The hemidiaphragm with phrenic nerve was pinned on its lateral side into a silicon-coated organ bath containing oxygenated Krebs-Ringer (K-R) solution. Then, the tendon part was linked to the lever of an isometric mechano-electrical transducer (Grass Instruments, West Warwick, RI, USA) via a stainless-steel hook and silk thread. The equal stimulus protocol, equipment, and software were used as previously described by Ristovski et al. [9 (link)]. The concentrations of C1 studied were 30, 60, 90, and 120 μM and their effect on muscle contraction was continuously measured during 60 min after application. The muscle twitch and tetanic contraction blockade produced by C1 was shown as the percentage of the initial maximal response. The well-known reversible AChE inhibitor neostigmine methyl sulphate (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) was used as a positive control, and a corresponding solution of 2.5% DMSO in deionized water as a negative control.
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