title: Ecto-5'-nucleotidase and intestinal ion secretion by enteropathogenic Escherichia coli.
abstract: Enteropathogenic Escherichia coli (EPEC) triggers a large release of adenosine triphosphate (ATP) from host intestinal cells and the extracellular ATP is broken down to adenosine diphosphate (ADP), AMP, and adenosine. Adenosine is a potent secretagogue in the small and large intestine. We suspected that ecto-5'-nucleotidase (CD73, an intestinal enzyme) was a critical enzyme involved in the conversion of AMP to adenosine and in the pathogenesis of EPEC diarrhea. We developed a nonradioactive method for measuring ecto-5'-nucleotidase in cultured T84 cell monolayers based on the detection of phosphate release from 5'-AMP. EPEC infection triggered a release of ecto-5'-nucleotidase from the cell surface into the supernatant medium. EPEC-induced 5'-nucleotidase release was not correlated with host cell death but instead with activation of phosphatidylinositol-specific phospholipase C (PI-PLC). Ecto-5'-nucleotidase was susceptible to inhibition by zinc acetate and by alpha,beta-methylene-adenosine diphosphate (alpha,beta-methylene-ADP). In the Ussing chamber, these inhibitors could reverse the chloride secretory responses triggered by 5'-AMP. In addition, alpha,beta-methylene-ADP and zinc blocked the ability of 5'-AMP to stimulate EPEC growth under nutrient-limited conditions in vitro. Ecto-5'-nucleotidase appears to be the major enzyme responsible for generation of adenosine from adenine nucleotides in the T84 cell line, and inhibitors of ecto-5'-nucleotidase, such as alpha,beta-methylene-ADP and zinc, might be useful for treatment of the watery diarrhea produced by EPEC infection.
authors.affiliation: Department of Medicine, Division of Infectious Diseases, University at Buffalo, Room 317 Biomedical Research Bldg., 3435 Main St., Buffalo, NY, 14214, USA, jcrane@acsu.buffalo.edu.
authors.forename: John K
authors.initials: JK
authors.lastname: Crane
authors.forename: Irina
authors.initials: I
authors.lastname: Shulgina
authors.forename: Tonniele M
authors.initials: TM
authors.lastname: Naeher