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 (3)H-thymidine incorporation. Protein biochemistry was by western blot analysis.


RESULTS
Transient exposure to peroxynitrite stimulated the proliferation of PASMC (peroxynitrite 0.2 nM-2 μM) and PAEC (peroxynitrite 0.2 μM). Peroxynitrite 0.2 μM 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 μM. 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 μM. Peroxynitrite-induced proliferation and extracellular signal-regulated kinase (ERK) phosphorylation in PASMC was prevented by selective inhibitors of MAP kinase kinase (MEK) (U0126 5 μM, PD98059 50 μM), Raf-1 (Raf-1 kinase inhibitor 10 μM), Ras (FPT II and FPT III 10 μM) and protein kinase C (PKC) (GF109203X 10 μM). Inhibition of EGF or PDGF receptor signaling using AG-1296, AG-1478 or imatinib prevented peroxynitrite-induced cell proliferation and ERK phosphorylation in PASMC.


CONCLUSIONS
Peroxynitrite can stimulate proliferation of pulmonary artery cells, involving ERK, PKC and EGF or PDGF receptors.
Agbani
E O
EO
Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow G40NR, Scotland, UK.
Coats
P
P
Mills
A
A
Wadsworth
R M
RM
D000818:Animals; D002417:Cattle; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000187:drug effects* / Q000502:physiology; D066246:ErbB Receptors / Q000502:physiology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000502:physiology*; D005260:Female; D020929:Mitogen-Activated Protein Kinase Kinases / Q000502:physiology; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects*; D032389:Myocytes, Smooth Muscle / Q000187:drug effects* / Q000502:physiology; D030421:Peroxynitrous Acid / Q000494:pharmacology*; D011493:Protein Kinase C / Q000502:physiology*; D019908:Proto-Oncogene Proteins c-raf / Q000502:physiology; D011651:Pulmonary Artery / Q000166:cytology / Q000187:drug effects*; D017479:Receptors, Platelet-Derived Growth Factor / Q000502:physiology; D018631:ras Proteins / Q000502:physiology
D030421:Peroxynitrous Acid; D066246:ErbB Receptors; D017479:Receptors, Platelet-Derived Growth Factor; D019908:Proto-Oncogene Proteins c-raf; D011493:Protein Kinase C; D048049:Extracellular Signal-Regulated MAP Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D018631:ras Proteins