Pseudoapoptosis Induced by Brief Activation of ATP-gated P2X7 Receptors

Abstract

P2X7 receptors are ATP-gated ion channels primarily expressed on antigen-presenting immune cells where they play a role in the acute inflammatory response. These ion channels couple not only to influx of cations, including calcium, but also to rapid alterations in cell morphology (membrane blebbing, phosphatidylserine exposure, microvesicle shedding). These features resemble the extranuclear events associated with end stages of apoptosis but cell death does not occur if receptor activation is brief. Here we delineate two signaling pathways underlying these apoptotic-like processes. Loss of membrane asymmetry occurs within seconds, which directly triggers cytoskeletal disruption and zeiotic membrane blebbing; this is readily reversible and requires both calcium influx through P2X7 channels and mitochondrial calcium increase but is not associated with cytochrome c release. A slower, calcium-independent, ROCK-1-dependent cascade that does not involve rapid loss of membrane asymmetry but is associated with cytochrome c release is secondarily activated. The ROCK-1 pathway appears largely responsible for cell death, which occurs after prolonged stimulation of P2X7 receptors. We suggest that the former mechanism underlies the reversible pseudoapoptotic events induced by brief activation of P2X7 receptors. P2X7 receptors are ATP-gated ion channels primarily expressed on antigen-presenting immune cells where they play a role in the acute inflammatory response. These ion channels couple not only to influx of cations, including calcium, but also to rapid alterations in cell morphology (membrane blebbing, phosphatidylserine exposure, microvesicle shedding). These features resemble the extranuclear events associated with end stages of apoptosis but cell death does not occur if receptor activation is brief. Here we delineate two signaling pathways underlying these apoptotic-like processes. Loss of membrane asymmetry occurs within seconds, which directly triggers cytoskeletal disruption and zeiotic membrane blebbing; this is readily reversible and requires both calcium influx through P2X7 channels and mitochondrial calcium increase but is not associated with cytochrome c release. A slower, calcium-independent, ROCK-1-dependent cascade that does not involve rapid loss of membrane asymmetry but is associated with cytochrome c release is secondarily activated. The ROCK-1 pathway appears largely responsible for cell death, which occurs after prolonged stimulation of P2X7 receptors. We suggest that the former mechanism underlies the reversible pseudoapoptotic events induced by brief activation of P2X7 receptors. In antigen-presenting cells of the immune system, prolonged application of high concentrations of extracellular ATP leads to apoptotic and/or necrotic cell death via activation of the ATP-gated ionotropic P2X7 receptor (1Di Virgilio F. Chiozzi P. Falzoni S. Ferrari D. Sanz J.M. Venketaraman V. Baricordi O.R. Cell Death Differ. 1998; 5: 191-199Crossref PubMed Scopus (229) Google Scholar, 2Burnstock G. Arterioscler. Thromb. Vasc. Biol. 2002; 22: 364-373Crossref PubMed Scopus (360) Google Scholar, 3North R.A. Physiol. Rev. 2002; 82: 1013-1067Crossref PubMed Scopus (2490) Google Scholar). P2X7 receptor coupling has also been associated with classical apoptotic signaling cascades such as activation of stress-activated kinases/c-Jun N-terminal kinases and caspase-3 (4Wen L.T. Caldwell C.C. Knowles A.F. Mol. Pharmacol. 2003; 63: 706-713Crossref PubMed Scopus (17) Google Scholar, 5Coutinho-Silva R. Persechini P.M. Biosaggio R.D. Perfettini J.L. Neto A.C. Kanellopoulos J.M. Motta-Ly I. Dautry-Varsat A. Ojcius D.M. Am. J. Physiol. 1999; 276: C1139-C1147Crossref PubMed Google Scholar, 6Ferrari D. Los M. Bauer M.K. Vandenabeele P. Wesselborg S. Schulze-Osthoff K. FEBS Lett. 1999; 447: 71-75Crossref PubMed Scopus (243) Google Scholar, 7Humphreys B.D. Rice J. Kertesy S.B. Dubyak G.R. J. Biol. Chem. 2000; 275: 26792-26798Abstract Full Text Full Text PDF PubMed Google Scholar). However, P2X7 receptors in the immune system can also couple to responses distinct from cell death including cell fusion, cell proliferation, release of pro-inflammatory cytokines, and bone formation (8Chiozzi P. Sanz J.M. Ferrari D. Falzoni S. Aleotti A. Buell G.N. Collo G. Di Virgilio F. J. Cell Biol. 1997; 138: 697-706Crossref PubMed Scopus (155) Google Scholar, 9Falzoni S. Chiozzi P. Ferrari D. Buell G. Di Virgilio F. Mol. Biol. Cell. 2000; 9: 3169-3176Crossref Scopus (55) Google Scholar, 10MacKenzie A.B. Wilson H.L. Kiss-Toth E. Dower S.K. North R.A. Surprenant A. Immunity. 2001; 8: 825-835Abstract Full Text Full Text PDF Scopus (713) Google Scholar, 11Ke H.Z. Qi H. Weidema A.F. Zhang Q. Panupinthu N. Crawford D.T. Grasser W.A. Paralkar V.M. Li M. Audoly L.P. Gabel C.A. Jee W.S. Dixon S.J. Sims S.M. Thompson D.D. Mol. Endocrinol. 2003; 17: 1356-1367Crossref PubMed Scopus (255) Google Scholar). Indeed, brief activation of P2X7 receptors, on the order of sec