Abstract 321: Reactive Oxygen Species (ROS) Induce Phosphodiesterase 5 (PDE5) Expression and Activity in Ovine Fetal Pulmonary Artery Smooth Muscle Cells (FPASMC)
Background: In the pulmonary vasculature, cGMP concentrations are regulated in part by a cGMP-dependent phosphodiesterase, PDE5. Little is known about the mechanisms that regulate PDE5 activity and expression in the pulmonary vasculature after birth. Objective: To determine the effects of oxygen and ROS on PDE5 expression and activity. Design/Methods: Ovine fetal PASMC (FPASMC) were incubated in 21% or 95% O2 for 24 hours with or without the NO donor DETANONOate (100 μM), or in 21% O2 with H2O2 (50 μM). Oxidative stress was measured with a Ro-GFP fluorescent probe and flow cytometry. cGMP was measured via EIA. PDE5 protein was quantified by Western blot, mRNA by real-time PCR, and activity via colorimetric assay. Late gestation fetal lambs were ventilated for 24 hours with 100% O2; lung and PA samples were harvested and analyzed for PDE5 protein and activity. Results: Hyperoxia increased Ro-GFP oxidation relative to untreated FPASMC (33.7± 10.9% vs. 15.7± 5.7%), indicating increased ROS. Treatment with DETANONOate led to significantly greater cGMP levels in 21% O2 versus 95% O2-exposed cells (4.1 vs 2.0 fold). Hyperoxia significantly increased PDE5 mRNA (270± 50%), protein (198± 67%), phosphorylation (457± 205%), and activity (201± 72%) relative to normoxia-exposed cells. H2O2 treatment of normoxic FPASMC significantly increased PDE5 protein (311± 87%), phosphorylation (197± 10%), and activity (163± 32%) compared to untreated cells. Ventilation of intact lambs with 100% O2 significantly increased PDE5 protein (179± 23%) and activity (263± 60%), relative to 21% O2 exposed controls. Conclusions: Exposure to either hyperoxia or exogenous oxidants increases both PDE5 expression and activity, leading to a diminished cGMP response to exogenous NO, which may result in impaired vasorelaxation. Increased oxidative stress in hyperoxia-exposed FPASMC implicates ROS in the regulation of PDE5 expression and activity. These data suggest that PDE5 expression and activity play a critical role in modulating neonatal pulmonary vascular tone, and modulate the response to common therapies for neonatal respiratory failure and pulmonary hypertension, such as oxygen and inhaled NO.