Gangliosides sensitize unresponsive fibroblasts to Escherichia coli heat-labile enterotoxin.

Moss, Joel; Garrison, S; Fishman, Peter H.; Richardson, Stephen H.

doi:10.1172/jci109472

Cited by 53 publications

(23 citation statements)

References 29 publications

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“…initial experiments with crude LT were consistent with the possibility that this enterotoxin interacted with GM1 as well as with a cell surface receptor distinct from that utilized by choleragen (20,23,24,35,38,47,48,55,(248)(249)(250)(251). In further studies using ganglioside-deficient transformed fibroblasts that lack chemically detectable ganglioside GM1 (NCTC 2071), it was observed that after incubation with GM1, but not GM2 or GM3 , their sensitivity to a relatively impure LT preparation was increased; similar results were obtained with these cells using choleragen (56,60,251).…”

Section: Similarities Betwen E Coli Heat-labile Enterotoxin and Cholsupporting

confidence: 61%

Mechanism of action of choleragen andE. coli heat-labile enterotoxin: activation of adenylate cyclase by ADP-ribosylation

Moss

Vaughan

1981

Mol Cell Biochem

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SummaryCholeragen exerts its effects on cells through the activation of adenylate cyclase. The initial event appears to be the binding of the B subunit of the toxin to ganglioside GM1 on the cell surface, following which there is a delay prior to activation of adenylate cyclase. Patching and capping of the toxin on the cell surface, perhaps involved in the internalization of the enzymatically active subunit, may be occuring during this time. The activation of adenylate cyclase, which is catalyzed by the A! peptide of choleragen, does not require the B subunit or ganglioside GMI. The A 1 peptide catalyzes the transfer of ADP-ribose from NAD to an amino acid, probably arginine, in a 42 000 dalton membrane protein. This protein appears to be the GTP-binding component (or G/F factor) of the adenylate cyclase system and is crucial to the regulation of cyclase activity by hormones such as epinephrine. ADP-ribosylation of the G/F factor is enhanced by GTP and, in some systems, by a cytosolic factor. GTP is also required for stabilization and optimal catalytic function of the choleragen-activated cyclase. Calmodulin, a calcium-binding protein, is necessary for expression of catalytic activity of the toxin-activated adenylate cyclase in brain and other tissues.The ADP-ribosyltransferase activity required for activation of the cyclase is an intrinsic property of the A 1 peptide of choleragen which is expressed only after the peptide is released from the holotoxin by reduction of a single disulfide bond. In the absence of cellular components, choleragen catalyzes the ADP-ribosylation of small guanidino compounds such as arginine as well as peptides and proteins that contain arginine. It is assumed, therefore, that the site of ADP-ribosylation in the natural acceptor protein is an arginine or similar amino acid. When guanidino compounds are not present as ADP-ribose acceptors, choleragen hydrolyzes NAD to ADP-ribose and nicotinamide at a considerably slower rate.E. coli heat-labile enterotoxin (LT) is very similar to choleragen in structure and function. It consists of two types of subunits, A and B, with sizes comparable to those of the A and B subunits of choleragen. Binding of LT to the cell surface is enhanced by prior incorporation of GM1 but not other gangliosides; the oligosaccharide of GMI specifically interacts with LT and its B subunit. The A subunit of LT exhibits ADP-ribosyltransferase activity following activation by thiol to release the A 1 peptide. The A subunit of LT can be isolated in an 'unnicked' form and thus requires, in addition to reduction by a thiol, proteolytic cleavage to generate the active A 1 peptide. Like choleragen, LT uses guanidino compounds as model ADP-ribose acceptors and catalyzes the ADP-ribosylation of a 42 000 dalton protein in cell membrane preparations.ADP-ribosyltransferases that use arginine as ADP-ribose acceptors are not restricted to bacterial systems; such an enzyme has been purified to apparent homogeneity (>500 000-fold) from turkey erythrocytes. Based on a subunit molecular...

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Section: Similarities Betwen E Coli Heat-labile Enterotoxin and Cholsupporting

confidence: 61%

Mechanism of action of choleragen andE. coli heat-labile enterotoxin: activation of adenylate cyclase by ADP-ribosylation

Moss

Vaughan

1981

Mol Cell Biochem

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“…Ganglioside GM, is the site of CT binding (9,36); LT-Ih is believed to use GM, as well as glycoproteins (31,(37)(38)(39)(40)(41)(42). LT-IIa and LT-IIb may also bind to gangliosides, but their specificities differ from those of CT and LT, in agreement with the differences in primary structure of the B subunits (17,21,22,(43)(44)(45).…”

Section: Discussionmentioning

confidence: 76%

Activation of Escherichia coli heat-labile enterotoxins by native and recombinant adenosine diphosphate-ribosylation factors, 20-kD guanine nucleotide-binding proteins.

Lee

Chang²,

Tsai³

et al. 1991

J. Clin. Invest.

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“…E. coli enterotoxins which are known to evoke secretion through different intracellular signal transduction pathways. Following an initial step, which is an interaction with specific G M1 ganglioside receptors on the brush border membrane of the intestinal epithelium, 15,16) LT causes activation of adenyl cyclase and accumulation of intracellular cAMP. 17,18) The increase in intracellular cAMP activates Cl Ϫ secretion most likely acting through a cAMP-dependent protein kinase (cAK).…”

Section: Discussionmentioning

confidence: 99%

In Vitro Effects of Wood Creosote on Enterotoxin-Induced Secretion Measured Electrophysiologically in the Rat Jejunum and Colon.

Kuge

Venkova

Meerveld

2001

Biological & Pharmaceutical Bulletin

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Wood creosote, a mixture of guaiacol, creosol and related phenolic compounds, has been used in Japan as a traditional drug for the treatment of diarrhea for more than a century. 1)However, only during the last decade, have the mechanisms of antidiarrheal action of wood creosote become the subject of thorough investigation. The therapeutic effect of wood creosote was first linked to its ability to reduce intestinal motility. 2,3) In addition, an antisecretory and/or pro-absorptive effect of wood creosote was suggested by findings in animal models of diarrhea. Wood creosote was found to prevent castor oil-induced diarrhea in rats 4) and to reduce fluid secretion induced by an Escherichia coli (E. coli) enterotoxin applied into loops of rabbit jejunum in vivo. 5) In previous studies, using in vitro electrophysiologic measurements of electrogenic ion transport in isolated intestinal mucosal sheets, we demonstrated that, while having a moderate effect on basal secretion, wood creosote completely inhibited secretory responses to exogenous acetylcholine. 6,7) The goal of the present investigation was to extend our knowledge about the mechanism of antisecretory action of wood creosote using an in vitro model of enterotoxin-induced hypersecretory conditions. Since secretory diarrhea occurs when the balance between intestinal absorption and secretion is disturbed by excessive secretion caused by enterotoxins produced by the pathogen, we consider the experiments relevant to the clinical cases of infectious secretory diarrhea. The active net electrogenic transport was measured electrophysiologically as the short circuit current passed via a voltage clamp to eliminate the transmucosal potential difference. MATERIALS AND METHODS AnimalsMale Sprague-Dawley rats (body weight 250-300 g) were used in the experiments. The animals were purchased from Charles River Laboratories (Wilmington, Massachusetts, U.S.A.) and were housed under standard conditions (21°C and a 12-h light/dark cycle) at the animal facility for at least one week prior to initiating the experiments. On the day of experiment the rats were killed by decapitation and the jejunum and mid portion of the colon were immediately removed and placed in aerated ice-cold Krebs solution. The experimental protocol of the study was approved by the Oklahoma City V.A. Medical Center Animal Care Committee.Electrophysiological Measurement of Net Electrogenic Transport Small intestinal and colonic segments (20 mm long) were used for the preparation of mucosal sheets as described previously.7) Muscle-stripped mucosal sheets were placed in modified Ussing chambers (0.6 cm 2 exposed surface area) that were filled with Krebs bicarbonate buffer (pH 7.2), containing 10 mM mannitol on the mucosal (intraluminal) side and 10 mM glucose at the serosal side. The lack of glucose at the intraluminal side avoided alterations in mucosal transport due to glucose absorption. The Krebs buffer was maintained at 37°C and was continuously aerated with 95% O 2 and 5% CO 2 . An equilibration period of at ...

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Gangliosides sensitize unresponsive fibroblasts to Escherichia coli heat-labile enterotoxin.

Cited by 53 publications

References 29 publications

Mechanism of action of choleragen andE. coli heat-labile enterotoxin: activation of adenylate cyclase by ADP-ribosylation

Mechanism of action of choleragen andE. coli heat-labile enterotoxin: activation of adenylate cyclase by ADP-ribosylation

Activation of Escherichia coli heat-labile enterotoxins by native and recombinant adenosine diphosphate-ribosylation factors, 20-kD guanine nucleotide-binding proteins.

In Vitro Effects of Wood Creosote on Enterotoxin-Induced Secretion Measured Electrophysiologically in the Rat Jejunum and Colon.

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