ISSN:
0886-1544
Keywords:
vinculin
;
PDGF
;
cell growth
;
vascular smooth muscle
;
Life and Medical Sciences
;
Cell & Developmental Biology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Medicine
Notes:
Exposure of porcine vascular smooth muscle cells to platelet-derived growth factor (PDGF; 18-180 ng/ml) but not epidermal growth factor (EGF; 30ng/ml), somatomedin C (SmC; 30 ng/ml), or insulin (10 μM), results in a rapid, reversible, time- and concentration-dependent disapperance of vinculin staining in adhesion plaques; actin-containing stress fibers also become disrupted following exposure of cells to PDGF. Disapperance 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 disapperance 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-diethylamin) octy1-3,4,5-trimethoxybenzoate (TMA-8; 0.25-4 μM) and leupepetin (2-300 μM), and by n-α-rosyl-L-lysine chloromethylketone (TLCK; 100 μM) and trifluoperazine (TFP; 2.5 μM). Addition of PDGF to vascular smooth muscle cells caused a rapid, tranient 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 leupeption. This rise in cytosolic free calcium was found to occur in ∼ 80% of the sample population and dispalyed 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. Our data also suggest that cytosolic vinculin distribution is a sensitive indicator of the response of vascular smooth muslce cells to PDGF.
Additional Material:
9 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/cm.970080202
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