Interactions of cholera toxin with isolated hepatocytes. Effects of low pH, chloroquine and monensin on toxin internalization, processing and action.
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.
Janicot
M
M
Institut National de la Santé et de la Recherche Médicale, Unité 30, Hôpital des Enfants Malades, Paris, France.
Clot
J P
JP
Desbuquois
B
B
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002738:Chloroquine / Q000494:pharmacology; D002772:Cholera Toxin / Q000378:metabolism / Q000494:pharmacology*; D000242:Cyclic AMP / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D007106:Immune Sera; D066298:In Vitro Techniques; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D008985:Monensin / Q000494:pharmacology; D010455:Peptides / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains
D007106:Immune Sera; D010455:Peptides; D002738:Chloroquine; D002772:Cholera Toxin; D008985:Monensin; D000242:Cyclic AMP; D000262:Adenylyl Cyclases