cell biochemistry and function

Cell Biochem Funct 2005; 23: 307–313.

Published online 18 January 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1027/cbf.1208

Differential susceptibility of C2C12 myoblasts and myotubesto group II phospholipase A2 myotoxins from crotalidsnake venoms

Yamileth Angulo1,2 and Bruno Lomonte1*

1Instituto Clodomiro Picado, Facultad de Microbiologıa, Universidad de Costa Rica, San Jose, Costa Rica2Departamento de Bioquımica, Escuela de Medicina, Universidad de Costa Rica, San Jose, Costa Rica

Group II phospholipase A2 (PLA2) myotoxins isolated from Viperidae/Crotalidae snake venoms induce a rapid cytolyticeffect upon diverse cell types in vitro. Previous studies suggested that this effect could be more pronounced on skeletalmuscle myotubes than on other cell types, including undifferentiated myoblasts. This study utilized the murine skeletal mus-cle C2C12 cell line to investigate whether differentiated myotubes are more susceptible than myoblasts, and if this charac-teristic is specific for the group II myotoxic PLA2s. The release of lactic dehydrogenase was quantified as a measure ofcytolysis, 3 h after cell exposure to different group II PLA2s purified from Bothrops asper, Atropoides nummifer, Cerrophi-dion godmani, and Bothriechis schlegelii venoms. In addition, susceptibility to lysis induced by synthetic melittin and groupIII PLA2 from bee (Apis mellifera) venom, as well as by anionic, cationic, and neutral detergents, was comparatively eval-uated on the two cultures. Myotubes were significantly more susceptible to group II PLA2 myotoxins, but not to the otheragents tested, under the same conditions. Moreover, the increased susceptibility of myotubes over myoblasts was alsodemonstrated with two cytolytic synthetic peptides, derived from the C-terminal region of Lys49 PLA2 myotoxins, thatreproduce the action of their parent proteins. These results indicate that fusion and differentiation of myoblasts into myo-tubes induce changes that render these cells more susceptible to the toxic mechanism of group II PLA2 myotoxins, but not togeneral perturbations of membrane homeostasis. Such changes are likely to involve myotoxin acceptor site(s), whichremain(s) to be identified. Copyright # 2005 John Wiley & Sons, Ltd.

key words—myoblast; myotube; skeletal muscle; phospholipase A2; myotoxin; snake venom

INTRODUCTION

Phospholipases A2 (PLA2; EC 3.1.1.4) are major com-ponents of snake venoms that have acquired a varietyof toxic activities during evolution, including myo-toxic, neurotoxic, cytotoxic, anticoagulant, andinflammatogenic effects.1,2 Two structural groups ofPLA2s have been distinguished in snake venoms:those from species of the Elapidae family belong togroup I, which also includes the pancreatic PLA2sof mammals, whereas those from Viperidae/Crotali-

dae are classified as group II, together with mamma-lian secreted inflammatory PLA2s.

3,4

Skeletal muscle necrosis is a drastic and frequentconsequence of snakebites.5 Myotoxic PLA2s havebeen shown to play a main role in the pathogenesisof this effect, inducing a common pattern of degenera-tive effects leading to cell death.6–9 Current evidenceindicates that PLA2 myotoxins act primarily on thesarcolemma, rapidly altering its permeability by eithercatalytically-dependent or -independent mechan-isms.10–12 However, the nature of the membraneacceptor site(s) involved in these mechanisms, andthe detailed molecular events that follow toxin bind-ing, are still unknown.

Studies using a variety of cell lines in culture haveshown that myotoxic group I PLA2s are not cytolytic,

Received 17 August 2004Revised 4 October 2004

Copyright # 2005 John Wiley & Sons, Ltd. Accepted 14 October 2004

*Correspondence to: Dr B. Lomonte, Instituto Clodomiro Picado,Facultad de Microbiologıa, Universidad de Costa Rica, San Jose,Costa Rica. Fax: (þ506) 292-0485.E-mail: blomonte@cariari.ucr.ac.cr

whereas group II PLA2s exert a broad cytolytic activ-ity,7,13–18 which correlates with their in vivo myotoxicaction.19 Several of the group II myotoxins studied incell culture models belong to the subgroup of Lys49PLA2 homologues, protein variants that are enzymi-cally inactive due to a number of amino acid substitu-tions, including the critical Asp49 to Lys49 change(reviewed by Lomonte et al.12). Among the group IIPLA2s evaluated, no major differences were observedin the cytolytic activity induced by either Asp49 orLys49 variants, indicating that this effect can developin the absence of an intrinsic enzymatic activity of themyotoxins.19 Further work using synthetic peptideshas shown that the cytotoxic activity of Lys49 PLA2

myotoxins depends on a cationic/hydrophobic effec-tor site located at their C-terminal region, comprisingresidues 115–129.20–22

Previous studies showed that in vitro differentiatedmyotubes are highly susceptible targets for the cytoly-tic activity of some group II PLA2 myotox-ins.14,17,19,23 This observation, added to the knownvariations between different cell types to the cytolyticaction of myotoxic PLA2s, posed the question: are thedifferentiated myotubes specifically more sensitivethan myoblasts to these myotoxins, or are the myo-tubes intrinsically more susceptible to any type ofmembrane perturbation? The present study comparedthe in vitro susceptibility of muscle cells in these twodifferentiation stages, when exposed to different cyto-lytic agents, including a panel of group II myotoxicPLA2s, two synthetic peptides derived from these pro-teins, detergents, as well as melittin and the group IIIPLA2 from bee (Apis mellifera) venom.

MATERIALS AND METHODS

Myotoxic/cytolytic agents

The crude venoms came from pools obtained fromsnakes collected in Costa Rica and kept at the serpen-tarium of the Instituto Clodomiro Picado. MyotoxicPLA2s were purified by cation-exchange chromato-graphy on carboxymethyl-Sephadex C-25 (Pharma-cia, Sweden) as previously described: B. aspermyotoxins I,24 II,25 III26 and IV;27 C. godmani myo-toxin II;28 A. nummifer myotoxins I29 and II;30 andB.schlegelii myotoxin I.31 Toxin homogeneity wasassessed by urea-polyacrylamide gel electrophoresisfor basic proteins,32 and by reverse-phase high perfor-mance liquid chromatography (RP-HPLC) on a C4column (25� 4.6mm; Vydac), eluted at 1.0mlmin�1

with a gradient from 0 to 60% acetonitrile in 0.1%trifluoroacetic acid (v/v), using an Agilent model1100 HPLC system.

Two synthetic peptides (Ba-p115–129, KKYR-YYLKPFCKK, and Acl-p115–129, KKY-KAYFKFKCKK) derived from B. asper myotoxin IIand Agkistrodon contortrix laticinctus myotoxin,respectively,20,22 were obtained from SynPep, Inc.(Dublin, CA, USA). They were synthesized by Fmocchemistry, with native endings. Their final purity wasat least 95% by RP-HPLC analysis, and their observedmass spectrometry values corresponded to theexpected formula values.

Neutral (Triton X-100), anionic (sodium dodecylsulphate), or cationic (cetyl-trimethyl ammonium bro-mide) detergents, as well as purified group III PLA2

and synthetic melittin from bee (Apis mellifera)venom, were obtained from Sigma-Aldrich (St. Louis,MO, USA).

Cell cultures

The cell line utilized as the target in this study was themurine skeletal muscle C2C12, obtained from theAmerican Type Culture Collection (CRL-1772,ATCC). Cytolysis was assessed either on undifferen-tiated myoblasts, or on the differentiated myotubesoriginating after their fusion. Myoblasts were grownin 25 cm2 bottles using Dulbecco’s Modified Eagle’sMedium (DMEM; Sigma D-5796), supplementedwith 15% fetal calf serum (FCS; Sigma F-2442),2mM glutamine, 1mM pyruvic acid, penicillin(100Uml�1), streptomycin (0.1mgml�1), andamphotericin B (0.25 mgml�1), in a humified atmo-sphere with 7% CO2, at 37

�C. Cells were harvestedfrom subconfluent monolayers after their detachmentby exposure to trypsin (1500Uml�1) containing5.3mM EDTA, for 5min at 37�C. The resuspendedcells were seeded in 96-well microplates, at anapproximate initial density of 1–4� 104 cells perwell, in the same growth medium. After reachingabout 75% confluence, undifferentiated myoblastswere utilized directly in the cytotoxicity assaydescribed below. In order to differentiate the cells tomyotubes, after reaching near confluence in the 96-well plates, the growth medium was aspirated andreplaced by medium containing 1% FCS.33 After 3to 5 additional days of culture, when a large propor-tion of long multinucleated myotubes was observedamong the myoblasts, cells were utilized in the cyto-toxicity assay, as described below.

Cytotoxicity assay

Cytolysis was determined as previouslydescribed.16,19 In brief, myoblasts or myotubes were

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Copyright # 2005 John Wiley & Sons, Ltd. Cell Biochem Funct 2005; 23: 307–313.

exposed to variable amounts of the differentmyotoxic/cytolytic agents (purified group II myotoxicPLA2s, synthetic peptides, detergents, group III beevenom PLA2, and synthetic melittin), in order to gen-erate dose–response curves. All agents were diluted inDMEM containing 1% FCS, and added to the cells(myoblasts or myotubes) after aspirating their med-ium, in a total volume of 150 ml per well. After 3 hof incubation at 37�C, supernatant aliquots wereobtained for quantification of lactate dehydrogenase(LDH; EC 1.1.1.27) released into the medium, usinga colorimetric end-point procedure (Sigma N�500).Reference controls for 0 and 100% cytolysis consistedof medium alone or medium containing 0.1% (v/v)Triton X-100, respectively. All assays were carriedout in triplicate.

RESULTS

Under the culture conditions described, a large pro-portion of C2C12 myoblasts fused into myotubeswithin a few days of lowering the FCS concentrationin the medium to 1%. The morphology of cells uti-lized as targets in this study, myoblasts and myotubes,is shown in Figure 1. Dose–response curves showedthat the cytolytic effect of the different group II myo-toxic PLA2s tested, in all cases, was significantlyhigher on differentiated myotubes than on myoblasts,as summarized in Figure 2. Differences in susceptibil-ity to lysis were more evident at submaximal doses ofmyotoxin, i.e. 5–10 mg per well, corresponding toapproximate protein concentrations of 2–4 mM(Figure 2).

In contrast to the group II PLA2 myotoxins fromsnakes, both the bee venom group III PLA2 and melit-tin had similar cytolytic activities on myoblasts andmyotubes, resulting in nearly superimposable dose–response curves (Figure 3). In similarity with thesetwo myotoxic proteins from bee venom, all three typesof detergents tested (neutral, anionic, or cationic)caused the same lytic effect on both types of cell cul-tures (Figure 4). On the other hand, the two syntheticpeptides corresponding to the C-terminal regionsLys49 myotoxic PLA2s from B. asper and A. c. lati-cinctus, respectively, were significantly more toxicto myotubes than to myoblasts (Figure 5), thusmimicking the action of the whole proteins.

DISCUSSION

The use of skeletal muscle myoblasts/myotubes as tar-gets for group II PLA2s has been proposed as a usefulin vitro model to study their myotoxic mechanism(s),

Figure 1. Light micrographs showing the cell morphology ofundifferentiated C2C12 myoblasts (A), and differentiated myotubes(B) utilized as targets in the experiments. An example of thecytolytic effect induced on myoblasts after exposure to theC-terminal synthetic peptide (sequence 115–129) of Agkistrodoncontortrix laticinctus myotoxin (100mg per well) is shown in C

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as it correlates with the muscle-damaging activityobserved in vivo.14,17,19 This study investigatedwhether the fusion and differentiation of C2C12 myo-blasts, renders them more susceptible to the cytolyticaction of group II myotoxic PLA2s.An increased susceptibility of the myotubes was

clearly observed using a panel of eight different snakemyotoxins, including both Asp49 PLA2 and Lys49PLA2 variants. Interestingly, other types of proteinsfor which myotoxic action has been documented, suchas the bee venom (group III) PLA2, and the cationicpeptide melittin,34 affected both types of cells in cul-

ture to the same extent. The use of various detergentsalso demonstrated a similar cytolytic effect in bothmyoblasts and myotubes. Thus, the increased suscept-ibility of myotubes to the group II PLA2s cannot beattributed to a possibly lower ability of these cells tocope with general perturbations of membrane home-ostasis. The differentiation of myoblasts into myo-tubes must lead to changes that specifically favourthe action of group II PLA2 myotoxins. It is likely thatthese changes involve the expression of either a highernumber, or a higher affinity type, of acceptor sites forthe myotoxic PLA2s on the plasma membrane. How-ever, the possible participation of intracellular pro-cesses arising after myoblast fusion, as seen in theenhanced damage to myotubes, cannot be excludedat this time.

Figure 2. Comparison of the cytolytic activity of different group IIphospholipase A2 myotoxins on C2C12 myoblasts (*) or myotubes(*). (A) Bothrops asper myotoxin I; (B) B. asper myotoxin II; (C)B. asper myotoxin III; (D) B. asper myotoxin IV; (E) Atropoidesnummifer myotoxin I; (F) A. nummifer myotoxin II; (G)Cerrophidion godmani myotoxin II; (H) Bothriechis schlegeliimyotoxin I. Cytolysis was estimated by the release of lacticdehydrogenase (LDH) into supernatants after 3 h. Maximalmyotoxin doses (20mg per well) correspond to approximate proteinconcentrations of 8mM. Each point represents mean� SD oftriplicate cultures

Figure 3. Comparison of the cytolytic activity of Apis melliferagroup III phospholipase A2 (A) and synthetic melittin (B) on C2C12myoblasts (*) or myotubes (*). Cytolysis was estimated by therelease of lactic dehydrogenase (LDH) into supernatants after 3 h.Each point represents mean� SD of triplicate cultures

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The selectivity of the myotoxic effect of venomPLA2s in vivo has been ascribed to the existence ofspecific binding sites on the plasma membrane of ske-letal muscle cells, but their nature is completelyunknown. A number of high affinity protein acceptorsfor presynaptically active neurotoxic PLA2s havebeen identified in neuronal tissue.35,36 On the otherhand, a multidomain membrane protein of 180 kDa,known as the M-type receptor, has been characterizedas a binding site for some group I PLA2s in skeletal

muscle.37 However, no evidence for the involvementof M-type receptors in the myotoxic effect of groupII PLA2s has been reported.

Alternatively, group II PLA2 myotoxins could beacting through the recognition of membrane lipids,such as glycerophospholipids or glycolipids, thatmight be differentially expressed, or differentiallyclustered, in myoblasts and myotubes. Several obser-vations suggest that negatively-charged phospholi-pids38 may be important acceptors for the myotoxicmechanism of these proteins, as recentlyreviewed.11,12 In particular, the cytolytic activitydemonstrated for an all-D amino acid form of a syn-thetic peptide derived from Agkistrodon piscivoruspiscivorus Lys49 PLA2, strongly suggests that themechanism of action of these myotoxins does notinvolve recognition of a proteinaceous acceptor siteon muscle cells.21

In the subgroup of Lys49 PLA2 myotoxins, theC-terminal region has been identified as central formyotoxic/cytolytic activity.20–22,39 Synthetic peptidescorresponding to the sequence 115–129 of some ofthese proteins can reproduce their membrane-dama-ging actions and, therefore, it was also of interest tocompare the susceptibility of myoblasts and myotubesto such peptides. The results demonstrated a highercytolytic effect of synthetic peptides from Lys49PLA2s on the myotubes than on the myoblasts. Thus,

Figure 4. Comparison of the cytolytic activity of differentdetergent types on C2C12 myoblasts (*) or myotubes (*). (A)Triton X-100, neutral; (B) sodium dodecylsulfate, anionic; and (C)cetyl-trimethyl ammonium bromide, cationic. Cytolysis wasestimated by the release of lactic dehydrogenase (LDH) intosupernatants after 3 h. Each point represents mean�SD of triplicatecultures

Figure 5. Comparison of the cytolytic activity of two syntheticpeptides corresponding to the C-terminal regions (sequence115–129) of Bothrops asper myotoxin II (KKYRYYLKPFCKK)and Agkistrodon contortrix laticinctus myotoxin (KKYKAY-FKFKCKK), respectively, on C2C12 myoblasts (&) or myotubes(&). Cytolysis was estimated by the release of lactic dehydrogenase(LDH) into supernatants after 3 h. Peptides were assayed at 100mgper well, corresponding to approximate protein concentrations of360mM. Bars represent mean�SD of three determinations

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these C-terminal peptides behaved in a qualitativelysimilar fashion to their parent proteins, in agreementwith their proposed effector role in the membranedamaging mechanism of Lys49 PLA2 myotoxins,12

and further supporting the notion of a specificincreased susceptibility of myotubes to group IIPLA2 myotoxins.

ACKNOWLEDGEMENTS

This work was supported by the International Founda-tion for Science (F/2766-2), University of Costa Rica(VI-741-99269 and VI-741-A35013), CONICIT-FORINVES (FV058-02), and NeTropica Sweden-Central America network.We thank Dr J. M. Gutierrez for critically reading

this manuscript.This study was performed in partial fulfilment of

the doctoral degree of Y. Angulo at the University ofCosta Rica.

REFERENCES

1. Ogawa T, Kitajima M, Nakashima K, Sakaki Y, Ohno M.Molecular evolution of group II phospholipases A2. J Mol Evol1995; 41: 867–877.

2. Kini RM. Phospholipase A2—a complex multifunctional pro-tein puzzle. In Venom Phospholipase A2 Enzymes: Structure,Function and Mechanism, Kini RM (ed.). John Wiley & Sons:England, 1997; 1–28.

3. Kudo I, Murakami M. Phospholipase A2 enzymes. ProstaglLipid Med 2002; 68–69: 3–58.

4. Arni RK, Ward RJ. Phospholipase A2—a structural review.Toxicon 1996; 34: 827–841.

5. Warrell DA. Clinical features of envenoming from snake bites.In Envenomings and their Treatment, Bon C, Goyffon M (eds).Editions Fondation Marcel Merieux: Lyon, 1996; 63–76.

6. Mebs D, Ownby CL. Myotoxic components of snake venoms:their biochemical and biological activities. Pharmac Ther 1990;48: 223–236.

7. Harris JB. Phospholipases in snake venoms and their effects onnerve and muscle. In Snake Toxins, Harvey AL (ed.). PergamonPress: New York, 1991; 91–129.

8. Gutierrez JM, Lomonte B. Phospholipase A2 myotoxins fromBothrops snake venoms. Toxicon 1995; 33: 1405–1424.

9. Gutierrez JM, Lomonte B. Phospholipase A2 myotoxins fromBothrops snake venoms. In Venom Phospholipase A2 Enzymes:Structure, Function, and Mechanism, Kini RM (ed.). JohnWiley and Sons: England, 1997; 321–352.

10. Dixon RW, Harris JB. Myotoxic activity of the toxic phospho-lipase, notexin, from the venom of the Australian tiger snake.J Neuropathol Exp Neurol 1996; 55: 1230–1237.

11. Gutierrez JM, Ownby CL. Skeletal muscle degenerationinduced by venom phospholipases A2: insights into the mechan-isms of local and systemic myotoxicity. Toxicon 2003; 42: 915–931.

12. Lomonte B, Angulo Y, Calderon L. An overview of Lysine-49phospholipase A2 myotoxins from crotalid snake venoms and

their structural determinants of myotoxic action. Toxicon 2003;42: 885–901.

13. Krizaj I, Bieber AL, Ritonja A, Gubensek F. The primarystructure of ammodytin L, a myotoxic phospholipase A2 homo-logue from Vipera ammodytes venom. Eur J Biochem 1991;202: 1165–1168.

14. Bruses JL, Capaso J, Katz E, Pilar G. Specific in vitro biologicalactivity of snake venom myotoxins. J Neurochem 1993; 60:1030–1042.

15. Bultron E, Thelestam M, Gutierrez JM. Effects on culturedmammalian cells of myotoxin III, a phospholipase A2 isolatedfrom Bothrops asper (terciopelo) venom. Biochim Biophys Acta1993; 1179: 253–259.

16. Lomonte B, Tarkowski A, Hanson LA. Broad cytolytic speci-ficity of myotoxin II, a lysine-49 phospholipase A2 of Bothropsasper snake venom. Toxicon 1994; 32: 1359–1369.

17. Incerpi S, de Vito P, Luly P, Rufini S. Effect of ammodytin Lfrom Vipera ammodytes on L-6 cells from rat skeletal muscleBiochim Biophys Acta 1995; 1268: 137–142.

18. Andriao-Escarso SH, Soares AM, Rodrigues VM, et al. Myo-toxic phospholipases A2 in Bothrops snake venoms: effect ofchemical modifications on the enzymatic and pharmacologicalproperties of bothropstoxins from Bothrops jararacussu.Biochimie 2000; 82: 755–763.

19. Lomonte B, Angulo Y, Rufini S, et al. Comparative study of thecytolytic activity of myotoxic phospholipases A2 on mouseendothelial (tEnd) and skeletal muscle (C2C12) cells in vitro.Toxicon 1999; 37: 145–158.

20. Lomonte B, Moreno E, Tarkowski A, Hanson LA, MaccaranaM. Neutralizing interaction between heparins andmyotoxin II, aLysine 49 phospholipase A2 from Bothrops asper snake venom.Identification of a heparin-binding and cytolytic toxin region bythe use of synthetic peptides and molecular modeling. J BiolChem 1994; 269: 29867–29873.

21. Lomonte B, Angulo Y, Santamarıa C. Comparative study ofsynthetic peptides corresponding to region 115–129 in Lys49myotoxic phospholipases A2 from snake venoms. Toxicon 2003;42: 307–312.

22. Nunez CE, Angulo Y, Lomonte B. Identification of the myo-toxic site of the Lys49 phospholipase A2 from Agkistrodonpiscivorus piscivorus snake venom: synthetic C-terminal pep-tides from Lys49, but not from Asp49 myotoxins, exert mem-brane-damaging activities. Toxicon 2001; 39: 1587–1594.

23. Bieber AL, Ziolkowski C, d’Avis PA. Rattlesnake toxins alterdevelopment of muscle cells in culture. Ann NY Acad Sci 1994;710: 126–145.

24. Gutierrez JM, Ownby CL, Odell GV. Isolation of a myotoxinfrom Bothrops asper venom: partial characterization and actionon skeletal muscle. Toxicon 1984; 22: 115–128.

25. Lomonte B, Gutierrez JM. A new muscle damaging toxin,myotoxin II, from the venom of the snake Bothrops asper(terciopelo). Toxicon 1989; 27: 725–733.

26. Kaiser II, Gutierrez JM, Plummer D, Aird D, Odell GV. Theamino acid sequence of a myotoxic phospholipase from thevenom of Bothrops asper. Arch Biochem Biophys 1990; 278:319–325.

27. Dıaz C, Lomonte B, Zamudio F, Gutierrez JM. Purification andcharacterization of myotoxin IV, a phospholipase A2 variant,from Bothrops asper snake venom. Natural Toxins 1995; 3:26–31.

28. Dıaz C, Gutierrez JM, Lomonte B. Isolation and characteriza-tion of basic myotoxin phospholipase A2 from Bothropsgodmani (Godman’s pit viper) snake venom. Arch BiochemBiophys 1992; 298: 135–142.

312 y. angulo and b. lomonte

Copyright # 2005 John Wiley & Sons, Ltd. Cell Biochem Funct 2005; 23: 307–313.

29. Gutierrez JM, Lomonte B, Cerdas L. Isolation and partialcharacterization of a myotoxin from the venom of the snakeBothrops nummifer. Toxicon 1986; 24: 885–894.

30. Angulo Y, Olamendi-Portugal T, Possani LD, Lomonte B.Isolation and characterization of myotoxin II from Atropoides(Bothrops) nummifer snake venom, a new Lys49 phospholipaseA2 homologue. Int J Biochem Cell Biol 2000; 32: 63–71.

31. Angulo Y, Chaves E, Alape A, Rucavado A, Gutierrez JM,Lomonte B. Isolation and characterization of a myotoxic phos-philpase A2 from the venom of the arboreal snake Bothriechis(Bothrops) schlegelii from Costa Rica. Arch Biochem Biophys1997; 339: 260–267.

32. Traub P,Mizushima S, Lowry CV, NomuraM. Reconstitution ofribosomes from subribosomal components. Methods Enzymol1971; 20: 391–417.

33. Ebisui C, Tsujinaka T, Morimoto T, et al. Interleukin-6 inducesproteolysis by activating intracellular proteases (cathepsins Band L, proteasome) in C2C12 myotubes. Clin Sci 1995; 89:431–439.

34. Ownby CL, Powell JR, Jiang MS, Fletcher JE. Melittin andphospholipase A2 from bee (Apis mellifera) venom cause

necrosis of murine skeletal muscle in vivo. Toxicon 1997; 35:67–80.

35. Lambeau G, Barhanin J, Schweitz H, Qar J, Lazdunski M.Identification and properties of very high affinity brain mem-brane-binding sites for a neurotoxic phospholipase from thetaipan venom. J Biol Chem 1989; 264: 11503–11510.

36. Krizaj I, Gubensek F. Neuronal receptors for phospholipases A2

and �-neurotoxicity. Biochimie 2000; 82: 807–814.37. Lambeau G, Schmid-Alliana A, Lazdunski M, Barhanin J.

Identification and purification of a very high affinity bindingprotein for toxic phospholipases A2 in skeletal muscle. J BiolChem 1990; 265: 9526–9532.

38. Dıaz C, Leon G, Rucavado A, Rojas N, Schroit AJ, GutierrezJM. Modulation of the susceptibility of human erythrocytes tosnake venom myotoxic phospholipases A2; role negativelycharged phospholipids as potential membrane binding sites.Arch Biochem Biophys 2001; 391: 56–64.

39. Chioato L, Ward RJ. Mapping structural determinants of bio-logical activities in snake venom phospholipases A2 bysequence analysis and site directed mutagenesis. Toxicon2003; 42: 869–883.

differential susceptibility of myoblasts and myotubes 313

Copyright # 2005 John Wiley & Sons, Ltd. Cell Biochem Funct 2005; 23: 307–313.