Stiffness changes in cultured airway smooth muscle cells
Stiffness changes in cultured airway smooth muscle cells
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DOI：
10.1152/ajpcell.00425.2001
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发表时间：
2002-09-01
影响因子：
5.5
通讯作者：
Fredberg, JJ
中科院分区：
文献类型：
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作者：
An, SS;Laudadio, RE;Fredberg, JJ
培养中的呼吸道平滑肌(ASM)细胞在受到收缩激动剂的作用时会变硬。这种细胞僵硬可能反映了收缩装置的激活以及细胞骨架生物聚合物的聚合。在此，我们评估了在有或没有抑制肌球蛋白收缩或丝状肌动蛋白聚合的药物存在或不存在的情况下，这些机制在5-羟色胺(5-羟色胺；5-羟色胺)刺激的培养ASM细胞中的相对贡献。用磁扭转细胞术测量细胞硬度，用共聚焦显微镜观察肌动蛋白细胞骨架结构的变化。我们发现，5-羟色胺以剂量依赖的方式增加细胞僵硬，并诱导F-肌动蛋白的快速形成，其显著特征是FITC-Palloidin染色强度增加。钙调素拮抗剂(W-7)、肌球蛋白轻链激酶抑制剂(ML-7)和肌球蛋白ATPase抑制剂(BDM)均可阻断5-羟色胺诱导的肌动蛋白F-肌动蛋白聚合，但不能阻断肌球蛋白ATPase抑制剂(BDM)。抑制F-肌动蛋白形成的药物(细胞松弛素D、马立克菌素A、C3外酶和Y-27632)可同时减弱基线僵硬和细胞对5-羟色胺反应的僵硬程度。总之，这些数据表明，激动剂诱导的ASM细胞僵硬需要肌动蛋白聚合和肌球蛋白激活，而且肌动蛋白聚合和肌球蛋白激活本身都不足以解释细胞僵硬反应。
Airway smooth muscle (ASM) cells in culture stiffen when exposed to contractile agonists. Such cell stiffening may reflect activation of the contractile apparatus as well as polymerization of cytoskeletal biopolymers. Here we have assessed the relative contribution of these mechanisms in cultured ASM cells stimulated with serotonin (5-hydroxytryptamine; 5-HT) in the presence or absence of drugs that inhibit either myosin-based contraction or polymerization of filamentous (F) actin. Magnetic twisting cytometry was used to measure cell stiffness, and associated changes in structural organization of actin cytoskeleton were evaluated by confocal microscopy. We found that 5-HT increased cell stiffness in a dose-dependent fashion and also elicited rapid formation of F-actin as marked by increased intensity of FITC-phalloidin staining in these cells. A calmodulin antagonist (W-7), a myosin light chain kinase inhibitor (ML-7) and a myosin ATPase inhibitor (BDM) each ablated the stiffening response but not the F-actin polymerization induced by 5-HT. Agents that inhibited the formation of F-actin (cytochalasin D, latrunculin A, C3 exoenzyme, and Y-27632) attenuated both baseline stiffness and the extent of cell stiffening in response to 5-HT. Together, these data suggest that agonist-evoked stiffening of cultured ASM cells requires actin polymerization as well as myosin activation and that neither actin polymerization nor myosin activation by itself is sufficient to account for the cell stiffening response.