Isolated aortic ring preparation

All experiments were approved by the local institutional animal welfare committee at UFG (CEUA/UFG, protocol number 084/2018) and were conducted in accordance with the Brazilian Federal Law No. 11.794. We made all efforts to minimize the number of animals used. The rats were maintained in controlled temperature (22±2°C) with a 12-h light/dark cycle and had free access to water and food. Isolated aortic rings from male Wistar rats (250-300 g) were used to evaluate the vascular effects of PV3, as follows.

Following decapitation, the descending thoracic aorta was removed and sectioned into 4-mm rings. The aortic rings were placed in 10 mL of organ bath at 37°C containing gassed (95% O₂ and 5% CO₂) Krebs-Henseleit solution (KHS) with the following composition: NaCl (118.06 mM), KCl (4.6 mM), NaHCO₃ (24.9 mM), MgSO₄·7H₂O (2.4 mM), CaCl₂·2H₂O (3.3 mM), KH₂PO₄ (0.9 mM), and glucose (11.1 mM), pH 7.4. The rings were fixed to an isometric force transducer under a tension of 1.5 g for 1 h for stabilization. The mechanical activity was recorded using a data acquisition system (DATAQ Instruments, USA). The vasorelaxant effect of PV3 was investigated in rings with intact (E+) or denuded (E-) endothelium. Endothelial integrity was assessed by the degree of relaxation induced by acetylcholine (ACh, 10^-5 mol/L) in rings pre-contracted with 10^-7 mol/L phenylephrine (Phe). Functional endothelium was considered when Ach-induced relaxation was greater than 80%. In denuded endothelium rings, the absence of functional endothelium was verified by the absence of Ach-induced relaxation rings pre-contracted with Phe. After an equilibration period (30 min), the aortic rings were pre-constricted with Phe (10^-7 mol/L), and PV3 (10^-7, 10^-6, or 10^-5 g/L) was added to the KHS. In some experiments, the rings were pre-incubated with the L-NAME (10^-6 mol/L), a nitric oxide synthase (NOS) inhibitor.