4.5. Organ Bath Experiments to Study Relaxation of Aortic Rings

Animals were euthanized by inhalation of CO₂ and thoracic aortas were isolated after gently perfusion through heart punction with 1–2 mL Krebs-Henseleit buffer (118.3 mM NaCl; 4.69 mM KCl; 1.87 mM CaCl₂; 1.0 mM MgSO₄; 1.03 mM KH₂PO₄; 25 mM NaHCO₃; 11.1 mM D-glucose, pH 7.35) supplied with 5 IE/mL Heparin. Concentration-relaxation curves of aortic tissue were measured as described previously [99]. In brief, the thoracic part of aortas isolated from CRP4 WT and CRP4 KO mice were liberated from fatty tissue, rinsed, and cut into 4 mm segments. The aortic rings were mounted between stainless triangles in an organ bath chamber connected to force transducers to measure isometric tension through a computerized acquisition system (Kent scientific corporation, Torrington, CT, USA; Powerlab, ADInstruments, Spechbach, Germany) and a computer running Labchart 4.0 software (AD Instruments, UK). The organ chambers with a capacity of 20 mL were gassed with 95% O₂/5% CO₂ and filled with Krebs-Henseleit buffer. The aortic rings were stretched progressively to achieve a resting tension of 1.0 g. To analyze agonist-induced relaxation responses, the vessels were pre-constricted with 7.5 µM Prostaglandin F2α (PGF_2α; Sigma Aldrich) to assess their maximal tension. Relaxation responses were evoked by concentration-response curves to cumulatively increasing concentrations of cinaciguat (0.1 nM–250 nM), glyceryl trinitrate (1 nM–30 µM), and carbachol (10 nM–50 µM). The respective tension to each concentration was recorded, and the relaxation was finally calculated with respect to the maximal contraction.