scholarly journals Enterohemorrhagic E. coli (EHEC)—Secreted Serine Protease EspP Stimulates Electrogenic Ion Transport in Human Colonoid Monolayers

Toxins ◽  
2018 ◽  
Vol 10 (9) ◽  
pp. 351 ◽  
Author(s):  
C. Tse ◽  
Julie In ◽  
Jianyi Yin ◽  
Mark Donowitz ◽  
Michele Doucet ◽  
...  
Keyword(s):  
Ion Transport ◽  
Serine Protease ◽  
Shiga Toxin ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Adult Stem Cell ◽  
Short Circuit Current ◽  
Watery Diarrhea ◽  
E Coli ◽  
Electrogenic Ion Transport

One of the characteristic manifestations of Shiga-toxin-producing Escherichia coli (E. coli) infection in humans, including EHEC and Enteroaggregative E. coli O104:H4, is watery diarrhea. However, neither Shiga toxin nor numerous components of the type-3 secretion system have been found to independently elicit fluid secretion. We used the adult stem-cell-derived human colonoid monolayers (HCM) to test whether EHEC-secreted extracellular serine protease P (EspP), a member of the serine protease family broadly expressed by diarrheagenic E. coli can act as an enterotoxin. We applied the Ussing chamber/voltage clamp technique to determine whether EspP stimulates electrogenic ion transport indicated by a change in short-circuit current (Isc). EspP stimulates Isc in HCM. The EspP-stimulated Isc does not require protease activity, is not cystic fibrosis transmembrane conductance regulator (CFTR)-mediated, but is partially Ca2+-dependent. EspP neutralization with a specific antibody reduces its potency in stimulating Isc. Serine Protease A, secreted by Enteroaggregative E. coli, also stimulates Isc in HCM, but this current is CFTR-dependent. In conclusion, EspP stimulates colonic CFTR-independent active ion transport and may be involved in the pathophysiology of EHEC diarrhea. Serine protease toxins from E. coli pathogens appear to serve as enterotoxins, potentially significantly contributing to watery diarrhea.

scholarly journals Cytoskeletal Effects Induced by Pet, the Serine Protease Enterotoxin of Enteroaggregative Escherichia coli

1999 ◽  
Vol 67 (5) ◽  
pp. 2184-2192 ◽  
Author(s):  
Fernando Navarro-García ◽  
Cynthia Sears ◽  
Carlos Eslava ◽  
Alejandro Cravioto ◽  
James P. Nataro
Keyword(s):  
Escherichia Coli ◽  
Serine Protease ◽  
Protease Activity ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
T84 Cells ◽  
Ussing Chambers ◽  
E Coli ◽  
Cytopathic Effects

ABSTRACT We have previously described enteroaggregative Escherichia coli (EAEC) strains that induce cytotoxic effects on T84 cells, ligated rat ileal loops, and human intestine in culture. Such strains secrete a 104-kDa protein termed Pet (for plasmid-encoded toxin). We have also shown previously that the Pet toxin induces rises in short-circuit current and decreases the electrical resistance in rat jejunum mounted in an Ussing chamber. The nucleotide sequence of thepet gene revealed that Pet is a member of the autotransporter class of secreted proteins. Here we show that a concentrated supernatant of E. coli HB101 harboring the minimal pet clone pCEFN1 induces temperature-, time- and dose-dependent cytopathic effects on HEp-2 cells and HT29 C1 cells in culture. The effects were characterized by release of the cellular focal contacts from the glass substratum, followed by complete rounding of the cells and detachment from the glass. Staining of the Pet-treated cells with Live/Dead viability stain revealed that >90% of rounded cells were viable. Pet-intoxicated HEp-2 and HT29 cells stained with fluorescein-labeled phalloidin revealed contraction of the cytoskeleton and loss of actin stress fibers. However, the effects of Pet were not inhibited by cytoskeleton-altering drugs, including colchicine, taxol, cytochalasin D, and phallicidin. The Pet protein induced proteolysis in zymogram gels, and preincubation with the serine protease inhibitor phenylmethylsulfonyl fluoride resulted in complete abrogation of Pet cytopathic effects. We introduced a mutation in a predicted catalytic serine residue and found that the mutant (Pet S260I) was deficient in protease activity and did not produce cytopathic effects, cytoskeletal damage, or enterotoxic effects in Ussing chambers. These data suggest that Pet is a cytoskeleton-altering toxin and that its protease activity is involved in each of the observed phenotypes.

Basolateral Cl− uptake mechanisms in Xenopus laevis lung epithelium

2010 ◽  
Vol 299 (1) ◽  
pp. R92-R100 ◽  
Author(s):  
Jens Berger ◽  
Martin Hardt ◽  
Wolfgang G. Clauss ◽  
Martin Fronius
Keyword(s):  
Xenopus Laevis ◽  
Ion Transport ◽  
Anion Exchanger ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Transport Processes ◽  
Lung Epithelium ◽  
Active Ion Transport ◽  
Uptake Mechanisms

A thin liquid layer covers the lungs of air-breathing vertebrates. Active ion transport processes via the pulmonary epithelial cells regulate the maintenance of this layer. This study focuses on basolateral Cl− uptake mechanisms in native lungs of Xenopus laevis and the involvement of the Na+/K+/2 Cl− cotransporter (NKCC) and HCO3−/Cl− anion exchanger (AE), in particular. Western blot analysis and immunofluorescence staining revealed the expression of the NKCC protein in the Xenopus lung. Ussing chamber experiments demonstrated that the NKCC inhibitors (bumetanide and furosemide) were ineffective at blocking the cotransporter under basal conditions, as well as under pharmacologically stimulated Cl−-secreting conditions (forskolin and chlorzoxazone application). However, functional evidence for the NKCC was detected by generating a transepithelial Cl− gradient. Further, we were interested in the involvement of the HCO3−/Cl− anion exchanger to transepithelial ion transport processes. Basolateral application of DIDS, an inhibitor of the AE, resulted in a significantly decreased the short-circuit current (ISC). The effect of DIDS was diminished by acetazolamide and reduced by increased external HCO3− concentrations. Cl− secretion induced by forskolin was decreased by DIDS, but this effect was abolished in the presence of HCO3−. These experiments indicate that the AE at least partially contributes to Cl− secretion. Taken together, our data show that in Xenopus lung epithelia, the AE, rather than the NKCC, is involved in basolateral Cl− uptake, which contrasts with the common model for Cl− secretion in pulmonary epithelia.

Dantrolene and basal ileal sodium and chloride transport: involvement of calcium stores

1983 ◽  
Vol 245 (6) ◽  
pp. G780-G785
Author(s):  
M. Donowitz ◽  
S. Cusolito ◽  
L. Battisti ◽  
G. W. Sharp
Keyword(s):  
Ion Transport ◽  
Intracellular Calcium ◽  
Chloride Transport ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Calcium Stores ◽  
Ionophore A23187 ◽  
Rabbit Ileum ◽  
Intracellular Calcium Stores

The effect of dantrolene on active ion transport in rabbit ileum was determined using the Ussing chamber short-circuiting technique. Dantrolene prevents the release of calcium from intracellular stores in skeletal muscle and was used to probe the role of intracellular calcium stores in intestinal ion transport. A saturated solution of dantrolene (approx 25 microM) decreased ileal short-circuit current and potential difference, increased conductance and mucosal-to-serosal and net Na and Cl fluxes, but did not alter serosal-to-mucosal Na and Cl fluxes. The dantrolene stimulation of active Na and Cl absorption was specific since it did not alter glucose-dependent Na absorption, transport changes caused by Ca2+ ionophore A23187, or the increase in short-circuit current caused by dibutyryl cAMP or theophylline. These effects were associated with an increase in total ileal calcium content and a decreased rate of 45Ca2+ efflux without any change in 45Ca2+ influx from the serosal or mucosal surfaces. These findings are consistent with an effect of dantrolene to stimulate active ileal Na and Cl absorption by a mechanism involving lowered cytosol Ca2+ levels and compatible with trapping calcium in intracellular stores. It thus appears as if intracellular calcium stores have an important role in the control of basal ion transport in the intestine.

756 - Enterohemorrhagic E. Coli (EHEC) Secreted Serine Protease Espp, Stimulates Active Electrogenic Ion Transport in Human Colonoid Monolayers

2018 ◽  
Vol 154 (6) ◽  
pp. S-160
Author(s):  
Ming Tse ◽  
Julie In ◽  
Michele Doucet ◽  
Jianyi Yin ◽  
Jennifer D. Foulke-Abel ◽  
...  
Keyword(s):  
Ion Transport ◽  
Serine Protease ◽  
E Coli ◽  
Electrogenic Ion Transport

Mast cells in the regulation of intestinal electrolyte transport

1986 ◽  
Vol 251 (2) ◽  
pp. G253-G262 ◽  
Author(s):  
D. A. Russell
Keyword(s):  
Mast Cells ◽  
Ion Transport ◽  
Trichinella Spiralis ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Antigen Challenge ◽  
Intracellular Camp ◽  
Camp Content ◽  
Ionic Basis

Experiments were performed to determine the ionic basis and physiological messengers of transepithelial ion transport alterations (short-circuit current, Isc) measured during the induction of intestinal anaphylaxis in an Ussing chamber. Antigens derived from Trichinella spiralis, an intestinal parasite, were used to challenge jejunal tissue from guinea pigs immunized by infection with the parasite. Histamine (10(-4) M) caused an increased in Isc that was similar to that induced by antigen. Diphenhydramine (10(-5) M) inhibited the epithelial electrical responses to histamine by 100% and to antigen by 60-70%. Indomethacin (10(-5) M), in combination with diphenhydramine, completely inhibited the antigen-induced rise in Isc. Furosemide (10(-4) M) caused 50-60% inhibition of the increase in Isc induced by antigen and histamine. Antigen challenge of isolated enterocytes did not alter intracellular cAMP content. However, antigen challenge of jejunal segments in which epithelial cells were in contact with sensitized mast cells increased mucosal cAMP content. These results suggest that electrogenic Cl- secretion, mediated in part by cAMP, contributes to antigen-induced jejunal ion transport changes and that histamine and prostaglandins are involved in eliciting these epithelial responses.

Effects of carbon monoxide on ion transport across rat distal colon

2011 ◽  
Vol 300 (2) ◽  
pp. G207-G216 ◽  
Author(s):  
Julia Steidle ◽  
Martin Diener
Keyword(s):  
Carbon Monoxide ◽  
Ion Transport ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Distal Colon ◽  
Disulfonic Acid ◽  
Maximal Response ◽  
Colonic Crypts ◽  
Acid Sodium Salt

The aim of the present study was to investigate whether carbon monoxide (CO) induces changes in ion transport across the distal colon of rats and to study the mechanisms involved. In Ussing chamber experiments, tricarbonyldichlororuthenium(II) dimer (CORM-2), a CO donor, evoked a concentration-dependent increase in short-circuit current ( Isc). A maximal response was achieved at a concentration of 2.5·10−4 mol/l. Repeated application of CORM-2 resulted in a pronounced desensitization of the tissue. Anion substitution experiments suggest that a secretion of Cl− and HCO3− underlie the CORM-2-induced current. Glibenclamide, a blocker of the apical cystic fibrosis transmembrane regulator channel, inhibited the Isc induced by the CO donor. Similarly, bumetanide, a blocker of the basolateral Na+-K+-2Cl− cotransporter, combined with 4-acetamido-4′-isothiocyanato-stilbene-2,2′-disulfonic acid sodium salt, an inhibitor of the basolateral Cl−/HCO3− exchanger, inhibited the CORM-2-induced Isc. Membrane permeabilization experiments indicated an activation of basolateral K+ and apical Cl− channels by CORM-2. A partial inhibition by the neurotoxin, tetrodotoxin, suggests the involvement of secretomotor neurons in this response. In imaging experiments at fura-2-loaded colonic crypts, CORM-2 induced an increase of the cytosolic Ca2+ concentration. This increase depended on the influx of extracellular Ca2+, but not on the release of Ca2+ from intracellular stores. Both enzymes for CO production, heme oxygenase I and II, are expressed in the colon as observed immunohistochemically and by RT-PCR. Consequently, endogenous CO might be a physiological modulator of colonic ion transport.

Enteropathogenic E. coli attenuates secretagogue-induced net intestinal ion transport but not Cl− secretion

1999 ◽  
Vol 276 (3) ◽  
pp. G781-G788 ◽  
Author(s):  
Gail Hecht ◽  
Athanasia Koutsouris
Keyword(s):  
Ion Transport ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Transport Processes ◽  
Intestinal Epithelial ◽  
Cell Monolayers ◽  
E Coli ◽  
Ion Secretion ◽  
Dependent Transport ◽  
Intestinal Ion Transport

Enteric bacterial pathogens often increase intestinal Cl− secretion. Enteropathogenic Escherichia coli(EPEC) does not stimulate active ion secretion. In fact, EPEC infection decreases net ion transport in response to classic secretagogues. This has been presumed to reflect diminished Cl− secretion. The aim of this study was to investigate the influence of EPEC infection on specific intestinal epithelial ion transport processes. T84 cell monolayers infected with EPEC were used for these studies. EPEC infection significantly decreased short-circuit current ( I sc) in response to carbachol and forskolin, yet125I efflux studies revealed no difference in Cl− channel activity. There was also no alteration in basolateral K+ channel or Na+-K+-2Cl−cotransport activity. Furthermore, net36Cl−flux was not decreased by EPEC. No alterations in either K+ or Na+ transport could be demonstrated. Instead, removal of basolateral bicarbonate from uninfected monolayers yielded an I sc response approximating that observed with EPEC infection, whereas bicarbonate removal from EPEC-infected monolayers further diminished I sc. These studies suggest that the reduction in stimulated I sc is not secondary to diminished Cl−secretion. Alternatively, bicarbonate-dependent transport processes appear to be perturbed.

scholarly journals Segment-specific effects of epinephrine on ion transport in the colon of the rat

1998 ◽  
Vol 275 (6) ◽  
pp. G1367-G1376 ◽  
Author(s):  
Silke Hörger ◽  
Gerhard Schultheiß ◽  
Martin Diener
Keyword(s):  
Ion Transport ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Distal Colon ◽  
Proximal Colon ◽  
Rat Colon ◽  
Second Phase ◽  
Stimulatory Action ◽  
Β Receptor

The effect of epinephrine on transport of K+, Na+, Cl−, and[Formula: see text] across the rat colon was studied using the Ussing chamber technique. Epinephrine (5 × 10−6mol/l) induced a biphasic change in short-circuit current ( Isc) in distal and proximal colon: a transient increase followed by a long-lasting decay. The first phase of the Iscresponse was abolished in Cl−-poor solution or after bumetanide administration, indicating a transient induction of Cl−secretion. The second phase of the response to epinephrine was suppressed by apical administration of the K+channel blocker, quinine, and was concomitant with an increase in serosal-to-mucosal Rb+flux, indicating that epinephrine induced K+secretion, although this response was much smaller than the change in Isc. In addition, the distal colon displayed a decrease in mucosal-to-serosal and serosal-to-mucosal Cl−fluxes when treated with epinephrine. In the distal colon, indomethacin abolished the first phase of the epinephrine effect, whereas the second phase was suppressed by TTX. In the proximal colon, indomethacin and TTX were ineffective. The neuronally mediated response to epinephrine in the distal colon was suppressed by the nonselective β-receptor blocker, propranolol, and by the β2-selective blocker, ICI-118551, whereas the epithelial response in the proximal colon was suppressed by the nonselective α-blocker, phentolamine, and by the selective α2-blocker, yohimbine. These results indicate a segment-specific action of epinephrine on ion transport: a direct stimulatory action on epithelial α2-receptors in the proximal colon and an indirect action on secretomotoneurons via β2-receptors in the distal colon.

Interleukin-13 induces a hypersecretory ion transport phenotype in human bronchial epithelial cells

2002 ◽  
Vol 282 (2) ◽  
pp. L226-L236 ◽  
Author(s):  
Henry Danahay ◽  
Hazel Atherton ◽  
Gareth Jones ◽  
Robert J. Bridges ◽  
Christopher T. Poll
Keyword(s):  
Epithelial Cells ◽  
Ion Transport ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Fluid Secretion ◽  
Bronchial Epithelial Cells ◽  
Human Bronchial Epithelial Cells ◽  
Bronchial Epithelial ◽  
Anion Conductance

Interleukin (IL)-13 has been associated with asthma, allergic rhinitis, and chronic sinusitis, all conditions where an imbalance in epithelial fluid secretion and absorption could impact upon the disease. We have investigated the effects of IL-13 on the ion transport characteristics of human bronchial epithelial cells cultured at an apical-air interface. Ussing chamber studies indicated that 48 h pretreatment with IL-13 or IL-4 significantly reduced the basal short-circuit current ( I sc) and inhibited the amiloride-sensitive current by >98%. Furthermore, the I scresponses were increased by more than six- and twofold over control values when stimulated with UTP or forskolin, respectively, after cytokine treatment. The IL-13-enhanced response to UTP/ionomycin was sensitive to bumetanide and DIDS and was reduced in a low-chloride, bicarbonate-free solution. Membrane permeablization studies indicated that IL-13 induced the functional expression of an apical Ca2+-activated anion conductance and that changes in apical or basolateral K+ conductances could not account for the increased I sc responses to UTP or ionomycin. The results indicate that IL-13 converts the human bronchial epithelium from an absorptive to a secretory phenotype that is the result of loss of amiloride-sensitive current and an increase in a DIDS-sensitive apical anion conductance.

scholarly journals Pet, an Autotransporter Enterotoxin from Enteroaggregative Escherichia coli

1998 ◽  
Vol 66 (7) ◽  
pp. 3155-3163 ◽  
Author(s):  
Carlos Eslava ◽  
Fernando Navarro-García ◽  
John R. Czeczulin ◽  
Ian R. Henderson ◽  
Alejandro Cravioto ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Nucleotide Sequence Analysis ◽  
E Coli ◽  
Eaec Strain ◽  
Heat Labile ◽  
Tissue Resistance

ABSTRACT Enteroaggregative Escherichia coli (EAEC) is an emerging cause of diarrheal illness. Clinical data suggest that diarrhea caused by EAEC is predominantly secretory in nature, but the responsible enterotoxin has not been described. Work from our laboratories has implicated a ca. 108-kDa protein as a heat-labile enterotoxin and cytotoxin, as evidenced by rises in short-circuit current and falls in tissue resistance in rat jejunal tissue mounted in an Ussing chamber. Here we report the genetic cloning, sequencing, and characterization of this high-molecular-weight heat-labile toxin. The toxin (designated the plasmid-encoded toxin [Pet]) is encoded on the 65-MDa adherence-related plasmid of EAEC strain 042. Nucleotide sequence analysis suggests that the toxin is a member of the autotransporter class of proteins, characterized by the presence of a conserved C-terminal domain which forms a β-barrel pore in the bacterial outer membrane and through which the mature protein is transported. The Pet toxin is highly homologous to the EspP protease of enterohemorrhagic E. coli and to EspC of enteropathogenicE. coli, an as yet cryptic protein. In addition to its potential role in EAEC infection, Pet represents the first enterotoxin within the autotransporter class of secreted proteins. We hypothesize that other closely related members of this class may also produce enterotoxic effects.

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