Herman B, Roe M W, Harris C, Wray B, Clemmons D
Department of Anatomy, Lineberger Cancer Research Center, University of North Carolina Medical School, Chapel Hill 27514.
Cell Motil Cytoskeleton. 1987;8(2):91-105. doi: 10.1002/cm.970080202.
Exposure of porcine vascular smooth muscle cells to platelet-derived growth factor (PDGF; 18-180 ng/ml) but not epidermal growth factor (EGF; 30 ng/ml), somatomedin C (SmC; 30 ng/ml), or insulin (10 microM), results in a rapid, reversible, time- and concentration-dependent disappearance of vinculin staining in adhesion plaques; actin-containing stress fibers also become disrupted following exposure of cells to PDGF. Disappearance of vinculin staining from adhesion plaques is also caused by 12-O-tetradecanoyl-phorbol-13-acetate (TPA; 200-400 nM), though the time course of the disappearance of vinculin staining under these conditions takes longer than in cells exposed to PDGF. The PDGF-induced removal of vinculin from adhesion plaques was inhibited in a concentration-dependent fashion by 8-(N,N-diethylamino) octyl-3,4,5-trimethoxybenzoate (TMB-8; 0.25-4 microM) and leupepetin (2-300 microM), and by n-alpha-tosyl-L-lysine chloromethylketone (TLCK; 100 microM) and trifluoperazine (TFP; 2.5 microM). Addition of PDGF to vascular smooth muscle cells caused a rapid, transient increase in cytosolic free calcium, from a basal resting level of 146 +/- 6.9 nM (SEM, n = 62) to 414 +/- 34 nM (SEM, n = 22) as determined using the calcium-sensitive indicator Fura-2 and Digitized Video Microscopy. This increase in cellular calcium preceded the disappearance of vinculin from adhesion plaques and was partially blocked by pretreatment of cells with TMB-8 but not leupeptin. This rise in cytosolic free calcium was found to occur in approximately 80% of the sample population and displayed both spatial and temporal subcellular heterogeneity. Exposure of cells to TPA (100 nM) did not result in a change in cytosolic free calcium. Both PDGF (20 ng/ml) and TPA (100 nM) caused cytosolic alkalinization which occurred after PDGF-induced disruption of vinculin from adhesion plaques, as determined using the pH-sensitive indicator BCECF and Digitized Video Microscopy. PDGF stimulated DNA synthesis and vinculin disruption in a similar dose-dependent fashion. Both could be inhibited by leupeptin or TMB-8. These results suggest that 1) exposure of vascular smooth muscle cells to PDGF is associated with the disruption of vinculin from adhesion plaques, 2) PDGF-induced vinculin disruption is regulated by an increase in cytosolic calcium (but not cytosolic alkalinization), and involves proteolysis; 3) activation of protein kinase C also causes vinculin removal from adhesion plaques but by a calcium-independent mechanism, and 4) the cellular response to PDGF-stimulated increases in cytosolic free calcium is heterogeneous.(ABSTRACT TRUNCATED AT 400 WORDS)
将猪血管平滑肌细胞暴露于血小板衍生生长因子（PDGF；18 - 180 ng/ml）而非表皮生长因子（EGF；30 ng/ml）、生长调节素C（SmC；30 ng/ml）或胰岛素（10 μM）时，粘着斑中纽蛋白染色会迅速、可逆、呈时间和浓度依赖性地消失；细胞暴露于PDGF后，含肌动蛋白的应力纤维也会受到破坏。12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯（TPA；200 - 400 nM）也会导致粘着斑中纽蛋白染色消失，不过在这些条件下纽蛋白染色消失的时间进程比细胞暴露于PDGF时更长。8 - （N，N - 二乙氨基）辛基 - 3,4,5 - 三甲氧基苯甲酸酯（TMB - 8；0.25 - 4 μM）、亮抑酶肽（2 - 300 μM）、N - α - 对甲苯磺酰 - L - 赖氨酸氯甲基酮（TLCK；100 μM）和三氟拉嗪（TFP；2.5 μM）能以浓度依赖性方式抑制PDGF诱导的粘着斑中纽蛋白的去除。向血管平滑肌细胞中添加PDGF会导致胞质游离钙迅速、短暂升高，使用钙敏感指示剂Fura - 2和数字视频显微镜测定，基础静息水平从146±6.9 nM（SEM，n = 62）升至414±（SEM，n = 22）。这种细胞内钙的升高先于粘着斑中纽蛋白的消失，并且用TMB - 8预处理细胞可部分阻断，但亮抑酶肽不能。发现约80%的样本群体中会出现这种胞质游离钙的升高，且表现出亚细胞水平的空间和时间异质性。细胞暴露于TPA（100 nM）不会导致胞质游离钙发生变化。使用pH敏感指示剂BCECF和数字视频显微镜测定发现，PDGF（20 ng/ml）和TPA（100 nM）都会导致胞质碱化，且发生在PDGF诱导粘着斑中纽蛋白破坏之后。PDGF以类似的剂量依赖性方式刺激DNA合成和纽蛋白破坏。两者都可被亮抑酶肽或TMB - 8抑制。这些结果表明：1）血管平滑肌细胞暴露于PDGF与粘着斑中纽蛋白的破坏有关；2）PDGF诱导的纽蛋白破坏受胞质钙升高（而非胞质碱化）调节，且涉及蛋白水解；3）蛋白激酶C的激活也会导致粘着斑中纽蛋白去除，但通过与钙无关的机制；4）细胞对PDGF刺激引起的胞质游离钙升高的反应是异质性的。（摘要截断于400字）
