Extracellular ATP regulation of Caco-2 cells

ZAHER K BAZZI, C L ALVAREZ, M F LEAL DENIS, I MARGINEDAS-FREIXA, M A. OSTUNI, V HERLAX, P J. SCHWARZBAUM

Congreso; Reunión Conjunta de Sociedades de Biociencias; 2017

In eukaryotic cells, intracellular ATP can be released by mechanicaland oxidative stress, exposure to toxins and calcium influx.These treatments mimic several conditions to which epithelial cellslining the intestine are exposed.In the intestinal lumen, extracellular ATP (ATPe) can be hydrolyzedby ectonucleotidases located on the apical domain of the epithelium.We analyzed the effect of several stimuli on ATPe regulation of thehuman intestinal Caco-2 cell line, a model of epithelial enterocytes.Real time luminometry was used to measure ATPe kinetics, ATPrelease and ATPase activity.In the absence of stimuli, Caco-2 cells displayed a regulated[ATPe] at approx. 20 nM, suggesting negligible ATP release. Additionof exogenous ATP (640 nM) led to an acute [ATPe] increase,followed by a non linear decay caused by ectoATPase activity, whichamounted to 320 nM/min.Next, we determined the effect of calcium influx (i.e., exposure toionophore A231287), mechanical perturbation and exposure to E.coli suspensions on ATP release of Caco-2 cells.All these stimuli led to similar ?but quantitatively different- ATPekinetics, with variable degrees of [ATPe] increase to a maximum(2-6 fold over basal levels), followed by an acute exponential decay.Results were compatible with fast activation of ATP exit, where therate of [ATPe] increase was partially balanced by ATPe hydrolysisdue to ectoATPase activity.In conclusion, ATPe kinetics of Caco-2 cells depends on the dynamicbalance between ATP release and ATPe hydrolysis. ATP exitis transient in nature, and sensitive to various physiologically relevantstimuli. EctoATPase activity is significant, but nevertheless muchslower that ATP exit during the acute phase of ATPe kinetics. Withgrants from CONICET (PIPC0459), UBA (20020130100027BA),ANPCYT (PICT0327) AND ECOS-Sud-MINCYT (A15S01).Keywords: extracellular ATP, ectoATPase, ATP efflux.