Wang Q, Patton W F, Chiang E T, Hechtman H B, Shepro D
Microvascular Research Laboratory, Boston University, Massachusetts 02215, USA.
J Cell Biochem. 1996 Sep 1;62(3):383-96. doi: 10.1002/(SICI)1097-4644(199609)62:3%3C383::AID-JCB8%3E3.0.CO;2-N.
Endothelial cell (EC) cytoskeletal proteins are one of the earliest primary targets of second messenger cascades generated in response to inflammatory agonists. Actin binding proteins, by modulating actin gelation-solation state and membrane-cytoskeleton interactions, in part regulate cell motility and cell-cell apposition. This in turn can also modulate interendothelial junctional diameter and permeability. Nonmuscle filamin (ABP-280), a dimeric actin-crosslinking protein, promotes orthogonal branching of F-actin and links microfilaments to membrane glycoproteins. In the present study, immunoblot analysis demonstrates that filamin protein levels are low in sparse EC cultures, increase once cell-cell contact is initiated and then decrease slightly at post-confluency. Both bradykinin and ionomycin cause filamin redistribution from the peripheral cell border to the cytosol of confluent EC. Forskolin, an activator of adenylate cyclase, blocks filamin translocation. Bradykinin activation of EC is not accompanied by significant proteolytic cleavage of filamin. Instead, intact filamin is recycled back to the membrane within 5-10 min of bradykinin stimulation. Inhibitors of calcium/calmodulin dependent protein kinase (KT-5926 and KN-62) attenuate bradykinin-induced filamin translocation. H-89, an inhibitor of cAMP-dependent protein kinase, causes translocation of filamin in unstimulated cells. Calyculin A, an inhibitor of protein phosphatases, also causes translocation of filamin in the absence of an inflammatory agent. ML-7, an inhibitor of myosin light chain kinase and phorbol myristate acetate, an activator of protein kinase C, do not cause filamin movement into the cytosol, indicating that these pathways do not modulate the translocation. Pharmacological data suggest that filamin translocation is initiated by the calcium/calmodulin-dependent protein kinase whereas the cAMP-dependent protein kinase pathway prevents translocation. Inflammatory agents therefore may increase vascular junctional permeability by increasing cytoplasmic calcium, which disassembles the microfilament dense peripheral band by releasing filamin from F-actin.
内皮细胞（EC）细胞骨架蛋白是炎症激动剂引发的第二信使级联反应最早的主要靶点之一。肌动蛋白结合蛋白通过调节肌动蛋白凝胶化 - 溶胶化状态以及膜 - 细胞骨架相互作用，部分调节细胞运动性和细胞间附着。这反过来又可调节内皮细胞间连接直径和通透性。非肌肉细丝蛋白（ABP - 280），一种二聚体肌动蛋白交联蛋白，促进F - 肌动蛋白的正交分支并将微丝连接到膜糖蛋白。在本研究中，免疫印迹分析表明，在稀疏的内皮细胞培养物中细丝蛋白水平较低，一旦开始细胞间接触便会升高，然后在汇合后略有下降。缓激肽和离子霉素都会导致汇合的内皮细胞中细丝蛋白从细胞周边边界重新分布到细胞质中。腺苷酸环化酶激活剂福斯可林可阻止细丝蛋白易位。内皮细胞的缓激肽激活并不伴随着细丝蛋白的显著蛋白水解切割。相反，完整的细丝蛋白在缓激肽刺激后5 - 10分钟内被循环回到细胞膜。钙/钙调蛋白依赖性蛋白激酶抑制剂（KT - 5926和KN - 62）减弱缓激肽诱导的细丝蛋白易位。cAMP依赖性蛋白激酶抑制剂H - 89导致未受刺激细胞中的细丝蛋白易位。蛋白磷酸酶抑制剂花萼海绵诱癌素A在没有炎症因子的情况下也会导致细丝蛋白易位。肌球蛋白轻链激酶抑制剂ML - 7和蛋白激酶C激活剂佛波酯肉豆蔻酸酯不会导致细丝蛋白移入细胞质，表明这些途径不会调节易位。药理学数据表明，细丝蛋白易位由钙/钙调蛋白依赖性蛋白激酶启动，而cAMP依赖性蛋白激酶途径阻止易位。因此，炎症因子可能通过增加细胞质钙来增加血管连接通透性，细胞质钙通过从F - 肌动蛋白释放细丝蛋白来分解微丝密集的周边带。
