Extracellular ATP causes ROCK I-dependent bleb formation in P2X7-transfected HEK293 cells
Extracellular ATP causes ROCK I-dependent bleb formation in P2X7-transfected HEK293 cells
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DOI：
10.1091/mbc.02-04-0061
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发表时间：
2003-07-01
影响因子：
3.3
通讯作者：
Di Virgilio, F
中科院分区：
文献类型：
--
作者：
Morelli, A;Chiozzi, P;Di Virgilio, F
P2 X（7）ATP受体介导细胞外ATP的细胞毒性作用。P2 X（7）依赖性细胞死亡的预兆是显著的质膜泡形成。膜起泡是一种复杂的现象，涉及到尚未充分表征的细胞内途径。我们研究了细胞外ATP对转染细胞毒性/成孔P2 X（7）受体的HEK 293细胞的影响。向转染P2 X（7）的HEK 293细胞中加入ATP，而不是野生型P2 X（7）较少的HEK 293细胞，在1-2分钟内引起大量膜起泡。UTP，一种不能激活P2 X（7）的核苷酸，对细胞形状和水泡形成没有早期影响。由ATP触发的水泡形成是可逆的，需要细胞外Ca 2+和完整的细胞骨架。此外，通过与P2 X（7）阻断剂氧化ATP预孵育，它被完全阻止。最近观察到ROCK蛋白是水泡形成的关键决定因素。用ROCK阻断剂Y-27632预孵育HEK 293-P2 X（7）细胞完全阻止了P2 X（7）依赖性起泡。尽管ATP在P2 X（7）转染的HEK 293细胞中触发ROCK I亚型的裂解，但广泛的半胱天冬酶抑制剂z-VAD-氟甲基酮没有影响。这些观察结果表明，P2 X（7）依赖性质膜起泡依赖于丝氨酸/苏氨酸激酶ROCK I的激活。
The P2X(7) ATP receptor mediates the cytotoxic effect of extracellular ATP. P2X(7)-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 P2X(7) receptor. Addition of ATP to P2X(7)-transfected, but not to wt P2X(7)-less, HEK293 cells caused massive membrane blebbing within 1-2 min. UTP, a nucleotide incapable of activating P2X(7), 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(7) blocker oxidized ATP. It was recently observed that the ROCK protein is a key determinant of bleb formation. Preincubation of HEK293-P2X(7) cells with the ROCK blocker Y-27632 completely prevented P2X(7)-dependent blebbing. Although ATP triggered cleavage of the ROCK I isoform in P2X(7)- transfected HEK293 cells, the wide range caspase inhibitor z-VAD-fluoromethylketone had no effect. These observations suggest that P2X(7)-dependent plasma membrane blebbing depends on the activation of the serine/threonine kinase ROCK I.