title: The neuromuscular activity of Bothriopsis bilineata smaragdina (forest viper) venom and its toxin Bbil-TX (Asp49 phospholipase A2) on isolated mouse nerve-muscle preparations.
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 (3μg/ml) caused a quadriphasic response in PND twitch height whilst at 10μg/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 [210nM (3μg/ml)] caused a progressive increase in PND twitch amplitude but no change in the decay time constant. Venom (10μg/ml) and Bbil-TX (210nM) 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 (3.1nM) blocked the CAP at the end of the experiments. In mouse triangularis sterni nerve-muscle (TSn-m) preparations, venom (10μg/ml) and Bbil-TX (210nM) 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 (210nM) caused a progressive increase in the quantal content of TSn-m preparations maintained in low Ca(2+) solution. Venom (3μg/ml) and toxin (210nM) 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 (10μg/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.
authors.affiliation: Department of Pharmacology, Faculty of Medical Sciences, State University of Campinas (UNICAMP), Rua Tessália Vieira de Camargo, 126, 13083-887, Campinas, SP, Brazil; Centre for Venom and Toxin Drug Discovery, Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Cathedral Street, 161, G4 0RE, Glasgow, UK.
authors.forename: Rafael Stuani
authors.initials: RS
authors.lastname: Floriano
authors.affiliation: Department of Histology and Embryology, Institute of Biology, State University of Campinas (UNICAMP), Rua Monteiro Lobato, 255, 13083-862, Campinas, SP, Brazil; Campus Bragança Paulista, São Francisco University (USF), Avenida São Francisco de Assis, 218, 12916-900, Bragança Paulista, SP, Brazil.
authors.forename: Thalita
authors.initials: T
authors.lastname: Rocha
authors.affiliation: Department of Biochemistry, Institute of Biology, State University of Campinas (UNICAMP), Rua Monteiro Lobato, 255, 13083-862, Campinas, SP, Brazil.
authors.forename: Victor Corasolla
authors.initials: VC
authors.lastname: Carregari
authors.affiliation: Department of Biochemistry, Institute of Biology, State University of Campinas (UNICAMP), Rua Monteiro Lobato, 255, 13083-862, Campinas, SP, Brazil.
authors.forename: Sergio
authors.initials: S
authors.lastname: Marangoni
authors.affiliation: Department of Histology and Embryology, Institute of Biology, State University of Campinas (UNICAMP), Rua Monteiro Lobato, 255, 13083-862, Campinas, SP, Brazil.
authors.forename: Maria Alice
authors.initials: MA
authors.lastname: da Cruz-Höfling
authors.affiliation: Department of Pharmacology, Faculty of Medical Sciences, State University of Campinas (UNICAMP), Rua Tessália Vieira de Camargo, 126, 13083-887, Campinas, SP, Brazil.
authors.forename: Stephen
authors.initials: S
authors.lastname: Hyslop
authors.affiliation: Department of Pharmacology, Faculty of Medical Sciences, State University of Campinas (UNICAMP), Rua Tessália Vieira de Camargo, 126, 13083-887, Campinas, SP, Brazil.
authors.forename: Léa
authors.initials: L
authors.lastname: Rodrigues-Simioni
authors.affiliation: Centre for Venom and Toxin Drug Discovery, Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Cathedral Street, 161, G4 0RE, Glasgow, UK. Electronic address: e.g.rowan@strath.ac.uk.
authors.forename: Edward G
authors.initials: EG
authors.lastname: Rowan
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D007150:Immunohistochemistry; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D009119:Muscle Contraction / Q000187:drug effects; D018482:Muscle, Skeletal / Q000187:drug effects; D009469:Neuromuscular Junction / Q000187:drug effects*; D054467:Phospholipases A2 / Q000633:toxicity*; D010791:Phrenic Nerve / Q000187:drug effects; D014757:Viper Venoms / Q000633:toxicity*; D017819:Viperidae / Q000378:metabolism*
chemical_list: D014757:Viper Venoms; D054467:Phospholipases A2
keywords: Calcium mobilization; Ion channel; Neuromuscular blockade; Presynaptic activity; Quantal content; SNAP-25; Snake venom phospholipase A(2); Synaptophysin