Extracellular ATP regulation of Caco-2 cells. Zaher K. Bazzi, Cora L. Alvarez, Paula Faillace, Débora Rinaldi, Pablo J. Schwarzbaum, Julieta Schachter.

ZAHER K. BAZZI,; CORA L. ALVAREZ; M. PAULA FAILLACE; DÉBORA RINALDI; PABLO J. SCHWARZBAUM; JULIETA SCHACHTER

Rosario

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

Sociedad Argentina de Fisiología (SAFIS)

In many eukaryotic cells, intracellular ATP can be releasedby several stimuli as mechanical stress, calcium influx and exposure to osmotic change. Candidate conduits for ATP exit include anionic channels and exocytosis. In the intestinal lumen, extracellular ATP (ATPe) can activate purinergic receptors, which can modulate ATP release. In addition, ATPe can be hydrolyzed by ectonucleotidases located on the apical domain of the epithelium. Thus, ATP release, purinergic activation and ectonucleotidases can regulate ATPe concentration.To gain insight into ATPe regulation in the intestine epithelium, we analyzed the effects of a hypotonic shock on ATP efflux, purinergic activation and ATPe hydrolysis of the human intestinal Caco-2 cell line. ATPe kinetics and ectoATPase activity were assessed by real time luminometric detection of [ATPe] and colorimetric and radioactive techniques to quantify Pi release from ATP and other nucleotides. The presence of ectonucleotidases was assessed by immunocytochemistry.In the absence of stimuli, Caco-2 cells displayed a stableATPe concentration at approx. 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. Using pharmacological tools, we observed that ATP release exhibited exocytotic and conductive components, the latter being mediated by Pannexin-1. Blockage of P2X receptors reduced ATP release by 45-50%. On the other hand, Caco-2 cells exhibited significant 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 activation of ATP exit. Simultaneously, ectoATPase activity partially compensated these increases in [ATPe]. Thus, ATPe kinetics of Caco-2 cells resulted from the dynamic balance between ATP release, purinergic activation and ATPe hydrolysis.