Liau G, Janat M F, Wirth P J
American Red Cross, Jerome H. Holland Laboratory for Biomedical Sciences, Rockville, Maryland 20855.
J Cell Physiol. 1990 Feb;142(2):236-46. doi: 10.1002/jcp.1041420204.
We have examined alpha-smooth muscle actin (alpha-SM actin) protein and mRNA levels in proliferating and density-arrested rabbit vascular smooth muscle cells (SMC) and also studied overall polypeptide synthesis in these cells by two-dimensional (2-D) gel electrophoresis. Of the approximately 1,000 cellular polypeptides resolved by 2-D gel analysis, we consistently detected increased expression of 12 polypeptides in growth-arrested SMC. These polypeptides, with apparent molecular weights of 24,000 to 55,000 exhibited relative increases of between fourfold to greater than tenfold. Three of these polypeptides were expressed at undetectable levels in proliferating SMC. We also detected 12 secreted polypeptides that were expressed at higher levels in growth-arrested SMC. More changes were associated with the secreted polypeptides, since they represented approximately 4% of the total resolved secreted polypeptides, while only 1% of the cellular polypeptides were increased in high-density growth-arrested cells. Under these conditions we observed no change in relative alpha-SM actin protein content as determined by 2-D gel analysis and Western blots. This was corroborated by high levels of alpha-SM actin mRNA levels in both proliferating and high-density growth-arrested SMC. These results indicate rabbit vascular SMC maintain a high level of expression of a smooth muscle differentiation marker (alpha-SM actin) in a proliferation- and density-independent manner. We also examined polypeptide synthesis in SMC isolated by enzymatic digestion of the aorta vs. cells isolated by the explant method. We found that although overall protein patterns were remarkably similar, several differences were observed. These differences were not due to increased contamination by fibroblasts, since both enzymatically- and explant-derived SMC contained high levels of alpha-SM actin as determined by immunofluorescence and by Northern analysis.
我们检测了增殖的和密度抑制的兔血管平滑肌细胞（SMC）中α-平滑肌肌动蛋白（α-SM肌动蛋白）的蛋白质和mRNA水平，并且还通过二维（2-D）凝胶电泳研究了这些细胞中的整体多肽合成。在通过二维凝胶分析分辨出的大约1000种细胞多肽中，我们始终检测到生长抑制的SMC中12种多肽的表达增加。这些多肽的表观分子量为24,000至55,000，相对增加了4倍至10倍以上。其中三种多肽在增殖的SMC中表达水平不可检测。我们还检测到12种分泌型多肽，它们在生长抑制的SMC中表达水平更高。与分泌型多肽相关的变化更多，因为它们约占总分辨分泌型多肽的4％，而在高密度生长抑制细胞中仅1％的细胞多肽增加。在这些条件下，通过二维凝胶分析和蛋白质印迹法测定，我们观察到α-SM肌动蛋白的相对蛋白质含量没有变化。增殖的和高密度生长抑制的SMC中高水平的α-SM肌动蛋白mRNA水平证实了这一点。这些结果表明兔血管SMC以增殖和密度独立的方式维持平滑肌分化标志物（α-SM肌动蛋白）的高水平表达。我们还研究了通过主动脉酶消化分离的SMC与通过外植体方法分离的细胞中的多肽合成。我们发现，尽管总体蛋白质模式非常相似，但仍观察到一些差异。这些差异不是由于成纤维细胞污染增加所致，因为通过免疫荧光和Northern分析确定，酶促来源和外植体来源的SMC均含有高水平的α-SM肌动蛋白。
