CONICET | Buscador de Institutos y Recursos Humanos

ATP induce ATP release in hepatoma cells M. V. Espelt*, G. Sanchez Alberti*, O. Chara&, P. J. Schwarzbaum*- *IQUIFIB-CONICET, &CONICET -UNLP Background: Extracellular nucleotides regulate different physiological responses. In hepatic cells extracellular ATP regulates biological processes in a paracrine or autocrine way. The aim of our study was to analyze how ATPe activates ATP release in HepG2 cells. Methods: ATPe was quantified using the luciferin-luciferase method. Intracellular calcium was measured by fluorescence microscopy. Results: After addition of exogenous ATP, ATPe rised to a maximum followed 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 (n=5). When cells were preincubated with cibacron blue and suramin, 400 nM ATP induced 426.0±33.02 nM ATPe. ATP release was further increased when cells were preincubated with PI3K inhibitors and stimulated with 400 nM ATP to 823.4±36.16 nM (n=4), this release was 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 ATP release in HepG2 cells in concentrations higher than 100 nM. The release is further stimulated with PI3K inhibitors and inhibited with cibacron blue and suramin. ATPe increases Ca2+ spike frequency, which in principle could lead to ATP secretion.