Peroxynitrite stimulates pulmonary artery endothelial and smooth muscle cell proliferation: Involvement of ERK and PKC

Background: There is evidence that peroxynitrite is generated in pulmonary hypertension and we have therefore investigated whether peroxynitrite can cause proliferation of pulmonary artery cells. Methods: Bovine pulmonary artery endothelial (PAEC) and smooth muscle cells (PASMC) were exposed to peroxynitrite solution or to the peroxynitrite generating compound, 3-morpholinosydnonimine (SIN-1). Vascular cell proliferation was determined by cell count and H-3-thymidine incorporation. Protein biochemistry was by western blot analysis. Results: Transient exposure to peroxynitrite stimulated the proliferation of PASMC (peroxynitrite 0.2 nM -2 mu M) and PAEC (peroxynitrite 0.2 mu M). Peroxynitrite 0.2 mu M stimulated DNA synthesis in PASMC cell by 200 +/- 22% and in PAEC by 137 +/- 4%. DNA synthesis in PAEC and PASMC was also stimulated by the peroxynitrite generator SIN-1 2 mu M. Cell proliferation was accompanied by activation of ERK, which peaked at 15 min and remained elevated for 12 h in PASMC. However peroxynitrite at the concentrations used in this study did not activate the stress pathways p38 mitogen activated protein kinase (MAPK) or Jun N-terminal kinase (JNK). Peroxynitrite-induced proliferation and ERK phosphorylation in PASMC were abolished by the peroxynitrite scavenger ebselen 5 mu M. Peroxynitrite-induced proliferation and extracellular signal-regulated kinase (ERK) phosphorylation in PASMC was prevented by selective inhibitors of MAP kinase kinase (MEK) (U0126 5 mu M, PD98059 50 mu M), Raf-1 (Raf-1 kinase inhibitor 10 mu M), Ras (FPT II and FPT III 10 mu M) and protein kinase C (PKC) (GF109203X 10 mu M). Inhibition of EGF or PDGF receptor signaling using AG-1296. AG-1478 or imatinib prevented peroxynitrite-induced cell proliferation and ERK phosphorylation in PASMC. Conclusion: Peroxynitrite can stimulate proliferation of pulmonary artery cells, involving ERK, PKC and EGF or PDGF receptors. (C) 2010 Elsevier Ltd. All rights reserved.