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. 1988 Oct;85(19):7260–7264. doi: 10.1073/pnas.85.19.7260

Cholera toxin potentiates IgE-coupled inositol phospholipid hydrolysis and mediator secretion by RBL-2H3 cells.

M A McCloskey 1
PMCID: PMC282165  PMID: 2845404

Abstract

Binding of polyvalent antigens to IgE present in Fc epsilon receptors on the surface of mast cells and the RBL-2H3 cell line triggers the exocytotic release of allergic mediators. Preincubation of RBL-2H3 cells with cholera toxin (CT) was found to potentiate greater than or equal to 2- to 3-fold the rate and final amount of antigen-induced secretion of [3H]serotonin and N-acetyl beta-D-glucosaminidase. This was accompanied by a more variable increase in the initial rate of antigen-triggered formation of inositol phosphates. The holotoxin was required for potentiation, as neither the A nor the B subunit was effective when added separately. Four observations indicate that cAMP was not the primary effector of the augmentation of secretion caused by CT: (i) culture conditions were found in which CT caused large increases in secretion but very modest (or no) increases in cAMP; (ii) under other conditions, progressive increase in [CT] caused a maximum 2.5- to 3-fold increase in cAMP followed by a return to basal levels, whereas the secretory response saturated and remained stable; (iii) permeant cAMP analogs consistently enhanced secretion at low doses and inhibited at higher doses, but the peak enhancement was always much less than that achieved by an optimal dose of CT; (iv) the selective phosphodiesterase inhibitor Ro 20-1724 exhibited similar biphasic dose-response curves, the maximum enhancement again being small compared to that caused by CT itself. Both in vitro and in vivo, CT catalyzed transfer of ADP-ribose from NAD to two membrane proteins that comigrated on NaDodSO4/polyacrylamide gel electrophoresis with two CT substrates in other cell types, and these were identified by immunoblotting as Gs alpha. These results suggest that ADP-ribosylation of a cholera toxin substrate potentiates IgE-mediated secretion from RBL-2H3 cells by a largely cAMP-independent route.

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