Extracellular ATP causes ROCK I-dependent bleb formation in P2X7-transfected HEK293 cells

• Published in: Molecular biology of the cell 2003-07, Vol.14 (7), p.2655-2664
• Main Authors: Morelli, Anna, Chiozzi, Paola, Chiesa, Anna, Ferrari, Davide, Sanz, Juana M, Falzoni, Simonetta, Pinton, Paolo, Rizzuto, Rosario, Olson, Michael F, Di Virgilio, Francesco
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - metabolism; Adenosine Triphosphate - physiology; Amides - pharmacology; Amino Acid Chloromethyl Ketones - pharmacology; Calcium - metabolism; Caspase Inhibitors; Cell Surface Extensions - metabolism; Cell Surface Extensions - physiology; Cells, Cultured; Cytoskeleton - metabolism; Cytoskeleton - physiology; Enzyme Inhibitors - pharmacology; Extracellular Space - metabolism; Humans; Intracellular Signaling Peptides and Proteins; Protein Serine-Threonine Kinases - antagonists & inhibitors; Protein Serine-Threonine Kinases - metabolism; Protein Serine-Threonine Kinases - physiology; Pyridines - pharmacology; Receptors, Purinergic P2 - metabolism; Receptors, Purinergic P2 - physiology; Receptors, Purinergic P2X7; rho-Associated Kinases; Transfection
• ISSN: 1059-1524
• DOI: 10.1091/mbc.02-04-0061

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.