Is the receptor‐mediated endocytosis of cholera toxin a pre‐requisite for its activation of adenylate cyclase in intact rat hepatocytes?

Housley, Miles D.; Elliott, Keith

doi:10.1016/0014-5793(81)80101-9

Cited by 36 publications

(24 citation statements)

References 42 publications

(23 reference statements)

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“…2) Others have speculated that choleragen undergoes receptor-mediated endocytosis, is processed in the lysosomes and recycled back to the plasma membranes [68,82]. This idea was developed based on the ability of lysomotropic agents such as chloroquine, NH4C1, dansylcadeverine, and methylamine to inhibit the action of choleragen on intact cells [68,82].…”

Section: The Remaining Mystery In Cholera Toxin Actionmentioning

confidence: 99%

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Role of membrane gangliosides in the binding and action of bacterial toxins

1982

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Gangliosides are complex glycosphingolipids that contain from one to several residues of sialic acid. They are present in the plasma membrane of vertebrate cells with their oligosaccharide chains exposed to the external environment. They have been implicated as cell surface receptors and several bacterial toxins have been shown to interact with them. Cholera toxin, which mediates its effects on cells by activating adenylate cyclase, bind with high affinity and specificity to ganglioside GM1. Toxin-resistant cells which lack GM1 can be sensitized to cholera toxin by treating them with GM1. Cholera toxin specifically protects GM1 from cell surface labeling procedures and only GM1 is recovered when toxin-receptor complexes are isolated by immunoadsorption. These results clearly demonstrate that GM1 is the specific and only receptor for cholera toxin. Although cholera toxin binds to GM1 on the external side of the plasma membrane, it activates adenylate cyclase on the cytoplasmic side of the membrane by ADP-ribosylation of the regulatory component of the cyclase. GM1 in addition to functioning as a binding site for the toxin appears to facilitate its transmembrane movement. The heat-labile enterotoxin of E. coli is very similar to cholera toxin in both form and function and can also use GM1 as a cell surface receptor. The potent neurotoxin, tetanus toxin, has a high affinity for gangliosides GD1b and GT1b and binds to neurons which contain these gangliosides. It is not yet clear whether these gangliosides are the physiological receptors for tetanus toxin. By applying the techniques that established GM1 as the receptor for cholera toxin, the role of gangliosides as receptors for tetanus toxin as well as physiological effectors may be elucidated.

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Section: The Remaining Mystery In Cholera Toxin Actionmentioning

confidence: 99%

“…This idea was developed based on the ability of lysomotropic agents such as chloroquine, NH4C1, dansylcadeverine, and methylamine to inhibit the action of choleragen on intact cells [68,82]. Choleragen undergoes partial endocytosis [53,70] and is slowly degraded by intact cells [51,91].…”

Section: The Remaining Mystery In Cholera Toxin Actionmentioning

confidence: 99%

Role of membrane gangliosides in the binding and action of bacterial toxins

1982

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“…These observations, however, were not correlated with the activation of adenylate cyclase . Other groups have suggested that CT must first be degraded by the cell in order to generate a fragment that can then activate the cyclase (18,21,22) .…”

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confidence: 99%

Internalization and degradation of cholera toxin by cultured cells: relationship to toxin action.

Fishman¹

1982

The Journal of Cell Biology

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Using anticholeragen antibodies and '25 1-protein A, we developed a specific and quantitative assay for measuring choleragen on the surfaces of cultured cells. When neuroblastoma cells containing bound toxin were incubated at 37°C, surface toxin disappeared with a half-life of -2 h and a significant loss was detected by 10 min. When cells were incubated with ' 25 1-choleragen in order to measure toxin degradation, cell-associated radioactivity disappeared with time and a corresponding amount of TCA-soluble label appeared in the culture medium with a half-life of 4-6 h. No degradation was detected until 45 min. Although there was a lag of 15 min before bound choleragen activated adenylate cyclase, the enzyme became maximally activated between 45 and 60 min . Similar results were obtained with Friend erythroleukemia cells. Internalization, degradation, and activation all were blocked when the cells were maintained at 4°C. At 22°C, internalization and activation occurred, albeit at a slower rate, whereas degradation was effectively inhibited . These results indicated that choleragen does not have to be degraded by intact cells in order for it to activate adenylate cyclase. Some internalization of the toxin, however, appears to precede the activation process.Choleragen (CT), the active agent of cholera, is composed of two components, A and B (4) . The B component binds to specific receptors on the cell surface that are believed to be the ganglioside GM, (see reference 6 for a recent review) . The A component consists of two polypeptide chains, A l and A2, connected by a single disulfide bond. The A l peptide is an ADP-ribosyltransferase (23) and catalyzes the transfer of ADPribose from nicotinamide adenine dinucleotide (NAD) to the regulatory component of adenylate cyclase (2, 15). This modification results in a persistent activation of adenylate cyclase . With intact cells, there is a definite lag period before cyclase activity begins to rise in response to the toxin (1, 5). When membranes are incubated with A 1 plus NAD, there is no lag (14). It is believed that during the lag period CT or some part of it is translocated across the membrane, that Al is formed, and that adenylate cyclase is then activated by A l (6, 9, 12, 16). Several groups, using immunochemistry or cytochemistry and electron microscopy, have reported that CT becomes internalized in a time-and temperature-dependent manner (17,19,20). These observations, however, were not correlated with the activation of adenylate cyclase . Other groups have suggested that CT must first be degraded by the cell in order to generate a fragment that can then activate the cyclase (18,21,22) .The present studies were initiated in order to develop a specific and quantitative method for assaying surface bound CT and to follow the kinetics of CT internalization, degradation, and activation of adenylate cyclase . MATERIALS AND METHODS Cells and Cell CultureMouse neuroblastoma clone NB41A3 was obtained from the American Type Culture Collection (Rockville, MD) and ...

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“…were judged to be viable [as described previously (Smith et al, 1978;]. A washed membrane fraction was obtained as described by Houslay & Elliot (1979, 1981. In all cases membranes, which were kept on ice, were used within 2 h of their preparation.…”

Section: Introductionmentioning

confidence: 99%

Resensitization of hepatocyte glucagon-stimulated adenylate cyclase can be inhibited when cyclic AMP phosphodiesterase inhibitors are used to elevate intracellular cyclic AMP concentrations to supraphysiological values

Murphy

Houslay

1988

Biochemical Journal

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Treatment of intact hepatocytes with glucagon led to the rapid desensitization of adenylate cyclase, which reached a maximum around 5 min after application of glucagon, after which resensitization ensued. Complete resensitization occurred some 20 min after the addition of glucagon. In hepatocytes which had been preincubated with the cyclic AMP phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine (IBMX), glucagon elicited a stable desensitized state where resensitization failed to occur even 20 min after exposure of hepatocytes to glucagon. Treatment with IBMX alone did not elicit desensitization. The action of IBMX in stabilizing the glucagon-mediated desensitized state was mimicked by the non-methylxanthine cyclic AMP phosphodiesterase inhibitor Ro-20-1724 [4-(3-butoxy-4-methoxylbenzyl)-2-imidazolidinone]. IBMX inhibited the resensitization process in a dose-dependent fashion with an EC50(concn. giving 50 % ofmaximal effect) of 26 + 5 ,M, which was similar to the EC50 value of 22 + 6,M observed for the ability of IBMX to augment the glucagon-stimulated rise in intracellular cyclic AMP concentrations. Pre-treatment of hepatocytes with IBMX did not alter the ability of either angiotensin or the glucagon analogue TH-glucagon, ligands which did not increase intracellular cyclic AMP concentrations, to cause the rapid desensitization and subsequent resensitization of adenylate cyclase. It is suggested that, although desensitization of glucagon-stimulated adenylate cyclase is elicited by a cyclic AMP-independent process, the resensitization of adenylate cyclase can be inhibited by a process which is dependent on elevated cyclic AMP concentrations. This action can be detected by attenuating the degradation of cyclic AMP by using inhibitors of cyclic AMP phosphodiesterase.

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Is the receptor‐mediated endocytosis of cholera toxin a pre‐requisite for its activation of adenylate cyclase in intact rat hepatocytes?

Cited by 36 publications

References 42 publications

Role of membrane gangliosides in the binding and action of bacterial toxins

Role of membrane gangliosides in the binding and action of bacterial toxins

Internalization and degradation of cholera toxin by cultured cells: relationship to toxin action.

Resensitization of hepatocyte glucagon-stimulated adenylate cyclase can be inhibited when cyclic AMP phosphodiesterase inhibitors are used to elevate intracellular cyclic AMP concentrations to supraphysiological values

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