Extracellular ATP regulation of Caco-2 cells

CORA LILIA ALVAREZ; MARIANO OSTUNI; ZAHER K BAZZI; IRENE MARGINEDAS-FREIXA; PABLO SCHWARZBAUM,; MARIA FLORENCIA LEAL DENIS; VANESA HERLAX

Cuidad Autónoma de Buenos Aires

Congreso; Reunión Conjunta de Sociedades de BioCiencias; 2017

In eukaryotic cells, intracellular ATP can be released by mechanical and oxidative stress, exposure to toxins and calcium influx. These treatments mimic several conditions to which epithelial cells lining the intestine are exposed.In the intestinal lumen, extracellular ATP (ATPe) can be hydrolyzed by ectonucleotidases located on the apical domain of the epithelium. We analyzed the effect of several stimuli on ATPe regulation of the human intestinal Caco-2 cell line, a model of epithelial enterocytes.Real time luminometry was used to measure ATPe kinetics, ATP release and ATPase activity.In the absence of stimuli, Caco-2 cells displayed a regulated [ATPe] at approx. 20 nM, suggesting negligible ATP release. Addition of exogenous ATP (640 nM) led to an acute [ATPe] increase, followed by a non linear decay caused by ectoATPase activity, which amounted to 320 nM/min.Next, we determined the effect of calcium influx (i.e., exposure to ionophore 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- ATPe kinetics, 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 the rate of [ATPe] increase was partially balanced by ATPe hydrolysis due to ectoATPase activity.In conclusion, ATPe kinetics of Caco-2 cells depends on the dynamic balance between ATP release and ATPe hydrolysis. ATP exit is transient in nature, and sensitive to various physiologically relevant stimuli. EctoATPase activity is significant, but nevertheless much slower that ATP exit during the acute phase of ATPe kinetics. With grants from CONICET (PIPC0459), UBA (20020130100027BA), ANPCYT (PICT0327) AND ECOS-Sud-MINCYT (A15S01).Keywords: extracellular ATP, ectoATPase, ATP efflux.