Peroxynitrite formed during a transient episode of brain ischaemia increases endothelium-derived hyperpolarization-type dilations in thromboxane/prostaglandin receptor-stimulated rat cerebral arteries

Acta Physiol (Oxf). 2017 May;220(1):150-166. doi: 10.1111/apha.12809. Epub 2016 Oct 16.

Abstract

Aim: Increased thromboxane A2 and peroxynitrite are hallmarks of cerebral ischaemia/reperfusion (I/R). Stimulation of thromboxane/prostaglandin receptors (TP) attenuates endothelium-derived hyperpolarization (EDH). We investigated whether EDH-type middle cerebral artery (MCA) relaxations following TP stimulation are altered after I/R and the influence of peroxynitrite.

Methods: Vascular function was determined by wire myography after TP stimulation with the thromboxane A2 mimetic 9,11-dideoxy-9α, 11α -methano-epoxy prostaglandin F2α (U46619) in MCA of Sprague Dawley rats subjected to MCA occlusion (90 min)/reperfusion (24 h) or sham operation, and in non-operated (control) rats. Some rats were treated with saline or the peroxynitrite decomposition catalyst 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron (III) (20 mg kg-1 ). Protein expression was evaluated in MCA and in human microvascular endothelial cells submitted to hypoxia (overnight)/reoxygenation (24 h) (H/R) using immunofluorescence and immunoblotting.

Results: In U46619-pre-constricted MCA, EDH-type relaxation by the proteinase-activated receptor 2 agonist serine-leucine-isoleucine-glycine-arginine-leucine-NH2 (SLIGRL) was greater in I/R than sham rats due to an increased contribution of small-conductance calcium-activated potassium channels (SKCa ), which was confirmed by the enlarged relaxation to the SKCa activator N-cyclohexyl-N-2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-4-pyrimidinamine. I/R and H/R induced endothelial protein tyrosine nitration and filamentous-actin disruption. In control MCA, either cytochalasin D or peroxynitrite disrupted endothelial filamentous-actin and augmented EDH-type relaxation. Furthermore, peroxynitrite decomposition during I/R prevented the increase in EDH-type responses.

Conclusion: Following TP stimulation in MCA, EDH-type relaxation to SLIGRL is greater after I/R due to endothelial filamentous-actin disruption by peroxynitrite, which prevents TP-induced block of SKCa input to EDH. These results reveal a novel mechanism whereby peroxynitrite could promote post-ischaemic brain injury.

Keywords: SKC a; KCa2.3; actin; calcium-activated potassium channels; ischaemia/reperfusion; stroke; thromboxane.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Blotting, Western
  • Cell Line
  • Disease Models, Animal
  • Endothelium, Vascular / metabolism
  • Endothelium, Vascular / physiopathology*
  • Fluorescent Antibody Technique
  • Humans
  • Infarction, Middle Cerebral Artery / metabolism
  • Infarction, Middle Cerebral Artery / physiopathology*
  • Ischemic Attack, Transient / metabolism
  • Ischemic Attack, Transient / physiopathology
  • Male
  • Middle Cerebral Artery
  • Oligopeptides / pharmacology
  • Peroxynitrous Acid / metabolism*
  • Rats
  • Rats, Sprague-Dawley
  • Receptors, Prostaglandin / metabolism
  • Receptors, Thromboxane / metabolism
  • Reperfusion Injury / metabolism
  • Reperfusion Injury / physiopathology*
  • Vasodilation / drug effects
  • Vasodilation / physiology*

Substances

  • Oligopeptides
  • Receptors, Prostaglandin
  • Receptors, Thromboxane
  • seryl-leucyl-isoleucyl-glycyl-arginyl-leucine
  • Peroxynitrous Acid