Peroxynitrite impairs pulmonary artery cell function through JNK and p38 MAP kinase

Ejaife Agbani1, Paul Coats1, Andrew Mills2, Roger Wadsworth1. 1Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde., Glasgow, Scotland, United Kingdom, 2Department of Pure and Applied Chemistry, University of Strathclyde., Glasgow, Scotland, United Kingdom.

Rationale: Peroxynitrite is up-regulated in lung sections of infants with pulmonary hypertension as well as in patients with long standing and severe forms of the disease. We hypothesize that if ONOO- has a significant role in the etiopathogenesis of pulmonary hypertension, the non-cytotoxic biological effects of the radical such as the stimulation of pulmonary artery cells proliferation must occur at concentrations not more than the lower limits of toxicity. We therefore sought to establish the cytotoxic limit of peroxynitrite in pulmonary artery endothelial cells.

Methods: Cells were primary isolates of bovine pulmonary arteries identified as endothelial and smooth muscle cells by standard technologies. 24 and 48h cell viability was assessed by Trypan blue dye exclusion. Concentration dependent effects of ONOO- on cell proliferation were investigated by [3H] thymidine incorporation. Signal transduction investigation was by western blot analysis. Pulmonary endothelial artery cells (PAEC) were exposed to authentic or SIN-1 generated ONOO-. Peroxynitrite concentration and time course were determined by absorbance spectroscopy and by quenching of NADH fluorescence.

Results: Relative to untreated and vehicle treated PAEC, cell viability did not decline following exposure to 0.02 or 0.2μM ONOO- for 24h. However 24h exposure to 2μM ONOO- significantly reduced PAEC viability. While PAEC proliferated in response to 0.02 or 0.2μM ONOO-, 2μM caused significant reduction in cell number (p<0.01) due to cell lift-off at 48h. The ONOO- generator caused significant membrane impairment after 24h exposure period. The effect was similar between 0.2, 2, or 20μM SIN-1 treated cells (p<0.05). PAEC exposed to 2nM, 0.02μM or 0.2μM ONOO- for 15min or 3h did not stimulate the phosphorylation of p38. However, 20μM ONOO- caused a 3-fold increase in p38 activation relative to untreated cells at 15min. This was associated with severe reduction in DNA synthesis and further decrease in cell viability. The decrease in DNA synthesis and cell viability caused by 2μM ONOO- was neither associated with p38 activation nor attenuated by 10-30μM p38 inhibitor (SB230580); in addition, 2 and 20μM ONOO- significantly activated JNK.

Conclusion: The results confirm the cytotoxic effects of ≥ 2μM peroxynitrite; preliminary data linked this to activated JNK but not phosphorylated p38. We however report a lower threshold of 2μM for this effect in pulmonary artery endothelial cells.