Neuromuscular and phospholipase activities of venoms from three subspecies of Bothrops neuwiedi (B. n. goyazensis, B. n. paranaensis and B. n. diporus)

Abstract

The Bothrops neuwiedi (Neuwied's lancehead) species complex consists of a variety of subspecies with a wide distribution in South America. In this work, we compared the neuromuscular blockade caused by venoms from three subspecies (B. n. goyazensis, B. n. paranaensis and B. n. diporus) of this complex using chick biventer cervicis (BC) and mouse phrenic nerve-diaphragm (PND) preparations and investigated their phospholipase A₂ (PLA₂) activities and electrophoretic profiles. The order of potency of PLA₂ activity was B. n. diporus > B. n. paranaensis > B. n. goyazensis. In BC preparations, B. n. goyazensis venom (50 μg/mL) was significantly (p < 0.05) more active than B. n. paranaensis and B. n. diporus venoms, which did not produce a significant blockade at this time interval; after 120 min, B. n. goyazensis, B. n. paranaensis and B. n. diporus venoms (100 μg/mL) produced blockades of 57.4 ± 5%, 30 ± 3% and 17.4 ± 7% (n = 3–6 each), respectively. The three venoms inhibited contractures in response to ACh, indicating interference with postsynaptic neurotransmission. Only B. n. goyazensis and B. n. paranaensis venoms caused a long-lasting, concentration-dependent muscle contracture prior to blockade. In PND preparations, all of the venoms blocked the twitch-tension responses within 45–100 min, indicating that these preparations were more sensitive than avian preparations. There was a correlation between PLA₂ activity and the time for 50% blockade in PND but not in BC preparations. SDS-PAGE showed quantitative rather than qualitative differences among the venoms. These results indicate that the venoms of the three subspecies had similar profiles of neuromuscular activity, although the relationship with PLA₂ activity varied with the preparation used.

Introduction

Snake species with a wide geographical distribution may pose a taxonomical problem because of their marked variation in coloration and morphology (Wüster et al., 1997, Jorge da Silva and Sites, 1999). In addition, venoms of the same snake species from different geographical regions may produce different pharmacological/pathophysiological effects (Warrell, 1986, Straight et al., 1992, Rodrigues et al., 1998, Daltry et al., 1999, Santoro et al., 1999, Saravia et al., 2002).

The pitviper Bothrops neuwiedi (Neuwied's lancehead) occurs in Brazil, Bolivia, Paraguay, Argentina, Uruguay and Peru (Peters and Orejas-Miranda, 1986, Campbell and Lamar, 1989, Reyes and Onofre, 1997), and is the third most common Bothrops species in Brazil (Belluomini, 1968). B. neuwiedi is a large species complex that currently contains 12 subspecies (Campbell and Lamar, 1989, Silva, 2004). This large number of subspecies represents a particular taxonomic challenge because of their morphological variation and extensive but often uncertain geographical ranges. Based on an extensive taxonomic analysis of this complex, Silva (2004) concluded that B. n. goyazensis and B. n. diporus should be raised to the species level as Bothrops marmoreado and Bothrops diporus, respectively, whereas B. n. paranaensis should be retained as a subspecies of B. neuwiedi.

B. neuwiedi venom contains a variety of enzymes (Tan and Ponnudurai, 1991) including hemorrhagic proteinases (Mandelbaum et al., 1984, Queiroz et al., 1985), a prothrombin activator (Govers-Riemslag et al., 1987), a fibrino(geno)lytic metalloproteinase (Rodrigues et al., 2000, Rodrigues et al., 2001), phospholipase A₂ (PLA₂) (Vidal et al., 1966, Soares et al., 2000, Rodrigues et al., 2004) and a variety of bradykinin-potentiating peptides (Ferreira et al., 1998, Wermelinger et al., 2005) that can contribute to the biological actions (edema, hemorrhage, necrosis, and (anti)coagulant and platelet-aggregating activities) of this venom (Furtado et al., 1991, Moura-da-Silva et al., 1990, Moura-da-Silva et al., 1991, Ferreira et al., 1992, Francischetti et al., 1998). Envenomation by B. neuwiedi in humans (Dempfle et al., 1990, Nishioka and Silveira, 1992, Jorge and Ribeiro, 2000) and domestic animals (Mendez and Riet-Correa, 1995) produces local and systemic effects similar to those of other Bothrops species (França and Málaque, 2003).

The venoms of various Brazilian Bothrops species (B. jararaca, B. jararacussu, B. moojeni, B. erythromelas and B. neuwiedi) cause neuromuscular blockade as well as muscle contracture and inhibition of the contracture to acetylcholine (ACh) in avian and mammalian nerve-muscle preparations (Zamunér et al., 2004). The neuromuscular blockade caused by B. neuwiedi venom in avian (Borja-Oliveira et al., 2003, Rodrigues-Simioni et al., 2004) and mammalian (Durigon et al., 2005) preparations involves pre- and postsynaptic sites, with the presynaptic action being similar to that of South American rattlesnake (Crotalus durissus terrificus) venom (Rodrigues-Simioni et al., 2004). B. neuwiedi venoms from different geographic regions vary in their myotoxin content, with basic toxins occurring in venom of B. n. pauloensis from the Brazilian state of São Paulo but not in that of B. n. urutu from the state of Minas Gerais (Rodrigues et al., 1998). Similarly, Borja-Oliveira et al. (2002) observed intraspecific variation in the neuromuscular activity of 16 lots of B. neuwiedi venom in chick biventer cervicis preparations, although in several of these cases the precise origin of the venoms and the subspecies involved were uncertain.

Here, we investigated the intraspecific variation in the neuromuscular and PLA₂ activities and electrophoretic profiles of venom from three subspecies of B. neuwiedi (B. n. goyazensis, B. n. paranaensis and B. n. diporus) and examined the relationship between these activities and the taxonomic reorganization proposed by Silva (2004).