Title
Selective loss of basal but not receptor-stimulated relaxation by endothelial nitric oxide synthase after isolation of the mouse aorta Selective loss of basal but not receptor-stimulated relaxation by endothelial nitric oxide synthase after isolation of the mouse aorta
Author
Faculty/Department
Faculty of Pharmaceutical, Biomedical and Veterinary Sciences. Pharmacy
Faculty of Medicine and Health Sciences
Publication type
article
Publication
Amsterdam ,
Subject
Pharmacology. Therapy
Source (journal)
European journal of pharmacology. - Amsterdam
Volume/pages
696(2012) :1-3 , p. 111-119
ISSN
0014-2999
ISI
000310582100016
Carrier
E
Target language
English (eng)
Full text (Publishers DOI)
Affiliation
University of Antwerp
Abstract
Bioavailability of nitric oxide (NO) is mostly studied in isolated blood vessels. We investigated changes in basal and receptor-stimulated endothelial NO synthase (eNOS) activity after isolation of wild-type and Marfan mouse aorta. Starting 1 h after dissection, basal NO release was assessed at hourly intervals by its ability to suppress isometric contractions in aortic segments. Relaxation induced by acetylcholine or α2-adrenoceptor agonist 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK14304) was used to study stimulated NOS activity. One hour after dissection, phenylephrine- or prostaglandin F2α-induced force attained only 17±4% or 31±7% of maximum tension in the presence of NΩ-nitro-l-arginine-methylesther (l-NAME), and contractions increased to 63±6% and 82±11%, respectively at 5 h. In contrast, acetylcholine or UK14304 relaxation curves changed minimally. l-NAME and eNOS-deficiency abolished basal NO production, unlike inhibitors of neuronal (NΩ-propyl-l-arginine) or inducible (1400 W) NOS. Acetylcholine-induced relaxation was abolished by l-NAME, strongly suppressed by eNOS-deficiency and attenuated by NΩ-propyl-l-arginine. In a bioassay based on diethylamine NONOate concentration-response curves the suppression of contractile forces was interpolated into NO equivalents. This showed exponential decay of basal NO, which occurred three times faster in aortas from mice with Marfan syndrome, while acetylcholine-induced relaxation remained unaltered. Immunoblotting showed unchanged eNOS expression, or phosphorylation at Ser1177, Ser617 or Thr495 between 1 h and 4 h, but Akt phosphorylation declined gradually. The dramatic loss of basal NO release after tissue isolation shows that timing is crucial when studying NO responses. The preservation of receptor-induced relaxation implies differential regulation of basal and stimulated eNOS activity, and phosphoinositide-3-kinase/Akt signalling seems specifically associated with basal eNOS activity.
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