Elevated endothelin-1 levels impair nitric oxide homeostasis through a PKC-dependent pathway

Circulation. 2006 Jul 4;114(1 Suppl):I319-26. doi: 10.1161/CIRCULATIONAHA.105.001503.


Background: Endothelin-1 (ET-1) plays an important role in the maintenance of vascular tone and pathological states such as ischemia/reperfusion (I/R) injury, coronary vasospasm, and cardiac allograft vasculopathy. We assessed the effects of elevated ET-1 levels as seen after I/R to determine if ET-1 modulates nitric oxide (NO) production via the translocation of specific protein kinase C (PKC) isoforms.

Methods and results: Human saphenous vein endothelial cells (HSVECs) (n=8) were incubated with ET-1 or phosphate-buffered saline (PBS) for 24 hours. NO production was determined in the supernatant by measuring nitrate/nitrite levels. Protein expression of endothelial nitric oxide synthase (eNOS), inducible NOS (iNOS), caveolin-1 and PKC were determined. Lastly, PKC translocation and activity were assessed after exposure to the drug of interest. HSVECs exposed to ET-1 displayed decreased NO production. PKC inhibition reduced NO production, whereas PKC activation increased production. NO production was maintained when HSVECs exposed to ET-1 were treated with the PKC agonist, PMA. eNOS protein expression was reduced after ET-1 treatment. PKC inhibition also downregulated eNOS protein expression, whereas PMA upregulated expression. ET-1 exposure led to a significant increase in PKCdelta and PKCalpha translocation compared with control, whereas translocation of PKClambda was inhibited. ET-1 exposure significantly reduced overall PKC activity compared with control.

Conclusions: Our study demonstrates that high levels of ET-1 impair endothelial NO production via an isoform-specific PKC-mediated inhibition of eNOS expression. ET-1 antagonism with bosentan stimulates translocation of PKClambda and leads to increased PKC activity and NO production. ET-1 antagonism may provide a novel therapeutic strategy to improve vascular homeostasis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alkaloids
  • Benzophenanthridines
  • Bosentan
  • Caveolin 1 / biosynthesis
  • Caveolin 1 / genetics
  • Cell Membrane / enzymology
  • Cells, Cultured / drug effects
  • Cells, Cultured / enzymology
  • Endothelial Cells / drug effects
  • Endothelial Cells / enzymology*
  • Endothelin-1 / antagonists & inhibitors
  • Endothelin-1 / pharmacology*
  • Endothelium, Vascular / cytology
  • Enzyme Activation / drug effects
  • Enzyme Induction / drug effects
  • Homeostasis / drug effects
  • Isoenzymes / antagonists & inhibitors
  • Isoenzymes / physiology*
  • Naphthalenes / pharmacology
  • Nitric Oxide / biosynthesis*
  • Nitric Oxide Synthase Type II / biosynthesis
  • Nitric Oxide Synthase Type II / genetics
  • Nitric Oxide Synthase Type III / biosynthesis*
  • Nitric Oxide Synthase Type III / genetics
  • Phenanthridines / pharmacology
  • Protein Kinase C / antagonists & inhibitors
  • Protein Kinase C / physiology*
  • Protein Kinase C-alpha / metabolism
  • Protein Kinase C-delta / metabolism
  • Protein Transport / drug effects
  • Saphenous Vein / cytology
  • Sulfonamides / pharmacology
  • Tetradecanoylphorbol Acetate / pharmacology


  • Alkaloids
  • Benzophenanthridines
  • CAV1 protein, human
  • Caveolin 1
  • Endothelin-1
  • Isoenzymes
  • Naphthalenes
  • Phenanthridines
  • Sulfonamides
  • Nitric Oxide
  • chelerythrine
  • Nitric Oxide Synthase Type II
  • Nitric Oxide Synthase Type III
  • Protein Kinase C
  • Protein Kinase C-alpha
  • Protein Kinase C-delta
  • protein kinase C lambda
  • calphostin C
  • Tetradecanoylphorbol Acetate
  • Bosentan