Abstract 1472: PDE5A Reciprocally Regulates NOS3 Expression and Activity in Vascular Endothelial Cells.
It has been well demonstrated that PDE5A is expressed in smooth muscle cells (SMCs) and plays an important role in regulation of vascular tone. However, recently, PDE5A expression in non-traditional cell types has elucidated a critical role for the protein in regulating responses to physical and neurohormonal stress. The present study was undertaken to determine the presence, localization and potential physiologic significance of PDE5A within vascular endothelial cells. PDE5A expression was identified by both qPCR and western blot in isolated mouse pulmonary vascular endothelial cells (mPVEC) as well as cultured bovine aortic endothelial cells (bAEC). PDE5A activity was measured and found to comprise ~70% of cGMP-dependent PDE activity in mPVEC and bAEC. PDE5A immunolocalization in mPVEC and bAEC was confirmed by confocal microscopy and immuno electron microscopy where PDE5A appeared to be localized at/near caveolae. Caveolar localization of PDE5A was confirmed using a sucrose gradient fractionation method in order to isolate fractions rich in lipid raft caveolae which showed high expression of caveolin-1, and NOS3. PDE5A was also found to be expressed in cytosolic fractions of endothelium as well. Given the spatial localization of PDE5A at the level of caveolae, we sought to determine whether PDE5A may reciprocally regulate NOS3 activity. Under basal conditions, treatment with sildenafil citrate (1μM) for 48 hours resulted in a 20% increase (P<0.05) in NO2/NO3 in culture medium and a 15% increase in NOS3 activity by DAF2 fluorescence (P<0.05). Overexpression of bovine PDE5A using an adenoviral vector which produced a 3–5 fold increase in PDE5A protein expression and activity, resulted in a 30% reduction in NOS3 activity (P<0.05), 23% reduction in medium NO2/NO3 (P<0.05), and a reduction in pNOS3 expression by western blot analysis and compared to cells transfected with reporter gene only. These findings demonstrate that PDE5A is highly expressed in vascular endothelium and reciprocally regulates NOS3 activity. These data may suggest another potential target for PDE5A inhibition in endothelial dysfunction and other vascular diseases.