Mechanism of the cytoskeletal regulation of store-operated Ca^<2+> entry

用微摩尔浓度的ATP刺激P2受体不仅引起细胞内游离Ca^<2+>浓度的短暂增加（[Ca^<2+>]_i），这主要是由于细胞内储存的Ca^<2+>的释放；这种刺激也几乎完全抑制了thapsigargin在大鼠棕色脂肪细胞中通过储存操作的Ca^<2+>进入途径介导的持续[Ca^<2+>]_i升高。我们研究了细胞骨架肌动蛋白在细胞外ATP对储存操作的Ca^<2+>进入的抑制作用中的作用，使用fura-2荧光连续测量[Ca^<2+>] i_，并使用Alexa氟488-phalloidin染色肌动蛋白。细胞松弛素D （1 μM）或latrunculin A （3 μM）对肌动蛋白网络的破坏可阻止ATP （10 μM）对thapsigargin （100 μM）诱发的储存操作Ca^<2+>的抑制作用，但不改变ATP提高[Ca^<2+>]_i的作用。在正常细胞中，尽管激动剂持续存在，但ATP的溶液应用诱导了短暂的[Ca^<2+>]_i升高，包括快速增加（上升期）和随后的下降（下降期）至较低的稳定水平。肌动蛋白组装的破坏对上升期没有显著影响，但阻止了下降期。10 μM ATP作用4 min后，细胞周围有明显的肌动蛋白丝堆积，细胞表面呈突起；这个动作蛋白组装过程是通过P2受体介导的。在细胞松弛素D或拉runculin A处理的细胞中，细胞外ATP不诱导肌动蛋白再分布。这些结果表明，肌动蛋白重组在atp诱导的大鼠棕色脂肪细胞储存操作的Ca^<2+>进入抑制中起作用。

Stimulation of P2 receptors with micromolar concentration of ATP not only evokes a transient increase in intracellular free Ca^<2+> concentration ([Ca^<2+>]_i), primarily due to release of Ca^<2+> from intracellular stores ; such stimulation also triggers almost complete suppression of thapsigargin-evoked sustained [Ca^<2+>]_i elevation mediated through a store-operated Ca^<2+> entry pathway in rat brown adipocytes. We investigated the role of cytoskeletal actin in the inhibitory effect which extracellular ATP has on store-operated Ca^<2+> entry, using fura-2 fluorescence for continuous measurement of [Ca^<2+>]_i_, and using Alexa fluor 488-phalloidin staining of actin. Disassembly of actin networks by cytochalasin D (1 μM) or latrunculin A (3 μM) prevented the inhibitory effect of ATP (10 μM) on the thapsigargin (100 μM)-evoked store-operated Ca^<2+>entry, without changing the effect of ATP in raising [Ca^<2+>]_i. In normal cells, bath application of ATP induced a transient [Ca^<2+>]_i elevation consisting of a rapid increase (the rising phase) and the subsequent decrease (the declining phase) to a lower steady level despite the continued presence of the agonist. Disruption of actin assemblies did not significantly affect the rising phase, but prevented the declining phase. Cells incubated with 10 μM ATP for 4 min demonstrated marked accumulations of actin filaments at the cell periphery, showing protrusions at the cell surface ; this actin-assembly process is mediated through P2 receptors. In cells treated with cytochalasin D or latrunculin A, extracellular ATP did not induce actin redistribution. These results suggest the actin reorganization plays a role in ATP-induced inhibition of store-operated Ca^<2+> entry in rat brown adipocytes.