The neuromuscular activity of Bothriopsis bilineata smaragdina (forest viper) venom and its toxin Bbil-TX (Asp49 phospholipase A2) on isolated mouse nerve-muscle preparations. (15th March 2015)

• Record Type: Journal Article
• Authors: Floriano, Rafael Stuani; Rocha, Thalita; Carregari, Victor Corasolla; Marangoni, Sergio; da Cruz-Höfling, Maria Alice; Hyslop, Stephen; Rodrigues-Simioni, Léa; Rowan, Edward G.
• Is Part Of: Toxicon. Volume 96(2015)
• Page Start: 24
• Page End: 37
• Publication Date: 2015-03-15
• Subjects: Calcium mobilization -- Ion channel -- Neuromuscular blockade -- Snake venom phospholipase A2 -- Presynaptic activity -- Quantal content -- SNAP-25 -- Synaptophysin; Toxins -- Periodicals; Venom -- Periodicals; 615.9
• DOI: 10.1016/j.toxicon.2015.01.001
• Languages: English
• ISSNs: 0041-0101

Abstract

Abstract: The presynaptic action of Bothriopsis bilineata smaragdina (forest viper) venom and Bbil-TX, an Asp49 PLA2 from this venom, was examined in detail in mouse phrenic nerve-muscle (PND) preparations in vitro and in a neuroblastoma cell line (SK-N-SH) in order to gain a better insight into the mechanism of action of the venom and associated Asp49 PLA2 . In low Ca 2+ solution, venom (3 μg/ml) caused a quadriphasic response in PND twitch height whilst at 10 μg/ml the venom additionally induced an abrupt and marked initial contracture followed by neuromuscular facilitation, rhythmic oscillations of nerve-evoked twitches, alterations in baseline and progressive blockade. The venom slowed the relaxation phase of muscle twitches. In low Ca 2+, Bbil-TX [210 nM (3 μg/ml)] caused a progressive increase in PND twitch amplitude but no change in the decay time constant. Venom (10 μg/ml) and Bbil-TX (210 nM) caused minor changes in the compound action potential (CAP) amplitude recorded from sciatic nerve preparations, with no significant effect on rise time and latency; tetrodotoxin (3.1 nM) blocked the CAP at the end of the experiments. In mouse triangularis sterni nerve-muscle (TSn-m) preparations, venom (10 μg/ml) and Bbil-TX (210 nM) significantly reduced the perineural waveform associated with the outward K + current while the amplitude of the inward Na + current was not significantly affected. Bbil-TX (210 nM) caused a progressive increase in the quantal content of TSn-m preparations maintained in low Ca 2+ solution. Venom (3 μg/ml) and toxin (210 nM) increased the calcium fluorescence in SK-N-SH neuroblastoma cells loaded with Fluo3 AM and maintained in low or normal Ca 2+ solution. In normal Ca 2+, the increase in fluorescence amplitude was accompanied by irregular and frequent calcium transients. In TSn-m preparations loaded with Fluo4 AM, venom (10 μg/ml) caused an immediate increase in intracellular Ca 2+ followed by oscillations in fluorescence and muscle contracture; Bbil-TX did not change the calcium fluorescence in TSn-m preparations. Immunohistochemical analysis of toxin-treated PND preparations revealed labeling of junctional ACh receptors but a loss of the presynaptic proteins synaptophysin and SNAP25. Together, these data confirm the presynaptic action of Bbil-TX and show that it involves modulation of K + channel activity and presynaptic protein expression. Highlights: The activity of Bothriopsis bilineata smaragdina (Bbs) venom was studied in detail. Quadriphasic changes in twitch amplitude were observed. Bbs-venom increased calcium fluorescence in SK-N-SH neuroblastoma cells. Bbs-venom caused a loss of synaptophysin and SNAP25 from nerve terminals. The complex pharmacology of Bbs-venom suggests the presence of a number toxins.