Mechanism of extracellular ATP-induced proliferation of vascular smooth muscle cells

The mitogenic effect of extracellular ATP was examined in cultured rat aortic smooth muscle cells (VSMCs). ATP, 2-methylthio-ATP, and ADP stimulated [³H]thymidine and [³H]leucine incorporation and cell growth, AMP, adenosine, UTP, and P(2X) agonists showed little of these effects. Reactive blue 2, a P(2Y) purinoceptor antagonist, was effective in suppressing the mitogenic effect of ATP and 2-methylthio-ATP, indicating that extracellular ATP-induced VSMC proliferation is mediated by P(2Y) purinoceptors. The P(2Y) purinoceptor activation was coupled to a pertussis toxin (PTX)-insensitive G protein (G(q)) and triggered phosphoinositide hydrolysis with subsequent activation of protein kinase C (PKC), Raf-1, and mitogen-activated protein kinase (MAPK) in VSMCs. In response to ATP, both 42- and 44-kDa MAPKs were activated, and tyrosine was phosphorylated. Western blot analysis using PKC isozyme-specific antibodies indicated that VSMCs express PKC-α, PKC-δ, and PKC-ζ. A complete down-regulation of PKC-α and PKC-δ was seen after 24-hr treatment with 12-O-tetradecanoylphorbol-13-acetate. When cells were pretreated with 12-O-tetradecanoyl-phorbol-13-acetate for 24 hr and subsequently challenged with ATP, Raf-1 activation and 42-kDa as well as 44- kDa MAPK tyrosine phosphorylation failed to be induced. These results demonstrate that ATP-induced Raf-1 and MAPK activations involve the activation of PKC-α and PKC-δ P(2Y) purinoceptor stimulation with ATP also caused accumulation of c-fos and c-myc mRNAs. Both Reactive blue 2 and staurosporine significantly blocked this increase by ATP. In conclusion, the mitogenic effect of ATP seemed to be triggered by activation of the G(q) protein-coupled P(2Y) purinoceptor that led to the formation of inositol trisphosphate and activation of PKC. PKC and, in turn, Raf-1 and MAPK were then activated, leading eventually to DNA synthesis and cell proliferation.