Depending on the number of phosphate groups, diadenosine polyphosphates (ApnA, Ap3A, Ap4A, Ap5A and Ap6A) differ in properties such as proliferation, apoptosis, vasoconstriction and vasodilatation of vascular smooth muscle cells (VSMCs). Possible signaling pathways leading to effects such as proliferation are still unknown. This study examined the proliferative effects of diadenosine polyphosphates on VSMCs and their intracellular pathways. Proliferation of VSMCs was measured by the cell count and [(3)H] thymidine incorporation. Phosphorylation of the MAP kinases ERK1/2 was determined by Western blotting. Single-cell [Ca(2+)](i) measurements were done to determine the influence of [Ca(2+)](i) on intracellular signaling. Stress fiber formation was assessed by fluorescence microscopy to detect an influence of G alpha(12). Ap3A and Ap4A, but not Ap5A or Ap6A, were shown to increase proliferation of VSMCs by activating P2Y receptors, which leads to stimulation of the Ras-Raf-MEK-ERK1/2 cascade. Ap3A- and Ap4A-induced activation of the MAP kinases ERK1/2 was dependent on a signaling pathway that included the EGF receptor, PKC, PLCbeta and the increase of [Ca(2+)](i). In conclusion, Ap3A and Ap4A, but not Ap5A or Ap6A, induce proliferation of VSMCs by a signaling pathway that begins with activation of P2Y receptors and leads to stimulation of the MAP kinases ERK1/2.
