Verhoef Philip A, Estacion Mark, Schilling William, Dubyak George R
Department of Pharmacology, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA.
J Immunol. 2003 Jun 1;170(11):5728-38. doi: 10.4049/jimmunol.170.11.5728.
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.
作为对ATP结合的反应，P2X7受体促进阳离子通道激活、非特异性孔形成、质膜形态的快速变化以及脂多糖预处理的巨噬细胞分泌白细胞介素-1β。为了研究P2X7受体依赖性质膜组织变化与白细胞介素-1β释放之间的关系，我们制作了ATP刺激的BAC1小鼠巨噬细胞的延时电影，并结合白细胞介素-1β释放的生化分析。在表达重组P2X7受体的人胚肾293细胞（HEK-P2X7）中进行类似的图像分析，以便比较巨噬细胞与非巨噬细胞背景下P2X7受体依赖性效应。虽然HEK-P2X7细胞在ATP处理后5分钟内出现凋亡性气泡形成，但BAC1巨噬细胞在添加ATP后10分钟开始出现独特的“拴系”气泡形成。这种气泡形成由P2X7受体选择性激动剂BzATP类似地诱导，并被P2X7受体抑制剂KN-62和氧化ATP阻断。气泡形成在ATP浓度≥3 mM时开始，但最佳白细胞介素-1β释放在1 mM ATP时发生。在存在Rho效应激酶抑制剂法舒地尔和Y-27632的情况下，P2X7受体依赖性气泡形成被消除，但ATP诱导的白细胞介素-1β释放不受影响。在HEK-P2X7细胞和BAC1小鼠巨噬细胞中都能检测到ATP诱导的RhoA激活。因此，P2X7受体激活引发不同的、新的膜气泡形成事件（依赖于RhoA激活和Rho效应激酶活性），并同时启动白细胞介素-1β的释放。我们关于气泡形成和白细胞介素-1β释放可分离的观察结果表明，这些事件是通过平行而非汇聚的信号通路发生的。
