Mlt0420

For muscle contraction measurements, the left and right diaphragm were suspended under a constant tension of 5 g in a 20‐mL organ bath (15 mL/min perfusion rate) containing aerated (5% CO₂ and 95% O₂) physiological solution (pH 7.4, 25℃). Supramaximal pulses (0.1 Hz, 0.3 millisecond, 3‐6 V) and tetanic stimulation (50 Hz, 0.3 millisecond, 30 pulses) delivered by a DS3 Digitimer Ltd., were applied by electrodes placed on the motor nerve. Isometric muscle tension was recorded using a force transducer (MLT0420; AD Instruments). One end of the diaphragm muscle (lower edge) was tied to a fixed nail, and the other end was linked to a force transducer. A constant passive tension was kept. The preparations were allowed to stabilize for at least 20 minutes before onset of drug applications (Power‐Lab installation, AD Instruments). The signals were recorded and analysed using LabChart Pro software.

Cylindrical segments (rings) of the thoracic aorta (2 mm in length), free of adipose and connective tissue, were mounted in an isolated tissue chamber containing Krebs-Henseleit solution (118 mM NaCl, 4.7 mM KCl, 25 mM NaHCO3, 2.5 mM CaCl2-2H2O, 1.2 mM KH2PO4, 1.2 mM MgSO4-7H2O, 11 glucose, and 0.01 mM EDTA) and gassed with 95% O₂ and 5% CO₂. Aortic rings were maintained at a resting tension of 0.5 g at 37 °C, pH 7.4 as previously described [16 (link)]. Isometric tension was recorded using an isometric force transducer (MLT0420, ADInstruments, Sydney, Australia) connected to an acquisition system (PowerLab 8/30, ADInstruments). After a 60-min equilibration period, aortic rings were exposed to 125 mM KCl to assess maximal tension. After a washout period, concentration-response curves to the α₁-adrenoceptor agonist phenylephrine (0.1 nM-10 µM) were obtained and there were no differences. Relaxation concentration–response curves to acetylcholine (1 nM–30 µM) and to the NO-donor sodium nitroprusside (10 pM–3 µM) were generated for the aortic rings contracted with phenylephrine until 50–70% of maximum contraction with 125 mM KCl. Some aortic rings were pre-incubated for 30 min with the nonselective nitric oxide synthase (NOS) inhibitor N-nitro-L-arginine methyl ester (L-NAME, 100 µM, Sigma-Aldrich, St. Louis, MO, USA) before acetylcholine-induced relaxation curves.

Colonic tissue samples were dissected, weighed and placed in an oxygenated Krebs solution. Tissue strips were suture-mounted in tissue baths (10 ml, Panlab Two Chamber Compact Organ Bath, ML0126/10-220; ADInstruments Ltd, UK) connected to force transducers (MLT0420, ADInstruments Ltd, UK) and bridge amplifiers (FE221, ADInstruments Ltd, UK). Tissues were equilibrated in oxygenated Krebs solution for 60 min at 37 °C and an initial tension of 0.5 g was maintained. During equilibration, the tissues were perfused with three washes of oxygenated Krebs solution. Basal activity was recorded and collected using PowerLab 2/26 data acquisition system (PL2602/P, ADInstruments Ltd, UK), and analysed using LabChartPro v8.