Factors influencing the restitution of the duodenal and colonic mucosa after damage

Rapid epithelial restitution is an important protective mechanism which enables the gastrointestinal mucosa to reestablish epithelial integrity following superficial injury within hours. In this study we examined the influence of an acidic luminal pH, removal of the necrotic layer, nutrient bicarbonate, calcium and sodium desoxycholate (Na-DOC) on restitution in the rabbit duodenum in vitro and the role of Na-DOC and calcium for rapid restitution of the human colon in vitro. Transmucosal potential difference (PD), short-circuit current (lsc) were measured and resistance against passive ion flux (R) was calculated. Electrophysiological changes paralleled morphological injury but did not necessarily reflect restitution in all experiments. The extent of mucosal injury was assessed by computerized real-time morphometry. 5 hrs after luminal exposure to 10 mH HCl for 10 min residual damage (RD) was 14% in the duodenum. Luminal pH of 3.0 (RD of 30%), removal of necrotic layer at acidic luminal pH (RD of 66%), absence of bicarbonate from the serosal solution (RD of 35% at neutral luminal pH; RD of 96% at acidic luminal pH) and removal of calcium from the serosal solution (RD of 58%) impaired restitution in the duodenum. Continuous postinjury luminal Na-DOC exposure did not influence restitution in the duodenum (RD of 19%). 5 hrs after luminal exposure to 0.5 mM Na-DOC for 10 min RD was 26% in the human colon. Continuous postinjury luminal Na-DOC exposure (RD of 51%) and removal of calcium from the nutrient solution (RD of 65%) impaired restitution in the human colon. Thus we conclude that restitution of the rabbit duodenum in vitro requires a necrotic layer and bicarbonate flux to withstand acidic luminal pH, while restitution is not affected by Na-DOC. In the human colon Na-DOC inhibits restitution. Both the duodenum and colon require calcium for rapid restitution.     

Ammonia-induced apoptosis is accelerated at higher pH in gastric surface mucous cells

Gastric luminal ammonia produced by Helicobacter pylori has been shown to cause gastric mucosal injury. This study was conducted to examine the mechanisms by which gastric luminal ammonia causes apoptosis of gastric epithelial cells. Monolayers of GSM06 cells, developed from murine gastric surface mucous cells, were cultured in the absence or presence of 10-30 mM NH(4)Cl at ambient pH of 5.0, 6.0, and 7.0. In the presence of luminal NH(4)Cl, GSM06 cells showed 1) cell shrinkage and nuclear chromatin condensation, 2) DNA fragmentation into oligonucleosomes, 3) leakage of cytochrome c into cytosolic fraction without affecting bax expression, and 4) increases in activity of caspases-3 and -9. These changes were accentuated when the cells were cultured at pH 7.0. In the absence of NH(4)Cl, none of these changes was detected at any pH examined. These results suggest that gastric luminal ammonia, at concentrations detected in H. pylori-infected subjects, induces apoptosis of gastric epithelial cells by release of cytochrome c from mitochondria, followed by activation of caspases-9 and -3, especially at higher ambient pH.     

EGF promotes gastric mucosal restitution by activating Na(+)/H(+) exchange of epithelial cells

This study was conducted to determine whether the contributions of epidermal growth factor (EGF) to gastric mucosal restitution after injury are mediated by stimulation of Na(+)/H(+) exchangers in surface mucous cells (SMC). Intact sheets of guinea pig gastric mucosae were incubated in vitro. Intracellular pH (pH(i)) in SMC was measured fluorometrically, using 2',7'- bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein. Restitution after Triton X-100-induced injury was evaluated by recovery of electrical resistance. At neutral luminal pH, exogenous EGF (ex-EGF) increased pH(i) and enhanced restitution in the absence but not in the presence of serosal HCO. During exposure to luminal acid, ex-EGF not only prevented intracellular acidosis but also promoted restitution. These effects of ex-EGF were blocked by serosal amiloride or anti-EGF-receptor antibody. In the absence of ex-EGF, restitution was inhibited by replacement of luminal and serosal solutions with fresh solutions and was blocked more completely by serosal anti-EGF-receptor antibody. These results suggest that both endogenous and ex-EGF contribute to restitution via basolateral EGF receptors, with effects mediated, at least in part, by stimulation of basolateral Na(+)/H(+) exchangers.     

Permeability of the urinary bladder of the turtle, Pseudemys scripta elegans, to ammonia

1. The permeability of the isolated turtle urinary bladder to ammonia was investigated by varying the ratio of NH3 to NH4+ concentration in the serosal bath and measuring the flux of ammonia into the mucosal bath. 2. The permeability of the turtle bladder to NH3 was in the range of 5.4-6.1 x 10(-3) cm/sec and the permeability of the unstirred layer to NH4+ was in the range of 1.1-2.1 x 10(-5) cm/sec. 3. The flux of ammonia calculated from the permeability of NH3 can account for the decrease in mucosal H+ secretion observed with addition of NH4Cl to the serosal bath.     

Effect of E. coli heat-stable enterotoxin on colonic transport in guanylyl cyclase C receptor-deficient mice

We studied the functional importance of the colonic guanylyl cyclase C (GCC) receptor in GCC receptor-deficient mice. Mice were anesthetized with pentobarbital sodium, and colon segments were studied in Ussing chambers in HCO3- Ringer under short-circuit conditions. Receptor-deficient mouse proximal colon exhibited similar net Na+ absorption, lower net Cl- absorption, and a negative residual ion flux (J(R)), indicating net HCO3- absorption compared with that in normal mice. In normal mouse proximal colon, mucosal addition of 50 nM Escherichia coli heat-stable enterotoxin (STa) increased the serosal-to-mucosal flux of Cl- (J(s-->m)(Cl)) and decreased net Cl- flux (J(net)(Cl)) accompanied by increases in short-circuit current (I(sc)), potential difference (PD), and tissue conductance (G). Serosal STa had no effect. In distal colon neither mucosal nor serosal STa affected ion transport. In receptor-deficient mice, neither mucosal nor serosal 500 nM STa affected electrolyte transport in proximal or distal colon. In these mice, 1 mM 8-bromo-cGMP produced changes in proximal colon J(s-->m)(Cl) and J(net)(Cl), I(sc), PD, G, and J(R) similar to mucosal STa addition in normal mice. We conclude that the GCC receptor is necessary in the mouse proximal colon for a secretory response to mucosal STa.     

Differential effects of apical and basolateral uridine triphosphate on intestinal epithelial chloride secretion

Our goal was to examine the sidedness of effects of the purinergic agonist, uridine 5'-triphosphate (UTP), on Cl(-) secretion in intestinal epithelial cells. We hypothesized that UTP might exert both stimulatory and inhibitory effects. All studies were conducted with T84 intestinal epithelial cells. UTP induced Cl(-) secretion in a concentration-dependent fashion. Responses to serosally added UTP were smaller and more transient than those evoked by mucosal addition, but there was no evidence that mucosal responses involved cAMP-dependent mechanisms. Pretreatment with serosal UTP inhibited subsequent Ca(2+)-dependent Cl(-) secretion induced by carbachol or thapsigargin, or secretion induced by mucosal UTP, in a manner that was reversed by a tyrosine kinase inhibitor. The inhibitory effect of serosal UTP on Cl(-) secretion was not additive with that of carbachol, known to exert its inhibitory effects through the tyrosine kinase-dependent generation of inositol 3,4,5,6-tetrakisphosphate [Ins(3,4,5,6)P(4)]. Moreover, responses to both serosal and mucosal UTP were reduced by prior treatment of T84 cells with carbachol. Finally, serosal, but not mucosal, UTP evoked an increase in Ins(3,4,5,6)P(4). We conclude that different signaling mechanisms lie downstream of apical and basolateral UTP receptors in epithelial cells, at least in the intestine. These differences may be relevant to the use of UTP as a therapy in cystic fibrosis.     

Over-expression of 70-kDa heat shock protein confers protection against monochloramine-induced gastric mucosal cell injury

The major heat shock protein, HSP70, is known to be involved in cytoprotection against environmental stresses mediated by their function as a "molecular chaperone". Monochloramine (NH(2)Cl) is a potent cytotoxic oxidant generated by neutrophil-derived hypochlorous acid and Helicobacter pylori urease-induced ammonia. In this study, to evaluate the cytoprotective effect of HSP70 against NH(2)Cl-induced gastric mucosal cell injury, rat gastric mucosal cells (RGM-1) were stably transfected with pBK-CMV containing the human HSP70 gene (7018-RGM-1) or pBK-CMV alone (pBK-CMV-12) as control cells. These cells were treated with various concentrations of NH(2)Cl. Cell Viability was determined by MTT assay and the direct plasma membrane damage was analyzed by lactate dehydrogenase (LDH) release assay. Apoptosis was determined by DNA fragmentation analysis. NH(2)Cl caused injury to pBK-CMV-12 cells in a concentration-dependent manner. NH(2)Cl-induced gastric cell injury was significantly diminished in HSP70 over-expressing cell line (7018-RGM-1) both necrosis and apoptosis compared to the control cell line (pBK-CMV-12) transfected with CMV vector alone. These result suggest that overexpression of HSP70 plays an important role in protecting gastric cells against NH(2)Cl-induced injury.     

Ion transport across the isolated intestinal mucosa of the winter flounder,Pseudopleuronectes americanus

The isolated intestinal mucosa of the flounder, Pseudopleuronectes americanus, when bathed in a 20 mM HCO3-Ringer's solution bubbled with 1% CO2 in O2, generated a serosa-negative PD and, when short-circuited, absorbed Cl at almost 3 times the rate of Na. Reducing HCO3 to 5 mM decreased the net Cl flux by more than 60%. The following results suggest that, despite the PD, Na and Cl transport processes are nonelectrically coupled: replacing all Na with choline abolished both the PD and net Cl flux; replacing all Cl with SO4 and mannitol abolished the PD and the net Na flux; and adding ouabain (to 0.5 mM) abolished the PD and the net Cl flux. Nearly all of the unidirectional serosa-to-mucosa Cl flux (JClsm) seemed to be paracellular since it varied with PD and Cl concentration in a manner consistent with simple diffusion. JClsm was only about one-fourth of JNasm, suggesting that the paracellular pathway is highly cation-selective. The data can be explained by the following model: (i) Na and Cl uptake across the brush border are coupled 1 : 1; Na is pumped into the lateral space and Cl follows passively, elevating the salt concentration there; (ii) the tight junction is permeable to Na but relatively impermeable to Cl; and (iii) resistance to Na diffusion is greater in the lateral space (considered in its entirety) than in the tight junction. If these assumptions are correct, the serosa-negative transmural PD is due mainly to a salt diffusion potential across the tight junction and, under short-circuit condition, most of the Na pumped into the lateral space diffuses back into the luminal solution, whereas most of the Cl enters the serosal solution. Morphological features of the epithelium support this interpretation: the cells are unusually long (60 micrometer); there is little distension of the apical 12 micrometer of the lateral space during active fluid absorption; and distension distal to this region is intermittently constricted by desmosomes.     

The neurosecretory effect of ouabain on isolated rabbit ileal mucosa

Ouabain, when added to fluid bathing rabbit ileal mucosa mounted in a flux chamber, transiently increases short circuit current, implying a paradoxical secretory response. To determine the cause of this change, we studied unidirectional fluxes of 36Cl and 23Na and the effects of ion substitution, of reduced Ca concentration, verapamil, tetrodotoxin and atropine. Ouabain 0.1 mM, transiently increased the serosal to mucosal flux of Cl and Na, increased Isc and PD and reduced ion conductance. The Isc response to ouabain was diminished by reducing the bath fluid concentration of Cl, of Ca, and by adding verapamil. Tetrodotoxin both delayed and reduced the maximal Isc response; atropine had no effect. We conclude that ouabain acts by releasing a neurotransmitter of unknown identity and by increasing the serosal to mucosal flux of Cl.     

Effect of monochloramine on recovery of gastric mucosal integrity and blood flow response in rat stomachs--relations to capsaicin-sensitive sensory neurons.

Gastric mucosal blood flow (GMBF) response and the recovery of gastric mucosal integrity were investigated in anesthetized rat stomachs after damage by monochloramine (NH2Cl), in comparison with 20 mM taurocholate Na (TC). A rat stomach was mounted in an ex-vivo chamber, and the mucosa was exposed to 50 mM HCl during a test period. Mucosal application of 20 mM TC for 10 min caused a marked reduction of transmucosal potential difference (PD), but the PD recovered rapidly without development of gross lesions 90 min later. In contrast, the exposure of the mucosa to NH2Cl (5 to approximately 20 mM) produced a concentration-dependent decrease in gastric PD, and the values remained lowered even 90 min after removal of the agent, resulting in severe hemorrhagic damage in the stomach. TC caused a considerable H+ back-diffusion, followed by an increase in the GMBF. In the mucosa damaged by NH2Cl, such GMBF responses were not observed, except for the temporal increase during the exposure, although similar degrees of H+ back-diffusion were observed following NH2Cl treatment. In addition, the prior exposure of the mucosa to NH2Cl significantly attenuated gastric hyperemic response induced by capsaicin but not by misoprostol (a PGE1 derivative) or NOR-3 (a NO donor). Chemical ablation of capsaicin-sensitive sensory neurons had no effect on the PD reduction caused by TC but totally attenuated the GMBF response, resulting in hemorrhagic damage in the stomach. These results suggest that NH2Cl delayed the recovery of the mucosal integrity in the stomach after damage, and this effect may be attributable, at least partly, to the impairment of gastric hyperemic response associated with H+ back-diffusion, probably due to dysfunction of capsaicin-sensitive sensory neurons.     

Adaptative increase of ornithine production and decrease of ammonia metabolism in rat colonocytes after hyperproteic diet ingestion

Chronic high-protein consumption leads to increased concentrations of NH(4)(+)/NH(3) in the colon lumen. We asked whether this increase has consequences on colonic epithelial cell metabolism. Rats were fed isocaloric diets containing 20 (P20) or 58% (P58) casein as the protein source for 7 days. NH(4)(+)/NH(3) concentration in the colonic lumen and in the colonic vein blood as well as ammonia metabolism by isolated surface colonic epithelial cells was determined. After 2 days of consumption of the P58 diet, marked increases of luminal and colonic vein blood NH(4)(+)/NH(3) concentrations were recorded when compared with the values obtained in the P20 group. Colonocytes recovered from the P58 group were characterized at that time and thereafter by an increased capacity for l-ornithine and urea production through arginase (P < 0.05). l-Ornithine was mostly used in the presence of NH(4)Cl for the synthesis of the metabolic end product l-citrulline. After 7 days of the P58 diet consumption, however, the ammonia metabolism into l-citrulline was found lower (P < 0.01) when compared with the values measured in the colonocytes recovered from the P20 group despite any decrease in the related enzymatic activities (i.e., carbamoyl-phosphate synthetase I and ornithine carbamoyl transferase). This decrease was found to coincide with a return of blood NH(4)(+)/NH(3) concentration in colonic portal blood to values close to the one recorded in the P20 group. In response to increased NH(4)(+)/NH(3) concentration in the colon, the increased capacity of the colonocytes to synthesize l-ornithine is likely to correspond to an elevated l-ornithine requirement for the elimination of excessive blood ammonia in the liver urea cycle. Moreover, in the presence of NH(4)Cl, colonocytes diminished their synthesis capacity of l-citrulline from l-ornithine, allowing a lower cellular utilization of this latter amino acid. These results are discussed in relationship with an adaptative process that would be related to both interorgan metabolism and to the role of the colonic epithelium as a first line of defense toward luminal NH(4)(+)/NH(3) concentrations.     

Role of glutamine and arginase in protection against ammonia-induced cell death in gastric epithelial cells

Ammonia is a cytotoxic factor produced during Helicobacter pylori infection that may reduce the survival of surface epithelial cells. Here we examine whether ammonia kills cells and whether L-glutamine (L-Gln) protects against cell death by stimulating ammonia detoxification pathways. Cell viability and vacuolation were quantified in rat gastric epithelial (RGM1) cells incubated with ammonium chloride at pH 7.4 in the presence or absence of L-Gln. Incubation of RGM1 cells with ammonium chloride caused a dose-dependent increase in cell death and vacuolation, which were both inhibited by L-Gln. We show that RGM1 cells metabolize ammonia to urea via arginase, a process that is stimulated by L-Gln and results in reduced ammonia cytotoxicity. L-Gln also inhibits the uptake and facilitates the extrusion of ammonia from cells. Blockade of glutamine synthetase did not reduce the survival of RGM1 cells, demonstrating that the conversion of L-glutamate and ammonia to L-Gln is not involved in ammonia detoxification. Thus our data support a role for L-Gln and arginase in protection against ammonia-induced cell death in gastric epithelial cells.     

Physiological and morphological effects of alendronate on rabbit esophageal epithelium

Alendronate, an aminobisphosphonate, produces as a side effect a topical (pill induced) esophagitis. To gain insight into this phenomenon, we assessed the effects of luminal alendronate on both esophageal epithelial structure and function. Sections of rabbit esophageal epithelium were exposed to luminal alendronate at neutral or acidic pH while mounted in Ussing chambers to monitor transmural electrical potential difference (PD), short-circuit current (I(sc)), and resistance (R). Morphological changes were sought by light microscopy in hematoxylin and eosin-stained sections. Impedance analysis was used for localization of alendronate-induced effects on ion transport. Luminal, but not serosal, alendronate (pH 6.9-7.2), increased PD and I(sc) in a dose- and time-dependent manner, with little change in R and mild edema of surface cell layers. The changes in I(sc) (and PD) were reversible with drug washout and could be prevented either by inhibition of Na,K-ATPase activity with serosal ouabain or by inhibition of apical Na channels with luminal acidification to pH 2.0 with HCl. An effect on apical Na channel activity was also supported by impedance analysis. Luminal alendronate at acidic pH was more damaging than either alendronate at neutral pH or acidic pH alone. These data suggest that alendronate stimulates net ion (Na) transport in esophageal epithelium by increasing apical membrane sodium channel activity and that this occurs with limited morphological change and no alteration in barrier function. Also alendronate is far more damaging at acidic than at neutral pH, suggesting its association with esophagitis requires gastric acid for expression. This expression may occur either by potentiation between the damaging effects of (refluxed) gastric acid and drug or by acid-induced conversion of the drug to a more toxic form.     

Peptide fragments of AMP-18, a novel secreted gastric antrum mucosal protein,are mitogenic and motogenic

Antrum mucosal protein (AMP)-18 is a novel 18-kDa protein synthesized by cells of the gastric antrum mucosa. The protein is present in secretion granules of murine gastric antrum epithelial cells and is a component of canine antrum mucus, suggesting that it is secreted into the viscoelastic gel layer on the mucosal surface. Release of the protein appears to be regulated because forskolin decreased the amount of immunoreactive AMP-18 in primary cultures of canine antrum mucosal epithelial cells, and indomethacin gavaged into the stomach of mice reduced AMP-18 content in antrum mucosal tissue before inducing histological injury. A functional domain of the protein was identified by preparing peptides derived from the center of human AMP-18. A 21-mer peptide stimulated growth of gastric and intestinal epithelial cells, but not fibroblasts, and increased restitution of scrape-wounded gastric epithelial monolayers. These functions of AMP-18 suggest that its release onto the apical cell surface is regulated and that the protein and/or peptide fragments may protect the antral mucosa and promote healing by facilitating restitution and proliferation after injury.     

NH(4)Cl inhibition of acid secretion: possible involvement of an apical K(+) channel in bullfrog oxyntic cells

This study was undertaken to determine the mechanism by which ammonium chloride (NH(4)Cl) inhibits stimulated acid secretion in the bullfrog gastric mucosa. To this end, four possible pathways of inhibition were studied: 1) blockade of basolateral K(+) channel, 2) blockade of ion transport activity, 3) neutralization of secreted H(+) in the luminal solution, or 4) ATP depletion. Addition of nutrient 10 mM NH(4)Cl (calculated NH(3) concentration = 92.5 microM and NH(4)(+) concentration = 9.91 mM) inhibited acid secretion within 30 min. Inhibition of acid secretion did not occur by blockade of basolateral K(+) channel activity or ion transport activity or by neutralization of the luminal solution. Although ATP depletion occurred in the presence of NH(4)Cl, the magnitude of ATP depletion in 30 min was not sufficient to inhibit stimulated acid secretion. By comparing the effect of NH(4)Cl on the resistance of inhibited or stimulated tissues, we demonstrate that NH(4)Cl acts specifically on stimulated tissues. We propose that NH(4)Cl blocks activity of an apical K(+) channel present in stimulated oxyntic cells. Our data suggest that the activity of this channel is important for the regulation of acid secretion in bullfrog oxyntic cells.     

Purinergic stimulation induces Ca2+-dependent activation of Na+-K+-2Cl- cotransporter in human nasal epithelia

Increasing evidence suggests that P2 receptors (P2Rs) in airway epithelial cells perform critical functions in auto- or paracrine regulation of fluid and mucus secretion. In the present study, we characterized the effects of P2R stimulation on Na(+)-K(+)-2Cl(-) cotransporter (NKCC) activity in normal human nasal epithelial (NHNE) cells. [Ca(2+)](i) and pH(i) were measured in primary cultures of NHNE cells using a double perfusion chamber, which enabled us to analyze membrane-specific transporter activities. NKCC activities were estimated by the pH(i) reduction due to Na(+)-dependent and bumetanide-sensitive intracellular uptake of NH(4)(+). NKCC activities were observed in the basolateral membrane, but not in the luminal membrane, of NHNE cells. Interestingly, P2Rs were expressed in both membranes, and the stimulation of either luminal or basolateral P2R increased NKCC activity. Blockades of luminal Cl(-) channels, basolateral K(+) channels, or protein kinase C did not affect the activation of NKCC by basolateral P2R stimulation. The effects of luminal P2R stimulation were partially reduced by Cl(-) channel blockers. However, chelation of intracellular Ca(2+) by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) treatment completely blocked the stimulatory effects of luminal and basolateral P2Rs on NKCC. In addition, increasing [Ca(2+)](i) by treatment with ionomycin-stimulated NKCC activity. These results provide evidence that stimulation of P2Rs directly activates basolateral NKCC by Ca(2+)-dependent pathways in NHNE cells, which is an important aspect of the purinergic regulation of ion and fluid secretions in human airway epithelia under physiologic and pathologic conditions.     

Adrenomedullin promotes epithelial restitution of rat and human gastric mucosa in vitro

We have investigated the effect of adrenomedullin (AM) on restitution of mucosal integrity following damage in rat and human gastric mucosa, measuring the potential difference (PD) on a mucosal strip mounted on an Ussing chamber. Mucosal damage was induced by 0.5, 1.0, and 2.0 M NaCl solution, and it caused an immediate and significant decrease in PD. In the rat AM group, PD recovered significantly more than in control group at 120 min after exposure to 0.5 M (p < 0.01) and 1.0 M (p < 0.05) NaCl solution. In the human AM group, PD completely recovered at 120 min after exposure to 0.5 M (p < 0.05) NaCl solution. In rat mucosa damaged by 0.5 M NaCl solution, the effect was inhibited by human (h)-CGRP(8-37) and there was no significant difference between the h-CGRP(8-37) group and control group. On immunohistochemical examination of rat gastric mucosa, AM was detected within the chief cell. AM probably promotes epithelial restitution primarily through the CGRP receptor, but it does not ameliorate more severe damage of gastric mucosa in vitro.     

Responses of frog skin Na(+) and Cl(-) transport to guanylin

A possible role for the peptide hormone guanylin was investigated in frog skin (Rana pipiens) epithelium. Sodium and chloride fluxes in response to this peptide were evaluated in Ussing-type chambers. Net and unidirectional Na(+) fluxes were measured by using (22)Na(+) and atomic absorption analysis of total [Na(+)], whereas net Cl(-) fluxes were measured by using electrometric titration for [Cl(-)]. Mucosal application of guanylin (0.5-2.0 micromol/l) caused marked increases in serosal to mucosal net flux and efflux of Na(+). Serosal application of guanylin over the same dose range caused similar large increases in net serosal to mucosal (S-->M) Na(+) and Cl(-) flux as well as Na(+) efflux. Responses of Na(+) influx were small and inconsistent. When frog skin was bathed on the serosal side with Cl(-)-free Ringer's solution mucosal application of guanylin stimulated large efflux and S-->M net fluxes of Na(+). Serosal treatment yielded large Na(+) effluxes and S-->M Na(+) and Cl(-) net fluxes. When frog skin serosal surfaces were bathed with Na(+)- free Ringer's solution mucosal guanylin treatment had no effect but serosal treatment produced large S-->M Cl(-) net fluxes.     

Ion transport across cat and ferret tracheal epithelia

Sheets of trachea from ferret and cat were mounted in Ussing chambers and continuously short circuited. Under resting conditions, in both the cat and ferret there was little or no Cl secretion, and Na absorption accounted for most of the short-circuit current (Isc). Ouabain (10(-4) M, serosal bath) reduced Isc to zero in 30-60 min. This decline was matched by a decrease in net Na absorption. Amiloride (10(-4) M, luminal bath) caused a significant decrease in Isc and conductance (G) in both species. Bumetanide (10(-4) M, serosal bath) had negligible effects on Isc and G. In both species, isoproterenol increased Isc by stimulating Cl secretion. Methacholine induced equal amounts of Na and Cl secretion, with little change in Isc. In the cat, prostaglandins E2 and F2 alpha and bradykinin increased Isc, responses which were abolished in Cl-free medium. In open-circuited cat tissues, Na flux from the serosal to mucosal side was measured simultaneously with the secretion of nondialyzable 35S. Prostaglandins E1, E2, and F2 alpha, histamine, bradykinin, methacholine and isoproterenol all increased both Na and 35S-mucin secretion.     

NSAIDs counteract H. pylori VacA toxin-induced cell vacuolation in MKN 28 gastric mucosal cells

The relationship between nonsteroidal anti-inflammatory drugs (NSAIDs) and Helicobacter pylori-induced gastric mucosal injury is still under debate. VacA toxin is an important H. pylori virulence factor that causes cytoplasmic vacuolation in cultured cells. Whether and how NSAIDs affect VacA-induced cytotoxicity is unclear. This study was designed to evaluate the effect of NSAIDs on H. pylori VacA toxin-induced cell vacuolation in human gastric mucosal cells in culture (MKN 28 cell line). Our data show that 1) NSAIDs (indomethacin, aspirin, and NS-398) inhibit VacA-induced cell vacuolation independently of inhibition of cell proliferation and prostaglandin synthesis; 2) NSAIDs impair vacuole development/maintenance without affecting cell binding and internalization of VacA; and 3) NSAIDs, as well as the chloride channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid, also inhibit cell vacuolation induced by ammonia. We thus hypothesize that NSAIDs might protect MKN 28 cells against VacA-induced cytotoxicity by inhibiting VacA channel activity required for vacuole genesis.