Hypotonic stress activates BK channels in clonal kidney cells via purinergic receptors, presumably of the P2Y1 subtype
Hypotonic stress activates BK channels in clonal kidney cells via purinergic receptors, presumably of the P2Y1 subtype
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DOI：
10.1111/j.1748-1716.2006.01601.x
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发表时间：
2006-09-01
影响因子：
6.3
通讯作者：
Sand, O.
中科院分区：
文献类型：
--
作者：
Hafting, T.;Haug, T. M.;Sand, O.
目的：细胞肿胀引起的细胞膜牵张可引起三磷酸腺苷（ATP）的微小泄漏，从而刺激内源性嘌呤能受体。细胞内游离Ca 2+浓度（[Ca 2 +]（i））的随后升高可能参与细胞体积调节。本研究的目的是在克隆肾细胞中检测嘌呤能受体和大电导钙激活钾通道（BK）是否在低渗应激反应中被激活方法：采用Fura-2显微荧光法、细胞贴附式膜片钳技术和逆转录聚合酶链反应（RT-PCR）技术。通过暴露于渗透压降低45%的溶液使细胞经受低渗应激10 s，诱导[Ca 2 +]（i）瞬时升高。这种反应持续在几乎无钙的细胞外溶液中，表明Ca 2+主要从细胞内储存释放。低渗诱导的[Ca ~（2+）]（i）升高可被P2受体拮抗剂苏拉明（100 μ M）和磷酸吡哆醛-6-偶氮苯基-2 '4 '-二磺酸盐（PPADS; 20 μ M）完全抑制，表明细胞外ATP对[Ca ~（2+）]（i）升高至关重要。RT-PCR显示Vero细胞中有P2 Y（1）受体mRNA的表达。P2 Y（1）受体拮抗剂PPADS完全阻断了ATP和低渗应激引起的Ca ~（2+）反应，提示P2 Y（1）受体介导了这种反应。结论：Vero细胞表达功能性嘌呤受体，推测为P2 Y（1）亚型。这些受体负责由低渗应激诱发的[Ca 2 +]（i）升高。BK通道的同时激活允许K+流出，这可能有助于调节性体积减少。
Aim: Membrane stretch due to cell swelling may cause a minute leakage of adenosine triphosphate (ATP) that stimulates endogenous purinergic receptors. The following elevation of the cytosolic-free Ca2+ concentration ([Ca2+](i)) may then participate in cell volume regulation. The aim of the present study was to test if purinergic receptors and large conductance Ca2+ activated K+ (BK) channels are activated in response to hypotonic stress in clonal kidney cells (Vero cells).Methods: The methods used are fura-2 microfluorometry, cell-attached patch clamp and reverse-transcriptase polymerase chain reaction (RT-PCR).Results: Subjecting cells to hypotonic stress for 10 s by exposure to a solution with 45% reduced osmolality induced a transient rise in [Ca2+](i). This response persisted in virtually Ca2+-free extracellular solution, demonstrating that Ca2+ was mainly released from intracellular stores. The hypotonically induced elevation of [Ca2+](i) was completely inhibited by the P2 receptor antagonists suramine (100 mu M) and pyridoxalphosphate-6-azophenyl-2'4'-disulphonate (PPADS; 20 mu M), indicating that extracellular ATP is crucial for the [Ca2+](i) increase. RT-PCR revealed the expression of mRNA for P2Y(1) receptors in Vero cells. The putatively selective P2Y(1) antagonist PPADS did completely block Ca2+ responses to both ATP and hypotonic stress, suggesting that P2Y(1) receptors are mediating the response. Furthermore, patch clamp recordings in cell-attached configuration revealed that BK channels are activated in response to hypotonic stress.Conclusion: Vero cells express functional purinergic receptors, presumably of the P2Y(1) subtype. These receptors are responsible for the elevation of [Ca2+](i) evoked by hypotonic stress. The concurrent activation of BK channels permits K+ efflux that may contribute to regulatory volume decrease.