title: Phosphodiesterase-5A (PDE5A) is localized to the endothelial caveolae and modulates NOS3 activity.
abstract: OBJECTIVE
  It has been well demonstrated that phosphodiesterase-5A (PDE5A) is expressed in smooth muscle cells and plays an important role in regulation of vascular tone. The role of endothelial PDE5A, however, has not been yet characterized. The present study was undertaken to determine the presence, localization, and potential physiologic significance of PDE5A within vascular endothelial cells.
  
  
  RESULTS
  We demonstrate primary location of human, mouse, and bovine endothelial PDE5A at or near caveolae. We found that the spatial localization of PDE5A at the level of caveolin-rich lipid rafts allows for a feedback loop between endothelial PDE5A and nitric oxide synthase (NOS3). Treatment of human endothelium with PDE5A inhibitors resulted in a significant increase in NOS3 activity, whereas overexpression of PDE5A using an adenoviral vector, both in vivo and in cell culture, resulted in decreased NOS3 activity and endothelium-dependent vasodilation. The molecular mechanism responsible for these interactions is primarily regulated by cGMP-dependent second messenger. PDE5A overexpression also resulted in a significant decrease in protein kinase 1 (PKG1) activity. Overexpression of PKG1 rapidly activated NOS3, whereas silencing of the PKG1 gene with siRNA inhibited both NOS3 phosphorylation (S1179) and activity, indicating a novel role for PKG1 in direct regulation of NOS3.
  
  
  CONCLUSIONS
  Our data collectively suggest another target for PDE5A inhibition in endothelial dysfunction and provide another physiologic significance for PDE5A in the modulation of endothelial-dependent flow-mediated vasodilation. Using both in vitro and in vivo models, as well as human data, we show that inhibition of endothelial PDE5A improves endothelial function.
authors.affiliation: Division of Cardiology, Department of Medicine, Johns Hopkins University School of Medicine, Rutland Avenue, Baltimore, MD 21205, USA.
authors.forename: Milena A
authors.initials: MA
authors.lastname: Gebska
authors.forename: Blake K
authors.initials: BK
authors.lastname: Stevenson
authors.forename: Anna R
authors.initials: AR
authors.lastname: Hemnes
authors.forename: Trinity J
authors.initials: TJ
authors.lastname: Bivalacqua
authors.forename: Azeb
authors.initials: A
authors.lastname: Haile
authors.forename: Geoffrey G
authors.initials: GG
authors.lastname: Hesketh
authors.forename: Christopher I
authors.initials: CI
authors.lastname: Murray
authors.forename: Ari L
authors.initials: AL
authors.lastname: Zaiman
authors.forename: Marc K
authors.initials: MK
authors.lastname: Halushka
authors.forename: Nispa
authors.initials: N
authors.lastname: Krongkaew
authors.forename: Travis D
authors.initials: TD
authors.lastname: Strong
authors.forename: Carol A
authors.initials: CA
authors.lastname: Cooke
authors.forename: Hazim
authors.initials: H
authors.lastname: El-Haddad
authors.forename: Rubin M
authors.initials: RM
authors.lastname: Tuder
authors.forename: Dan E
authors.initials: DE
authors.lastname: Berkowitz
authors.forename: Hunter C
authors.initials: HC
authors.lastname: Champion
mesh_terms: D000818:Animals; D001011:Aorta / Q000166:cytology / Q000201:enzymology; D002417:Cattle; D021941:Caveolae / Q000378:metabolism*; D002478:Cells, Cultured; D003331:Coronary Vessels / Q000166:cytology / Q000201:enzymology; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5 / Q000235:genetics / Q000378:metabolism*; D042783:Endothelial Cells / Q000166:cytology / Q000201:enzymology*; D006801:Humans; D006976:Hypertension, Pulmonary / Q000378:metabolism / Q000473:pathology; D021962:Membrane Microdomains / Q000378:metabolism; D051379:Mice; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D011651:Pulmonary Artery / Q000166:cytology / Q000201:enzymology; D015398:Signal Transduction / Q000502:physiology; D014471:Umbilical Veins / Q000166:cytology / Q000201:enzymology; D014664:Vasodilation / Q000502:physiology*
chemical_list: C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases; C569718:PRKG1 protein, human; C569720:Prkg1 protein, mouse; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; C517381:PDE5A protein, human; C517382:Pde5a protein, mouse