Characterization of the synergistic interaction of Escherichia coli heat-stable toxin and carbachol

1991 ◽  
Vol 261 (4) ◽  
pp. G592-G601 ◽  
Author(s):  
S. A. Levine ◽  
M. Donowitz ◽  
A. J. Watson ◽  
G. W. Sharp ◽  
J. K. Crane ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Short Circuit ◽  
Second Messengers ◽  
Short Circuit Current ◽  
Ussing Chambers ◽  
Simultaneous Addition ◽  
Heat Stable ◽  
Cholinergic Agent ◽  
Synergistic Response ◽  
Intracellular Mediators

STa, the heat-stable enterotoxin of Escherichia coli, is a specific activator of membrane-bound guanylyl cyclase and stimulates secretion of Cl- in a human colonic carcinoma cell line (T84). We investigated the effect of the cholinergic agent carbachol on the secretory response to STa. T84 cell monolayers were studied under voltage-clamped conditions in modified Ussing chambers. Simultaneous addition of STa and carbachol resulted in a biphasic synergistic response characterized by a brief peak in short-circuit current (Isc) followed by a prolonged plateau phase lasting up to 90 min. A synergistic response was also seen with sequential addition of the agonists, and was altered by the order and timing of agonist addition. Pretreatment with STa enhanced the synergistic response to carbachol, while the reverse order of additions produced synergy only when STa was added during or immediately after the Isc response to carbachol. Synergy occurred only with a concentration of STa sufficient to produce an Isc response alone. However, a concentration of carbachol that caused neither an increase in Isc nor intracellular Ca2+ mobilization was sufficient to evoke a synergistic response. Addition of 8-bromoguanosine 3',5'-cyclic monophosphate also produced a synergistic Isc response with carbachol, although maximal synergism was seen with simultaneous addition. Augmentation of the intracellular Ca2+ response to carbachol by STa is not the mechanism of synergy. Although the mechanism of synergy is not understood, these studies suggest that STa-induced cGMP interacts with other second messengers to produce the synergistic response, and that multiple intracellular mediators may influence the ability of STa to cause disease.

scholarly journals Spontaneous water secretion in T84 cells: effects of STa enterotoxin, bumetanide, VIP, forskolin, and A-23187

2001 ◽  
Vol 281 (3) ◽  
pp. G816-G822 ◽  
Author(s):  
Roxana Toriano ◽  
Arlinet Kierbel ◽  
Marco Antonio Ramirez ◽  
Gerhard Malnic ◽  
Mario Parisi
Keyword(s):  
Short Circuit ◽  
Second Messengers ◽  
Short Circuit Current ◽  
Rt Pcr ◽  
T84 Cells ◽  
Pharmacological Agents ◽  
Heat Stable ◽  
A 23187 ◽  
Water Secretion ◽  
Secretory Apparatus

The regulated Cl− secretory apparatus of T84 cells responds to several pharmacological agents via different second messengers (Ca2+, cAMP, cGMP). However, information about water movements in T84 cells has not been available. In the absence of osmotic or chemical gradient, we observed a net secretory transepithelial volume flux ( J w = −0.16 ± 0.02 μl · min−1 · cm−2) in parallel with moderate short-circuit current values ( I sc = 1.55 ± 0.23 μA/cm2). The secretory J wreversibly reverted to an absorptive value when A-23187 was added to the serosal bath. Vasoactive intestinal polypeptide increased I sc, but, unexpectedly, J w was not affected. Bumetanide, an inhibitor of basolateral Na+-K+-2Cl−cotransporter, completely blocked secretory J wwith no change in I sc. Conversely, serosal forskolin increased I sc, but J w switched from secretory to absorptive values. Escherichia coli heat-stable enterotoxin increased secretory J w and I sc. No difference between the absorptive and secretory unidirectional Cl−fluxes was observed in basal conditions, but after STa stimulation, a significant net secretory Cl− flux developed. We conclude that, under these conditions, the presence of secretory or absorptive J w values cannot be shown by I sc and ion flux studies. Furthermore, RT-PCR experiments indicate that aquaporins were not expressed in T84 cells. The molecular pathway for water secretion appears to be transcellular, moving through the lipid bilayer or, as recently proposed, through water-solute cotransporters.

Prostaglandin-induced recovery of barrier function in porcine ileum is triggered by chloride secretion

1999 ◽  
Vol 276 (1) ◽  
pp. G28-G36 ◽  
Author(s):  
Anthony T. Blikslager ◽  
Malcolm C. Roberts ◽  
Robert A. Argenzio
Keyword(s):  
Short Circuit ◽  
Second Messengers ◽  
Short Circuit Current ◽  
Chloride Secretion ◽  
Ringer Solution ◽  
Signal Recovery ◽  
Osmotic Gradient ◽  
Current Increase ◽  
Ussing Chambers

We have previously shown that PGI2 and PGE2 have a synergistic role in restoring electrical transepithelial resistance ( R) in ischemia-injured porcine ileum via the second messengers Ca2+ and cAMP. Because Ca2+ and cAMP stimulate Cl− secretion, we assessed the role of PG-induced Cl−secretion in recovery of R. Mucosa from porcine ileum subjected to ischemia for 45 min was mounted in Ussing chambers and bathed in indomethacin and Ringer solution. Addition of PGs stimulated a twofold increase in R, which was preceded by elevations in short-circuit current (increase of 25 μA/cm2). The PG-induced effect on R was partially inhibited with bumetanide, an inhibitor of Cl− secretion. The remaining elevations in R were similar in magnitude to those induced in ischemic tissues by amiloride, an inhibitor of Na+ absorption. Treatment with 10−4 M 8-bromo-cGMP or 300 mosM mucosal urea resulted in elevations in R similar to those attained with PG treatment. PGs signal recovery of Rvia induction of Cl−secretion and inhibition of Na+absorption, possibly by establishing a transmucosal osmotic gradient.

Bradykinin stimulates airway epithelial Cl- secretion via two second messenger pathways

1990 ◽  
Vol 258 (6) ◽  
pp. L369-L377 ◽  
Author(s):  
J. J. Smith ◽  
J. D. McCann ◽  
M. J. Welsh
Keyword(s):  
Short Circuit ◽  
Second Messengers ◽  
Second Messenger ◽  
Short Circuit Current ◽  
Cytosolic Calcium ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Ussing Chambers ◽  
Cl Secretion ◽  
Second Messenger Pathways

In canine airway epithelial cells, bradykinin increases intracellular concentrations of D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], cytosolic calcium concentration ([Ca2+]c), and adenosine 3',5'-cyclic monophosphate (cAMP). To determine the role of these second messengers in bradykinin-stimulated Cl- secretion, we studied the secretory response to this peptide using canine tracheal monolayers mounted in Ussing chambers. Bradykinin stimulated Cl- secretion [measured as short-circuit current (Isc)] when added to submucosal or mucosal surfaces; however, secretory responses differed substantially. Submucosal addition of bradykinin induced a biphasic increase in secretion; mucosal addition induced a monophasic increase in secretion. Both responses were mediated by B2 receptors. We show that activation of bradykinin receptors can stimulate Cl- secretion in two ways. 1) Bradykinin added to either surface stimulates prostaglandin synthesis and release at the basolateral surface. This leads to activation of prostaglandin E2-sensitive receptors on the basolateral surface that are coupled to cAMP production and an increase in apical membrane Cl- conductance. 2) In addition, bradykinin added to the submucosal surface increases Ins(1,4,5)P3 and [Ca2+]c levels, which enhance basolateral K+ conductance and the electrical driving force for apical Cl- exit. Whereas secretion requires activation of apical Cl- channels, the data show that Cl- secretion can also be modulated by activation of basolateral K+ channels. These data indicate that bradykinin-induced transepithelial Cl- secretion is mediated by two independent, second messenger pathways. These results provide the first evidence for expression of both pathways in a polar fashion in an epithelial monolayer.

Effect of theophylline and heat-stable enterotoxin of Escherichia coli on transcellular and paracellular ion movement across isolated porcine colon

1983 ◽  
Vol 61 (10) ◽  
pp. 1138-1148 ◽  
Author(s):  
R. A. Argenzio ◽  
S. C. Whipp
Keyword(s):  
Escherichia Coli ◽  
Ion Transport ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Secretory Process ◽  
Anion Conductance ◽  
Heat Stable ◽  
Ion Movement ◽  
Transport Response

The effect of theophylline and a heat-stable enterotoxin of Escherichia coli (ST) on ion transport was examined using an in vitro, short-circuited preparation of the porcine colon. Theophylline abolished net Na absorption and elicited net Cl secretion, which quantitatively accounted for the increase in short-circuit current (Isc) observed. In contrast, a maximal dose of ST elicited an Isc response about one-half that of theophylline and only partially reduced the net absorption of Na and Cl. A significant residual ion flux, consistent with HCO3 secretion, was elicited by ST and was sustained after theophylline addition. Ion replacement experiments showed that the Isc and net ion transport response to ST was abolished when either Cl or HCO3 were removed from the bathing solutions. Voltage clamp experiments to evaluate the contribution of the paracellular and transcellular transepithelial pathways from serosa to mucosa showed that approximately one-half of the total serosa-to-mucosa flux (Jsm) of both Na and Cl was through the cells. Theophylline and ST both significantly reduced transcellular.[Formula: see text], but did not affect [Formula: see text]. Theophylline, but not ST, caused an increase in paracellular conductance of both ions. These results demonstrate significant differences in the effects of ST or theophylline on both transcellular and paracellular ion movement, and suggest that ST induces a Cl-dependent HCO3 secretion which is unobserved under control or theophylline-stimulated conditions. In addition, results are consistent with the operation of a neutral NaCl secretory process which is normally masked by the greater net rates of the neutral Na and Cl absorptive mechanisms. Thus, both ST and theophylline appear to reduce or abolish the neutral processes and convert the neutral secretory process into an electrogenic one. This latter effect could be explained simply by an increase in the anion conductance of the mucosal membranes.

cGMP modulation of ileal ion transport: in vitro effects of Escherichia coli heat-stable enterotoxin

1982 ◽  
Vol 243 (1) ◽  
pp. G36-G41 ◽  
Author(s):  
S. Guandalini ◽  
M. C. Rao ◽  
P. L. Smith ◽  
M. Field
Keyword(s):  
Escherichia Coli ◽  
Ion Transport ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Rabbit Ileum ◽  
Heat Stable ◽  
Transport Effects ◽  
In Vitro Effects ◽  
Nonadditive Effects

Diarrheagenic strains of Escherichia coli have been shown to produce a heat-stable enterotoxin (ST) that simulates guanylate cyclase, increases short-circuit current (Isc), and inhibits active Cl absorption in the intestine. In rabbit ileum, the ion transport effects are smaller than those produced by cAMP-related agonists. Because ST may be a selective cGMP agonist, we further explored its mode of action in rabbit ileum. ST inhibits net Na and net Cl absorption. ST also inhibits the same fraction of Cl influx across the brush border that theophylline inhibits. At maximal doses, ST and 8-bromo-cGMP (8-Br-cGMP) had nearly equal, nonadditive effects of Isc that were about 66% of that produced by 8-Br-cAMP. ST increased mucosal cGMP concentration 16-fold, whereas epinephrine, an inhibitor of secretion, increased cGMP concentration by only 30%. This is insufficient to alter ion transport because doses of ST that increased cGMP concentration by 100% failed to alter Cl fluxes. Furthermore, epinephrine did not increase cGMP concentration in isolated enterocytes. We conclude that 1) cGMP mediates ST effects on ion transport, and 2) although ST and cAMP-related agonists have the same antiabsorptive effects, ST is less effective in stimulating electrogenic Cl secretion.

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.

Effect ofE. coliheat-stable enterotoxin on colonic transport in guanylyl cyclase C receptor-deficient mice

2001 ◽  
Vol 280 (2) ◽  
pp. G216-G221 ◽  
Author(s):  
Alan N. Charney ◽  
Richard W. Egnor ◽  
Jesline T. Alexander-Chacko ◽  
Valentin Zaharia ◽  
Elizabeth A. Mann ◽  
...  
Keyword(s):  
Guanylyl Cyclase ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Distal Colon ◽  
Proximal Colon ◽  
Guanylyl Cyclase C ◽  
Ussing Chambers ◽  
Heat Stable ◽  
Deficient Mice ◽  
Colonic Transport

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 ( JR), 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−( Js→mCl) and decreased net Cl−flux ( JnetCl) accompanied by increases in short-circuit current ( Isc), 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 Js→mCland JnetCl, Isc, PD, G, and JRsimilar 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.

Fasting alters basal and stimulated ion transport in piglet jejunum

1994 ◽  
Vol 267 (1) ◽  
pp. R156-R163 ◽  
Author(s):  
H. V. Carey ◽  
U. L. Hayden ◽  
K. E. Tucker
Keyword(s):  
Ion Transport ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Short Term ◽  
Ussing Chambers ◽  
Cl Secretion ◽  
Heat Stable ◽  
Active Ion Transport ◽  
Total Tissue ◽  
Net Fluxes

Three-week-old piglets were used to study the effects of short-term fasting on jejunal ion transport. A 48-h fast significantly reduced mucosal weight, villus height, and crypt depth. Fasting increased basal short-circuit current (Isc), which reflects active ion transport, and total tissue conductance (Gt) of muscle-stripped jejunal sheets mounted in Ussing chambers. Increases in Isc evoked by carbachol, serotonin, histamine, prostaglandin E2, or Escherichia coli heat-stable enterotoxin were significantly greater in the fasted piglets. Isc responses to mucosal D-glucose were also enhanced by the fast. Under basal conditions, unidirectional and net fluxes of Na+ and Cl-, as well as serosal-to-mucosal inulin fluxes, were significantly increased in fasted piglets. In fed piglets, carbachol increased net Cl- secretion by stimulating serosal-to-mucosal Cl- flux; Gt was not affected. In fasted piglets, carbachol increased net Cl- secretion by inhibiting mucosal-to-serosal fluxes with no effect on serosal-to-mucosal fluxes. In addition, carbachol significantly inhibited mucosal-to-serosal Na+ fluxes and reduced Gt in this group. Thus a 48-h fast increased unidirectional and net ion fluxes in piglet jejunum and enhanced ion transport responses to secretory agonists. The mechanism by which carbachol stimulated net Cl- secretion was also altered by the fast. These results suggest that the absence of luminal nutrition changes the ion transport characteristics of the jejunal epithelium.

Induction of Na+/K+/2Cl− cotransporter expression mediates chronic potentiation of intestinal epithelial Cl− secretion by EGF

2008 ◽  
Vol 294 (6) ◽  
pp. C1362-C1370 ◽  
Author(s):  
Fiona O'Mahony ◽  
Ferial Toumi ◽  
Magdalena S. Mroz ◽  
Gail Ferguson ◽  
Stephen J. Keely
Keyword(s):  
Time Course ◽  
Egf Receptor ◽  
Epithelial Transport ◽  
Short Circuit ◽  
Second Messengers ◽  
Short Circuit Current ◽  
Pcr Analysis ◽  
Intestinal Epithelial ◽  
Ussing Chambers ◽  
Intracellular Ca

Alterations in EGF receptor (EGFR) signaling occur in intestinal disorders associated with dysregulated epithelial transport. In the present study, we investigated a role for the EGFR in the chronic regulation of intestinal epithelial secretory function. Epithelial Cl− secretion was measured as changes in short-circuit current ( Isc) across voltage-clamped monolayers of T84 cells in Ussing chambers. Acute treatment of T84 cells with EGF (100 ng/ml, 15 min) chronically enhanced Isc responses to a broad range of secretagogues. This effect was apparent within 3 h, maximal by 6 h, and sustained for 24 h after treatment with EGF. The Na+/K+/2Cl− cotransporter (NKCC1) inhibitor bumetanide (100 μM) abolished the effect of EGF, indicating increased responses are due to potentiated Cl− secretion. Neither basal nor agonist-stimulated levels of intracellular Ca2+ or PKA activity were altered by EGF, implying that the effects of the growth factor are not due to chronic alterations in levels of second messengers. EGF increased the expression of NKCC1 with a time course similar to that of its effects on Cl− secretion. This effect of EGF was maximal after 6 h, at which time NKCC1 expression in EGF-treated cells was 199.9 ± 21.9% of that in control cells ( n = 21, P < 0.005). EGF-induced NKCC1 expression was abolished by actinomycin D, and RT-PCR analysis demonstrated EGF increased expression of NKCC1 mRNA. These data increase our understanding of mechanisms regulating intestinal fluid and electrolyte transport and reveal a novel role for the EGFR in the chronic regulation of epithelial secretory capacity through upregulation of NKCC1 expression.

scholarly journals Production of Enterotoxin by Yersinia bercovieri, a Recently Identified Yersinia enterocolitica-Like Species

1999 ◽  
Vol 67 (2) ◽  
pp. 968-971 ◽  
Author(s):  
Alexander Sulakvelidze ◽  
Arnold Kreger ◽  
Aaron Joseph ◽  
Roy M. Robins-Browne ◽  
Alessio Fasano ◽  
...  
Keyword(s):  
Yersinia Enterocolitica ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Ussing Chambers ◽  
Heat Stable ◽  
Biologic Activity ◽  
Genetic Probes

ABSTRACT Yersinia bercovieri, a recently identified Y. enterocolitica-like species, produces a heat-stable enterotoxin (designated YbST) which has biologic activity in infant mice and increases short circuit current in Ussing chambers. Although YbST has some properties in common with the heat-stable enterotoxins of Y. enterocolitica (YST I and YST II), it appears to be a novel toxin because (i) it was not neutralized by anti-YST I antiserum, (ii) YbST-neutralizing antiserum did not neutralize YST I, and (iii)Y. bercovieri strains did not hybridize with genetic probes for yst I, yst II, and other known enterotoxins.

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