Platelet‐derived growth factor‐induced alterations in vinculin distribution in porcine vascular smooth muscle cells

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Abstract
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.