Abstract 20720: H2O2 from Mitochondrial Sources Increases PDE5 Activity in Pulmonary Artery Smooth Muscle Cells (PASMC) from Persistent Pulmonary Hypertension (PPHN) Lambs
Background: In the pulmonary vasculature, phosphodiesterase 5 (PDE5) degrades cGMP and inhibits NO-mediated vasorelaxation. We previously reported that 24 h of 100% O2 ventilation increases PDE5 activity in pulmonary arteries (PA) of neonatal lambs in an oxidant-dependent manner and that this response is amplified in PPHN lambs.
Objective: To determine mechanisms that regulate increased PDE5 activity in PASMC from PPHN lambs.
Design/Methods: PPHN was induced by ductus arteriosus ligation in fetal lambs at 126d gestation. PASMC generated from control and PPHN lambs were exposed to 21% or 95% O2 for 24 h. Mitochondrial matrix oxidative stress was assessed by a ratiometric, redox-sensitive protein sensor, Mito-RoGFP. PDE5 protein was measured by Western blot, cGMP by EIA, PDE5 activity by colorimetric assay, and MnSOD activity by commercially available assay. Some PPHN lambs were ventilated with 100% O2 for 24h. Five lambs received PEG-Catalase (15,000 units/kg), and 8 lambs were ventilated with O2 alone (PPHN-O2).
Results: At baseline, PPHN PASMC had decreased PDE5 protein (39±8%) but increased PDE5 activity (171±23%) vs. control PASMC. Hyperoxia increased PDE5 activity and blunted cGMP response to NO in both PPHN and control PASMC. Basal mitochondrial matrix oxidative stress (2.2±0.1-fold) and MnSOD activity (5.1±2-fold) were increased in PPHN vs. control PASMC. Hyperoxia increased mitochondrial matrix oxidant stress in PPHN and control PASMC, but increased MnSOD activity only in PPHN PASMC. Mitochondrially targeted catalase blocked hyperoxia-induced PDE5 activity in both PPHN (32±11%) and control (36±15%) PASMC. In intact lambs, catalase decreased PA PDE5 expression (52±12%) and activity (35±16%) and increased PA cGMP (458±100%).
Conclusions: Mitochondrial matrix H2O2 is increased in PPHN at baseline, likely due in part to increased MnSOD activity. This primes PASMC for an exaggerated hyperoxia response, as demonstrated by greater increases in PDE5 activity. Catalase restores normal postnatal patterns of PDE5 expression and activity and increases cGMP. These findings suggest that H2O2 generated by the mitochondria may mediate the abnormal pulmonary vascular signaling associated with PPHN, in part through activation of PDE5.
- © 2010 by American Heart Association, Inc.