Entry of cholera toxin into polarized human intestinal epithelial cells. Identification of an early brefeldin A sensitive event required for A1-peptide generation.
The effect of brefeldin-A (BFA), a reversible inhibitor of vesicular transport, on cholera toxin (CT)-induced Cl- secretion (Isc) was examined in the polarized human intestinal cell line, T84. Pretreatment of T84 monolayers with 5 microM BFA reversibly inhibited Isc in response to apical or basolateral addition of 120 nM CT (2.4 +/- 0.5 vs. 68 +/- 3 microA/cm2, n = 5). In contrast, BFA did not inhibit Isc responses to the cAMP agonist VIP (63 +/- 7 microA/cm2). BFA had no effect on cell surface binding and endocytosis of a functional fluorescent CT analog or on the dose dependency of CT induced 32P-NAD ribosylation of Gs alpha in vitro. In contrast, BFA completely inhibited (> 95%) the ability of T84 cells to reduce CT to the enzymatically active A1-peptide. BFA had to be added within the first 10 min of CT exposure to inhibit CT-elicited Isc. The early BFA-sensitive step occurred before a temperature-sensitive step essential for apical CT action. These studies show that sequential steps are required for a biological response to apical CT: (a) binding to cell surfaces and rapid endocytosis; (b) early, BFA-sensitive vesicular transport essential for reduction of the A1-peptide; and (c) subsequent temperature-sensitive translocation of a signal (the A1-peptide or possibly ADP-ribose-Gs alpha) to the basolateral domain.
Lencer
W I
WI
Combined Program in Pediatric Gastroenterology and Nutrition, Children's Hospital, Boston, Massachusetts 02115.
de Almeida
J B
JB
Moe
S
S
Stow
J L
JL
Ausiello
D A
DA
Madara
J L
JL
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000704:Analysis of Variance; D001692:Biological Transport / Q000187:drug effects; D020126:Brefeldin A; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism; D002772:Cholera Toxin / Q000378:metabolism* / Q000633:toxicity*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003517:Cyclopentanes / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism; D007700:Kinetics; D008954:Models, Biological; D009243:NAD / Q000378:metabolism; D011485:Protein Binding; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D013696:Temperature; D013997:Time Factors; D014660:Vasoactive Intestinal Peptide / Q000494:pharmacology
D002712:Chlorides; D003517:Cyclopentanes; D011500:Protein Synthesis Inhibitors; D009243:NAD; D020126:Brefeldin A; D015124:8-Bromo Cyclic Adenosine Monophosphate; D014660:Vasoactive Intestinal Peptide; D002772:Cholera Toxin; D017868:Cyclic AMP-Dependent Protein Kinases