2.4. Isolated Rat Aortic Ring Experiment

The experimental animal (one rat) was anesthetized with pentobarbital sodium (60 mg/kg i.p.). Once deep anesthesia was achieved, the thoracic aorta was dissected, placed in ice cold Krebs-Henseleit (K-H) buffer (content in mM: NaCl 118, KCl 4.7, CaCl₂ 2.5, MgCl₂ 1.64, NaHCO₃ 24.88, KH₂PO₄ 1.18, glucose 10.0, sodium pyruvate 5.0, and disodium salt of ethylenediaminetetraacetic acid 0.05), cleaned from connective and fatty tissues, and cut into 3-4 mm long rings. Endothelium was mechanically scraped off with the tips of forceps, and the aortic rings were mounted between two stainless steel hooks attached to an isometric force transducer in 20 ml organ bath in K-H buffer bubbled with 95% O₂ and 5% CO₂ at 37°C (one separate aortic ring was used for each tested compound). Passive tension was adjusted to approximately 40 mN over a 60 min equilibration period. The experiment was started after the equilibration period by adding KCl solution to obtain a concentration of 50 mM. Addition of KCl induced contraction of the aortic rings, and a plateau of contraction was reached about 80 mM concentration. Thereafter, the tested compound solutions in dimethyl sulfoxide were added to the aortic rings in order to obtain the final concentrations in the range of 10⁻¹⁰-10⁻⁵ M. Changes in the tension were recorded using LabChart Pro software from ADInstruments (https://www.adinstruments.com/products/labchart/labchart-for-research). The EC₅₀ values of the tested compounds were calculated using Prism 3.0 software from GraphPad (GraphPad Software Inc., USA) (https://www.graphpad.com/scientific-software/prism/) [20]. The experimental procedures involving experimental animals were carried out in accordance with the guidelines of the European Community, LR local laws and policies, and were approved by the Latvian Animal Protection Ethical Committee, the Food and Veterinary Service, Riga, Latvia.