Cholera toxin treatment of vascular smooth muscle cells decreases smooth muscle alpha-actin content and abolishes the platelet-derived growth factor-BB-stimulated DNA synthesis

1 The second messenger cyclic AMP regulates diverse biological processes su ch as cell morphology and cell growth. We examined the role of the second m essenger cyclic AMP on rat aortic vascular smooth muscle cell (VSMC) morpho logy and the intracellular transduction pathway mediated by platelet-derive d growth factor beta-receptor (PDGF-R beta). 2 The effect of PDGF-BB on VSMCs growth was assessed by [H-3]-thymidine inc orporation. Tyrosine phosphorylation of PDGF-R beta, PLC-gamma 1, ERK1 and ERK2, p125(FAK) and paxillin as well as Sm alpha-actin was examined by the chemiluminescence Western blotting method. Actin mRNA level was quantitated by Northern blotting. Visualization of Sm alpha-actin filaments, paxillin and PDGF-R beta was performed by immunofluorescence microscopy. 3 Cholera toxin (CTX; 10 nM) treatment lead to a large and sustained increa se in the cyclic AMP concentration after 2 h which correlated with change o f VSMC morphology including complete disruption of the Sm alpha-actin filam ent array and loss of focal adhesions. Treatment of VSMCs with CTX did not influence tyrosine phosphorylation of p125(FAK) and paxillin but decreased the content of a Sm alpha-actin protein. Maximal decrease of 70% was observ ed after 24 h of treatment. CTX also caused a 90% decrease of the actin mRN A level. CTX treatment completely abolished PDGF-BB stimulated DNA-synthesi s although PDGF-R beta level and subcellular distribution and translocation was not altered. Furthermore CTX attenuated the PDGF-BB-induced tyrosine p hosphorylation of the PDGF-R beta, PI 3'-K, PLC-gamma 1 and ERK1/2 indicati ng an action of cyclic AMP on PDGF-beta receptor. 4 We conclude that although cyclic AMP attenuates the PDGF-R beta mediated intracellular transduction pathway, an intact actin filament may be require d for the PDGF-BB-induced DNA synthesis in VSMCs.