Dynamic regulation of extracellular ATP in<i>Escherichia coli</i>

Abstract

We studied the kinetics of extracellular ATP (ATPe) in <i>Escherichia coli</i> and their outer membrane vesicles (OMVs) stimulated with amphipatic peptides melittin (MEL) and mastoparan 7 (MST7). Real-time luminometry was used to measure ATPe kinetics, ATP release, and ATPase activity. The latter was also determined by following [³²P]Pi released from [γ-³²P]ATP. <i>E. coli</i> was studied alone, co-incubated with Caco-2 cells, or in rat jejunum segments. In <i>E. coli</i>, the addition of [γ-³²P]ATP led to the uptake and subsequent hydrolysis of ATPe. Exposure to peptides caused an acute 3-fold (MST7) and 7-fold (MEL) increase in [ATPe]. In OMVs, ATPase activity increased linearly with [ATPe] (0.1-1 µM). Exposure to MST7 and MEL enhanced ATP release by 3-7 fold, with similar kinetics to that of bacteria. In Caco-2 cells, the addition of ATP to the apical domain led to a steep [ATPe] increase to a maximum, with subsequent ATPase activity. The addition of bacterial suspensions led to a 6-7 fold increase in [ATPe], followed by an acute decrease. In perfused jejunum segments, exposure to <i>E. coli</i> increased luminal ATP 2 fold. ATPe regulation of <i>E. coli</i> depends on the balance between ATPase activity and ATP release. This balance can be altered by OMVs, which display their own capacity to regulate ATPe. <i>E. coli</i> can activate ATP release from Caco-2 cells and intestinal segments, a response which <i>in vivo</i> might lead to intestinal release of ATP from the gut lumen.