Signalling Pathways Regulating Human Neutrophil Migration Induced By Secretory Phospholipases A2

This study was designed to elucidate the signalling pathways by which secretory phospholipases A2 (sPLA2s) induce in vitro neutrophil migration. The cell migration assays were performed with Naja mocambique venom PLA2 (sPLA2 with high catalytic activity), bothropstoxin-I (sPLA2 devoid of catalytic activity) and platelet-activating factor (PAF), using a 48-well microchemotaxis chamber. Both the non-selective protein kinase inhibitor staurosporine (30-300nM) and the selective protein kinase C (PKC) inhibitor 1-(5-isoquinolinesulfonyl)-2- methylpyperazine (H7; 50-200 μM) as well as the Gi inactivator pertussis toxin (30-300nM) caused a concentration-dependent inhibition of the neutrophil migration induced by either N. mocambique venom PLA2 (100 μg/ml) or bothropstoxin-I (100 μg/ml). Pertussis toxin nearly abolished PAF-induced migration, while staurosporine and H7 partly (but significantly) inhibited the chemotactic responses to PAF. The dual inhibitor of cytosolic PLA2 and Ca2+-independent PLA2 (iPLA2), arachidonil-trifluoromethyl-ketone (ATK; 0.2-20 μM), or the specific iPLA2 inhibitor bromoenol lactone (1-30 μM) caused a concentration-dependent inhibition of the migration induced by either sPLA 2s. At the maximal concentration used for each compound, the migration was almost suppressed. In contrast, both of these compounds caused only slight inhibitions of PAF-induced migration. No rise in intracellular Ca2+ was observed in neutrophil-stimulated sPLA2, as determined in cells preloaded with fura 2-AM. In the experimental condition used, pertussis toxin, staurosporine, H7, ATK or bromoenol lactone did not induce cytotoxic effects, according to MTT assay. Our results suggest that activation of an endogenous PLA2 through activation of GTP-binding protein and PKC is the main mechanism by which exogenous sPLA2s cause neutrophil migration. © 2004 Elsevier Ltd. All rights reserved.445473481Ackermann, E.J., Dennis, E.A., Mammalian calcium-independent phopholipase A2 (1995) Biochim. Biophys. Acta, 1259, pp. 125-136Alonso-Torre, S.R., Garcia-Sancho, J., Arachidonic acid inhibits capacitative calcium entry in rat thymocytes and human neutrophils (1997) Biochim. Biophys. Acta, 1328, pp. 207-213Balsinde, J., Dennis, E.A., Function and inhibition of intracellular calcium-independent phospholipase A2 (1997) J. Biol. Chem., 272, pp. 16069-16075Bokoch, G.M., Katada, T., Northup, K.K., Gilman, A.G., Identification of the predominant substrate for ADP-ribosylation by islet activating protein (1983) J. Biol. Chem., 258, pp. 2072-2077Cintra, A.C.O., Marangoni, S., Oliveira, B., Giglio, J.R., Bothropstoxin-I: Amino acid sequence and function (1993) J. Protein Chem., 12, pp. 57-64Clark, J.D., Lin, L., Kriz, R.W., Ramesha, C.S., Sultman, L.A., Lin, A.Y., Milona, N., Knopf, J.L., A novel arachidonic acid-selective cytosolic PLA2 contains a Ca2+-dependent translocation domain with homology to PKC and GAP (1991) Cell, 65, pp. 1043-1051Codina, J., Hildebrandt, J., Iyengar, R., Birnbaumer, L., Sekura, R.D., Manclark, C.R., Pertussis toxin substrate, the putative Ni component of adenylyl cyclases, is an alpha-beta heterodimer regulated by guanine nucleotide and magnesium (1983) Proc. Natl Acad. Sci. USA, 80, pp. 4276-4283Flower, R.J., Blackwell, G.J., The importance of phospholipase A2 in prostaglandin biosynthesis (1976) Biochem. Pharmacol., 25, pp. 285-291Fonteh, A.N., Differential effects of arachidonoyl trifluoromethyl ketone on arachidonic acid release and lipid mediator biosynthesis by human neutrophils. Evidence for different arachidonate pools (2002) Eur. J. 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The Amino Acid Sequence Of Bothropstoxin-ii, An Asp-49 Myotoxin From Bothrops Jararacussu (jararacucu) Venom With Low Phospholipase A2 Activity

The complete amino acid sequence of bothropstoxin-II (BthTX-II), a myotoxin isolated from Bothrops jararacussu snake venom, is reported. The results show that BthTX-II is an Asp-49 phospholipase A2 (PLA2)-like protein composed of a single polypeptide chain of 120 amino acid residues (Mr = 13,976), containing one methionine and 14 half-cystines. Despite a high degree of homology with other PLA2's and the presence of the strategic residues known to compose the Ca2+-binding loop, namely Tyr-28, Gly-30, Gly-32, and especially Asp-49, besides His-48, Tyr-52, and Asp-99, all of them directly or indirectly involved in catalysis, BthTX-II revealed a very low PLA2 activity when assayed on egg yolk phosphatidylcholine. We attribute this low catalytic activity to the existence of extra mutations, e.g., Trp-5 for Phe-5, which points to the need of considering other strategic positions, since only Lys-49 PLA2's have been considered to be devoid of this enzymatic activity. © 1998 Plenum Publishing Corporation.174381386Arni, R.K., Ward, R.J., Phospholipase a2 - A structural review (1996) Toxicon, 34, pp. 827-841Cintra, A.C.O., Marangoni, S., Oliveira, B., Giglio, J.R., Bothropstoxin-I: Amino acid sequence and function (1993) J. Protein Chem., 12, pp. 57-64De Haas, G.H., Postema, N.M., Nieuwenhuizem, W., Van Deenen, L.L.M., Purification and properties of phospholipase a from porcine pancreas (1968) Biochim. Biophys. Acta, 159, pp. 103-117Dupureur, C.M., Yu, B.-Z., Ramone, A., Jain, M.K., Tsai, M.-D., Phospholipase a2 engineering. Structural and functional roles of aromaticity and hydrophobicity in the conserved phenylalanine-22 and phenylalanine-106 aromatic sandwich (1992) Biochemistry, 31, pp. 10576-10583Edman, P., Begg, G., A protein sequenator (1967) Eur J. Biochem., 1, pp. 80-91Fleer, E.A., Verheij, H.M., De Haas, G.H., The primary structure of bovine pancreatic phospholipase a2 (1978) Eur. J. Biochem., 82, pp. 261-269Francis, B., Gutiérrez, J.M., Lomonte, B., Kaiser, I.I., Miotoxin II from Bothrops asper (terciopelo) venom is a lysine-49 phospholipase a2 (1991) Arch. Biochem. Biophys., 284, pp. 352-359Frangione, B., Moloshok, T., Soloman, A., Primary structure of the variable region of a human lambda VI light chain. Bence Jones Protein SUT (1983) J. Immunol., 131, pp. 2490-2493Gutiérrez, J.M., Lomonte, B., Phospholipase a2 myotoxins from Bothrops snake venoms (1995) Toxicon, 33, pp. 1405-1424Gutiérrez, J.M., Núñez, J., Diaz, C., Cintra, A.C.O., Homsi-Brandeburgo, M.I., Giglio, J.R., Skeletal muscle degeneration and regeneration after injection of bothropstoxin-II, a phospholipase a2 isolated from the venom of the snake Bothrops jararacussu (1991) Exp. Molec. Pathol., 55, pp. 217-229Harris, J.B., Phospholipases in snake venoms and their effects on nerve and muscle (1991) Snake Toxins, pp. 91-129. , (Harvey, A. L., ed.), Pergamon Press, New YorkHomsi-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. 605-627Houmard, J., Drapeau, G.R., Staphylococcal protease: A proteolytic enzyme specific for glutamyl bonds (1972) Proc. Nail. Acad. Sci. USA, 69, pp. 3506-3509Kaiser, I.I., Gutiérrez, J.M., Plummer, D., Aird, S.D., Odell, G., The amino acid sequence of a myotoxic phospholipase from the venom of Bothrops asper (1990) Arch. Biochem. Biophys., 278, pp. 319-325Krizaj, I., Bieber, A.L., Ritonja, A., Gubensek, F., The primary structure of ammodytin L, a myotoxic phospholipase a2 homologue from Vipera ammodytes venom (1991) Eur. J. Biochem., 202, pp. 1165-1168Kuipers, O.P., Kerver, J., Van Meersbergen, J., Vis Roel Dijkman, R., Verhjeij, H.M., De Haas, G., Influence of size and polarity of residue 31 in porcine pancreatic phospholipase a2 on catalytic properties (1990) Protein Eng., 3, pp. 599-603Laemmli, U.K., Cleavage of structural proteins among the assembly of the head of the bacteriophage T4 (1970) Nature, 277, pp. 680-685Lomonte, B., Gutiěrrez, J.M., A new muscle damaging toxin, myotoxin III, from the venom of the snake Bothrops asper (terciopelo) (1989) Toxicon, 27, pp. 725-733Lowry, O.H., Rosebrough, W.H., Farr, J.L., Randall, R.J., Protein measurement with Folin phenol reagent (1951) J. Biol. Chem., 193, p. 265Maraganore, J.M., Merutka, G., Cho, W., Weichest, W., Kézdy, F.J., Heinrikson, R.L., A new class of phospholipase a2 with lysine in place of aspartate-49 (1984) J. Biol. Chem., 259, pp. 13839-13843Marangoni, S., Ghiso, J., Sampaio, S.V., Arantes, E.C., Giglio, J.R., Oliveira, B., Frangione, B., The complete amino acid sequence of toxin TsTX-VI isolated from the venom of the scorpion Tityus serrulatus (1990) J. Protein Chem., 9, pp. 595-601Mebs, D., Samejima, J., Isolation and characterization of myotoxic phospholipases a2 from crotalid venoms (1986) Toxicon, 24, pp. 161-168Reisfeld, R.A., Lewis, V.J., Williams, D.E., Disc electrophoresis of basic proteins and peptides on polyacrylamide gels (1962) Nature, 4838, pp. 281-289Rosenberg, P., Phospholipases (1990) Handbook of Toxicology, pp. 67-277. , (Shier, W. T., and Mebs, E., eds.), Marcel Dekker, New YorkVan Den Bergh, C.J., Slotboom, A.J., Verheij, H.M., De Haas, G.H., The role of Asp-49 and other conserved amino acids in phospholipases a2 and their importance for enzymatic activity (1989) J. Cell. Biochem., 39, pp. 379-39

No Role For Enzymatic Activity Or Dantrolene-sensitive Ca2+ Stores In The Muscular Effects Of Bothropstoxin, A Lys49 Phospholipase A2 Myotoxin

The role of low levels of phospholipase A2 (PLA2) activity and intracellular Ca2+ stores in the pharmacological action of bothropstoxin (BthTX), a myotoxic Lys49 PLA2 homologue isolated from the venom of Bothrops jararacussu, was investigated. We examined the muscular effects of BthTX in the mouse diaphragm and its PLA2 activity in radiolabeled human and rat primary cultures of skeletal muscle. Although it is a Lys49 PLA2 homologue, BthTX had a low, but easily detectable, level of enzymatic activity relative to two Asp49 PLA2 enzymes from Naja naja kaouthia and Naja naja atra venoms, and this activity was reduced by about 85% in the presence of Sr2+ (4.0 mM). However, the replacement of 1.8 mM Ca2+ by 4 mM Sr2+ did not alter the BthTX-induced contracture and blockade of the muscle twitch tension. In addition, Sr2+ decreased by 50% the time required to cause 50% paralysis, and evoked approximately a four-fold increase in the number of spontaneous spikes. In isolated sarcoplasmic reticulum preparations, BthTX opened the intracellular Ca2+ release channel (ryanodine receptor) and lowered the threshold of Ca2+-induced Ca2+ release by a second, as yet unidentified, mechanism. However, in intact muscle, dantrolene, an antagonist of some forms of intracellular Ca2+ release, had no effect on the actions of BthTX. These findings do not support any role for the low levels of PLA2 activity, or dantrolene-sensitive intracellular Ca2+ stores, in the action of BthTX. The mechanism whereby Sr2+ stimulates the pharmacological activity of BthTX remains to be clarified. © 1995.331114791489Cintra, Marangoni, Oliveira, Giglio, Bothropstoxin-I: amino acid sequence and function (1993) J. Prot. Chem., 12, pp. 57-64Condrea, Comparison of enzymatic and pharmacological activities of lysine-49 and aspartate-49 phospholipases A2 from Agkistrodon piscivorus piscivorus snake venom. A reconsideration (1989) Toxicon, 27, pp. 705-706Dettbarn, Palade, Arachidonic acid-induced Ca2+ release from isolated sarcoplasmic reticulum (1993) Biochem. Pharmac., 45, pp. 1301-1309Dhillon, Condrea, Maraganore, Heinrikson, Benjamin, Rosenberg, Comparison of enzymatic and pharmacological activities of lysine-49 and aspartate-49 phospholipases A2 from Agkistrodon piscivorus piscivorus snake venom (1987) Biochem. Pharmac., 36, pp. 1723-1730Díaz, Gutiérrez, Lomonte, Nüñez, P-Bromophenacyl bromide modification of Bothrops asper myotoxin II, a lysine-49 phospholipase A2, affects its pharmacological activities (1993) Toxicon, 31, pp. 1202-1206Fletcher, Rapuano, Condrea, Yang, Rosenberg, Relationship between catalysis and toxicoloical properties of three phospholipases A2 from elapid snake venoms (1981) Toxic. appl. Pharmac., 59, pp. 375-388Fletcher, Tripolitis, Erwin, Hanson, Rosenberg, Conti, Beech, Fatty acids modulate calcium-induced calcium release from skeletal muscle heavy sarcoplasmic reticulum fractions: implications for malignant hyperthermia (1990) Biochem. cell. Biol., 68, pp. 1195-1201Fletcher, Jiang, Gong, Smith, Snake venom cardiotoxins and bee venom melittin activate phospholipase C activity in primary cultures of skeletal muscle (1991) Biochem. cell. Biol., 69, pp. 274-281Fletcher, Trioplitis, Beech, Species difference in modulation of calcium release by Naja naja kaouthia snake venom cardiotoxin in terminal cisternae from human and equine skeletal muscle (1993) Toxicon, 31, pp. 43-51Fletcher, Jiang, Middlebrook, Antibodies having markedly different effects in enzymatic activity and induction of acetylcholine release by two presynaptically-acting phospholipase A2 neurotoxins (1995) Biochem. Pharmac., 49, pp. 381-388Ghassemi, Dhillon, Rosenberg, β-Bungarotoxin-induced phospholipid hydrolysis in rat brain synaptosomes: effect of replacement of calcium by strontium (1988) Toxicon, 26, pp. 509-514Harris, Johnson, Further observations on the pathological responses of rat skeletal muscle to toxins isolated from the venom of the Australian tiger snake Notechis scutatus scutatus (1975) Clinical and Experimental Pharmacology and Physiology, 5, pp. 587-600Hawgood, Smith, The mode of action at the mouse neuromuscular function of the phopholipase A-crotapotin complex isolated from venom of the South American rattlesnake (1977) Br. J. 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Antibothropic action of Casearia sylvestris Sw. (flacourtiaceae) extracts

Casearia sylvestris Sw., popularly known in Brazil as 'guacatonga', has been used as antitumor, antiseptic, antiulcer, local anaesthetic and healer in folk medicine. Snakebite envenomation by Bothrops jararacussu (Bjssu) constitutes a relevant public health hazard capable of inducing serious local damage in victims. This study examined the pharmacological action of apolar and polar C sylvestris leaf extracts in reverting the neuromuscular blockade and myonecrosis, which is induced by Bjssu venom and its major toxin bothropstoxin-I on the mouse phrenic nerve-diaphragm preparations. The polar methanol extract (ME) was by far the most efficacious. ME not only prevented myonecrosis and abolished the blockade, but also increased ACh release. Such facilitation in neuromuscular transmission was observed with ME alone, but was accentuated in preparations incubated with ME plus venom or toxin. This established synergy opens an interesting point of investigation because the venom or toxin in contact with ME changes from a blocking to a facilitating effect. It is suggested that rutin, known to have potent antioxidant properties, and one of the components present in the ME, could have a role in the observed effects. Since commercial rutin did not reproduce the ME effects, it is likely that a rutin-containing phytocomplex is neutralizing the bothropic envenoming effects22678479

Antibothropic Action Of Casearia Sylvestris Sw. (flacourtiaceae) Extracts

Casearia sylvestris Sw., popularly known in Brazil as 'guaçatonga', has been used as antitumor, antiseptic, antiulcer, local anaesthetic and healer in folk medicine. Snakebite envenomation by Bothrops jararacussu (Bjssu) constitutes a relevant public health hazard capable of inducing serious local damage in victims. This study examined the pharmacological action of apolar and polar C. sylvestris leaf extracts in reverting the neuromuscular blockade and myonecrosis, which is induced by Bjssu venom and its major toxin bothropstoxin-I on the mouse phrenic nerve-diaphragm preparations. The polar methanol extract (ME) was by far the most efficacious. ME not only prevented myonecrosis and abolished the blockade, but also increased ACh release. Such facilitation in neuromuscular transmission was observed with ME alone, but was accentuated in preparations incubated with ME plus venom or toxin. This established synergy opens an interesting point of investigation because the venom or toxin in contact with ME changes from a blocking to a facilitating effect. It is suggested that rutin, known to have potent antioxidant properties, and one of the components present in the ME, could have a role in the observed effects. Since commercial rutin did not reproduce the ME effects, it is likely that a rutin-containing phytocomplex is neutralizing the bothropic envenoming effects. Copyright © 2008 John Wiley & Sons, Ltd.226784790Bjarnason, J.B., Fox, J.W., Hemorrhagic metalloproteinases from snake venoms (1994) Pharmacol Ther, 62, pp. 325-372Borges, M.H., Soares, A.M., Rodrigues, V.M., Effects of aqueous extract of Casearia sylvestris (Flacourtiaceae) on actions of snake and bee venoms and on activity of phospholipases A 2 (2000) Comp Biochem Physiol B Biochem Mol Biol, 127, pp. 21-30Borges, M.H., Soares, A.M., Rodrigues, V.M., Neutralization of proteases from Bothrops snake venoms by aqueous extract from Casearia sylvestris (Flacourtiaceae) (2001) Toxicon, 39, pp. 1863-1869Bülbring, E., Observation on the isolated phrenic nerve diaphragm preparation of the rat (1946) Br J Pharmacol, 1, pp. 38-61De Oliveira, M., Cavalcante, W.L., Arruda, E.A., Melo, P.A., Dal-Pai Silva, M., Gallaci, M., Antagonism of myotoxic and paralyzing activities of bothropstoxin-l by suramin (2003) Toxicon, 42, pp. 373-379Girish, K.S., Kemparaju, K., Inhibition of Naja naja venom hyaluronidase by plant-derived bioactive components and polysaccharides (2005) Biochemistry (Moscow), 70, pp. 948-952Gutierrez, J.M., Romero, M., Díaz, C., Borkow, G., Ovadia, M., Isolation and characterization of a metalloproteinase with weak hemorrhagic activity from the venom of the snake Bothrops asper (terciopelo) (1995) Toxicon, 33, pp. 19-29Harbone, J.B., (1998) Phytochemical Methods: A Guide to Modern Techniques of Plants Analysis, , 3rd edn, Chapman & Hall: LondonHavsteen, B., Flavonoids, a class of natural products of high pharmacological potency (1983) Biochem Pharmacol, 32, pp. 1141-1148Heluany, N.F., Homsi-Brandeburgo, M., Giglio, J.R., Prado-Franceschi, J., Rodrigues-Simioni, L., Effects induced by bothrop-stoxin, a component from Bothrops jararacussu snake venom, on mouse and chick muscle preparations (1992) Toxicon, 30, pp. 1203-1210Iwanaga, S., Suzuki, T., Enzymes in snake venoms (1979) Snake Venoms. Handbook of Experimental Pharmacology, pp. 61-158. , Lee CY ed, Springer: New YorkJin, G.Z., Yamagata, Y., Tomita, K., Structure of rutin pentamethanol (1990) Chem Pharm Bull, 38, pp. 297-300Khan, A.A., Ashfaq, A., Ali, M.N., (1979) Pharmacognostic Studies of Selected Indigenous Plants of Pakistan, , Pakistan Forest Institute. Spinezer Printers: PeshawarMahmood A, Ahmad M, Jabeen A, Zafar M, Nadeem S. 2005. Pharmacognostic studies of some indigenous medicinal plants of Pakistan. Available in http://www.siu.edu/∼ebl/leaflets/abid.htm. Access in 06/03/2005Maistro, E.L., Carvalho, J.C., Mantovani, M.S., Evaluation of the genotoxic potential of the Casearia sylvestris extract on HTC and V79 cells by the comet assay (2004) Toxicol In Vitro, 18, pp. 337-342Markland, F.S., Snake venoms and the hemostatic system (1998) Toxicon, 36, pp. 1749-1800Matrisian, L.M., The matrix-degrading metalloproteinases (1992) BioEssays, 14, pp. 455-463Milani Junior, R., Jorge, M.T., de Campos, F.P., Snake bites by the jararacuçu (Bothrops jararacussu): Clinicopatho-logical studies of 29 proven cases in Sao Paulo State, Brazil (1997) Q J Med, 90, pp. 323-334(2001) Manual de Diagnóstico e Tratamento de Acidentes por Animais Pegonhentos, , Ministério da Saúde do Brasil, 2nd edn. 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(guaçatonga) (2005) J Venom Anim Toxins Trop Dis, 11, pp. 465-478Oshima-Franco, Y., Hyslop, S., Cintra, A.C.O., Giglio, J.R., Cruz-Höfling, M.A., Rodrigues-Simioni, L., Neutralizing capacity of commercial bothropic antivenom against Bothrops jararacussu venom and bothropstoxin-l (2000) Muscle Nerve, 23, pp. 1832-1839Oshima-Franco, Y., Leite, G.B., Dal Belo, C.A., The presynaptic activity of bothropstoxin-l, a myotoxin from Bothrops jararacussu snake venom (2004) Basic Clin Pharmacol Toxicol, 95, pp. 175-182Oshima-Franco, Y., Leite, G.B., Silva, G.H., Neutralization of the pharmacological effects of bothropstoxin-l from Bothrops jararacussu (jararacuçu) venom by crotoxin antiserum and heparin (2001) Toxicon, 39, pp. 1477-1485Oshima-Franco, Y., Leite, G.B., Valério, A.A., Rabbit antivenom efficacy against myotoxic and neurotoxic activities of Bothrops jararacussu venom and bothropstoxin-l (2002) J Venom Anim Toxins, 8, pp. 226-243Pessini, A.C., Takao, T.T., Cavalheiro, E.C., A hyaluronidase from Tityus serrulatus scorpion venom: Isolation, characterization and inhibition by flavonoids (2001) Toxicon, 39, pp. 1495-1504Randazzo-Moura, P., Leite, G.B., Silva, G.H., Study of myotoxicity of bothropstoxin-l (BthTX-l) using manganese (Mn 2+) in mouse phrenic nerve-diaphragm (PND) and extensor digitorum longus (EDL) preparations (2006) Braz J Morphol Sci, 23, pp. 171-184Rates, S.M.K., Plants as source of drugs (2001) Toxicon, 39, pp. 603-613Rodrigues-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., Prado-Franceschi, J., Cintra, A.C.O., Giglio, J.R., Jiang, M.S., Fletcher, J.E., No role for enzymatic activity or dantrolene-sensitive Ca 2+ stores in the muscular effects of bothropstoxin, a Lys49 phospholipase A 2 myotoxin (1995) Toxicon, 33, pp. 1479-1489Rosenfeld, G., Symptomatology, pathology and treatment of snakebites in South America (1971) Venomous Animals and their Venoms, pp. 345-384. , Bucherl W, Buckley E eds, Academic Press: New YorkRucavado, A., Escalante, T., Gutiérrez, J.M., Effect of the metalloproteinase inhibitor batimastat in the systemic toxicity induced by Bothrops asper snake venom: Understanding the role of metalloproteinases in envenomation (2004) Toxicon, 43, pp. 417-424Selistre, H.S., Queiroz, L.S., Cunha, O.A., De Souza, G.E., Giglio, J.R., Isolation and characterization of hemorrhagic, myonecrotic and edema-inducing toxins from Bothrops insularis (jararaca ilhoa) snake venom (1990) Toxicon, 28, pp. 261-273Simōes CMO, Schenkel EP, Gosmann G, Mello JCP, Mentz LA, Petrovick PR. 2004. 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Mast cell degranulation induced by two phospholipase A(2) homologues: Dissociation between enzymatic and biological activities

Bothropstoxin-I and bothropstoxin-II are phospholipase A(2) homologues isolated from Bothrops jararacussu snake venom. The former is devoid of phospholipase A(2) activity whereas the latter has very low enzymatic activity. In this study, we have investigated the in vivo (rat paw and skin oedema) and in vitro (mast cell degranulation) inflammatory effects caused by bothropstoxin-I and bothropstoxin-II. Bothropstoxin-I (25-100 mu g/paw) and bothropstoxin-II (12.5-50 mu g/paw) caused dose-dependent rat paw oedema. The intradermal injection of bothropstoxin-I (0.125-5 mu g/site) and bothropstoxin-II (0.125-5 mu g/site) into rat skin also resulted in dose-dependent oedema formation. These oedematogenic activities were largely reduced in animals pretreated with the histamine/5-hydroxytryptamine (5-HT) receptor antagonist cyproheptadine (2 mg/kg, i.p. 0.5 h before). Similarly, p-bromophenacyl bromide, a compound known to inhibit phospholipase A(2) activity, significantly inhibited rat paw and skin oedema induced by both phospholipase A(2) homologues. The polyanion heparin (5 IU/site) significantly reduced the rat skin oedema induced by either bothropstoxin-I or bothropstoxin-II as well as the paw oedema (50 IU/site) induced by the former. When assayed in the rat peritoneal mast cells in vitro, both bothropstoxin-I (10 and 100 mu g/ml) and bothropstoxin-II (3 and 10 mu g/ml) significantly caused [C-14]5-HT release. The [C-14]5-HT release caused by these phospholipase A(2) homologues were reduced by p-bromophenacyl bromide and heparin (50 IU/ml). Our results indicate that oedema formation induced by bothropstoxin-I and bothropstoxin-II is mostly dependent on in vivo mast cell degranulation. Since heparin greatly reduced the oedematogenic activity of these phospholipase A(2) homologues, it is likely that the cationic charge of these substances plays a major role in the mast cell activation. Our results also indicate that p-bromophenacyl bromide may not be a suitable pharmacological tool to investigate the correlation between enzymatic activity and the inflammatory effects of phospholipases A(2)3432-325726

Mast Cell Degranulation Induced By Two Phospholipase A2 Homologues: Dissociation Between Enzymatic And Biological Activities

Bothropstoxin-I and bothropstoxin-II are phospholipase A2 homologues isolated from Bothrops jararacussu snake venom. The former is devoid of phospholipase A2 activity whereas the latter has very low enzymatic activity. In this study, we have investigated the in vivo (rat paw and skin oedema) and in vitro (mast cell degranulation) inflammatory effects caused by bothropstoxin-I and bothropstoxin-II. Bothropstoxin-I (25-100 μg/paw) and bothropstoxin-II (12.5-50 μg/paw) caused dose-dependent rat paw oedema. The intradermal injection of bothropstoxin-I (0.125-5 μg/site) and bothropstoxin-II (0.125-5 μg/site) into rat skin also resulted in dose-dependent oedema formation. These oedematogenic activities were largely reduced in animals pretreated with the histamine/5-hydroxytryptamine (5-HT) receptor antagonist cyproheptadine (2 mg/kg, i.p. 0.5 h before). Similarly, p-bromophenacyl bromide, a compound known to inhibit phospholipase A2 activity, significantly inhibited rat paw and skin oedema induced by both phospholipase A2 homologues. The polyanion heparin (5 IU/site) significantly reduced the rat skin oedema induced by either bothropstoxin-I or bothropstoxin-II as well as the paw oedema (50 IU/site) induced by the former. When assayed in the rat peritoneal mast cells in vitro, both bothropstoxin-I (10 and 100 μg/ml) and bothropstoxin-II (3 and 10 μg/ml) significantly caused [14C]5-HT release. The [14C]5-HT release caused by these phospholipase A2 homologues were reduced by p-bromophenacyl bromide and heparin (50 IU/ml). Our results indicate that oedema formation induced by bothropstoxin-I and bothropstoxin-II is mostly dependent on in vivo mast cell degranulation. Since heparin greatly reduced the oedematogenic activity of these phospholipase A2 homologues, it is likely that the cationic charge of these substances plays a major role in the mast cell activation. Our results also indicate that p-bromophenacyl bromide may not be a suitable pharmacological tool to investigate the correlation between enzymatic activity and the inflammatory effects of phospholipases A2.3432-3257263Antunes, E., Mariano, M., Cirino, G., Levi, S., De Nucci, G., Pharmacological characterisation of polycation-induced rat hind-paw oedema (1990) Br. J. Pharmacol., 101, pp. 986-990Barker, R.L., Gundel, R.H., Gleich, G.J., Checkel, J.L., Loegering, D.A., Pease, L.R., Hamann, K.J., Acid polyamino acids inhibit human eosinophil granule major basic protein toxicity (1991) J. Clin. Invest., 88, pp. 798-805Blackwell, G.J., Flower, R.J., Inhibition of phospholipase (1983) Br. Med. 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