Extracellular ATP causes ROCK I-dependent bleb formation in P2X7-transfected HEK293 cells.
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.
Morelli
Anna
A
Department of Experimental and Diagnostic Medicine, Section of General Pathology, and Interdisciplinary Center for the Study of Inflammation, University of Ferrara, I-44100 Ferrara, Italy.
Chiozzi
Paola
P
Chiesa
Anna
A
Ferrari
Davide
D
Sanz
Juana M
JM
Falzoni
Simonetta
S
Pinton
Paolo
P
Rizzuto
Rosario
R
Olson
Michael F
MF
Di Virgilio
Francesco
F
D000255:Adenosine Triphosphate / Q000378:metabolism* / Q000502:physiology; D000577:Amides / Q000494:pharmacology; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D061945:Caspase Inhibitors; D022081:Cell Surface Extensions / Q000378:metabolism / Q000502:physiology; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000378:metabolism / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005110:Extracellular Space / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000502:physiology; D011725:Pyridines / Q000494:pharmacology; D018048:Receptors, Purinergic P2 / Q000378:metabolism* / Q000502:physiology; D058486:Receptors, Purinergic P2X7; D014162:Transfection; D054460:rho-Associated Kinases
D000577:Amides; D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; C546468:P2RX7 protein, human; D011725:Pyridines; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C108830:Y 27632; D000255:Adenosine Triphosphate; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D002118:Calcium