Extracellular ATP regulation of Caco-2 cells

BASSI, Z.; ALVAREZ, C.L.; FAILLACE, P.; RINALDI, D.E; SCHWARZBAUM, P.J.; SCHACHTER,J.

Rosario

Congreso; Sociedad Argentina de Fisiología. Reunión Anual 2019; 2019

In many eukaryotic cells, intracellular ATP can be released by several stimuli asmechanical stress, calcium influx and exposure to osmotic change. Candidate conduitsfor ATP exit include anionic channels and exocytosis. In the intestinal lumen,extracellular ATP (ATPe) can activate purinergic receptors, which can modulate ATPrelease. In addition, ATPe can be hydrolyzed by ectonucleotidases located on theapical domain of the epithelium. Thus, ATP release, purinergic activation andectonucleotidases can regulate ATPe concentration.To gain insight into ATPe regulation in the intestine epithelium, we analyzed the effectsof a hypotonic shock on ATP efflux, purinergic activation and ATPe hydrolysis of thehuman intestinal Caco-2 cell line. ATPe kinetics and ectoATPase activity wereassessed by real time luminometric detection of [ATPe] and colorimetric andradioactive techniques to quantify Pi release from ATP and other nucleotides. Thepresence of ectonucleotidases was assessed by immunocytochemistry.In the absence of stimuli, Caco-2 cells displayed a stable ATPe concentration atapprox. 20 ± 5 nM. Addition of exogenous ATP (0.5 - 8 µM) led to an acute [ATPe]increase, followed by a non linear decay caused by ectoATPase activity.Next, we determined the effect of hypo-osmotic stress on ATP release. Usingpharmacological tools, we observed that ATP release exhibited exocytotic andconductive components, the latter being mediated by Pannexin-1. Blockage of P2Xreceptors reduced ATP release by 45-50 %. On the other hand, Caco-2 cells exhibitedsignificant ectoATPase and ectoAMPase activities, but low ectoADPase activity.Accordingly, we detected the presence of NTPDase 1, 2 and ecto-5′-nucleotidase(ectoAMPase) in Caco-2 cells.ATP efflux was fast and sensitive to osmotic shock. Upon activation of ATP release,the accumulated ATPe activated purinergic receptors that promoted further activationof ATP exit. Simultaneously, ectoATPase activity partially compensated theseincreases in [ATPe]. Thus, ATPe kinetics of Caco-2 cells resulted from the dynamicbalance between ATP release, purinergic activation and ATPe hydrolysis.