scholarly journals Interactions of cholera toxin with isolated hepatocytes. Effects of low pH, chloroquine and monensin on toxin internalization, processing and action

1988 ◽  
Vol 253 (3) ◽  
pp. 735-743 ◽  
Author(s):  
M Janicot ◽  
J P Clot ◽  
B Desbuquois
Keyword(s):  
Plasma Membrane ◽  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Acidic Medium ◽  
Isolated Hepatocytes ◽  
Surface Generation ◽  
Lag Phase ◽  
Single Class ◽  
Endosomal Membrane ◽  
A Subunit

The major steps in cholera-toxin action, i.e. binding, internalization, generation of A1 peptide and activation of adenylate cyclase, were examined in isolated hepatocytes. The binding of toxin involves a single class of high-affinity sites (KD congruent to 0.1 nM; Bmax. congruent to 10(7) sites/cell). At 37 degrees C, cell-associated toxin is progressively internalized, as judged by the loss of its accessibility to antibodies against whole toxin, A and B subunits (about 50, 75 and 30% of initially bound toxin after 40 min respectively). Two distinct pathways are involved in this process: endocytosis of the whole toxin, and selective penetration of the A subunit into the plasma membrane. Exposure of hepatocytes to an acidic medium (pH 5) results in a rapid and marked disappearance of the A subunit from the cell surface. Generation of A1 peptide and activation of adenylate cyclase by the toxin occur after a lag phase (10 min at 37 degrees C), and increase with time in a parallel manner up to 2-3% A1 peptide generated; they are unaffected by exposure of cells to an acidic medium. Chloroquine and monensin, which elevate the pH in acidic organelles, inhibit by 2-4-fold both the generation of A1 peptide and the activation of adenylate cyclase. Unexpectedly, these drugs also inhibit the internalization of the toxin. These results suggest that an acidic pH facilitates the penetration of A subunit into the plasma membrane and presumably the endosomal membrane as well, and that endocytosis of cholera toxin is required for generation of A1 peptide and activation of adenylate cyclase.

scholarly journals Requirement for guanosine triphosphate for cholera-toxin-catalysed incorporation of adenosine diphosphate ribose into rat liver plasma membranes and for activation of adenylate cyclase

1980 ◽  
Vol 186 (3) ◽  
pp. 749-754 ◽  
Author(s):  
C A Doberska ◽  
A J S MacPherson ◽  
B R Martin
Keyword(s):  
Plasma Membrane ◽  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Adenosine Diphosphate ◽  
Activation Process ◽  
Liver Plasma Membrane ◽  
Adp Ribosylation ◽  
A Subunit

1. Cholera toxin was shown to require the presence of GTP to activate rat liver plasma-membrane adenylate cyclase. ATP did not affect the activation process. 2. Cholera toxin catalysed the incorporation of 32P from NAD labelled in the alpha-phosphate group of the ADP moiety into a rat liver plasma-membrane protein with a subunit mol.wt. of 42 500. This is taken to demonstrate ADP-ribosylation. The ADP-ribosylation of this protein also required GTP and was unaffected by ATP. 3. Nicotinamide inhibited both the activation of adenylate cyclase by cholera toxin and the ADP-ribosylation of the protein of 42 500 subunit mol wt. Neither the activation nor the ADP-ribosylation could be reversed by treatment with nicotinamide in the presence of cholera toxin.

scholarly journals Gangliosides That Associate with Lipid Rafts Mediate Transport of Cholera and Related Toxins from the Plasma Membrane to Endoplasmic Reticulm

2003 ◽  
Vol 14 (12) ◽  
pp. 4783-4793 ◽  
Author(s):  
Yukako Fujinaga ◽  
Anne A. Wolf ◽  
Chiara Rodighiero ◽  
Heidi Wheeler ◽  
Billy Tsai ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Cholera Toxin ◽  
Lipid Rafts ◽  
Intestinal Cells ◽  
B Subunit ◽  
High Affinity ◽  
A Subunit ◽  
Human Intestinal Cells

Cholera toxin (CT) travels from the plasma membrane of intestinal cells to the endoplasmic reticulum (ER) where a portion of the A-subunit, the A1 chain, crosses the membrane into the cytosol to cause disease. A related toxin, LTIIb, binds to intestinal cells but does not cause toxicity. Here, we show that the B-subunit of CT serves as a carrier for the A-subunit to the ER where disassembly occurs. The B-subunit binds to gangliosides in lipid rafts and travels with the ganglioside to the ER. In many cells, LTIIb follows a similar pathway, but in human intestinal cells it binds to a ganglioside that fails to associate with lipid rafts and it is sorted away from the retrograde pathway to the ER. Our results explain why LTIIb does not cause disease in humans and suggest that gangliosides with high affinity for lipid rafts may provide a general vehicle for the transport of toxins to the ER.

scholarly journals Lack of vasopressin receptors in liver, but not in kidney, of ob/ob mice

1983 ◽  
Vol 216 (2) ◽  
pp. 475-480 ◽  
Author(s):  
F Assimacopoulos-Jeannet ◽  
B Cantau ◽  
G van de Werve ◽  
S Jard ◽  
B Jeanrenaud
Keyword(s):  
Plasma Membrane ◽  
Adenylate Cyclase ◽  
Plasma Membranes ◽  
Metabolic Response ◽  
Isolated Hepatocytes ◽  
The Other ◽  
Kidney Medulla ◽  
Liver Plasma Membrane ◽  
Liver Plasma Membranes ◽  
Vasopressin Receptors

The activity of phosphorylase a was measured in isolated hepatocytes from fed lean and ob/ob mice after addition of vasopressin, angiotensin, phenylephrine and glucagon. The binding of these hormones to purified liver plasma membranes was also determined. In hepatocytes of ob/ob mice, no increase in phosphorylase a was measured after addition of vasopressin, whereas the other hormones promoted an increase in the activity of the enzyme. No specific vasopressin receptors could be measured on purified liver plasma membrane of ob/ob mice. A decrease in the number of receptors for angiotensin and glucagon, without modification of the affinity, was also observed. No restoration of the number of vasopressin receptors was observed in liver of ob/ob mice starved for 3 days or in younger (5-6 weeks) animals. Vasopressin receptors and vasopressin-stimulated adenylate cyclase, measured on purified kidney medulla membranes, were similar in both lean and ob/ob mice. The data indicate a selective lack of vasopressin receptors and metabolic response in liver of the ob/ob mouse.

The hysteretic effect of Gpp(NH)p on adenylate cyclase is not altered by Mg2+ in adipocyte membranes of ob/ob mice

1986 ◽  
Vol 64 (9) ◽  
pp. 855-863 ◽  
Author(s):  
Nicole Bégin-Heick
Keyword(s):  
Adipose Tissue ◽  
Steady State ◽  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Incubation Medium ◽  
Time Course ◽  
Inhibitory Effect ◽  
Lag Phase ◽  
Guanine Nucleotide ◽  
Hysteretic Effect

We have established previously that the regulation of adenylate cyclase is abnormal in adipose tissue membranes of ob/ob mice. To help establish the nature of the defect, we studied the time course of guanine nucleotide activation and inhibition of adenylate cyclase. The activation of adenylate cyclase by Gpp(NH)p in adipocyte membranes of normal (+/+) and ob/ob mice proceeds with a lag phase. In +/+ membranes, this lag could be shortented by increasing the concentration of Mg2+ in the incubation medium or by pretreatment of the membranes with cholera toxin, and it could be abolished by isoproterenol in combination with 4 mM MgCl2. In contrast, in the ob/ob membranes, only pretreatment with cholera toxin was effective in shortening the lag phase. These results indicate an impediment in the activation of adenylate cyclase in ob/ob membranes. In the +/+ membranes, Gpp(NH)p inhibited forskolin-stimulated adenylate cyclase, following a short lag phase, producing lower steady-state velocities than those seen with forskolin alone. The inhibitory effect of Gpp(NH)p on forskolin-stimulated activity was abolished by pertussis but not by cholera toxin treatment. In the ob/ob membranes, neither Gpp(NH)p nor pertussis treatment had any effect on the steady-state velocity of the forskolin-stimulated activity. These data have been interpreted as meaning that an anomaly in Ni rather than in Ns is likely to be responsible for the impairment of adenylate cyclase activity in the membranes of the ob/ob mouse.

scholarly journals Activity of covalently cross-linked cholera toxin with the adenylate cyclase of intact and lysed pigeon erythrocytes

1977 ◽  
Vol 168 (3) ◽  
pp. 457-463 ◽  
Author(s):  
S van Heyningen
Keyword(s):  
Cell Surface ◽  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Dimethyl Ester ◽  
Lag Phase ◽  
Ganglioside Gm1 ◽  
Whole Cells ◽  
Subunit B

Reaction of cholera toxin with NN'-bis(carboximidomethyl)tartaramide dimethyl ester produced several cross-linked species that had subunit B (which binds to the cell surface) and peptides A1 (which activates adenylate cyclase) and A2 all covalently joined together. This cross-linded material had activity with pigeon erythrocytes that was comparable in all respects with that of native toxin. It activated the adenylate cyclase of whole cells, showing a characteristic lag phase, and this activation was increased if the cells had been preincubated with ganglioside GM1, but abolished if the protein had been preincubated with the ganglioside. It activated the enzyme in lysed cells more strongly and without the lag phase. These results show that the toxin is active even when peptide A1 cannot be released from the rest of the molecule.

PGE2, forskolin, and cholera toxin interactions in rabbit cortical collecting tubule

1986 ◽  
Vol 250 (1) ◽  
pp. F127-F135 ◽  
Author(s):  
S. P. Nadler ◽  
S. C. Hebert ◽  
B. M. Brenner
Keyword(s):  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Regulatory Protein ◽  
Inhibitory Effect ◽  
Lag Phase ◽  
Collecting Tubule ◽  
Dose Dependent Fashion ◽  
Pg E2 ◽  
Rabbit Cortical Collecting Tubule ◽  
Cortical Collecting Tubule

To define further the mechanism whereby prostaglandin (PG) E2 inhibits the hydroosmotic response to ADH, we studied the interactions of PGE2 with ADH and two nonhormonal activators of adenylate cyclase, forskolin and cholera toxin, in the isolated perfused rabbit cortical collecting tubule. Forskolin increased hydraulic conductivity (LP) in a dose-dependent fashion and to a degree comparable with ADH-stimulated LP. Forskolin also augmented maximal ADH-stimulated LP, from 135 +/- 15 (SE) to 174 +/- 7 . 10(-7) cm . s-1 . atm-1. Following a 45-min lag phase, 10(-9) M cholera toxin at 37 degrees C increased LP to 107 +/- 12 . 10(-7) cm . s-1 . atm-1, a response that was stable with time. In paired studies at both 25 and 37 degrees C, PGE2 reversibly inhibited ADH-stimulated LP by 45 and 47%, respectively. However, the same protocols with PGE2 and forskolin failed to reveal any inhibitory effect of PGE2 on forskolin-stimulated LP. PGE2 reversibly inhibited cholera toxin-stimulated LP, from 124 +/- 15 to 100 +/- 15 . 10(-7) cm . s-1 . atm-1. These results support the view that PGE2 inhibits ADH-stimulated LP by inhibiting the synthesis of cAMP and suggest that this inhibition occurs at a functional site at or distal to the nucleotide regulatory protein of adenylate cyclase.

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