Released nucleotides amplify the cilium-dependent, flow-induced [Ca2+]i response in MDCK cells
Released nucleotides amplify the cilium-dependent, flow-induced [Ca2+]i response in MDCK cells
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DOI：
10.1111/j.1748-1716.2009.02002.x
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发表时间：
2009-11-01
影响因子：
6.3
通讯作者：
Leipziger, J.
中科院分区：
文献类型：
--
作者：
Praetorius, H. A.;Leipziger, J.
目的：灌流液流量的变化导致肾上皮细胞[Ca 2 +]（i）增加。培养的肾上皮细胞需要初级纤毛来感知血流的细微变化。在灌注的肾小管中，这种流动反应是由通过P2 Y（2）受体的核苷酸信号传导引起的。然而，不知道核苷酸是否通过施加于肾初级纤毛的机械应力释放。方法：在37 ℃的半开放单室或封闭双室灌流条件下，用Fluo-4-AM负载的MDCK细胞，观察细胞内核苷酸的释放情况，结果：（1）ATP清除剂和P2受体拮抗剂可使纤毛依赖的[Ca ~（2+）]（i）信号降低55%;（2）ATP在阈下浓度下的加入使肾上皮细胞对流量变化敏感;（3）液体流量的增加瞬时增加了灌流液中的ATP浓度（由生物传感器细胞测量）。为了测试核苷酸是否以足够的量释放以刺激肾上皮，我们使用没有纤毛的非融合MDCK细胞作为报告细胞。我们证实，非融合细胞不响应流体流动的变化。将汇合的纤毛细胞放置在非汇合细胞的流入路径的上游，使它们对流体流动变化做出反应。如果将非融合或去纤毛的融合细胞置于上游，则未观察到这种现象。结论：初级纤毛感受到的微小流量变化可诱导核苷酸释放，从而放大上皮细胞的[Ca 2 +]（i）-反应。
Aim:Changes in perfusate flow produce increases in [Ca2+](i) in renal epithelial cells. Cultured renal epithelia require primary cilia to sense subtle changes in flow. In perfused kidney tubules this flow response is caused by nucleotide signalling via P2Y(2) receptors. It is, however, not known whether nucleotides are released by mechanical stress applied to renal primary cilia. Here we investigate whether nucleotides are released during the cilium-dependent flow response and contribute to the flow-induced, cilium-dependent [Ca2+](i) signal.Methods:MDCK cells loaded with Fluo-4-AM were observed at 37 degrees C in semi-open single or closed-double perfusion chambers.Results:Our data suggest a purinergic component of the cilium-dependent flow-response: (1) ATP scavengers and P2 receptor antagonists reduced (55%) the cilium-dependent flow-response; (2) ATP added at subthreshold concentration sensitized the renal epithelia to flow changes; (3) increases in fluid flow transiently enhanced the ATP concentration in the superfusate (measured by biosensor-cells). To test if nucleotides were released in sufficient quantities to stimulate renal epithelia we used non-confluent MDCK cells without cilia as reporter cells. We confirmed that non-confluent cells do not respond to changes in fluid flow. Placing confluent, ciliated cells upstream in the in-flow path of the non-confluent cells made them responsive to fluid flow changes. This phenomenon was not observed if either non-confluent or de-ciliated confluent cells were placed upstream. The [Ca2+](i)-response in the non-confluent cells with ciliated cells upstream was abolished by apyrase and suramin.Conclusion:This suggests that subtle flow changes sensed by the primary cilium induces nucleotide release, which amplifies the epithelial [Ca2+](i)-response.