ATP induce ATP relesae in hepatoma cells

ESPELT MV, SANCHEZ ALBERTI G, CHARA O, SCHWARZBAUM PJ

Barcelona

Congreso; International Purine Meeting; 2010

Background: In hepatic cells extracellular nucleotides act via P receptors regulating different physiological responses as:glucogenolysis, gluconeogenesis, canalicular contraction, etc. The aim of our study was to analyze how extracellular ATP(ATPe) activates ATP release in HepG2 cells. Methods: ATPe was quantified using the luciferin-luciferase method. Intracellularcalcium was measured by fluorescence microscopy. Results: After addition of exogenous ATP, ATPe rised to a maximumfollowed by an exponential decay. The maximum reached was 43.1±3.01 nM (with 50 nM ATP added; n=6), 135.0±19.25 nM(100 nM ATP; n=7), 269.0±26.78 nM (200 nM ATP; n=7), and 555.8±39.70 nM (400 nM, n=5). When cells werepreincubated with cibacron blue and suramin (P2 blockers), 400 nM ATP induced 426.0±33.02 nM ATPe. ATP releaseincreased to 823.4±36.16 nM when cells were stimulated with 400 nM ATP (n=4) and preincubated with PI3K inhibitors,being this release inhibited with P2 inhibitors (640.2±71.45 nM). Regarding potential 2nd messengers activating ATP release,we determined the percentage of cells showing at least one spike in Ca2+ after incubation with different ATPe concentrations.The spike frequency augmented hyperbolically with ATPe concentration with a K0.5=3.8 uM. Conclusion: ATP induce ATPrelease in HepG2 cells in concentrations between 100–400 nM. The release is further stimulated with PI3K inhibitors andinhibited with cibacron blue and suramin. ATPe increases Ca2+ spike frequency, which in principle could lead to ATP secretion.