Slow spontaneous [Ca2+]i oscillations reflect nucleotide release from renal epithelia
Slow spontaneous [Ca2+]i oscillations reflect nucleotide release from renal epithelia
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DOI：
10.1007/s00424-007-0366-4
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发表时间：
2008-03-01
影响因子：
4.5
通讯作者：
Praetorius, H. A.
中科院分区：
文献类型：
--
作者：
Geyti, C. S.;Odgaard, E.;Praetorius, H. A.
肾上皮细胞可被机械刺激释放核苷酸，随后通过激活嘌呤能（P2）受体增加细胞内Ca 2+浓度[Ca 2 +]（i）。培养的细胞通常显示自发的[Ca 2 +]（i）振荡，这是一种涉及核苷酸信号传导的特征。在本研究中，使用加载fluo-4的Madin-Darby犬肾（MDCK）细胞作为模型，用于定量和表征肾上皮细胞中自发性[Ca 2 +]（i）增加。自发性[Ca 2 +]（i）增加作为单细胞事件随机发生。在1分钟的观察期内，10.9 +/- 6.7%（n = 23）的细胞显示自发[Ca 2 +]（i）增加。MDCK细胞自发释放的三磷酸腺苷（ATP）直接通过荧光素酶检测。腺苷三磷酸双磷酸酶或己糖激酶清除ATP显著降低[Ca ~（2+）]（i）振荡活性，而抑制胞外ATP酶（ARL 67156）则增强[Ca ~（2+）]（i）振荡活性。在缺乏P2受体的132- 1 N1细胞中进一步测试了自发[Ca 2 +]（i）增加和核苷酸信号传导之间的关联。这些细胞几乎没有显示任何自发的[Ca 2 +]（i）增加。转染hP 2 Y（6）或hP 2 Y（2）受体后，显示出显著的振荡程度。在新鲜分离、灌注的小鼠髓质粗升支（mTAL）中观察到类似的自发性[Ca 2 +]（i）增加。振荡活性被基底外侧腺苷三磷酸双磷酸酶降低，并且与野生型（0.147 +/- 0.018事件/秒，n = 9）相比，来自P2 Y（2）敲除小鼠的mTAL（0.050 +/- 0.020事件/秒，n = 8）中的振荡活性显著较低。这些发现表明，肾上皮细胞自发释放核苷酸，导致P2受体依赖性[Ca 2 +]（i）振荡。因此，紧张性核苷酸释放可能会改变稳态肾功能。
Renal epithelia can be provoked mechanically to release nucleotides, which subsequently increases the intracellular Ca2+ concentration [Ca2+](i) through activation of purinergic (P2) receptors. Cultured cells often show spontaneous [Ca2+](i) oscillations, a feature suggested to involve nucleotide signalling. In this study, fluo-4 loaded Madin-Darby canine kidney (MDCK) cells are used as a model for quantification and characterisation of spontaneous [Ca2+](i) increases in renal epithelia. Spontaneous [Ca2+](i) increases occurred randomly as single cell events. During an observation period of 1 min, 10.9 +/- 6.7% (n = 23) of the cells showed spontaneous [Ca2+](i) increases. Spontaneous adenosine triphosphate (ATP) release from MDCK cells was detected directly by luciferin/luciferase. Scavenging of ATP by apyrase or hexokinase markedly reduced the [Ca2+](i) oscillatory activity, whereas inhibition of ecto-ATPases (ARL67156) enhanced the [Ca2+](i) oscillatory activity. The association between spontaneous [Ca2+](i) increases and nucleotide signalling was further tested in 132-1N1 cells lacking P2 receptors. These cells hardly showed any spontaneous [Ca2+](i) increases. Transfection with either hP2Y(6) or hP2Y(2) receptors revealed a striking degree of oscillations. Similar spontaneous [Ca2+](i) increases were observed in freshly isolated, perfused mouse medullary thick ascending limb (mTAL). The oscillatory activity was reduced by basolateral apyrase and substantially lower in mTAL from P2Y(2) knock out mice (0.050 +/- 0.020 events per second, n = 8) compared to the wild type (0.147 +/- 0.018 events per second, n = 9). These findings indicate that renal epithelia spontaneously release nucleotides leading to P2-receptor-dependent [Ca2+](i) oscillations. Thus, tonic nucleotide release is likely to modify steady state renal function.