Kinetic of extracellular ATP accumulation from hypotonically challenged hepatoma cells

SANCHEZ ALBERTI G, MV ESPELT, PJ SCHWARZBAUM

Congreso; First South American Spring Symposium in Signal Transduction and Molecular Medicine; 2010

In most animal cells, release of cytosolic ATP into the extracellular space occurs in response to osmotic gradients, with either cAMP and/or Ca2+i signaling such release. In this study, we evaluated the main processes governing the kinetic of extracellular ATP (ATPe) accumulation for HUH7 hepatoma cells exposed to hypotonicity. Exposure of cells to a 180 msoM hypotonic medium led to an exponential increase of [ATP]e to a maximum, followed by a slower non linear decrease. ATPe accumulation was inhibited 75% by carbenoxolone, an inhibitor of Pannexin1, whereas pre-treatment of cells with the purinergic receptor P2Y13 agonist 2-(Methylthio) adenosine 5’diphosphate (which inhibits adenylate cyclase) led to a 29% inhibition. Cells display ectoATPase activity ranging from 0.5 10-3 nmoles*sec-1*1e-6cells-1 to 4.9 10-3 nmoles*sec-1*1e-6cells-1. Since under these conditions cell mortality is negligible, the kinetic of ATPe is mainly controlled by ectoATPase activity (which consumes ATPe) and non lytic ATP efflux (which generates ATPe), with this latter efflux being partially inhibited by P2Y13 activation and partially mediated by Pannexin 1.