Molecular mechanism of the inhibitory effect of aldosterone on endothelial NO synthase activity

Hypertension. 2006 Jul;48(1):165-71. doi: 10.1161/01.HYP.0000226054.53527.bb. Epub 2006 Jun 5.

Abstract

Although the proinflammatory and profibrotic actions of aldosterone (Aldo) on the vasculature have been reported, the effects and molecular mechanisms of Aldo on endothelial function are yet to be determined. We investigated how Aldo regulates endothelial NO synthase (eNOS) function in human umbilical vein endothelial cells (HUVECs). HUVECs were incubated for 16 hours with Aldo 10(-7) mol/L. The concentration of reactive oxygen species was estimated by measuring 2',7'-dichlorodihydrofluorescein diacetate chemiluminescence. Signal transduction was estimated by Western immunoblots. Real-time RT-PCR was performed to measure expression of transcripts of endogenous GTP cyclohydrolase-1 and components of reduced nicotinamide-adenine dinucleotide phosphate oxidase. To eliminate the possible effect of the glucocorticoid receptor (GR) and to emphasize the role of mineralocorticoid receptor, we used GR small interfering RNA and knocked down GR expression in several experiments. NO output was estimated by intracellular cGMP concentration. Reactive oxygen species production increased significantly in Aldo-treated HUVECs but was abolished by pretreatment with eplerenone. Transcripts of p47(phox) were increased by Aldo treatment. Vascular endothelial growth factor-induced eNOS Ser 1177 but not Akt Ser 473 phosphorylation levels were reduced significantly by pretreatment with Aldo. Pretreatment with either eplerenone or okadaic acid restored phosphorylation levels of eNOS Ser 1177 in Aldo-treated cells, suggesting that protein phosphatase 2A was upregulated by Aldo via mineralocorticoid receptor. The decrease in NO output caused by Aldo pretreatment was reversed significantly by 5,6,7,8-tetrahydrobiopterin, GTP cyclohydrolase-1 overexpression, or p47(phox) knockdown. These results suggest that Aldo inhibits eNOS function through bimodal mechanisms of 5,6,7,8-tetrahydrobiopterin deficiency and protein phosphatase 2A activation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aldosterone / pharmacology*
  • Biopterins / analogs & derivatives*
  • Biopterins / deficiency
  • Biopterins / metabolism
  • Biopterins / pharmacology
  • Cells, Cultured
  • Endothelial Cells / drug effects
  • Endothelial Cells / enzymology*
  • Endothelium, Vascular
  • GTP Cyclohydrolase / genetics
  • GTP Cyclohydrolase / metabolism
  • Humans
  • NADPH Oxidases / genetics
  • NADPH Oxidases / metabolism
  • Nitric Oxide / biosynthesis*
  • Nitric Oxide Synthase Type III / drug effects*
  • Nitric Oxide Synthase Type III / metabolism
  • Phosphoprotein Phosphatases / metabolism
  • Phosphorylation
  • Protein Phosphatase 2
  • Reactive Oxygen Species / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Signal Transduction
  • Transcription, Genetic
  • Umbilical Veins / cytology

Substances

  • Reactive Oxygen Species
  • Biopterins
  • Nitric Oxide
  • Aldosterone
  • Nitric Oxide Synthase Type III
  • NADPH Oxidases
  • neutrophil cytosolic factor 1
  • Phosphoprotein Phosphatases
  • Protein Phosphatase 2
  • GTP Cyclohydrolase
  • sapropterin