Microtubule Disruption with BAPTA and Dimethyl BAPTA by a Calcium Chelation-Independent Mechanism in 3T3-L1 Adipocytes
Microtubule Disruption with BAPTA and Dimethyl BAPTA by a Calcium Chelation-Independent Mechanism in 3T3-L1 Adipocytes
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DOI：
10.1507/endocrj.k08e-321
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发表时间：
2009-04-01
影响因子：
2
通讯作者：
Shibata, Hiroshi
中科院分区：
文献类型：
--
作者：
Furuta, Ai;Tanaka, Marie;Shibata, Hiroshi
虽然钙在胰岛素作用油葡萄糖转运中的生理作用一直存在争议，但在最近的一项研究中，通过使用钙螯合剂1，2-双（邻氨基苯氧基）乙烷-N，N，N '，N'-四乙酸四（乙酰氧基甲基）酯（BAPTA-AM）对其进行了重新评估。虽然BAPTA已被广泛用于研究钙在各种细胞功能中的作用，但也有人认为它具有与钙螯合活性无关的性质。在此，我们研究了BAPTA和二甲基BAPTA油对3 T3-L1脂肪细胞细胞骨架的影响。这两种钙螯合剂都成功地加载在3 T3-L1脂肪细胞中，并抑制内皮素-I诱导的胞浆钙升高。共聚焦荧光显微镜显示，BAPTA和二甲基BAPTA引起微管的深度解聚，而不影响3 T3-L1脂肪细胞中的皮质肌动蛋白丝。生化定量也显示BAPTA和二甲基BAPTA显着降低了微管蛋白的聚合量，对丝状肌动蛋白的影响不大。与这些结果一致，GLUT 4阳性核周区室分散在整个细胞质中的BAPTA或二甲基BAPTA加载的脂肪细胞。有趣的是，这些钙螯合剂不会破坏未分化前脂肪细胞中的微管。BAPTA和二甲基BAPTA的微管解聚特性与钙螯合无关，因为微管通过使用钙离子载体A23187抵抗细胞溶质钙的耗尽。胰岛素刺激的葡萄糖转运不受A23187的胞质钙耗竭的影响，但BAPTA和二甲基BAPTA显着抑制的程度与诺考达唑相似。BAPTA及其衍生物应谨慎用于研究细胞凋亡相关的细胞功能。
While the physiological role for calcium in the insulin action oil glucose transport has been disputed, it was reassessed in a recent study by using a calcum chelator, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethyl) ester (BAPTA-AM). Although BAPTA has been widely used to study the role for calcium in a variety of cell functions, it has also been suggested to have properties unrelated to the calcium chelating activity. Here, we investigated the effects of BAPTA and dimethyl BAPTA oil the cytoskeletons in 3T3-L1 adipocytes. Both calcium chelators were successfully loaded in 3T3-L1 adipocytes and inhibited endothelin-l-induced cytosolic calcium elevation. Confocal fluorescence microscopy revealed that BAPTA and dimethyl BAPTA caused profound depolymerization of the microtubules Without affecting the cortical actin filaments in 3T3-L1 adipocytes. Biochemical quantification also showed that BAPTA and dimethyl BAPTA significantly decreased the amount of polymerized tubulin bill had little effect oil filamentous actin. Consistent with these results, GLUT4-positive perinuclear compartments were dispersed throughout the cytoplasm in BAPTA- or dimethyl BAPTA-loaded adipocytes. Intriguingly, these calcium chelators did not disrupt the microtubules in undifferentiated preadipocytes. The microtubule-depolymerizing property of BAPTA and dimethyl BAPTA is unrelated to calcium chelation, since the microtubules were resistant to depletion of cytosolic calcium by using a calcium ionophore A23187. Insulin-stimulated glucose transport was not affected by cytosolic calcium depletion with A23187, but significantly inhibited with BAPTA and dimethyl BAPTA to the extent similar to that with nocodazole. BAPTA and its derivatives should be used with caution in studies of cytoskeleton-related cell functions.