Peroxynitrite formed during a transient episode of brain ischaemia increases endothelium-derived hyperpolarization-type dilations in thromboxane/prostaglandin receptor-stimulated rat cerebral arteries.
Increased thromboxane A2and 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.



Vascular function was determined by wire myography after TP stimulation with the thromboxane A2mimetic 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.



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 SKCaactivator 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.



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 SKCainput to EDH. These results reveal a novel mechanism whereby peroxynitrite could promote post-ischaemic brain injury.
Departament de Farmacologia, de Terapèutica i de Toxicologia, Institut de Neurociències, Facultat de Medicina, Universitat Autònoma de Barcelona, Bellaterra, Spain.
Y
Y
Onetti
Institut Clínic Cardiovascular, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain.
A P
AP
Dantas
Departament de Farmacologia, de Terapèutica i de Toxicologia, Institut de Neurociències, Facultat de Medicina, Universitat Autònoma de Barcelona, Bellaterra, Spain.
B
B
Pérez
Reading School of Pharmacy, University of Reading, Reading, Berkshire, UK.
A J
AJ
McNeish
Departament de Farmacologia, de Terapèutica i de Toxicologia, Institut de Neurociències, Facultat de Medicina, Universitat Autònoma de Barcelona, Bellaterra, Spain.
E
E
Vila
Departament de Farmacologia, de Terapèutica i de Toxicologia, Institut de Neurociències, Facultat de Medicina, Universitat Autònoma de Barcelona, Bellaterra, Spain.
F
F
Jiménez-Altayó
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D004195:Disease Models, Animal; D004730:Endothelium, Vascular / Q000378:metabolism / Q000503:physiopathology*; D005455:Fluorescent Antibody Technique; D006801:Humans; D020244:Infarction, Middle Cerebral Artery / Q000378:metabolism / Q000503:physiopathology*; D002546:Ischemic Attack, Transient / Q000378:metabolism / Q000503:physiopathology; D008297:Male; D020768:Middle Cerebral Artery; D009842:Oligopeptides / Q000494:pharmacology; D030421:Peroxynitrous Acid / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011982:Receptors, Prostaglandin / Q000378:metabolism; D017482:Receptors, Thromboxane / Q000378:metabolism; D015427:Reperfusion Injury / Q000378:metabolism / Q000503:physiopathology*; D014664:Vasodilation / Q000187:drug effects / Q000502:physiology*
D009842:Oligopeptides; D011982:Receptors, Prostaglandin; D017482:Receptors, Thromboxane; C099570:seryl-leucyl-isoleucyl-glycyl-arginyl-leucine; D030421:Peroxynitrous Acid

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