Neuromuscular Activity Of Batx, A Presynaptic Basic Pla2 Isolated From Bothrops Alternatus Snake Venom

We have previously isolated a Lys49 phospholipase A2 homolog (BaTX) from Bothrops alternatus snake venom using a combination of molecular exclusion chromatography and reverse phase HPLC and shown its ability to cause neuromuscular blockade. In this work, we describe a one-step procedure for the purification of this toxin and provide further details of its neuromuscular activity. The toxin was purified by reverse phase HPLC and its purity and molecular mass were confirmed by SDS-PAGE, MALDI-TOF mass spectrometry, amino acid analysis and N-terminal sequencing. BaTX (0.007-1.4 μM) produced time-dependent, irreversible neuromuscular blockade in isolated mouse phrenic nerve-diaphragm and chick biventer cervicis preparations (time to 50% blockade with 0.35 μM toxin: 58 ± 4 and 24 ± 1 min, respectively; n = 3-8; mean ± S.E.) without significantly affecting the response to direct muscle stimulation. In chick preparations, contractures to exogenous acetylcholine (55 and 110 μM) or KCl (13.4 mM) were unaltered after complete blockade by all toxin concentrations. These results, which strongly suggested a presynaptic mechanism of action for this toxin, were reinforced by (1) the inability of BaTX to interfere with the carbachol-induced depolarization of the resting membrane, (2) a significant decrease in the frequency and amplitude of miniature end-plate potentials, and (3) a significant reduction (59 ± 4%, n = 12) in the quantal content of the end-plate potentials after a 60 min incubation with the toxin (1.4 μM). In addition, a decrease in the organ bath temperature from 37 °C to 24 °C and/or the replacement of calcium with strontium prevented the neuromuscular blockade, indicating a temperature-dependent effect possibly mediated by enzymatic activity. © 2009 Elsevier Inc. All rights reserved.1502291297Abreu, V.A., Dal Belo, C.A., Hernandes-Oliveira, S.S., Borja-Oliveira, C.R., Hyslop, S., Furtado, M.F., Rodrigues-Simioni, L., Neuromuscular and phospholipase activities of venoms from three subspecies of Bothrops neuwiedi (B. n. goyazensis, B. n. paranaensis and B. n. diporus) (2007) Comp. Biochem. Physiol. A Mol. Integr. Physiol., 148, pp. 142-149Acosta de Pérez, O., Koscinczuk, P., Negrette, S., Sánchez Negrette, M., Teibler, P., Ruiz, R., Efectos del veneno de Bothrops alternatus de Argentina sobre músculo y distintos organos de ratones (1996) Acta Physiol. Pharmacol. Ther. Latinoam., 46, pp. 97-102Borja-Oliveira, C.R., Durigon, A.M., Vallin, A.C., Toyama, M.H., Souccar, C., Marangoni, S., Rodrigues-Simioni, L., The pharmacological effect of Bothrops neuwiedii pauloensis (jararaca-pintada) snake venom on avian neuromuscular transmission (2003) Braz. J. Med. Biol. Res., 36, pp. 617-624Borja-Oliveira, C.R., Kassab, B.H., Soares, A.M., Toyama, M.H., Giglio, J.R., Marangoni, S., Re, L., Rodrigues-Simioni, L., Purification and N-terminal sequencing of two presynaptic neurotoxic PLA2, neuwieditoxin-I and neuwieditoxin-II, from Bothrops neuwiedi pauloensis (jararaca pintada) venom (2007) J. Venom. Anim. Toxins incl. Trop. Dis., 13, pp. 103-121Bülbring, E., Observations on the isolated phrenic nerve diaphragm preparation of the rat (1946) Br. J. Pharmacol., 1, pp. 38-61Chang, C.C., Neurotoxins with phospholipase A2 activity in snake venoms (1985) Proc. Natl. Sci. Counc. Repub. China [B], 9, pp. 126-142Chang, C.C., Chen, T.F., Lee, C.Y., Studies of the presynaptic effect of β-bungarotoxin on neuromuscular transmission (1973) J. Pharmacol. Exp. Ther., 184, pp. 339-345Cintra, A.C., Marangoni, S., Oliveira, B., Giglio, J.R., Bothropstoxin-I: amino acid sequence and function (1993) J. Protein Chem., 12, pp. 57-64Cogo, J.C., Prado-Franceschi, J., Cruz-Höfling, M.A., Corrado, A.P., Rodrigues-Simioni, L., Effect of Bothrops insularis venom on the mouse and chick nerve-muscle preparation (1993) Toxicon, 31, pp. 1237-1247Cogo, J.C., Prado-Franceschi, J., Giglio, J.R., Corrado, A.P., Cruz-Höfling, M.A., Donato, J.L., Leite, G.B., Rodrigues-Simioni, L., An unusual presynaptic action of Bothrops insularis snake venom mediated by phospholipase A2 fraction (1998) Toxicon, 36, pp. 1323-1332Cogo, J.C., Lilla, S., Souza, G.H., Hyslop, S., de Nucci, G., Purification, sequencing and structural analysis of two acidic phospholipases A2 from the venom of Bothrops insularis (jararaca ilhoa) (2006) Biochimie, 88, pp. 1947-1959Costa, P.D., Toyama, M.H., Marangoni, S., Rodrigues-Simioni, L., Cruz-Höfling, M.A., Effects of Bothrops pirajai venom on the mouse extensor digitorum longus (EDL) muscle preparation (1999) Toxicon, 37, pp. 1143-1153Dal Belo, C.A., Leite, G.B., Toyama, M.H., Marangoni, S., Corrado, A.P., Fontana, M.D., Southan, A., Rodrigues-Simioni, L., Pharmacological and structural characterization of a novel phospholipase A2 from Micrurus dumerilii carinicauda venom (2005) Toxicon, 46, pp. 736-750de Roodt, A.R., Dolab, J.A., Dokmetjian, J.C., Litwin, S., Segre, L., Vidal, J.C., A comparison of different methods to assess the hemorrhagic activity of Bothrops venoms (2000) Toxicon, 38, pp. 865-873de Roodt, A.R., Litwin, S., Vidal, J.C., Hemorrhagic activity of Bothrops venoms determined by two different methods and relationship with proteolytic activity on gelatin and lethality (2003) Toxicon, 41, pp. 949-958Elmqvist, D., Quastel, D.M.J., Presynaptic action of hemicholinium at the neuromuscular junction (1965) J. Physiol., 177, pp. 463-482Gay, C.C., Leiva, L.C., Maruñak, S., Teibler, P., Acosta de Pérez, O., Proteolytic, edematogenic and myotoxic activities of a hemorrhagic metalloproteinase isolated from Bothrops alternatus venom (2005) Toxicon, 46, pp. 546-554Ginsborg, B.L., Warriner, J., The isolated chick biventer cervicis nerve-muscle preparation (1960) Br. J. Pharm. Chemother., 15, pp. 410-411Gutiérrez, J.M., Lomonte, B., Efectos locales en el envenenamiento ofídico en América Latina (2003) Animais Peçonhentos no Brasil: Biologia, Clínica e Terapêutica dos Acidentes, pp. 310-323. , Cardoso J.L.C., França F.O.S., Wen F.H., Málaque C.M.S., and Haddad Jr. V. (Eds), Sarvier/FAPESP, São PauloGutiérrez, J.M., Ownby, C.L., Skeletal muscle degeneration induced by venom phospholipases A2: insights into the mechanisms of local and systemic myotoxicity (2003) Toxicon, 15, pp. 915-931Gutiérrez, J.M., Rucavado, A., Snake venom metalloproteinases: their role in the pathogenesis of local tissue damage (2000) Biochimie, 82, pp. 841-850Gutiérrez, J.M., Rucavado, A., Escalante, T., Díaz, C., Hemorrhage induced by snake venom metalloproteinases: biochemical and biophysical mechanisms involved in microvessel damage (2005) Toxicon, 45, pp. 997-1011Hawgood, B.J., Smith, J., The presynaptic action of crotoxin at the murine neuromuscular junction [proceedings] (1977) J. Physiol., 266, pp. 91P-92PHawgood, B.J., Smith, I.C., The importance of phospholipase A2 in the early induction by crotoxin of biphasic changes in endplate potentials at the frog neuromuscular junction (1989) Toxicon, 27, pp. 272-276Hawgood, B.J., Smith, I.C., Strong, P.N., Early induction by crotoxin of biphasic frequency changes and giant miniature endplate potentials in frog muscle (1988) Br. J. Pharmacol., 94, pp. 765-772Homsi-Brandeburgo, M.I., Queiroz, L.S., Santo-Neto, H., Rodrigues-Simioni, L., Giglio, J.R., Fractionation of Bothrops jararacussu snake venom: partial chemical characterization and biological activity of bothropstoxin (1988) Toxicon, 26, pp. 615-627Kini, R.M., Excitement ahead: structure, function and mechanism of snake venom phospholipase A2 enzymes (2003) Toxicon, 42, pp. 827-840Lomonte, B., Ângulo, Y., Calderón, L., An overview of lysine-49 phospholipase A2 myotoxins from crotalid snake venoms and their structural determinants of myotoxic action (2003) Toxicon, 42, pp. 885-901Maruñak, S.L., Acosta de Pérez, O., Ruíz de Torrent, R.M., Teibler, G.P., Koscinczuk, P., Sánchez Negrette, M., Actividades hemorrágica, edematizante, proteolítica y mionecrótica de venenos de Bothrops alternatus (víbora de la Cruz) (1999) Acta Physiol. Pharmacol. Ther. Latinoam., 49, pp. 149-154Nisenbom, H.E., Perazzo, J.C., Monserrat, A.J., Vidal, J.C., Contribution of phospholipase A2 to the lethal potency of Bothrops alternatus (vibora de la cruz) venom (1986) Toxicon, 24, pp. 807-817Nisenbom, H.E., Seki, C., Vidal, J.C., Phospholipase A2 from Bothrops alternatus (vibora de la cruz) venom. Purification and some characteristic properties (1986) Toxicon, 24, pp. 259-272Nisenbom, H.E., Perazzo, J.C., Monserrat, A.J., Vidal, J.C., Effect of chemical modification with p-bromophenacyl bromide on the enzymatic and lethal properties of phospholipase A2 from Bothrops alternatus (vibora de la cruz) venom (1988) Toxicon, 26, pp. 1137-1144Oberg, S.G., Kelly, R.B., The mechanism of beta-bungarotoxin action. I. Modification of transmitter release at the neuromuscular junction (1976) J. Neurobiol., 7, pp. 129-141Oshima-Franco, Y., Leite, G.B., Belo, C.A., Hyslop, S., Prado-Franceschi, J., Cintra, A.C., Giglio, J.R., Rodrigues-Simioni, L., The presynaptic activity of bothropstoxin-I, a myotoxin from Bothrops jararacussu snake venom (2004) Basic Clin. Pharmacol. Toxicol., 95, pp. 175-182Ponce-Soto, L.A., Bonfim, V.L., Rodrigues-Simioni, L., Novello, J.C., Marangoni, S., Determination of primary structure of two isoforms 6-1 and 6-2 PLA2 D49 from Bothrops jararacussu snake venom and neurotoxic characterization using in vitro neuromuscular preparation (2006) Protein J., 25, pp. 147-155Ponce-Soto, L.A., Lomonte, B., Gutiérrez, J.M., Rodrigues-Simioni, L., Novello, J.C., Marangoni, S., Structural and functional properties of BaTX, a new Lys49 phospholipase A2 homologue isolated from the venom of the snake Bothrops alternatus (2007) Biochim. Biophys. Acta, 1770, pp. 585-593Prianti Jr., A.C., Ribeiro, W., Lopes-Martins, R.A., Lira-Da-Silva, R.M., Prado-Franceschi, J., Rodrigues-Simioni, L., Cruz-Höfling, M.A., Cogo, J.C., Effect of Bothrops leucurus venom in chick biventer cervicis preparations (2003) Toxicon, 41, pp. 595-603Prior, C., Dempster, J., Marshall, G., Electrophysiological analysis of transmission at the skeletal neuromuscular junction (1993) J. Pharmacol. Toxicol. Methods, 30, pp. 1-17Rodrigues-Simioni, L., Borgese, N., Ceccarelli, B., The effects of Bothrops jararacussu venom and its components on frog nerve-muscle preparation (1983) Neuroscience, 10, pp. 475-489Rodrigues-Simioni, L., Hawgood, B.J., Smith, I.C., Properties of the early phases of crotoxin poisoning at frog neuromuscular junctions (1990) Toxicon, 28, pp. 1479-1489Rodrigues-Simioni, L., Zamunér, S.R., Cogo, J.C., Borja-Oliveira, C.R., Prado-Franceschi, J., Cruz-Höfling, M.A., Corrado, A.P., Pharmacological evidence for a presynaptic action of venoms from Bothrops insularis (jararaca ilhoa) and Bothrops neuwiedi (jararaca pintada) (2004) Toxicon, 43, pp. 633-638Sanchez, E.F., Freitas, T.V., Ferreira-Alves, D.L., Velarde, D.T., Diniz, M.R., Cordeiro, M.N., Agostini-Cotta, G., Diniz, C.R., Biological activities of venoms from South American snakes (1992) Toxicon, 30, pp. 95-103Soares, A.M., Guerra-Sá, R., Borja-Oliveira, C.R., Rodrigues, V.M., Rodrigues-Simioni, L., Rodrigues, V., Fontes, M.R., Giglio, J.R., Structural and functional characterization of BnSP-7, a Lys49 myotoxic phospholipase A2 homologue from Bothrops neuwiedi pauloensis venom (2000) Arch. Biochem. Biophys., 378, pp. 201-209Soares, A.M., Andrião-Escarso, S.H., Bortoleto, R.K., Rodrigues-Simioni, L., Arni, R.K., Ward, R.J., Gutiérrez, J.M., Giglio, J.R., Dissociation of enzymatic and pharmacological properties of piratoxins-I and -III, two myotoxic phospholipases A2 from Bothrops pirajai snake venom (2001) Arch. Biochem. Biophys., 387, pp. 188-196Teixeira, C.F., Landucci, E.C., Antunes, E., Chacur, M., Cury, Y., Inflammatory effects of snake venom myotoxic phospholipases A2 (2003) Toxicon, 42, pp. 947-962Van der Kloot, W., Nicotinic agonists antagonize quantal size increases and evoked release at frog neuromuscular junction (1993) J. Physiol., 468, pp. 567-589Veldesema-Currie, R.D., Van Wilgenburg, H., Labruyère, W.T., Langermeijer, M.W.E., Presynaptic facilitatory effects of the corticosteroid dexamethasone in rat diaphragm: modulation by beta-bungarotoxin (1984) Brain Res., 294, pp. 315-325Zamunér, S.R., Cruz-Höfling, M.A., Corrado, A.P., Hyslop, S., Rodrigues-Simioni, L., Comparison of the neurotoxic and myotoxic effects of Brazilian Bothrops venoms and their neutralization by commercial antivenom (2004) Toxicon, 44, pp. 259-27