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+>浓度的瞬时升高，（[Ca^2+] i），主要是由于胞内钙库释放Ca^2+所致;这种刺激也几乎完全抑制了毒胡萝卜素诱发的持续性[Ca^2+] i升高，这种升高是通过钙库操纵的Ca^2+通道介导的。大鼠棕色脂肪细胞的进入途径。我们研究了细胞骨架肌动蛋白在细胞外ATP对钙池操纵的Ca^2+内流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 fluor 488-鬼笔环肽染色肌动蛋白。细胞松弛素D（1 μM）或latrunculin A（3 μM）对肌动蛋白网络的阻断可阻止ATP（10 μM）对毒胡萝卜素（100 μ M）诱发的钙池操纵的Ca^2+内流的抑制作用，但不改变ATP升高[Ca^2+] i的作用。在正常细胞中，浴用ATP可引起短暂的[Ca^<2+>] i升高，包括迅速升高（上升期）和随后的下降（下降期），尽管激动剂持续存在，[Ca^<2 +>] i仍降至较低的稳定水平。肌动蛋白组装的破坏并没有显着影响上升阶段，但阻止了下降阶段。用10 μM ATP孵育4分钟的细胞显示肌动蛋白丝在细胞周边显著积聚，在细胞表面显示突起;该肌动蛋白组装过程通过P2受体介导。在细胞松弛素D或latrunculin 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诱导的抑制大鼠棕色脂肪细胞钙池操作的Ca^2+内流中起作用。

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.