ABSTRACT
AIMS: 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. METHODS AND 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. CONCLUSION: 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.
Subject(s)
Caveolae/metabolism , Cyclic Nucleotide Phosphodiesterases, Type 5/metabolism , Endothelial Cells/enzymology , Nitric Oxide Synthase Type III/metabolism , Vasodilation/physiology , Animals , Aorta/cytology , Aorta/enzymology , Cattle , Cells, Cultured , Coronary Vessels/cytology , Coronary Vessels/enzymology , Cyclic GMP-Dependent Protein Kinase Type I , Cyclic GMP-Dependent Protein Kinases/genetics , Cyclic GMP-Dependent Protein Kinases/metabolism , Cyclic Nucleotide Phosphodiesterases, Type 5/genetics , Endothelial Cells/cytology , Humans , Hypertension, Pulmonary/metabolism , Hypertension, Pulmonary/pathology , Membrane Microdomains/metabolism , Mice , Pulmonary Artery/cytology , Pulmonary Artery/enzymology , Signal Transduction/physiology , Umbilical Veins/cytology , Umbilical Veins/enzymologyABSTRACT
PURPOSE: To use the technique of differential gene display to analyze changes in gene expression that occur during the development of and recovery from form-deprivation myopia. METHODS: The differential display-reverse transcriptase-polymerase chain reaction technique was used to detect cDNAs that are differentially expressed after 24 h (including 12 h in the light) after fitting with a diffuser to induce form-deprivation myopia. Messenger RNA levels were determined by quantitative Northern blotting in retinas after 11 days of form deprivation or in retinas where the diffusers had been removed the previous day. RESULTS: Twenty-six differentially expressed genes were processed in our initial screen. Two of these, alphaB-crystallin and retinoic acid receptor-alpha, were studied further. Levels of alphaB-crystallin mRNA were increased on day 11 in retinas from form-deprived eyes relative to eyes of control chickens and were reduced to below those levels within 6 to 12 h after removal of the diffusers. Levels of retinoic acid receptor-alpha mRNA showed similar changes, except that after removal of the diffusers, the levels further increased. CONCLUSIONS: The technique of differential gene display can be used to detect changes in gene expression during the regulation of eye growth. The response of alphaB-crystallin is particularly interesting because expression increases when eye growth is high and decreases when eye growth slows.
