Abstract P20: Catalase Blocks Hyperoxia-Induced Mitochondrial Oxidative Stress and Decreases Phosphodiesterase 5 (PDE5) Activity in Ovine Fetal Pulmonary Artery Smooth Muscle Cells (FPASMC)
Background: In the pulmonary vasculature, cGMP is a central mediator for vasorelaxation. PDE5 degrades cGMP and inhibits nitric oxide-mediated, cGMP-dependent vasorelaxation. We previously reported that 24 hours of 100% O2 increases PDE5 activity in fetal PASMC and in ventilated neonatal lambs. However, the mechanisms underlying these responses, and the effect of shorter O2 exposures, are not fully understood.
Objective: To determine the mechanism by which hyperoxia initiates oxidative stress and regulates PDE5.
Design/Methods: Ovine FPASMC were exposed to normoxia (21% O2) or hyperoxia (95% O2) for 30 minutes. Redox status in specific cellular compartments was determined using recombinant adenoviruses expressing RoGFP, a ratiometric protein thiol redox sensor, targeted to the cytosol or the mitochondrial matrix. PDE5 activity was assessed via commercially-available colorimetric assay, and cGMP accumulation was assayed by EIA at baseline and following an NO donor, DETANONOate (100 μM). In some cells, MnSOD or catalase was overexpressed in the mitochondrial matrix using recombinant adenoviruses.
Results: Thirty minutes of hyperoxia significantly increased mitochondrial protein thiol oxidation compared with normoxic baseline (43.4±2.1% vs. 28.8±3.9%), but had no effect on cytosolic oxidative stress. Hyperoxia also increased PDE5 activity (157±18% of control) and decreased cGMP in response to NO (37±17% of control). MnSOD overexpression did not significantly alter RoGFP oxidation, but catalase overexpression significantly attenuated the hyperoxia-induced RoGFP oxidation in the matrix, compared to FPASMC infected with an empty adenoviral vector (mito-catalase: 33±2% of control). Catalase also decreased PDE5 activity (20±3% of control), and restored cGMP levels in response to NO (186±50% of control).
Conclusions: Acute hyperoxia exposure increases oxidative stress in the mitochondrial matrix, without altering oxidative stress in the cytosol. Acute hyperoxia also causes ROS-dependent increases in PDE5 activity, suggesting that oxidant signals, specifically H2O2, in the mitochondrial matrix initiate abnormal signaling in the pulmonary vascular smooth muscle cell.