1B). The incubation of mouse diaphragm muscle with P2 (30 μg/ml) resulted in a significant decrease in quantal content from 15 min onwards [from 70 ± 6 (basal) to 19 ± 3 after 60 min; n = 5; p < 0.05] ( Fig. 1C) and in the frequency of MEPPs from 5 min onwards [from 33 ± 3 (basal) to 16 ± 1 after 60 min; n = 5; p < 0.05] ( Fig. 2A). There was no change in MEPPs amplitude in PND preparations treated with

P2 (30 μg/ml) (0.8 ± 0.06 mV at t0 compared to 0.8 ± 0.04 mV at t60). In contrast to P2, P3 (30 μg/ml) produced an increase in quantal content from 5 min onwards although statistical significance BMS-907351 cost was seen only after 30 min [from 63 ± 6 (basal) to 85 ± 6 after 60 min; n = 5; p < 0.05] ( Fig. 1D). At this same concentration, P3 also increased the frequency of MEPPs from t30 onwards [from 20 ± 2 (basal) to 30 ± 3 after 60 min; n = 5; p < 0.05] ( Fig. 2B) without altering their amplitude (1.0 ± 0.2 mV

at t0 compared to 0.8 ± 0.1 mV at t60). Bbil-TX (0.5–10 μg/ml) produced irreversible time- and concentration-dependent neuromuscular blockade in indirectly stimulated BC preparations, with complete blockade occurring after 41 ± 2 min (n = 6) at the highest concentration (10 μg/ml); Bbil-TX reproduced the neuromuscular blockade seen with peak P2 (10 μg/ml) from which the toxin was purified ( Fig. 3A). The times required for 50% blockade were 87 ± 7, 41 ± 7 and 19 ± 2 min for Bbil-TX concentrations of 1, 5 and 10 μg/ml, respectively; the time required for 90% blockade was 37 ± 2 min for the highest Bbil-TX concentration. Fig. 3B1 (upper trace) shows a representative Carfilzomib order recording of the neuromuscular blockade produced by Bbil-TX (10 μg/ml) under indirect stimulation at 37 °C. There were no consistently significant changes in the contractures to exogenous ACh and KCl after complete neuromuscular blockade by Bbil-TX ( Fig. 3C). When the experiments were done at 22–24 °C Bbil-TX (5 μg/ml) caused only 21 ± 5% blockade

after 120 min whereas complete blockade was seen at 37 °C after 90 min (Fig. 3A). Pretreatment Cobimetinib price of Bbil-TX with p-BPB abolished the PLA2 activity (88 ± 6 units/mg vs. 2 ± 2 units/mg before and after p-BPB, respectively; n = 3) and also abolished the neuromuscular blockade by this toxin ( Fig. 3A; responses superposed on control preparations). The neuromuscular blockade normally caused by Bbil-TX (10 μg/ml) was absent in curarized (d-Tc, 10 μg/ml) directly stimulated BC preparations, with the twitch-tension response being similar to that of control preparations (Fig. 3B2, D). Bbil-TX caused partial time- and concentration-dependent neuromuscular blockade in indirectly stimulated PND preparations (maximum blockade of 15.2 ± 3%, 29.8 ± 3% and 52.2 ± 2% of the control for concentrations of 3, 10 and 30 μg/ml, respectively, after 120 min; n = 4–6).