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

• Autor(a) principal: Almeida, Jose Bruno de
• Data de Publicação: 1993
• Outros Autores: Lencer, W. I., Moe, S., Stow, Jennifer L., Ausiello, Dennis A., Madara, J. L.
• Tipo de documento: Artigo
• Idioma: por
• Título da fonte: Repositório Institucional da UFRN
• Texto Completo: https://repositorio.ufrn.br/handle/123456789/54382
• topic: cholera toxin; brefeldin-A; intestinal epithelial cells

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