Thirty days or 8 months after ulcer induction, tissue was mounted in an Ussing chamber. Transepithelial electrophysiologic parameters (short-circuit current, Isc. resistance, R) were compared between the regenerated healed ulcer region and the non-ulcerated contralateral region, in response to luminal hyperosmolar NaCl challenge (0.5 M).ResultsIn unperturbed stomach, luminal application of hyperosmolar NaCl transiently dropped Isc followed by gradual recovery over 2 h. Compared to the starting baseline Isc, percent Isc recovery was reduced in 30-day healing mucosa, but not at 8 months. Prior to NaCl challenge, a lower baseline Isc was observed in trefoil factor 2 (TFF2) knockout (KO) versus wild type (WT), with no Isc recovery in either non-ulcerated or healing mucosa of KO. Inhibiting Na/H exchanger (NHE) transport in WT mucosa inhibited Isc recovery in response to luminal challenge. NHE2-KO baseline Isc was reduced versus NHE2-WT. In murine gastric organoids, NHE inhibition slowed recovery of intracellular pH and delayed the repair of photic induced damage.ConclusionHealing gastric mucosa has deficient electrophysiological recovery in response to hypertonic NaCl. TFF2 and NHE2 contribute to Isc regulation, and the recovery and healing of transepithelial function.

Helicobacter pylori is a pathogen that chronically colonizes the stomachs of approximately half of the world's population and contributes to the development of gastric inflammation. We demonstrated previously in vivo that H. pylori uses motility to preferentially colonize injury sites in the mouse stomach. However, the chemoreceptor responsible for sensing gastric injury has not yet been identified. In this study, we utilized murine gastric organoids (gastroids) and mutant H. pylori strains to investigate the components necessary for H. pylori chemotaxis. High-intensity 730-nm light (two-photon photodamage) was used to cause single-cell damage in gastroids, and repair of the damage was monitored over time; complete repair occurred within ∼10 min in uninfected gastroids. Wild-type H. pylori accumulated at the damage site after gastric damage induction. In contrast, mutants lacking motility (ΔmotB) or chemotaxis (ΔcheY) did not accumulate at the injury site. Using mutants lacking individual chemoreceptors, we found that only TlpB was required for H. pylori accumulation, while TlpA, TlpC, and TlpD were dispensable. All strains that were able to accumulate at the damage site limited repair. When urea (an identified chemoattractant sensed by TlpB) was microinjected into the gastroid lumen, it prevented the accumulation of H. pylori at damage sites. Overall, our findings demonstrate that H. pylori colonizes and limits repair at damage sites via chemotactic motility that requires the TlpB chemoreceptor to sense signals generated by gastric epithelial cells.

Key pointsAn in vitro approach to study gastric development is primary mouse-derived epithelium cultured as three-dimensional spheroids known as organoids. We have devised two unique gastric fundic-derived organoid cultures: model 1 for the expansion of gastric fundic stem cells, and model 2 for the maintenance of mature cell lineages. Organoids maintained in co-culture with immortalized stomach mesenchymal cells express robust numbers of surface pit, mucous neck, chief, endocrine and parietal cells. Histamine induced a significant decrease in intraluminal pH that was reversed by omeprazole in fundic organoids and indicated functional activity and regulation of parietal cells. Localized photodamage resulted in rapid cell exfoliation coincident with migration of neighbouring cells to the damaged area, sustaining epithelial continuity. We report the use of these models for studies of epithelial cell biology and cell damage and repair.AbstractStudies of gastric function and disease have been limited by the lack of extended primary cultures of the epithelium. An in vitro approach to study gastric development is primary mouse-derived antral epithelium cultured as three-dimensional spheroids known as organoids. There have been no reports on the use of organoids for gastric function. We have devised two unique gastric fundic-derived organoid cultures: model 1 for the expansion of gastric fundic stem cells, and model 2 for the maintenance of mature cell lineages. Both models were generated from single glands dissociated from whole fundic tissue and grown in basement membrane matrix (Matrigel) and organoid growth medium. Model 1 enriches for a stem cell-like niche via simple passage of the organoids. Maintained in Matrigel and growth medium, proliferating organoids expressed high levels of stem cell markers CD44 and Lgr5. Model 2 is a system of gastric organoids co-cultured with immortalized stomach mesenchymal cells (ISMCs). Organoids maintained in co-culture with ISMCs express robust numbers of surface pit, mucous neck, chief, endocrine and parietal cells. Histamine induced a significant decrease in intraluminal pH that was reversed by omeprazole in fundic organoids and indicated functional activity and regulation of parietal cells. Localized photodamage resulted in rapid cell exfoliation coincident with migration of neighbouring cells to the damaged area, sustaining epithelial continuity. Thus, we report the use of these models for studies of epithelial cell biology and cell damage and repair.

BackgroundHelicobacter pylori (H. pylori) infection leads to acute induction of Sonic Hedgehog (Shh) in the stomach that is associated with the initiation of gastritis. The mechanism by which H. pylori induces Shh is unknown. Shh is a target gene of transcription factor Nuclear Factor-κB (NFκB). We hypothesize that NFκB mediates H. pylori-induced Shh.Materials and methodsTo visualize Shh ligand expression in response to H. pylori infection in vivo, we used a mouse model that expresses Shh fused to green fluorescent protein (Shh::GFP mice) in place of wild-type Shh. In vitro, changes in Shh expression were measured in response to H. pylori infection using 3-dimensional epithelial cell cultures grown from whole dissociated gastric glands (organoids). Organoids were generated from stomachs collected from the fundic region of control and mice expressing a parietal cell-specific deletion of Shh (PC-Shh(KO) mice).ResultsWithin 2 days of infection, H. pylori induced Shh expression within parietal cells of Shh::GFP mice. Organoids expressed all major gastric cell markers, including parietal cell marker H(+) ,K(+) -ATPase and Shh. H. pylori infection of gastric organoids induced Shh expression; a response that was blocked by inhibiting NFκB signaling and correlated with IκB degradation. H. pylori infection of PC-Shh(KO) mouse-derived organoids did not result in the induction of Shh expression.ConclusionGastric organoids allow for the study of the interaction between H. pylori and the differentiated gastric epithelium independent of the host immune response. H. pylori induces Shh expression from the parietal cells, a response mediated via activation of NFκB signaling.

Helicobacter pylori (H. pylori) is a pathogen contributing to peptic inflammation, ulceration, and cancer. A crucial step in the pathogenic sequence is when the bacterium first interacts with gastric tissue, an event that is poorly understood in vivo. We have shown that the luminal space adjacent to gastric epithelial damage is a microenvironment, and we hypothesized that this microenvironment might enhance H. pylori colonization. Inoculation with 106 H. pylori (wild-type Sydney Strain 1, SS1) significantly delayed healing of acetic-acid induced ulcers at Day 1, 7 and 30 post-inoculation, and wild-type SS1 preferentially colonized the ulcerated area compared to uninjured gastric tissue in the same animal at all time points. Gastric resident Lactobacillus spp. did not preferentially colonize ulcerated tissue. To determine whether bacterial motility and chemotaxis are important to ulcer healing and colonization, we analyzed isogenic H. pylori mutants defective in motility (ΔmotB) or chemotaxis (ΔcheY). ΔmotB (10(6)) failed to colonize ulcerated or healthy stomach tissue. ΔcheY (10(6)) colonized both tissues, but without preferential colonization of ulcerated tissue. However, ΔcheY did modestly delay ulcer healing, suggesting that chemotaxis is not required for this process. We used two-photon microscopy to induce microscopic epithelial lesions in vivo, and evaluated accumulation of fluorescently labeled H. pylori at gastric damage sites in the time frame of minutes instead of days. By 5 min after inducing damage, H. pylori SS1 preferentially accumulated at the site of damage and inhibited gastric epithelial restitution. H. pylori ΔcheY modestly accumulated at the gastric surface and inhibited restitution, but did not preferentially accumulate at the injury site. H. pylori ΔmotB neither accumulated at the surface nor inhibited restitution. We conclude that bacterial chemosensing and motility rapidly promote H. pylori colonization of injury sites, and thereby biases the injured tissue towards sustained gastric damage.

Gastric surface pH ([pH.sub.o]) transiently increases in response to focal epithelial damage. The sources of that increase, either from paracellular leakage of interstitial fluid or transcellular acid/base fluxes, have not been determined. Using in vivo microscopy approaches we measured pHo with Cl-NERF, tissue permeability with intravenous fluorescent-dextrans to label interstitial fluid (paracellular leakage), and gastric epithelial intracellular pH ([pH.sub.i]) with SNARF-5F (cellular acid/base fluxes). In response to two-photon photodamage, we found that cell-impermeant dyes entered damaged cells from luminal or tissue compartments, suggesting a possible slow transcellular, but not paracellular, route for increased permeability after damage. Regarding cytosolic acid/base status, we found that damaged cells acidified (6.63 [+ or -] 0.03) after photo-damage, compared with healthy surface cells both near (7.12 [+ or -] 0.06) and far (7.07 [+ or -] 0.04) from damage (P < 0.05). This damaged cell acidification was further attenuated with 20 [micro]M intravenous EIPA (6.34 [+ or -] 0.05, P < 0.05) but unchanged by addition of 0.5 mM luminal [H.sub.2]DIDS (6.64 [+ or -] 0.08, P > 0.05). Raising luminal pH did not realkalinize damaged cells, suggesting that the mechanism of acidification is not attributable to leakiness to luminal protons. Inhibition of apical HC[O.sup.-.sub.3] secretion with 0.5 mM luminal [H.sub.2]DIDS or genetic deletion of the solute-like cartier 26A9 (SLC26A9) [Cl.sup.-]/HC[O.sup.-.sub.3] exchanger blocked the [pH.sub.o] increase normally observed in control animals but did not compromise repair of damaged tissue. Addition of exogenous [PGE.sub.2] significantly increased [pH.sub.o] in wild-type, but not SLC26A9 knockout, animals, suggesting that prostaglandin-stimulated HC[O.sup.-.sub.3] secretion is fully mediated by SLC26A9. We conclude that cellular HC[O.sup.-.sub.3] secretion, likely through SLC26A9, is the dominant mechanism whereby surface pH transiently increases in response to photodamage. photodamage; NAD(P)H; autofluorescence; two-photon microscopy; confocal microscopy; intracellular pH; SNARF-5F; CI-NERF; permeability; solute-like carrier 26A9 doi: 10.1152/ajpgi.00037.2010.

The early events in an intestinal ischemic episode have been difficult to evaluate. Using in vivo microscopy we have analyzed in real-time the effects of short (15 min) and long (40-50 min) ischemia with subsequent reperfusion (IR), evaluating structure, integrity, and functioning of the mouse jejunal mucosa while monitoring blood flow by confocal microscopy. IR was imposed by inflation/deflation of a vascular occluder, and blood flow was monitored and confirmed with scanning confocal imaging. After short ischemia, villus tip cells revealed a rapid increase (23%) in the intracellular NAD(P)H concentration (confocal autofluorescence microscopy), and the pH-sensitive probe BCECF showed a biphasic response of the intracellular pH ([pH.sub.i]), quickly alkalinizing from the resting value of 6.8 [+ or -] 0.1 to 7.1 [+ or -] 0.1 but then strongly acidifying to 6.3 [+ or -] 0.1. Upon reperfusion, values returned toward control. In contrast, results were heterogeneous after long IR. During long ischemia, one-third of the epithelial cells remained viable with reversible changes upon reperfusion, but remaining cells lost membrane integrity (Lucifer Yellow uptake, LY) and had membrane blebs during ischemia. These effects became more pronounced as the reperfusion interval progressed when cells exhibited more severely affected NAD(P)H and [pH.sub.i] values, larger blebs, and more LY uptake and eventually were shed from the villus. Results from stereo microscopy suggest that these irreversible effects of IR may have occurred as a result of incomplete restorations of local blood flow, especially at the antimesenteric side of the intestine. We conclude that the adverse effects of short ischemia on the jejunum epithelium are fully reversible during the reperfusion interval. However, after long ischemia, reperfusion cannot restore normal structure and functioning of a majority of cells, which deteriorate further. Our results provide a basis for defining the cellular events that cause tissue to transit from reversible to irreversible damage during IR. jejunum; villus; microvasculature; pH; NAD(P)H; laser scanning confocal microscopy; stereo microscopy; BCECF; permeability

Cyclooxygenase-1 (Cox-1) contributes to gastric defense of healthy tissue, but the role in the protection of the gastric epithelium after minor, acute damage has been difficult to study in vivo. Using 710-nm two-photon light absorption to create microscopic gastric damage in anesthetized mice with the gastric mucosal surface surgically exposed and perfused on the microscope stage, the acute response of surface cells to injury could be monitored using in vivo microscopy within seconds after injury. Using exogenous (C1-NERF) and endogenous fluorophores, extracellular pH and cell death were monitored in real time during the entire damage and repair cycle. Two-photon damage was initiated by scanning ~200 [micro][m.sup.2] of gastric surface cells with high laser intensity, causing rapid bleaching of NAD(P)H fluorescence in optically targeted cells. In both Cox-[1.sup.+/-] and Cox-[1.sup.-/-] mice, a similar initial damage area expanded to include bystander epithelial cells over the next 2-5 min, with larger maximal damage noted in Cox-[1.sup.-/-] mice. The maximal damage size seen in Cox-[1.sup.-/-] mice could be reduced by exogenous dimethyl-[PGE.sub.2]. All damaged cells exfoliated, and the underlying epithelium was coincidently repaired over a time interval that was briefer in Cox-[1.sup.+/-] (12 [+ or -] 2 min, n = 12) than in Cox-[1.sup.-/-] (24 [+ or -] 4 min, n = 14) mice. Directly after damage, pH increased transiently in the juxtamucosal layer (maximal at 3-6 min). A smaller peak pH change was noted in Cox-[1.sup.-/-] mice ([DELTA]pH = 0.3 [+ or -] 0.04) than in Cox-[1.sup.+/-] mice ([DELTA]pH = 0.6 [+ or -] 0.2). Recovery to normal surface pH took longer in Cox-[1.sup.-/-] mice (27 [+ or -] 5 min) than in Cox-[1.sup.+/-] mice (12 [+ or -] 1 min). In conclusion, constitutive loss of Cox-1 leaves the gastric mucosa more prone to damage and slowed repair of microlesions. autofluorescence; photodamage; laser scanning microscopy; two-photon microscopy; reduced nicotinamide adenine dinucleotide; surface pH; cyclooxygenase

The mechanism of apical [Na.sup.+]-dependent [H.sup.+] extrusion in colonic crypts is controversial. With the use of confocal microscopy of the living mouse distal colon loaded with BCECF or SNARF-5F (fluorescent pH sensors), measurements of intracellular pH ([pH.sub.i]) in epithelial cells at either the crypt base or colonic surface were reported. After cellular acidification, the addition of luminal [Na.sup.+] stimulated similar rates of [pH.sub.i] recovery in cells at the base of distal colonic crypts of wild-type or [Na.sup.+]/[H.sup.+] exchanger isoform 2 (NHE2)-null mice. In wild-type crypts, 20 [micro]M HOE694 (NHE2 inhibitor) blocked 68-75% of the [pH.sub.i] recovery rate, whereas NHE2-null crypts were insensitive to HOE694, the NHE3-specific inhibitor S-1611 (20 [micro]M), or the bicarbonate transport inhibitor 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS; 1 mM). A general NHE inhibitor, 5-(N-ethyl-N-isopropyl)amiloride (EIPA; 20 [micro]M), inhibited [pH.sub.i] recovery in NHE2-null mice (46%) but less strongly than in wild-type mice (74%), suggesting both EIPA-sensitive and -insensitive compensatory mechanisms. Transepithelial [Na.sup.+] leakage followed by activation of basolateral NHE1 could confound the outcomes; however, the rates of [Na.sup.+]-dependent [pH.sub.i] recovery were independent of transepithelial leakiness to lucifer yellow and were unchanged in NHE1-null mice. NHE2 was immunolocalized on apical membranes of wild-type crypts but not NHE2-null tissue. NHE3 immunoreactivity was near the colonic surface but not at the crypt base in NHE2-null mice. Colonic surface cells from wild-type mice demonstrated S1611- and HOE694-sensitive [pH.sub.i] recovery in response to luminal sodium, confirming a functional role for both NHE3 and NHE2 at this site. We conclude that constitutive absence of NHE2 results in a compensatory increase in a [Na.sup.+]-dependent, EIPA-sensitive acid extruder distinct from NHE1, NHE3, or SITS-sensitive transporters. S1c9a2; S1c9a3; BCECF; SNARF-5F; intracellular pH; laser scanning confocal microscopy; colon; [Na.sup.+]/[H.sup.+] exchanger

Kempson, Stephen A., Vaibhave Parikh, Lixuan Xi, Shaoyou Chu, and Marshall H. Montrose. Subcellular redistribution of the renal betaine transporter during hypertonic stress. Am J Physiol Cell Physiol 285: C1091-Cl100, 2003.. First published July 2, 2003; 10.1152/ajpcell.00021. 2003.--The betaine transporter (BGT1) protects cells in the hypertonic renal inner medulla by mediating uptake and accumulation of the osmolyte betaine. Transcriptional regulation plays an essential role in upregulation of BGT1 transport when renal cells are exposed to hypertonic medium for 24 h. Posttranscriptional regulation of the BGT1 protein is largely unexplored. We have investigated the distribution of BGT1 protein in live cells after transfection with BGT1 tagged with enhanced green fluorescent protein (EGFP). Fusion of EGFP to the N[H.sub.2] terminus of BGT1 produced a fusion protein (EGFP-BGT) with transport properties identical to normal BGT1, as determined by ion dependence, inhibitor sensitivity, and apparent [K.sub.m] for GABA. Confocal microscopy of EGFP-BGT fluorescence in transfected Madin-Darby canine kidney (MDCK) cells showed that hypertonic stress for 24 h induced a shift in subcellular distribution from cytoplasm to plasma membrane. This was confirmed by colocalization with anti-BGT1 antibody staining. In fibroblasts, transfected EGFP-BGT caused increased transport in response to hypertonic stress. The activation of transport was not accompanied by increased expression of EGFP-BGT, as determined by Western blotting. Membrane insertion of EGFP-BGT protein in MDCK cells began within 2--3 h after onset of hypertonic stress and was blocked by cycloheximide. We conclude that posttranscriptional regulation of BGT1 is essential for adaptation to hypertonic stress and that insertion of BGT1 protein to the plasma membrane may require accessory proteins. Madin-Darby canine kidney cells; fibroblasts; enhanced green fluorescent protein; fluorescence; transfection; laser scanning confocal microscopy

Epithelial cells are gatekeepers that sit at the interface between two compartments. By controlling the flow of molecules and information between two compartments, epithelial cells provide unique benefit to the body. This article provides a brief appraisal of our current knowledge about the functions of gastrointestinal epithelial cells as a functionally diverse set of cells mediating transepithelial transport and as a continually renewing layer of cells. The convergence of new methodologies in laser capture microdissection, microarray analyses, microscopic analyses, and generation of mutant animals provides an exciting template for future research. microscopy; genomics; proteomics; stern cell; ion transport; gastrointestinal

Research has been conducted on green fluorescent protein. The study of pH-sensitive variants of this protein, constructed fo ratiomeric emission measurements in vivo, is presented.

Gastric secretion of hydrochloric acid requires protons and chloride, yet the mechanisms and regulation of gastric chloride secretion remain unclear. We developed an in vivo technique to simultaneously measure acid/base and chloride secretion into the gastric lumen of anesthetized rats. The cannulated stomach lumen was perfused with weakly pH-buffered chloride-free solution containing a chloride-sensitive fluorophore [5 [micro]M N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE)]. Gastric acid and chloride secretion was detected in gastric effluents by 1) flow-through pH electrode and 2) MQAE fluorescence. Gastric effluent was also collected at 1-min intervals for independent determination of chloride amount by chloridometer. In all conditions, both optical and chemical determinations of chloride report similar amounts of secreted chloride. During luminal perfusion with pH 5 solution, net acid and chloride secretion into the lumen was observed. Pentagastrin stimulated both secretions. In contrast, proton pump inhibition (omeprazole) caused alkalinization of the gastric effluent, but chloride secretion was not diminished. During luminal pH 3 perfusion, net alkali secretion was observed, and chloride secretion at luminal pH 3 was greater than pH 5. When tissue is pretreated with omeprazole at luminal pH 3, the addition of prostaglandin E2 synchronously stimulates both alkali and chloride secretion. Results suggest that both acid and alkali secretions are separately coupled with chloride secretion. proton pump, fluorescence, rat, stomach gastric luminal pH

Chu, Jingsong, Shaoyou Chu, and Marshall H. Montrose. Apical [Na.sup.+]/[H.sup.+] exchange near the base of mouse colonic crypts. Am J Physiol Cell Physiol 283: C358-C372, 2002; 10.1152/ajpcell.01380.2000.--Colonic crypts can absorb fluid, but the identity of the absorptive transporters remains speculative. Near the crypt base, the epithelial cells responsible for vectorial transport are relatively undifferentiated and often presumed to mediate only [Cl.sup.-] secretion. We have applied confocal microscopy in combination with an extracellular fluid marker [Lucifer yellow (LY)] or a pH-sensitive dye (2',7'-bis(2-carboxyethyl)-5(6)-carboxyfiuorescein) to study mouse colonic crypt epithelial cells directly adjacent to the crypt base within an intact mucosal sheet. Measurements of intracellular pH report activation of colonocyte [Na.sup.+]/[H.sup.+] exchange in response to luminal or serosal [Na.sup.+]. Studies with LY demonstrate the presence of a paracellular fluid flux, but luminal Na+ does not activate [Na.sup.+]/[H.sup.+] exchange in the nonepithelial cells of the lamina propria, and studies with LY suggest that the fluid bathing colonocyte basolateral membranes is rapidly refreshed by serosal perfusates. The apical [Na.sub.+]/[H.sub.+] exchange in crypt colonocytes is inhibited equivalently by luminal 20 [micro]M ethylisopropylamiloride and 20 [micro]M HOE-694 but is not inhibited by luminal 20 [micro]M S-1611. Immunostaining reveals the presence of epitopes from NHE1 and NHE2, but not NHE3, in epithelial cells near the base of colonic crypts. Comparison of apical Na+/H+ exchange activity in the presence of [Cl.sup.-] with that in the absence of [Cl.sup.-] (substitution by gluconate or nitrate) revealed no evidence of the [Cl.sup.-]-dependent [Na.sup.+]/[H.sup.+] exchange that had been previously reported as the sole apical [Na.sup.+]/[H.sup.+] exchange activity in the colonic crypt. Results suggest the presence of an apical [Na.sup.+]/[H.sup.+] exchanger near the base of crypts with functional attributes similar to those of the cloned NHE2 isoform. intracellular pH; NHE2; NHE3; NHE1; sodium absorption; epithelial polarity; laser scanning confocal microscopy; 2',7'bis(2-carboxyethyl)-5(6)-carboxyfluorescein; Lucifer yellow;immunoflorescence

The four Na+/H+ exchanger (NHE) isoforms have been shown to participate in the process of Na+ uptake by cells exposed to hypoosmotic media. Herein, PS120 cells transfected with cloned NHE1, NHE2 and NHE3 cDNAs were assessed for their responses when exposed to hyperosmolar solution. NHE activity of NHE2 and NHE3 was inhibited by hyperosmolarity. There was also a marked reduction in maximal reaction velocity, but no differences were observed in the Michaelis-Menten constant between the isoforms. Truncated forms of NHE3 cDNAs and NHE2 cDNA also inhibited NHE activity.

A study was conducted to examine how the three-dimensional structure of the colonic epithelium affects its complex absorptive and secretory functions. Confocal microscopy was used to measure the extracellular pH along the colonic crypt-to-axis of living perfused mouse colonic epithelium. The results show that exposure to short-chain fatty acids caused differential regulation of extracellular pH in the crypt lumen and in the lamina propria around the crypts.

Digital fluorescence imaging and dual excitation ratioing measurement from isolated murine colonocytes suggest the presence of a previously unreported short-chain fatty acid transporter (SCFA) which mediates sodium ion independent intracellular pH recovery. The SCFA transporter shows substrate specificity appropriate for transporting physiological SCFAs. SCFA transport mechanism can regulate pHi in more than 90% of isolated mouse colonocytes.