The preparation was established as previously mentioned for
the isolated phrenic nerve–hemidiaphragm. The phrenic nerve was
removed from the hemidiaphragm. The fan shaped hemidiaphragm
was then transferred into the organ bath containing 80mL Krebs
solution, aerated with 95% O2 and 5% CO2 at a temperature of 30 ◦C.
The muscle tension was set at 2 g. The needle platinum electrode
was passed through the basement of hemidiaphragm. The direct
muscle twitch was recorded by electrical stimulation of supramaximal
voltage at a frequency of 0.4 Hz and duration of 0.6 ms. The
preparation was completely curarized by adding 0.005mM pancuronium
(Chongrak, 1985). Thus, the contractile response was due
only to the skeletal muscle response.