Pharmacological properties of a pore induced by raising intracellular Ca2+
Pharmacological properties of a pore induced by raising intracellular Ca2+
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DOI：
10.1152/ajpcell.00476.2008
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发表时间：
2009-07-01
影响因子：
5.5
通讯作者：
Alves, L. A.
中科院分区：
文献类型：
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作者：
Faria, R. X.;Reis, R. A. M.;Alves, L. A.
Faria RX, Reis RA, Casabulho CM, Alberto AV, de Farias FP, Henriques-Pons A, Alves LA。提高细胞内Ca2+诱导的孔的药理特性。[J] .中国生物医学工程学报，2009,31(4):559 - 564。首次发表于2009年3月25日；doi: 10.1152 / ajpcell.00476.2008。-最近对2BH4细胞和腹膜巨噬细胞中P2X(7)受体的研究表明，细胞内Ca2+浓度的升高诱导了类似于P2X(7)受体孔的开孔。在这里，我们研究了细胞内Ca2+浓度升高激活的孔是否与P2X(7)受体有关。我们使用膜片钳在细胞贴壁、全细胞结构和染色摄取中测量了表达P2X(7)受体的细胞类型（2BH4细胞和腹膜巨噬细胞）和不表达该受体的细胞类型（HEK-293和IT45-RI细胞）的孔开度。在2BH4细胞中，离子霉素（5-10 μ M）刺激增加细胞内游离Ca2+浓度，诱导孔形成，电导为421 +/- 14 pS，溴化乙啶吸收的半衰期（t1/2）为118 +/- 17 s，路西法黄吸收的半衰期（t1/2）为122 +/- 11 s。P2X(7)受体拮抗剂不能阻断这些作用。对HEK-293和IT45-RI细胞的刺激导致气孔形成，其性质与2BH4细胞相似。连接蛋白半通道抑制剂（卡贝诺酮和庚醇）也没有抑制细胞内Ca2+浓度增加后的孔诱导效应。然而，P2X(7)受体孔阻滞剂5-（N，N-六亚甲基）阿米洛胺抑制了诱导的孔。此外，细胞内信号调节剂，如钙调蛋白、磷脂酶C、丝裂原活化蛋白激酶和细胞骨架成分对孔的形成也很重要。此外，我们证实了通过流式细胞术获得的电生理学结果，我们放弃了通过乳酸脱氢酶释放试验使用的剂量提高细胞内Ca2+诱导细胞死亡的可能性。综上所述，细胞内Ca+2浓度的增加诱导了一种不同于P2X(7)相关孔和半间隙连接孔的新型膜孔。
Faria RX, Reis RA, Casabulho CM, Alberto AV, de Farias FP, Henriques-Pons A, Alves LA. Pharmacological properties of a pore induced by raising intracellular Ca2+. Am J Physiol Cell Physiol 297: C28-C42, 2009. First published March 25, 2009; doi:10.1152/ajpcell.00476.2008.-Recent studies on the P2X(7) receptor in 2BH4 cells and peritoneal macrophages have demonstrated that the raise in intracellular Ca2+ concentration induces a pore opening similar to P2X(7) receptor pore. Herein, we have investigated whether the pore activated by the elevation of intracellular Ca2+ concentration is associated to P2X(7) receptor. Using patch clamp in cell attached, whole cell configuration, and dye uptake, we measured the pore opening in cell types that express the P2X(7) receptor (2BH4 cells and peritoneal macrophages) and in cells that do not express this receptor (HEK-293 and IT45-RI cells). In 2BH4 cells, the stimulation with ionomycin (5-10 mu M) increased intracellular free Ca2+ concentration and induced pore formation with conductance of 421 +/- 14 pS, half-time (t1/2) for ethidium bromide uptake of 118 +/- 17 s, and t1/2 for Lucifer yellow of 122 +/- 11 s. P2X(7) receptor antagonists did not block these effects. Stimulation of HEK-293 and IT45-RI cells resulted in pore formation with properties similar to those found for 2BH4 cells. Connexin hemichannel inhibitors (carbenoxolone and heptanol) also did not inhibit the pore-induced effect following the increase in intracellular Ca2+ concentration. However, 5-(N,N-hexamethylene)amiloride, a P2X(7) receptor pore blocker, inhibited the induced pore. Moreover, intracellular signaling modulators, such as calmodulin, phospholipase C, mitogen-activated protein kinase, and cytoskeleton components were important for the pore formation. Additionally, we confirmed the results obtained for electrophysiology by using the flow cytometry, and we discarded the possibility of cellular death induced by raising intracellular Ca2+ at the doses used by using lactate dehydrogenase release assay. In conclusion, increased concentration in intracellular Ca+2 induces a novel membrane pore pharmacologically different from the P2X(7) associated pore and hemigap-junction pore.