Phospholipase D activation in PDGF-stimulated vascular smooth muscle cells

PDGF is a potent mitogen and chemotactic factor in cells expressing PDGF receptors [ 1,2]. PDGF is also strongly implicated in the pathogenesis of a number of proliferative disorders including atherosclerosis and neoplasia [3,4]. The signal transduction mechanism through the PDGF receptor has therefore received much attention in recent years and involves a complex cascade of events initiated by receptor autophosphorylation. A number of proteins have been identified which associate with and are activated by the phosphorylated receptor. These include phospholipase Cy, phosphatidylinositol-3-kinase, GTPase activating protein of ras and pp60c-src [5,6] Activation of these proteins leads to further phosphorylation events which culminate in the induction and expression of several growth-related genes, notably c-fos and c-jun [7]. PDGF is also a potent stimulus for PLD in fibroblasts and smooth muscle cells [8,9] but the role of this pathway in PDGFsignalling is unclear. The aim of this study was to characterise PLD activation by PDGF in rat A10 vascular smooth muscle cells, in comparison with measurements of receptor autophosphorylation, and to assess certain inlubitors in both systems in an attempt to define more clearly a role for PLD in PDGF-signalling. A10 cells were grown to confluence in lOcm dishes in DMEM containing 10% foetal calf serum. Receptor autophosphorylation was measured by immunocytochemical labelling using a moose monoclonal antibody to phosphotyrosine (UBI) and detected by use of a second radiolabelled antibody (1% sheep anti-mouse Ig, Amersham). A protein of Mwt 180,000 was intensely labelled in a PDGF-dependent manner and was identified as the PDGF receptor using an anti-PDGF receptor antibody (UBI). The intensity of radiolabel was quantified using a Phosphorimager (Molecular Dynamics). PLD activity was measured as the incorporation of [3H]butanol into 13H]phosphatidylbutanol. Confluent cultures were incubated at