In vitro organ bath experiments

Isolated thoracic aortic rings from healthy control rats (in each group, 7–8 independent experiments) were mounted on stainless steel hooks under 2 g of resting tension in individual organ baths (Radnoti Glass Technology, Monrovia, CA, USA), containing 25 ml of Krebs–Henseleit solution. The solution was gassed continuously with 95 % O₂ − 5 % CO₂ and warmed to 37 °C. Special attention was paid during the aortic dissection to avoid damaging the endothelium. The tissue was equilibrated for 60 min. During this period, the tension was periodically adjusted to the desired level, and the Krebs–Henseleit solution was changed every 30 min as a precaution against interfering metabolites. Then, the rings were incubated for 30 min with Zn(ASA)₂ (10⁻³ M) or with a vehicle (dimethyl sulfoxide, DMSO). The maximal contraction force to potassium chloride (KCl, 80 mM) was determined, and the aortic rings were washed until the resting tension was again obtained. Aortic preparations were then again incubated with Zn(ASA)₂ or DMSO then preconstricted with an α-adrenergic receptor agonist, phenylephrine (10⁻⁹–10⁻⁵ M) until a stable plateau was reached. The integrity of the endothelium was verified by assessing the vasorelaxant response of the precontracted rings to the endothelium-dependent vasorelaxant acetylcholine (10⁻⁹–10⁻⁴ M).