Heimark R L, Degner M, Schwartz S M
Department of Pathology, University of Washington, Seattle 98195.
J Cell Biol. 1990 May;110(5):1745-56. doi: 10.1083/jcb.110.5.1745.
Confluent cultures of aortic endothelial cells contain two different cell-cell adhesion mechanisms distinguished by their requirement for calcium during trypsinization and adhesion. A hybridoma clone was isolated producing a monoclonal antibody Ec6C10, which inhibits Ca2(+)-dependent adhesion of endothelial cells. There was no inhibition of Ca2(+)-independent adhesion of endothelial cells and only a minor effect on Ca2(+)-dependent adhesion of smooth muscle cells. Immunoblotting analysis shows that the antibody Ec6C10 recognizes a protein in endothelial but not epithelial cells with an apparent molecular weight of 135,000 in reducing conditions and 130,000 in non-reducing conditions. Monoclonal antibody Ec6C10 reacts with an antigen at the cell surface as shown by indirect immunofluorescence of confluent endothelial cells in a junctional pattern outlining the cobblestone morphology of the monolayer. Removal of extracellular calcium increased the susceptibility of the antigen recognized by antibody Ec6C10 to proteolysis by trypsin. The role of the Ca2(+)-dependent cell adhesion molecule in organization of the dense peripheral microfilament band in confluent endothelial cells was examined by adjusting the level of extracellular calcium to modulate cell-cell contact. Addition of the monoclonal antibody Ec6C10 at the time of the calcium switch inhibited the extent of formation of the peripheral F-actin band. These results suggest an association between cell-cell contact and the peripheral F-actin band potentially through the Ca2(+)-dependent CAM.
主动脉内皮细胞的融合培养物包含两种不同的细胞间黏附机制，可根据它们在胰蛋白酶消化和黏附过程中对钙的需求来区分。分离出一个杂交瘤克隆，它产生一种单克隆抗体Ec6C10，该抗体可抑制内皮细胞的Ca2(+)依赖性黏附。对内皮细胞的Ca2(+)非依赖性黏附没有抑制作用，对平滑肌细胞的Ca2(+)依赖性黏附只有轻微影响。免疫印迹分析表明，抗体Ec6C10在还原条件下识别内皮细胞而非上皮细胞中的一种蛋白质，其表观分子量为135,000，在非还原条件下为130,000。如融合内皮细胞的间接免疫荧光所示，单克隆抗体Ec6C10以勾勒单层鹅卵石形态的连接模式与细胞表面的一种抗原发生反应。去除细胞外钙会增加抗体Ec6C10识别的抗原对胰蛋白酶蛋白水解的敏感性。通过调节细胞外钙水平以调节细胞间接触，研究了Ca2(+)依赖性细胞黏附分子在融合内皮细胞致密外周微丝带组织中的作用。在钙转换时添加单克隆抗体Ec6C10会抑制外周F-肌动蛋白带的形成程度。这些结果表明细胞间接触与外周F-肌动蛋白带之间可能通过Ca2(+)依赖性细胞黏附分子存在关联。
