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Articles in PresS, published online ahead of print March 13, 2002
Am J Physiol Gastrointest Liver Physiol, 10.1152/ajpgi.00484.2001
Submitted on November 15, 2001
Accepted on February 25, 2002
1 Departments of Medicine, University at Buffalo, Buffalo, NY, USA
2 Departments of Physiology and Biophysics, University at Buffalo, Buffalo, NY, USA
* To whom correspondence should be addressed. E-mail: jcrane{at}acsu.buffalo.edu.
Enteropathogenic E. coli (EPEC) causes severe, watery diarrhea in children. We investigated ATP release during EPEC-mediated killing of human cell lines, and whether released adenine nucleotides function as secretory mediators. Results. EPEC triggered a release of ATP from all human cell lines tested: HeLa, COS-7,and T84 (a colon cell line) as measured using a luciferase kit. Accumulation of ATP in the supernatant medium was enhanced if an inhibitor of 5'-ectonucleotidase was included, and further enhanced if an ATP regenerating system was added. In the presence of the inhibitor-regenerator, ATP concentrations in the supernatant medium reached 1.5 to 2 µM 4 h after infection with wild-type EPEC strains. ATP release by the cell death-deficient mutant espF was ~30% of wild-type in HeLa cells and ~60% of wildtype in T84 cells. In the absence of the inhibitor-regenerator system, extracellular ATP was rapidly broken down to ADP, AMP and adenosine. The amount of nucleotide release in conditioned media from EPEC-infected cells was sufficient to trigger a brisk chloride secretory response in intestinal tissues studied in the Ussing chamber. Conditioned medium from uninfected cells and sterile filtrates of EPEC bacteria produced no response, but conditioned medium from EPEC-infected cells triggered a short-circuit current (Isc) in naive monolayers. For example, 5 ml of EPEC-HeLa conditioned medium, collected in the absence of the inhibitor-regenerator, triggered an Isc of 13.8±5.4 µA/cm2 in rabbit colon; and 1 ml produced an Isc of 5.3±1.8 µA/cm2 in T84 cells grown on Snap-Well inserts, with the amplitude of the Isc being proportional to the amount of the test medium applied. The secretagogue had properties expected of AMP or adenosine, including stability to boiling, and passage through a low-MW cutoff filter. The Isc response of conditioned medium from EPEC-infected was completely reversed by adenosine receptor blockers such as 8-sulfophenyltheophylline and MRS1754. EPEC killing of host cells in vitro releases large amounts ATP, which is broken down to adenosine in the extracellular space. In the intestine, this adenosine activates adenosine receptors on the apical side of the cell, triggering a vigorous chloride secretion. These findings provide new insight into how EPEC causes watery diarrhea.
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