Entry of cholera toxin into polarized human intestinal epithelial cells: Identification of an early brefeldin A sensitive event required for A1-peptide generation

Abstract

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 μM BFA reversibly inhibited ISC in response to apical or basolateral addition of 120 nM CT (2.4±0.5 vs. 68±3 μA/cm2, n= 5). In contrast, BFA did not inhibit ISC responses to the cAMP agonist VIP (63±7 μA/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 Gsα 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-Gsα) to the basolateral domain.