The neuromuscular activity of Bothriopsis bilineata smaragdina (forest viper) venom and its toxin Bbil-TX (Asp49 phospholipase A(2)) on isolated mouse nerve-muscle preparations

Author(s)

Floriano, Rafael Stuani ^{[1, 2]} ; Rocha, Thalita ^{[3, 4]} ; Carregari, Victor Corasolla ^[5] ; Marangoni, Sergio ^[5] ; da Cruz-Hoefling, Maria Alice ^[4] ; Hyslop, Stephen ^[1] ; Rodrigues-Simioni, Lea ^[1] ; Rowan, Edward G. ^[2]

Affiliation

^[1] Univ Estadual Campinas, State Univ Campinas, Fac Med Sci, Dept Pharmacol, BR-13083887 Campinas, SP - Brazil

^[2] Univ Strathclyde, Ctr Venom & Toxin Drug Discovery, Strathclyde Inst Pharm & Biomed Sci, Glasgow G4 0RE, Lanark - Scotland

^[3] Univ Sao Francisco, BR-12916900 Braganca Paulista, SP - Brazil

^[4] Univ Estadual Campinas, State Univ Campinas, Inst Biol, Dept Histol & Embryol, BR-13083862 Campinas, SP - Brazil

^[5] Univ Estadual Campinas, State Univ Campinas, Inst Biol, Dept Biochem, BR-13083862 Campinas, SP - Brazil

• Document type: Journal article
• Source: Toxicon; v. 96, p. 24-37, MAR 2015.
• Web of Science Citations: 5

Abstract

The presynaptic action of Bothriopsis bilineata smaragdina (forest viper) venom and Bbil-TX, an Asp49 PLA(2) 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 PLA(2). In low Ca2+ solution, venom (3 mu g/ml) caused a quadriphasic response in PND twitch height whilst at 10 mu g/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 Ca2+, Bbil-TX {[}210 nM (3 mu g/ml)] caused a progressive increase in PND twitch amplitude but no change in the decay time constant. Venom (10 mu g/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 mu g/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 Ca2+ solution. Venom (3 mu g/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 Ca2+ solution. In normal Ca2+, the increase in fluorescence amplitude was accompanied by irregular and frequent calcium transients. In TSn-m preparations loaded with Fluo4 AM, venom (10 mu g/ml) caused an immediate increase in intracellular Ca2+ 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. (C) 2015 Elsevier Ltd. All rights reserved. (AU)

• FAPESP's process: 09/04127-0 - Biochemical characterization and study of the pharmacological activi in the junction neuromuscular of an isolated miotoxina from the Bothriopsis bilineata venom
• Grantee: Victor Corasolla Carregari
• Support Opportunities: Scholarships in Brazil - Master