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Extracellular ATP Causes ROCK I-dependent Bleb Formation in P2X 7 -transfected HEK293 Cells

2003; American Society for Cell Biology; Volume: 14; Issue: 7 Linguagem: Inglês

10.1091/mbc.02-04-0061

1939-4586

Anna Morelli, Paola Chiozzi, Anna Maria Chiesa, Davide Ferrari, Juana María Sanz, Simonetta Falzoni, Paolo Pinton, Rosario Rizzuto, Michael F. Olson, Francesco Di Virgilio,

Phagocytosis and Immune Regulation

The P2X7 ATP receptor mediates the cytotoxic effect of extracellular ATP. P2X7-dependent cell death is heralded by dramatic plasma membrane bleb formation. Membrane blebbing is a complex phenomenon involving as yet poorly characterized intracellular pathways. We have investigated the effect of extracellular ATP on HEK293 cells transfected with the cytotoxic/pore-forming P2X7 receptor. Addition of ATP to P2X7-transfected, but not to wt P2X7-less, HEK293 cells caused massive membrane blebbing within 1-2 min. UTP, a nucleotide incapable of activating P2X7, had no early effects on cell shape and bleb formation. Bleb formation triggered by ATP was reversible and required extracellular Ca2+ and an intact cytoskeleton. Furthermore, it was completely prevented by preincubation with the P2X blocker oxidized ATP. It was recently observed that the ROCK protein is a key determinant of bleb formation. Preincubation of HEK293-P2X7 cells with the ROCK blocker Y-27632 completely prevented P2X7-dependent blebbing. Although ATP triggered cleavage of the ROCK I isoform in P2X7-transfected HEK293 cells, the wide range caspase inhibitor z-VAD-fluoromethylketone had no effect. These observations suggest that P2X7-dependent plasma membrane blebbing depends on the activation of the serine/threonine kinase ROCK I.