Dept. of Anatomy and Cell Biology, Schulich School of Medicine & Dentistry, Univ. of Western Ontario, London, ON, Canada.
Am J Physiol Endocrinol Metab. 2012 Mar 1;302(5):E561-75. doi: 10.1152/ajpendo.00209.2011. Epub 2011 Dec 20.
The P2X₇ receptor is an ATP-gated cation channel expressed by a number of cell types, including osteoblasts. Genetically modified mice with loss of P2X₇ function exhibit altered bone formation. Moreover, activation of P2X₇ in vitro stimulates osteoblast differentiation and matrix mineralization, although the underlying mechanisms remain unclear. Because osteogenesis is associated with enhanced cellular metabolism, our goal was to characterize the effects of nucleotides on metabolic acid production (proton efflux) by osteoblasts. The P2X₇ agonist 2',3'-O-(4-benzoylbenzoyl)ATP (BzATP; 300 μM) induced dynamic membrane blebbing in MC3T3-E1 osteoblast-like cells (consistent with activation of P2X₇ receptors) but did not induce cell death. Using a Cytosensor microphysiometer, we found that 9-min exposure to BzATP (300 μM) caused a dramatic increase in proton efflux from MC3T3-E1 cells (∼2-fold), which was sustained for at least 1 h. In contrast, ATP or UTP (100 μM), which activate P2 receptors other than P2X₇, failed to elicit a sustained increase in proton efflux. Specific P2X₇ receptor antagonists A 438079 and A 740003 inhibited the sustained phase of the BzATP-induced response. Extracellular Ca²⁺ was required during P2X₇ receptor stimulation for initiation of sustained proton efflux, and removal of extracellular glucose within the sustained phase abolished the elevation elicited by BzATP. In addition, inhibition of phosphatidylinositol 3-kinase blocked the maintenance but not initiation of the sustained phase. Taken together, we conclude that brief activation of P2X₇ receptors on osteoblast-like cells triggers a dramatic, Ca²⁺-dependent stimulation of metabolic acid production. This increase in proton efflux is sustained and dependent on glucose and phosphatidylinositol 3-kinase activity.
P2X7 受体是一种由多种细胞类型表达的 ATP 门控阳离子通道，包括成骨细胞。P2X7 功能丧失的基因修饰小鼠表现出骨形成改变。此外，体外激活 P2X7 可刺激成骨细胞分化和基质矿化，尽管其潜在机制尚不清楚。由于成骨作用与细胞代谢增强有关，我们的目标是研究核苷酸对成骨细胞代谢性产酸（质子外排）的影响。P2X7 激动剂 2'，3'-O-(4-苯甲酰基苯甲酰基)ATP（BzATP；300μM）诱导 MC3T3-E1 成骨样细胞的动态膜泡形成（与 P2X7 受体的激活一致），但不会诱导细胞死亡。使用 Cytosensor 微生理仪，我们发现 9 分钟暴露于 BzATP（300μM）可使 MC3T3-E1 细胞的质子外排量显著增加（约 2 倍），并至少持续 1 小时。相比之下，激活除 P2X7 以外的 P2 受体的 ATP 或 UTP（100μM）未能引起质子外排量的持续增加。特异性 P2X7 受体拮抗剂 A 438079 和 A 740003 抑制 BzATP 诱导反应的持续相。在 P2X7 受体刺激期间需要细胞外 Ca²⁺才能启动持续的质子外排，并且在持续相中去除细胞外葡萄糖可消除 BzATP 引起的升高。此外，抑制磷脂酰肌醇 3-激酶可阻断维持但不阻断持续相的维持。综上所述，我们得出结论，短暂激活成骨样细胞上的 P2X7 受体可引发剧烈的、依赖 Ca²⁺的代谢性产酸刺激。这种质子外排量的增加是持续的，并且依赖于葡萄糖和磷脂酰肌醇 3-激酶活性。
