Measurement of isometric tension in isolated rings of rat PA

The intrapulmonary arteries were carefully dissected out from male SD rats under anaesthesia, with a dissecting microscope. Freshly isolated PA rings (2 mm long) were suspended between two tungsten wires to record isometric tension. The two tungsten wires were inserted into the lumen of the PA and fixed respectively to a force transducer and a micrometer and mounted on wire myographs (DMT, Aarhus, Denmark). PA rings were superfused with 5 mL Krebs solution including (in mM): 138 NaCl, 1.8 CaCl₂, 4.7 KCl, 1.2 MgSO₄, 1.2 NaH₂PO₄, 5 HEPES and 10 glucose (pH 7.4), maintained at 37°C and aerated with 95% O₂/5% CO₂.

The passive resting tension was set and maintained at an optimal tension of 300 mg. The rings were allowed to stabilize at the resting tension for approximately 1 h before experimentation. After the 1 h equilibration, the isolated PA rings were stimulated twice with 60 mM K⁺‐containing solution (60K) to stabilize the vessel and obtain a stable contractile response before further experimentation. The baseline tension of the isolated PA rings usually becomes stable after three challenges with a 60K‐containing solution. The ring was considered non‐viable if the ring could not provide reproducible contractions to 60K or phenylephrine, or because of poor recording quality. The ring was also excluded if it gave poor responses to phenylephrine, due to PA damage. For relaxation studies, rings were contracted with phenylephrine or high K⁺ solutions containing 40 mM (40K) or 80 mM (80K) of KCl. Selected experiments were performed using vessels with either intact or denuded endothelium. Endothelium was denuded mechanically using forceps before placing the PA rings on tungsten wires. Endothelium‐dependent vasodilation was assessed using ACh (endothelium‐dependent relaxation), while endothelium‐independent vasodilation was assessed using sodium nitroprusside (SNP, endothelium‐independent relaxation) after pre‐constriction of the artery with 1 μM phenylephrine. In some experiments, the rings were treated with l‐NG‐nitroarginine methyl ester (l‐NAME), indomethacin, apamin, paxilline, tetraethylammonium (TEA) and charybdotoxin (ChTX) for 30 min and continuously treated during the experiment to test the effect of chloroquine on phenylephrine‐induced contraction of the PA rings. Data are expressed as % relaxation of phenylephrine‐induced pre‐constriction.