This article presents experimental results performed following the standard procedures of scientific ethics. The study was funded by CNPq, FAPESP, INCTTox and Fundação Araucária. “
“Bothrops snake venoms contain a variety of Asp49 and Lys49 phospholipases A2, many of which are myotoxic ( Gutiérrez and Ownby, 2003; Lomonte et al., Trichostatin A manufacturer 2003). In addition, various Bothrops venoms ( Zamunér et al., 2004) and some of their PLA2 ( Gallacci and Cavalcante, 2010) cause neuromuscular blockade in avian and

mammalian nerve–muscle preparations in vitro. Several of these PLA2 (mainly Asp49 PLA2) appear to produce blockade via presynaptic mechanisms, generally at concentrations (5–50 μg/ml) lower than those required to produce blockade with the corresponding venom ( Cogo et al., 2006; Borja-Oliveira et al., 2007; Calgarotto et al., 2008; Ponce-Soto et al., 2009; Galbiatti et al., 2012). We have recently shown that the venom of Bothriopsis bilineata smargadina, an arboreal species of pitviper found in the Amazon basin ( Campbell and Lamar, JQ1 2004), causes neuromuscular blockade in avian and mammalian isolated neuromuscular

preparations ( Rodrigues-Simioni et al., 2011). In chick biventer cervicis preparations, the venom produced irreversible blockade without significantly affecting the responses to exogenous acetylcholine or KCl or stimulating creatine kinase release, while in mouse phrenic nerve–diaphragm preparations there was an initial facilitation followed by progressive blockade and a gradual decrease in quantal content; there was no change in the muscle membrane resting

potential or in the response to carbachol. Together, these findings suggested a presynaptic mechanism of action. In the present work, we show that this presynaptic activity is mediated at least partially by a basic Asp49 PLA2 (Bbil-TX) isolated from B. b. smargadina venom. Acetylcholine chloride was obtained from Sigma–Aldrich Chemical Co. (St. Louis, MO, USA) and d-tubocurarine chloride was from Abbott Laboratórios do Brasil Ltda. (São Paulo, SP, Brazil). All salts for the physiological solutions were of analytical grade. The B. b. smargadina venom used here was from the same pool used in a previous investigation of this venom ( Rodrigues-Simioni et al., 2011) and was obtained from adult snakes of both sexes captured in the Amazon region. The DNA Damage inhibitor venom was desiccated and stored at −20 °C until used. Male Swiss mice (25–30 g) obtained from the Multidisciplinary Center for Biological Investigation (CEMIB/Unicamp) were housed 10/cage at 23 °C on a 12 h light/dark cycle with lights on at 6 a.m. Male chicks (4–8 days old, HY-line) were provided by Globo Aves Agricola Ltda. (Campinas, SP, Brazil) and housed in metal cages with a sawdust substrate. The mice and chicks had free access to food and water. This study was approved by the institutional Committee for Ethics in Animal Use (CEUA/UNICAMP, protocol no. 2267-1).