Mechanism of extracellular ATP-induced proliferation of vascular smooth muscle cells
Yu, S.M.; Chen, S.F.; Lau, Y.T.; Yang, C.M.; Chen, J.C.
Molecular Pharmacology 50(4): 1000-1009
1996
ISSN/ISBN: 0026-895X PMID: 8863847 Document Number: 457331
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-alpha, PKC-delta, and PKC-zeta. A complete down-regulation of PKC-alpha and PKC-delta was seen after 24-hr treatment with 12-O-tetradecanoylphorbol-13-acetate. When cells were pretreated with 12-O-tetradecanoylphorbol-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-alpha and PKC-delta. 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 Gq 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.