Signal transduction by cholera toxin in polarized epithelia:
processing in vesicular compartments does not require
acidification.
Lencer, W. I., G. Strohmeier, S. Moe, S. L. Carlson, C. T. Constable,
and J. L. Madara.
Combined Program in Pediatric Gastroenterology and Nutrition,
Children's Hospital; Division of Gastrointestinal Pathology, Brigham
and Women's Hospital, the Departments of Pediatrics, and Pathology of
Harvard Medical School, and the Harvard Digestive Diseases Center
APStracts 2:0072G, 1995.
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 intra-vesicular pH may regulate signal transduction by
CT, we examined the cAMP-dependent Cl- secretory response (Isc) to CT
in T84 cell monolayers treated with chloroquine (500 [mu]M),
methylamine (50 mM), NH4Cl (10 mM), nigericin (4 [mu]M), or
bafilomycin A1 (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 specificly affected adenylate
cyclase itself or coupling of adenylate cyclase with the
heterotrimeric GTPase Gs as judged by the secretory response to the
cAMP agonists vasoactive intestinal peptide (VIP), forskolin, or 8Br
-cAMP. In vitro ELISA showed that CT binding to GM1 was not dependent
on pH between 5.0 and 10. Maximal Isc elicited by apical CT relative
to maximal Isc elicited by VIP was not affected by pretreatment with
chloroquine, methylamine, NH4Cl, or bafilomycin A1. Nigericin was the
only reagent to inhibit CT-induced Isc (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 GM1, or on
subsequent processing/signal transduction events.
Received 21 September 1994; accepted in final form 11 April 1995.
APS Manuscript Number G367-4.
Article publication pending Am. J. Physiol. (Gastrointest. Liver
Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 25 April 1995.