Pharmacological study of a new Asp49 phospholipase A2 (Bbil-TX) isolated from Bothriopsis bilineata smargadina (forest viper) venom in vertebrate neuromuscular preparations

Abstract

The neuromuscular activity of Bbil-TX, a PLA2 with catalytic activity isolated from Bothriopsis bilineata smargadina venom, was examined in chick biventer cervicis (BC) and mouse phrenic nerve-diaphragm (PND) preparations. In BC preparations, Bbil-TX (0.5-10 μg/ml) caused time- and concentration-dependent blockade that was not reversed by washing; the times for 50% blockade were 87 ± 7, 41 ± 7 and 19 ± 2 min (mean ± SEM; n = 4-6) for 1, 5 and 10 μg/ml, respectively. Muscle contractures to exogenous ACh and KCl were unaffected. The toxin (10 μg/ml) also did not affect the twitch-tension of directly-stimulated, curarized (10 μg/ml) BC preparations. However, Bbil-TX (10 μg/ml) produced mild morphological alterations (edematous and/or hyperchromic fibers) in BC; there was also a progressive release of CK (from 116 ± 17 IU/ml (basal) to 710 ± 91 IU/ml after 45 min). Bbil-TX (5 μg/ml)-induced blockade was markedly inhibited at 22-24 °C and pretreatment with p-bromophenacyl bromide (p-BPB) abolished the neuromuscular blockade. Bbil-TX (3-30 μg/ml, n = 4-6) caused partial time- and concentration-dependent blockade in PND preparations (52 ± 2% at the highest concentration). Bbil-TX (30 μg/ml) also markedly reduced the MEPPs frequency [from 26 ± 2.5 (basal) to 10 ± 1 after 60 min; n = 5; p < 0.05] and the quantal content [from 94 ± 14 (basal) to 24 ± 3 after 60 min; n = 5; p < 0.05] of PND preparations, but caused only minor depolarization of the membrane resting potential [from -80 ± 1 mV (basal) to -66 ± 2 mV after 120 min; n = 5; p < 0.05], with no significant change in the depolarizing response to exogenous carbachol. These results show that Bbil-TX is a presynaptic PLA2 that contributes to the neuromuscular blockade caused by B. b. smargadina venom.

Ultrastructural aspects of mouse nerve-muscle preparation exposed toBothrops jararacussuandBothrops bilineatusvenoms and their toxins BthTX-I and Bbil-TX: Unknown myotoxic effects

“…More recently, pharmacological and histological studies have shown that the venom of B. bilineatus and its Asp49 PLA 2 Bbil‐TX were neurotoxic too, but no significant muscle alterations was detected by histological analysis, thus indicating the absence of significant muscle effect. This was corroborated by findings showing that venom and toxin are ineffective in abolishing the muscle contracture induced by KCl and resting membrane potential, do not increase serum levels of CK and do not inhibit the response to direct electrical stimulation (Floriano et al, ; Rodrigues‐Simioni et al, ).…”

“…In vitro studies show that the venom of B. bilineatus causes neuromuscular blockade in avian and mammalian isolated nerve‐muscle preparations, and this effect has been attributed to a presynaptic mechanism of action PLA 2 ‐dependent (Rodrigues‐Simioni et al, ) with no apparent myotoxic effect. Recently, a presynaptic‐active Asp49‐PLA 2 (Bbil‐TX) was purified and characterized from this venom (Carregari et al, ), which appears to be responsible for the neurotoxicity shown by crude venom in vitro (Floriano et al, ).…”

Neuromuscular Activity of Micrurus laticollaris (Squamata: Elapidae) Venom in Vitro

“…Chick biventer cervicis nerve-muscle preparations (BC) were mounted (resting tension: 1 g) in Krebs solution (composition, in mM: NaCl 119, KCl 4.7, CaCl 2 1.9, KH 2 PO 4 1.2, MgSO 4 1.2, NaHCO 3 25 and glucose 11.7, pH 7.5 at 37 °C, after equilibration with 95% O 2 /5% CO 2 ) and allowed to stabilize for 20 min prior to use, as described elsewhere [47,48]. Field stimulation (0.1 Hz, 0.2 ms) were delivered from an LE 12406 TC stimulator (Panlab, Spain), and the muscle twitches were recorded using a TRI201AD force displacement transducer coupled to a Quad Bridge Amp and LabChart 6.0 software (all from ADInstruments Pty Ltd., Bella Vista, Australia).…”

“…Mouse phrenic nerve-diaphragm preparations (PND) were mounted in Tyrode solution (composition, in mM: NaCl 137, KCl 2.7, CaCl 2 1.8, MgCl 2 0.49, NaH 2 PO 4 0.42, NaHCO 3 11.9 and glucose 11.1, pH 7.0 at 37 °C, after equilibration with 95% O 2 /5% CO 2 ), as described elsewhere [48,50]. Supramaximal stimuli (0.1 Hz and 0.2 ms) were delivered to the nerve from a Grass S88 stimulator (Grass Instrument Co., Quincy, MA, USA), and the muscle twitches were recorded using a Load Cell BG-25 g force displacement transducer coupled to a Gould RS 3400 recorder (both from Gould Inc., Cleveland, OH, USA).…”

“…The effect of the venom on the frequency of miniature end-plate potentials (MEPPs) and membrane resting potential (RP) was recorded using mouse hemidiaphragm muscle mounted in a Lucite chamber containing Tyrode solution (composition shown above), as described previously elsewhere [24,48]. The MEPPs frequency was measured in multiple neuromuscular junctions at various intervals from control ( t 0 ) and venom-treated preparations ( t 5 – t 60 ).…”