Ft03 force displacement transducer

Mice were sacrificed by cervical dislocation. Subsequently, the ileum was gently removed and washed with Tyrode solution (NaCl 115.0 mM, KCl 8.0 mM, KH₂PO₄ 2.0 mM, NaHCO₃ 25.0 mM, MgCl₂ 2.4 mM, CaCl₂ 1.3 mM, glucose 10.0 mM). Full-thickness fragments of the ileum (0.5 cm) were kept in Tyrode solution. One end of each ileal fragment was attached using a silk thread to the bottom of the individual organ bath, another end to a FT03 force displacement transducer (Grass Technologies, West Warwick, RI, USA).
Each organ bath contained 25 ml of Tyrode solution oxygenated with 95% O₂ and 5% CO₂ at constant temperature (37 °C). The changes in tension were amplified by a P11T amplifier (Grass Technologies, West Warwick, RI, USA) and recorded using the POLYVIEW software (Polybytes Inc., Cedar Rapids, IA, USA).

Following euthanasia and blood sample collection, the thoracic aorta was removed and cleaned while submerged in cold Tyrode’s physiological salt solution (Tyrode’s PSS, mM: NaCl 136.9, KCl 5.4, MgCl ${}_2$ ·H ${}_2$ O 1.0, NaH ${}_2$ PO ${}_4$ ·2H ${}_2$ O 0.4, NaHCO ${}_3$ 22.6, CaCl ${}_2$ ·2H ${}_2$ O 1.8, glucose 5.5, ascorbic acid 0.3, Na ${}_2$ EDTA 0.05). Five millimetre-long ring segments were then threaded onto stainless steel hooks connected to an FT03 force displacement transducer (Grass Technologies, Middleton, WI, USA). The tissue was anchored in a 30-mL isolated organ bath, filled with warm (37 ${}^{\circ }$ C) Tyrode’s PSS and bubbled with carbogen (90% O ${}_2$ /10% CO ${}_2$ ). Concentration response curves were performed for NA, ACh and NaNO. Concentration response curves for each tissue sample were normalised as a percentage of their peak (providing a clear 100%), and non-parametric 4-parameter logistical fit analysis was performed to identify the curve and provide an accurate EC ${}_{50}$ .

Vasa deferentia segments were mounted vertically on perspex tissue holders so that contractions of the longitudinal muscle could be measured. The tissue holders were placed in separate 10-mL water-jacketed standard organ baths filled with Krebs–Henseleit solution maintained at 37°C and supplied with carbogen (95% oxygen; 5% CO₂) gas. Each tissue holder incorporated two platinum electrodes, which were connected to a Grass S88 stimulator (Grass Instruments, MA, USA). The epididymal end of the tissue was tied off and fastened to the tissue holders, and a needle was used to thread cotton ligature through the lumen and tissue wall at the prostatic end of the tissue, which was then attached to a FT03 force displacement transducer (Grass instruments, MA, USA), connected to a ML118 QUAD Bridge (ADInstruments, Australia). The signal was digitized and sent to a personal computer using a ML750 PowerLab/4SP (ADInstruments, Castle Hill, Australia), and data was recorded using LabChart software (version: Chart5 for Windows). The initial tension of the tissues was set at 1.0 g, and tissues were allowed to equilibrate for 1 h under electrical field stimulation (EFS, parameters: pulse duration = 0.5 ms; voltage = 60 V; frequency = 0.01 Hz) prior to experimentation.