Depolarization of mitochondria in endothelial cells promotes cerebral artery vasodilation by activation of nitric oxide synthase

Arterioscler Thromb Vasc Biol. 2013 Apr;33(4):752-9. doi: 10.1161/ATVBAHA.112.300560. Epub 2013 Jan 17.


Objective: Mitochondrial depolarization after ATP-sensitive potassium channel activation has been shown to induce cerebral vasodilation by the generation of calcium sparks in smooth muscle. It is unclear, however, whether mitochondrial depolarization in endothelial cells is capable of promoting vasodilation by releasing vasoactive factors. Therefore, we studied the effect of endothelial mitochondrial depolarization by mitochondrial ATP-sensitive potassium channel activators, BMS-191095 (BMS) and diazoxide, on endothelium-dependent vasodilation.

Approach and results: Diameter studies in isolated rat cerebral arteries showed BMS- and diazoxide-induced vasodilations that were diminished by endothelial denudation. Mitochondrial depolarization-induced vasodilation was reduced by inhibition of mitochondrial ATP-sensitive potassium channels, phosphoinositide-3 kinase, or nitric oxide synthase. Scavenging of reactive oxygen species, however, diminished vasodilation induced by diazoxide, but not by BMS. Fluorescence studies in cultured rat brain microvascular endothelial cells showed that BMS elicited mitochondrial depolarization and enhanced nitric oxide production; diazoxide exhibited largely similar effects, but unlike BMS, increased mitochondrial reactive oxygen species production. Measurements of intracellular calcium ([Ca(2+)]i) in cultured rat brain microvascular endothelial cells and arteries showed that both diazoxide and BMS increased endothelial [Ca(2+)]i. Western blot analyses revealed increased phosphorylation of protein kinase B and endothelial nitric oxide synthase (eNOS) by BMS and diazoxide. Increased phosphorylation of eNOS by diazoxide was abolished by phosphoinositide-3 kinase inhibition. Electron spin resonance spectroscopy confirmed vascular nitric oxide generation in response to diazoxide and BMS.

Conclusions: Pharmacological depolarization of endothelial mitochondria promotes activation of eNOS by dual pathways involving increased [Ca(2+)]i as well as by phosphoinositide-3 kinase-protein kinase B-induced eNOS phosphorylation. Both mitochondrial reactive oxygen species-dependent and -independent mechanisms mediate activation of eNOS by endothelial mitochondrial depolarization.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Benzopyrans / pharmacology
  • Blotting, Western
  • Calcium / metabolism
  • Cells, Cultured
  • Cerebral Arteries / drug effects
  • Cerebral Arteries / metabolism*
  • Cerebrovascular Circulation* / drug effects
  • Diazoxide / pharmacology
  • Dose-Response Relationship, Drug
  • Electron Spin Resonance Spectroscopy
  • Endothelial Cells / drug effects
  • Endothelial Cells / metabolism*
  • Enzyme Activation
  • Enzyme Inhibitors / pharmacology
  • Free Radical Scavengers / pharmacology
  • Imidazoles / pharmacology
  • Membrane Potential, Mitochondrial* / drug effects
  • Mitochondria / drug effects
  • Mitochondria / metabolism*
  • Nitric Oxide / metabolism
  • Nitric Oxide Synthase Type III / antagonists & inhibitors
  • Nitric Oxide Synthase Type III / metabolism*
  • Phosphatidylinositol 3-Kinase / metabolism
  • Phosphoinositide-3 Kinase Inhibitors
  • Phosphorylation
  • Potassium Channel Blockers / pharmacology
  • Potassium Channels / agonists
  • Potassium Channels / metabolism*
  • Proto-Oncogene Proteins c-akt / metabolism
  • Rats
  • Rats, Sprague-Dawley
  • Reactive Oxygen Species / metabolism
  • Signal Transduction
  • Vasodilation* / drug effects
  • Vasodilator Agents / pharmacology


  • BMS 191095
  • Benzopyrans
  • Enzyme Inhibitors
  • Free Radical Scavengers
  • Imidazoles
  • Phosphoinositide-3 Kinase Inhibitors
  • Potassium Channel Blockers
  • Potassium Channels
  • Reactive Oxygen Species
  • Vasodilator Agents
  • mitochondrial K(ATP) channel
  • Nitric Oxide
  • Nitric Oxide Synthase Type III
  • Nos3 protein, rat
  • Phosphatidylinositol 3-Kinase
  • Proto-Oncogene Proteins c-akt
  • Diazoxide
  • Calcium