SIGNAL-TRANSDUCTION BY CHOLERA-TOXIN - PROCESSING IN VESICULAR COMPARTMENTS DOES NOT REQUIRE ACIDIFICATION

In the polarized human intestinal epithelial cell line T84, signal transduction by cholera toxin (CT) follows a complex series of events in which CT enters the apical endosome and moves through multiple vesicular compartments before it activates adenylate cyclase. As with processing of many other surface ligands, it has been suggested that CT must enter acidic vesicles to exert its downstream effects. To determine if intravesicular pH may regulate signal transduction by CT, we examined the cAMP-dependent Cl- secretory response [short-circuit current (I-SC)] to CT in T84 cell monolayers treated with chloroquine (500 mu M), methylamine (50 mM), NH4Cl(10 mM), nigericin (4 mu M), or bafilomycin A(1) (1 mu M). Each of these reagents collapsed intravesicular pH gradients as confirmed by accumulation of acridine orange within subcellular compartments of living T84 cells imaged by confocal epifluorescence microscopy. Both acidotropic amines and nigericin inhibited the cAMP-dependent Cl secretory response in T84 cells. However, none of these reagents specifically affected adenylate cyclase itself or coupling of adenylate cyclase with the heterotrimeric guanosinetriphosphatase G(s) as judged by the secretory response to the adenosine 3',5'-cyclic monophosphate (cAMP) agonists vasoactive intestinal peptide (VIP), forskolin, or 8-bromo-cAMP. In vitro enzyme-linked immunosorbent assay showed that CT binding to ganglioside G(M1) was not dependent on pH between 5.0 and 10. Maximal I-SC elicited by apical CT relative to maximal I-SC elicited by VIP was not affected by pretreatment with chloroquine, methylamine, NH4Cl, or bafilomycin A(1). Nigericin was the only reagent to inhibit CT-induced I-SC (5 +/- 2% maximal response to VIP). The data indicate that low intravesicular pH will have little or no effect on CT association with its membrane receptor G(M1), or On subsequent processing/ signal transduction events.