Ca2+ mobilization in bovine corneal endothelial cells by P2 purinergic receptors.
Ca2+ mobilization in bovine corneal endothelial cells by P2 purinergic receptors.
复制标题
P2 嘌呤能受体对牛角膜内皮细胞中 Ca2 的动员。
DOI：
10.1076/ceyr.17.10.994.5242
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发表时间：
1998
影响因子：
2
通讯作者：
Bonanno,JA
中科院分区：
文献类型：
--
作者：
Srinivas,SP;Yeh,JC;Ong,A;Bonanno,JA
目的 表征牛角膜内皮细胞 (BCEC) 中 P2 受体对 Ca 2+ 的动员。方法通过负载 Ca 2+ 敏感染料 Fura-PE3 的培养和新鲜 BCEC 细胞的荧光成像来测量细胞内 Ca 2+ ([Ca 2+ ] i) 的变化。使用 Mn 2+ 作为 Ca 2+ 的替代物来测量 Ca 2+ 流入的相对速率。结果将培养的细胞暴露于尿苷 5'-三磷酸 (UTP)、2-甲硫基 ATP (msATP) 和 ATP 会引起 [Ca 2+ ] i 的双相变化，包括峰值和平台期。根据对 100 µM 激动剂的峰值响应，UTP 响应的幅度与 ATP 相似，但大于 msATP 或 ADP。 UTP 和 msATP 在 [Ca 2+ ] i 峰后刺激 Mn 2+ 流入，类似于对环匹阿尼酸 (CPA)（一种 ER Ca 2+ -ATP 酶抑制剂）的反应中观察到的情况。在无Ca 2+ 条件下，峰值响应与外部Ca 2+ 存在下的峰值响应相似，但当细胞预先暴露于CPA时会降低。 Reactive Blue-2 (RB2) 可抑制 msATP 反应 60.4 ± 18.8%，但 UTP 反应仅抑制 10.6 ± 9.5%。重复暴露于 UTP 或 msATP 减少了 [Ca 2+ ] i 动员，表明同源脱敏。对 UTP 的反应不受先前暴露于 msATP 的影响。然而，对 msATP 的反应因之前暴露于 UTP 而降低，表明混合异源脱敏。新鲜细胞对UTP (50 mM) 作出反应，[Ca 2+ ] i 动员的时间特征与培养细胞相似。结论BCEC 表达属于P2Y 亚家族的P2 受体。 IP3 敏感存储的清空导致 [Ca2+] i 响应的初始峰值，随后引起电容性 Ca 2+ 流入，导致平台期的开始。对UTP和msATP的显着同源脱敏、UTP和msATP之间的选择性异源脱敏以及RB2的选择性抑制表明多种P2Y受体的共存。
PURPOSETo characterize Ca 2+ mobilization by P2 receptors in the bovine corneal endothelial cells (BCEC).METHODSChanges in intracellular Ca 2+ ([Ca 2+ ] i) were measured by fluorescence imaging of cultured and fresh BCEC cells loaded with the Ca 2+ -sensitive dye Fura-PE3. Relative rates of Ca 2+ influx were measured employing Mn 2+ as a surrogate for Ca 2+.RESULTSExposure of cultured cells to uridine 5'-triphosphate (UTP), 2-methyl-thio ATP (msATP) and ATP caused biphasic changes in [Ca 2+ ] i consisting of a peak followed by a plateau phase. Based on the peak responses to 100 µM agonist, the magnitude of UTP responses were similar to that of ATP but greater than that of msATP or ADP. UTP and msATP stimulated Mn 2+ influx following [Ca 2+ ] i peak similar to that observed in response to cyclopiazonic acid (CPA), an inhibitor of ER Ca 2+ -ATPase. Under Ca 2+ -free conditions, peak responses were similar to those in the presence of external Ca 2+, but reduced when the cells were pre-exposed to CPA. Reactive Blue-2 (RB2), inhibited msATP responses by 60.4 ± 18.8% but UTP responses by only 10.6 ± 9.5%. Repeated exposures to UTP or msATP reduced [Ca 2+ ] i mobilization indicating homologous desensitization. Response to UTP was not affected by a prior exposure to msATP. However, response to msATP was reduced by a prior exposure to UTP indicating mixed heterologous desensitization. Fresh cells responded to UTP (50 mM) with temporal characteristics of [Ca 2+ ] i mobilization similar to that of cultured cells.CONCLUSIONBCEC express P2 receptors belonging to the P2Y subfamily. The emptying of the IP3-sensitive stores, leading to the initial peak in [Ca2+] i response, subsequently caused capacitative Ca 2+ influx leading to the onset of the plateau phase. A significant homologous desensitization to UTP and msATP, selective heterologous desensitization between UTP and msATP, and selective inhibition by RB2 indicate the coexistence of multiple P2Y receptors.