title: P2X7 receptor-dependent blebbing and the activation of Rho-effector kinases, caspases, and IL-1 beta release.
abstract: In response to ATP binding, the P2X7R facilitates cation channel activation, nonspecific pore formation, rapid changes in plasma membrane morphology, and secretion of IL-1 beta from LPS-primed macrophages. To investigate the relationship between the P2X7R-dependent changes in plasma membrane organization and the release of IL-1 beta, we generated time-lapse movies of ATP-stimulated BAC1 murine macrophages in conjunction with biochemical analyses of IL-1 beta release. Similar image analyses in human embryonic kidney 293 cells expressing recombinant P2X7R (HEK-P2X7) permitted comparison of P2X7R-dependent effects in macrophage vs nonmacrophage backgrounds. Whereas HEK-P2X7 cells exhibit zeiotic blebbing within 5 min of ATP treatment, BAC1 macrophages initiated a distinct "tethered" blebbing 10 min after ATP addition. This blebbing was comparably induced by the P2X7R-selective agonist BzATP and was blocked by P2X7R inhibitors KN-62 and oxidized ATP. Blebbing was initiated at ATP concentrations > or = 3 mM, but optimal IL-1 beta release occurred at 1 mM ATP. P2X7R-dependent blebbing was abrogated in the presence of Rho-effector kinase inhibitors Fasudil and Y-27632, but ATP-induced IL-1 beta release was unaffected. ATP-induced activation of RhoA could be detected in both HEK-P2X7 cells and BAC1 murine macrophages. Thus, P2X7R activation signals distinct, novel membrane blebbing events (dependent on RhoA activation and Rho-effector kinase activity) and simultaneously initiates release of IL-1 beta. Our observations that blebbing and IL-1 beta release are dissociable suggest these events occur via parallel rather than convergent signaling pathways.
authors.affiliation: Department of Pharmacology, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA.
authors.forename: Philip A
authors.initials: PA
authors.lastname: Verhoef
authors.forename: Mark
authors.initials: M
authors.lastname: Estacion
authors.forename: William
authors.initials: W
authors.lastname: Schilling
authors.forename: George R
authors.initials: GR
authors.lastname: Dubyak
mesh_terms: D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000037:antagonists & inhibitors / Q000502:physiology; D000577:Amides / Q000494:pharmacology; D000818:Animals; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D002460:Cell Line; D004789:Enzyme Activation / Q000187:drug effects; D005110:Extracellular Space / Q000502:physiology; D006801:Humans; D007375:Interleukin-1 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D008264:Macrophages / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D051379:Mice; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011554:Pseudopodia / Q000187:drug effects / Q000502:physiology; D011725:Pyridines / Q000494:pharmacology; D018048:Receptors, Purinergic P2 / Q000378:metabolism / Q000502:physiology*; D058486:Receptors, Purinergic P2X7; D015398:Signal Transduction / Q000502:physiology; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
chemical_list: D000577:Amides; D061945:Caspase Inhibitors; D007375:Interleukin-1; D047908:Intracellular Signaling Peptides and Proteins; C546468:P2RX7 protein, human; C546469:P2rx7 protein, mouse; D011725:Pyridines; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; C108830:Y 27632; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000255:Adenosine Triphosphate; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D020169:Caspases; D020742:rhoA GTP-Binding Protein; C049347:fasudil