Organ chamber experiments.

Thoracic aortas from adult mice were isolated after animal sacrifice under 2% isoflurane anesthesia. The aortic rings were mounted immediately in organ chambers (Multi Wire Myograph System, DMT, model 610M) filled with Krebs-Ringer solution ( 118.3 mmol/L NaCl, 4.7 mmol/L KCl, 1.2 mmol/L MgSO₄, 1.2 mmol/L KH₂PO₄, 1.25 mmol/L CaCl₂, 25.0 mmol/L NaHCO₃, 5.0 mmol/L, and glucose) and gassed with a mixture of 95% O₂ and 5% CO₂ (pH 7.4). The presence of functional endothelial cells was confirmed by relaxation to acetylcholine chloride (Sigma-Aldrich, A6625) (10^–5 mol/L) following a contraction evoked by phenylephrine (10^–7 mol/L), defined as a relaxation ≥70% of the precontraction as previously described (49). After extensive washout and equilibration, contraction to phenylephrine hydrochloride (concentration-response curve, 10^–9 to 10^–4 mol/L) (Sigma-Aldrich, P1250000), angiotensin II (concentration-response curve, 10⁻⁹ to 10⁻⁶ mol/L) (Sigma-Aldrich, A9525), or KCl (80 mmol/L) and relaxation to acetylcholine chloride (concentration-response curve, 10⁻⁹ to 10⁻⁴ mol/L) or SNAP (Sigma-Aldrich, N3398) (concentration-response curve, 10⁻¹⁰ to 10⁻⁵ mol/L) was studied. For NO synthase inhibition, aorta rings were preincubated for 45 minutes with 10^–4 mol/L l-NAME (Cayman, 80210) before concentration-response curve to phenylephrine without washout were performed. In some experiments, the endothelium was mechanically removed by inserting the tip of forceps within the lumen and gently rubbing the ring back and forth on a piece of wet tissue. For the NAC experiment (commercial HIDONAC, Zambon), NAC was added to the Krebs-Ringer solution at a final concentration of 20 mmol/L.