ENTRY OF CHOLERA-TOXIN INTO POLARIZED HUMAN INTESTINAL EPITHELIAL-CELLS - IDENTIFICATION OF AN EARLY BREFELDIN-A SENSITIVE EVENT REQUIRED FOR A(1)-PEPTIDE GENERATION

The effect of brefeldin-A (BFA), a reversible inhibitor of vesicular t ransport, on cholera toxin (CT)-induced Cl secretion (I(sc)) was exami ned in the polarized human intestinal cell line, T84. Pretreatment of T84 monolayers with 5 muM BFA reversibly inhibited I(sc) in response t o apical or basolateral addition of 120 nM CT (2.4+/-0.5 vs. 68+/-3 mu A/cm2, n = 5). In contrast, BFA did not inhibit I(sc) responses to the cAMP agonist VIP (63+/-7 muA/cm2). BFA had no effect on cell surface binding and endocytosis of a functional fluorescent CT analog or on th e dose dependency of CT induced P-32-NAD ribosylation of Gsalpha in vi tro. 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 Cr-elicited I(sc). The early BFA-sensitive step occurred before a temperature-sen sitive step essential for apical CT action. These studies show that se quential steps are required for a biological response to apical CT: (a ) binding to cell surfaces and rapid endocytosis; (b) early, BFA-sensi tive vesicular transport essential for reduction of the A1-peptide; an d (c) subsequent temperature-sensitive translocation of a signal (the A1-peptide or possibly ADP-ribose-Gsalpha) to the basolateral domain.