Selective loss of basal but not receptor-stimulated relaxation by endothelial nitric oxide synthase after isolation of the mouse aorta.

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 1h 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 α(2)-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 F(2α)-induced force attained only 17 ± 4% or 31 ± 7% of maximum tension in the presence of N(Ω)-nitro-l-arginine-methylesther (l-NAME), and contractions increased to 63 ± 6% and 82 ± 11%, respectively at 5h. In contrast, acetylcholine or UK14304 relaxation curves changed minimally. l-NAME and eNOS-deficiency abolished basal NO production, unlike inhibitors of neuronal (N(Ω)-propyl-l-arginine) or inducible (1400W) NOS. Acetylcholine-induced relaxation was abolished by l-NAME, strongly suppressed by eNOS-deficiency and attenuated by N(Ω)-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 1h and 4h, 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.