CONICET | Buscador de Institutos y Recursos Humanos

In most eukaryotic cells, intracellular ATP can be released by mechanical andoxidative stress, calcium influx and/or exposure to toxins. These treatments mimicseveral conditions to which epithelial cells lining the human intestine are exposed.In the intestinal lumen, extracellular ATP (ATPe) can activate purinergic receptors,which can modulate ATP release. Additionally, ATPe can be hydrolyzed byectonucleotidases located on the apical domain of the epithelium.To gain insight into ATPe regulation of the intestine, we analyzed the effects ofseveral stimuli on ATP efflux, purinergic activation and [ATPe] hydrolysis of thehuman intestinal Caco-2 cell line, a model of epithelial enterocytes.Real time luminometry was used to measure ATPe kinetics and ATPase activity.In the absence of stimuli, Caco-2 cells displayed a stable [ATPe] at approx. 20±5nM, suggesting negligible ATP release. Addition of exogenous ATP (0.5-8 μM) led toan acute [ATPe] increase, followed by a non linear decay caused by ectoATPaseactivity. This was a linear function of [ATP], with slope (KATP) being 0.044 min-1.Next, we determined the effect of calcium influx and the mechanical perturbationon ATP release. All these stimuli led to similar ATPe kinetics, with variable degreesof [ATPe] increase to a maximum (2-6 fold over basal levels), followed by an acuteexponential decay. Under mechanical stress in the presence of various blockers,ATP release exhibited exocytotic and conductive components, the latter beingmediated by Pannexin1. Blockage of P2X purinergic receptors reduced ATP releaseby 57-66%.Results shows that upon activation of ATP release, the accumulated ATPe activatedpurinergic receptors that promoted further activation of ATP exit. Simultaneously,EctoATPase activity partially compensated these increases in [ATPe]. Thus, ATPekinetics of Caco-2 cells resulted from the dynamic balance between ATP release,purinergic activation and ATPe hydrolysis.