Lysine 49 phospholipase A2 proteins

PERGAMON

Toxicon 37 (1999) 411±445

Review

Lysine 49 phospholipase A2 proteins Charlotte L. Ownby a, *, Heloisa S. Selistre de Araujo b, Steven P. White c, Je€rey E. Fletcher d a

Department of Anatomy, Pathology and Pharmacology, Oklahoma State University, Stillwater, OK 74078-0350, USA b Departamento de CieÂncias FisioloÂgicas, Universidade Federal de SaÄo Carlos, Sao Carlos, Brazil c Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, OK 74078-0350, USA d Department of Anesthesiology, Allegheny University of the Health Sciences, Philadelphia, PA, USA

Received 26 February 1998; accepted 30 June 1998

Abstract The structures of several K49 PLA2 proteins have been determined and these di€er as a group in several regions from the closely related D49 PLA2 enzymes. One outstanding di€erence is the presence of a high number of positively charged residues in the Cterminal region which combined with the overall high number of conserved lysine residues gives the molecule an interfacial adsorption surface which is highly positively charged compared to the opposite surface of the molecule. Although some nucleotide sequences have been reported, progress in obtaining active recombinant proteins has been slow. The K49 proteins exert several toxic activities, including myotoxicity, anticoagulation and edema formation. The most studied of these activities is myotoxicity. The myotoxicity induced by the K49 PLA2 proteins is histologically similar to that caused by the D49 PLA2 myotoxins, with some muscle ®ber types possibly more sensitive than others. Whereas it is clear that the K49 PLA2 myotoxins lyse the plasma membrane of the a€ected muscle cell in vivo, the exact mechanism of this lysis is not known. Also, it is not known whether the toxin is internalized before, during or after the initial lysis or ever. The K49 PLA2 toxins lyse liposomes and cells in culture and in

Abbreviations: ACL, Agkistrodon contortrix laticinctus, ACLM, Agkistrodon contortrix laticinctus myotoxin, APP, Agkistrodon piscivorus piscivorus, BthTX-1, Bothrops jararacussu myotoxin I, CK, creatine kinase, D49, aspartic acid at position 49, K49, lysine at position 49, S49, serine at position 49, FFA, free fatty acid, LDH, lactate dehydrogenase, MePDN, methylprednisolone, P-BPB, p-bromophenacyl bromide, PLA2, phospholipase A2, RACLMT, recombinant ACL myotoxin, TG, triacylglycerol, UTR, untranslated regions. * Corresponding author. Fax: +1-405-744-5275; E-mail: [email protected]. 0041-0101/99/$ - see front matter # 1999 Published by Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 1 - 0 1 0 1 ( 9 8 ) 0 0 1 8 8 - 3

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the latter, the PLA2 myotoxins exert at least two distinct mechanisms of action, neither of which is well-characterized. While the K49 PLA2 proteins are enzymatically inactive on arti®cial substrates, the toxins cause fatty acid production in cell cultures. Whether the fatty acid release is due to the enzymatic activity of the K49 PLA2 or stimulation of tissue lipases, is unknown. While there may be a role for fatty acid production in one mechanism of myotoxicity, a second mechanism appears to be independent of enzymatic activity. Although we are beginning to understand more about the structure of these toxins, we still know little about the precise mechanism by which they interact with the skeletal muscle cell in vivo. # 1999 Published by Elsevier Science Ltd. All rights reserved.

1. Introduction Many important tools for the study and understanding of the complexity of biological processes have been isolated from snake venoms, such as the bradykinin potentiating peptides (Aiken and Vane, 1970) and the inhibitors of platelet aggregation named disintegrins (Niewiarowski et al., 1994). Among them, the phospholipases A2 (PLA2) comprise one of the most intensely studied class of enzymes regarding structure and biological activity. Over ®fty di€erent PLA2 toxins have been isolated from snake venoms and characterized, and some have been cloned and expressed. The three dimensional structure of a few has been solved by both X-ray crystallography and nuclear magnetic resonance (Holland et al., 1990; Van den Bergh et al., 1995; Wang et al., 1996a). Secretory PLA2s can be divided into several types based on their primary sequences and disul®de bond arrangement. There are some very recent reviews on classi®cation and the di€erent types of PLA2s (Dennis, 1997; Kini, 1997), which is beyond the scope of this review. Here, we will address only the Type II secretory PLA2 enzymes which have a lysine residue at position 49 (Maraganore and Heinrikson, 1986). Type II PLA2 enzymes include those from viperid snake venoms and the mammalian secretory type II PLA2 which is found in in¯ammatory exudates. From sequence analysis of the primary structure of type II PLA2s, it was demonstrated that this group can also be subdivided into, at least, two subclasses: (a) the D49 enzymes which have an aspartic acid residue at position 49 and high catalytic activity on arti®cial phospholipid substrates and (b) the K49 enzymes, which have a lysine residue at position 49 and very low, or no hydrolytic activity on arti®cial substrates. The presence of an aspartic acid at position 49 is crucial for calcium binding and Ca2+ is essential for catalytic activity (Scott et al., 1992). Despite the lack of an aspartic acid residue at position 49, the K49 PLA2 proteins are very active in the induction of myonecrosis, by one or more still unknown mechanisms (Homsi-Brandeburgo et al., 1988; Johnson and Ownby, 1993). The K49 PLA2 myotoxins were ®rst described by Homsi-Brandeburgo and coauthors, with the isolation of Bothropstoxin I from the South American snake

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Fig. 1. Sequence comparison of K49 PLA2 proteins. Amino acid sequences of K49 PLA2 proteins: ACLMT, ACL myotoxin from A.c. laticinctus (Selistre de Araujo et al., 1996a); AppK49, from Agkistrodon piscivorus piscivorus (Maraganore and Heinrikson, 1986); Basper II, myotoxin II from Bothrops asper (Francis et al., 1991); BthTx-I, bothropstoxin I from Bothrops jararacussu (Cintra et al., 1993); TMK49, from Trimeresurus mucrosquamatus (Liu et al., 1991); TFBPI and II, basic protein I and II from Trimeresurus ¯avoviridis (Liu et al., 1990; Yoshizumi et al., 1990); TGR K49, from T. gramineus (Nakashima et al., 1995); D. acutus, K49 PLA2 from D. acutus (Wang et al., 1996b). Amino acid residues are numbered according to Renetseder et al. (1985). Asterisks indicate residues conserved in K49 PLA2 family and not conserved in the D49 PLA2 family. Deletions are indicated by dashes. Dots represent conserved residues in both D49 and K49 PLA2.

Bothrops jararacussu (Homsi-Brandeburgo et al., 1988) and by Lomonte and GutieÂrrez, with the puri®cation of myotoxin II from Bothrops asper (Lomonte and GutieÂrrez, 1989). Today there are about nine K49 PLA2 toxins described in the literature, and they all come from snakes from the Viperidae family (Fig. 1). These are the only known sources of the K49 PLA2 toxins in nature. Even in this family, they have not been reported in the Crotalus genus (Li et al., 1993). Evolutionary studies have suggested that K49 PLA2 proteins arose from gene duplication of the ancestral PLA2 gene (Davidson and Dennis, 1990; Moura da Silva et al., 1995), in

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an accelerated form of evolution. It is intriguing that evolution preserved a mutation at a position crucial for catalytic activity in the D49 enzyme structure. Two S49 PLA2 proteins have been reported (Krizaj et al., 1991; Polgar et al., 1996), thus suggesting the existence of another protein family of snake venom PLA2 proteins. It has been suggested that the presence of a serine residue at position 49 does not sterically block the binding of calcium ions, and the serine hydroxyl group could replace the Asp carboxylate in stabilization of Ca2+ binding (Polgar et al., 1996). One S49 PLA2 protein from Echis carinatus sochureki venom has signi®cant PLA2 activity (Polgar et al., 1996). The report of new forms of PLA2 proteins shows clearly that their group of subfamilies is still growing. In this review we will focus on the isolation of lysine 49 phospholipase A2 proteins, the biochemical characterization of their primary and tertiary structures, and their biological activities as studied in in vivo and in vitro systems.

2. K49 PLA2 puri®cation from crude venoms K49 PLA2 proteins can be easily puri®ed in large amounts from crude venoms due to their unusually high isoelectric point (r9.0). Their puri®cation can be conducted at room temperature with no loss of biological activity. Most procedures start with gel ®ltration on a Sephadex G-75 column or higher, followed by cation exchange chromatography. Bothropstoxin I has been puri®ed in a two-step protocol, which includes a Sephadex G-75 gel ®ltration of crude venom, followed by cation exchange chromatography on SP-Sephadex C-25 (Homsi-Brandeburgo et al., 1988). ACL myotoxin was puri®ed from Agkistrodon contortrix laticinctus venom by means of anion exchange HPLC followed by cation exchange HPLC (Johnson and Ownby, 1993). The puri®cation procedure must be followed by tests of myotoxic activity, since the K49 PLA2 proteins have no detectable enzymatic activity on the arti®cial substrates commonly used to monitor PLA2 activity in isolated fractions. The methods used to test myotoxic activity will be described in detail below. K49 PLA2s are reported to be non-glycosylated proteins of 121 amino acid residues, with a molecular weight of 14 kDa, usually estimated by SDS-PAGE in reduced conditions. K49 PLA2 proteins from Bothrops venoms are reported to be homodimers (Francis et al., 1991), while ACL myotoxin from A.c. laticinctus venom (Johnson and Ownby, 1993) and basic protein I from T. ¯avoviridis venom (Yoshizumi et al., 1990) were described as a single polypeptide chain. It is interesting that the crystal structures of bothropstoxin I and myotoxin II have shown the same pattern of dimerization (Arni et al., 1995a,b), while ACL myotoxin crystallizes in a monomeric form (Treharne et al., 1997), as does the K49 PLA2 from A.p. piscivorus (Scott et al., 1992). Based on heparin-binding properties, K49 PLA2s can also be puri®ed by anity chromatography on heparin-Sepharose columns (Farooqui et al., 1994). A one-

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step protocol for the puri®cation of K49 PLA2 proteins from crude venoms has also been reported, based on HPLC cation exchange chromatography (Toyama et al., 1995).

3. K49 PLA2 protein structure Amino acid sequences of K49 PLA2 proteins have been determined by both protein and DNA sequencing. Although more than 40 D49 PLA2 proteins have been sequenced, the sequences of only nine K49 PLA2 proteins have been reported (Fig. 1). Selistre de Araujo et al. (1996a) ®rst observed that K49 PLA2s comprise a highly conserved protein family very distinct from the D49 PLA2 protein family. Comparison of K49 PLA2 protein sequences reveals a high level of conservation within the K49 PLA2 protein family (70±95% identity). In contrast, identity within the D49 group can be as low as 47% for the D49 PLA2 from Crotalus adamanteus (Selistre de Araujo et al., 1996b). Although we do not present the amino acid sequences of D49 PLA2 proteins, an extensive comparison between the two groups has been reported elsewhere (Selistre de Araujo et al., 1996a,b). Therefore, we represent the conserved residues in both D49 and K49 protein families as dots in Fig. 1. As shown in Fig. 1, despite having homologous regions with the D49 PLA2 proteins, the K49 PLA2 protein family has some unique features, which strongly characterizes this class of PLA2 proteins. One striking characteristic of the K49 PLA2 group is the presence of a large number of basic residues, mostly lysines (about 15% of total residues). There is a lysine-rich region in the C-terminal domain, including two heparin-binding consensus sequences (Dua and Cho, 1994), which could explain why these toxins are neutralized by heparin (Melo et al., 1993; Lomonte et al., 1994c). ACL myotoxin and other members of the K49 protein family have some putative phosphorylation sites in the sequence. Residues 53±56 (KKLT) form a consensus sequence for phosphorylation by cAMP and cGMP-dependent protein kinase. This sequence is not present in the D49 family, due to the presence of a conserved G53 in those proteins (Fletcher et al., 1997), with the exception of the human secretory non-pancreatic PLA2. K49 PLA2 proteins also have a protein kinase C phosphorylation site (TGK, residues 13±15) which is present in some D49 PLA2 proteins. It is unknown if these proteins undergo phosphorylation after binding to target cells. Cytosolic PLA2 has been reported to be phosphorylated at S505 (Lin et al., 1992), and phosphorylation was reported to increase the enzymatic activity (Lin et al., 1993). An extensive discussion about the possible role of some speci®c residues in the myotoxic action of these proteins has recently been published (Fletcher et al., 1997) and it will not be repeated here. Brie¯y, there is evidence that the N-terminus and reverse turn (residues 1±17) are involved in myotoxicity (DõÂ az et al., 1994; Selistre de Araujo et al., 1996a,b). Residues K7, E12, T13 and K15 together with conserved lysines K78, K80, K115 and K116 from another part of the molecule have been proposed to form a myotoxic site for the K49 PLA2

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myotoxin from Agkistrodon contortrix laticinctus venom (Selistre de Araujo et al., 1996a,b). However, Arni et al. (1995a) proposed a role for residues E12 and K80 in the formation of dimers of myotoxin II, a K49 PLA2 from Bothrops asper venom. The consensus sequence Y25-G-C-Y/F-C-G-X-G-G33 in the calciumbinding loop (residues 25±33) of the D49 PLA2 enzymes is not conserved in the K49 PLA2 myotoxins. These changes in addition to the substitution of a lysine for the highly conserved aspartic acid residue at position 49 could explain the lack of Ca2+ binding reported for some K49 PLA2 myotoxins. Residues 74±84 make up the b-wing region of the molecule and Selistre de Araujo et al. (1996a,b) suggested that these residues may participate in the myotoxic action since lysines 78 and 80 are highly conserved in the K49 group and they are spatially closely related to the residues in the N-terminal region of the molecule which are also part of the proposed myotoxic site. It has also been suggested that basic residues in the region 54±77 could be involved in the anticoagulant activity of some PLA2 proteins (Kini, 1997). There is evidence that the C-terminus (residues 115±134) is involved in lytic action of Bothrops asper myotoxin II on cultured endothelial cells (Lomonte et al., 1994b), and Selistre de Araujo et al. (1996a,b) proposed K115 and K116 as part of the `myotoxic site' of ACL myotoxin. Also, Dua and Cho (1994) showed that K116 (and K10) in bovine pancreatic PLA2 were involved in the interfacial binding to anionic interfaces. K49 PLA2 proteins have similar three dimensional structure to the D49 enzymes. According to Han et al. (1997), the crystalline D49 PLA2 from Agkistrodon piscivorus piscivorus is approximately 22  30  42 A with 50% of the structure a-helix and 10% b-sheet. The characteristic b-turn is also found in the K49 PLA2 proteins. The two long anti-parallel a-helices that form the backbone of the molecule are ®rmly bonded by a series of disul®de bridges. The substrate binds to the enzyme by an external opening of a hydrophobic channel that leads to the active site. K49 PLA2 proteins have two residues involved in the catalytic center of D49 enzymes, i.e. H48 and D99. Molecular modeling of ACL myotoxin has shown that the highly conserved lysine residues together with other positively charged residues give the molecule a surface which is highly positively charged, which seems to be a feature common to the PLA2 myotoxins. Fig. 2 compares the surface charge distribution of a K49 PLA2 myotoxin (ACL myotoxin) with that of a D49 PLA2 from Agkistrodon piscivorus piscivorus venom which is also myotoxic (Ownby, unpublished results). The molecular modeling was done using Agkistrodon piscivorus piscivorus (APP) Fig. 2. Electrostatic surface potential map of phospholipase A2 myotoxins. (A and B) ACL myotoxin, a K49 PLA2 from Agkistrodon contortrix laticinctus venom modelled from the K49 PLA2 of Agkistrodon piscivorus piscivorus venom after changing V2L, L124F amd D131E. (C and D) D49 PLA2 myotoxin from Agkistrodon piscivorous piscivorous venom with Ca 2+ added. The N-terminus is labelled `N'. Views A and C show the interface binding surface whereas views B and D show the opposite surface obtained by rotating the molecule 1808 on the vertical axis as shown in the ®gure. The ®gure was generated using GRASP (Nicholls et al., 1993; Nicholls, 1993) and shown from ÿ1.4 (red) to +5.3 kT (blue).

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Fig. 2.

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K49 PLA2 coordinates submitted by Holland et al. (1990) to the protein data bank (entry 1 ppa) (Bernstein et al., 1977) after changing three residues in the APP structure to those present in the ACL molecule, i.e. V2L, L124F, D131E. Both molecules have a large number of cationic residues; ACL myotoxin has an isoelectric point of 9.6 (Ownby and Fletcher, unpublished results) whereas that of APP-D49 is 9.5 (Han et al., 1997). Fig. 2 shows that the distribution of positive charges is asymmetric on both molecules, with most of them occurring on the interfacial binding surface as de®ned by Han et al. (1997) (compare Fig. 2A and B; C and D). On this surface, there is extensive positive charge distribution which in ACL myotoxin (Fig. 2A) is especially concentrated at the upper part of the molecule (near the C-terminal region) whereas in the D49 molecule (Fig. 2C) the positive charged surface is more evenly distributed over the entire face. Fig. 2B also shows the concentration of positive charges near the C-terminal in the K49 PLA2 myotoxin (top of the molecule) which is lacking in the D49 molecule. Several D49 PLA2 enzymes were reported to be co-crystallized with transition state and substrate analogs (White et al., 1990), but the K49 PLA2 protein crystal structures reported so far (Scott et al., 1992; Arni et al., 1995a,b) were obtained in the absence of such substances. Even though they do bind the substrate, there is apparently no hydrolysis. Phospholipase A2 enzymes act at the lipid±water interface, with a preference for organized phospholipids in micelles and vesicles. It has been proposed that the interfacial adsorption is driven by electrostatic forces involving speci®c lysine residues. The arrangement of the critical positive charges would drive the orientation of the enzyme at the substrate interface (Han et al., 1997). The authors suggest that the variability in the topographic distribution of lysine residues corresponds to a ¯exibility in the orientation of the enzyme at the substrate interface. In the case of the K49 PLA2 group, which has at least 12 highly conserved lysine residues, it is suggested that charge interactions may facilitate interaction between these proteins and their target cells.

4. Molecular biology of the K49 PLA2 proteins Genomic and cDNA libraries of snake venom glands have been constructed for some species and several clones coding for PLA2 enzymes in general have been isolated and characterized. There are at least two very recent reviews on this subject (Danse et al., 1997; Gubensek and Kordis, 1997). Ogawa and co-authors ®rst observed the extremely high and unusual conservation of the introns in genomic DNA (Nakashima et al., 1993) and the 5 0 and 3 0 untranslated regions (UTRs) in the cDNAs (Ogawa et al., 1992) in comparison with the protein-coding region. Analysis of phylogenetic trees suggested that the mature protein coding region appears to have evolved in a more accelerated form than the 5 0 and 3 0 UTRs, probably due to the adaptive pressure to achieve new physiological and toxic activities (Ogawa et al., 1995). These activities would provide the animal with better tools for hunting and defense from predators. If so, K49 PLA2 proteins are one of the most interesting examples. All the reported K49 PLA2

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proteins are described to induce toxic e€ects such as membrane depolarization and blockade of the neuromuscular junction (Heluany et al., 1992; Kihara et al., 1992), myonecrosis (Homsi-Brandeburgo et al., 1988; Johnson and Ownby, 1993) and edema (Liu et al., 1991). The DNA sequences for some K49 PLA2 proteins have been reported, from both cDNA and genomic libraries (Nakashima et al., 1993; Ward et al., 1995; Selistre de Araujo et al., 1996a,b). To our knowledge, there is no report of a recombinant K49 PLA2 until now, although a few D49 PLA2 enzymes have been successfully expressed in bacteria (Kelley et al., 1992; Lathrop et al., 1992). Using the pET28a1 expression system, Selistre de Araujo has expressed recombinant ACL myotoxin (rACLMT) in BL21(DE3) E. coli strain as insoluble inclusion bodies (unpublished results). rACLMT was puri®ed from crude extract by means of Ni2+ chelating chromatography and partial cleavage with thrombin (Fig. 3). Refolding experiments are under way now to obtain the recombinant protein in an active form. Production of a recombinant K49 PLA2 protein, along with the production of mutants by site-directed mutagenesis would be very helpful in the understanding the complex relationship of structure and function of the K49 PLA2 molecules. Also, it should help to solve the question of K49 PLA2 catalytic activity.

5. Pathological, physiological and biochemical e€ects Since there are relatively few pharmacological studies of the K49 PLA2 myotoxins, occasional references will be made to the D49 PLA2 enzymes. Extensive reviews of D49 PLA2 myotoxins have appeared (Mebs and Ownby, 1990; Ownby, 1990; GutieÂrrez and Lomonte, 1995, 1997; Fletcher et al., 1997). In order to justify comparisons with D49 PLA2 myotoxins, it is essential to determine whether the K49 PLA2 molecules are enzymatically active, or whether they are devoid of phospholipid hydrolyzing activity. If they are devoid of enzymatic activity, do they activate tissue lipolytic enzymes that could then increase levels of free fatty acids? Currently, the role of PLA2 activity in the mechanisms of snake venom D49 PLA2 presynaptically-acting toxins and myotoxins is unresolved. The reported inactivity of the K49 PLA2 molecules may be an important contradictory ®nding, further negating a role for enzymatic activity in toxicity. However, more recent studies now suggest that, although inactive on arti®cial substrates, the K49 PLA2 molecules may be catalytically active on, or stimulate tissue lipolytic enzymes in, cell culture systems. This issue will be addressed in considerable detail in Sections 5.1, 5.2, 5.3 and 5.4. 5.1. Biological e€ects other than myotoxicity Most of the K49 PLA2 proteins have been isolated during the assay of venom fractions for myotoxic activity and thus are known to be myotoxic. However,

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Fig. 3. Expression of recombinant ACL myotoxin in E. coli. The coding region of the ACL myotoxin cDNA mature domain was subcloned into the pET28a1 expression vector and used to transform BL21(DE3) E. coli cells. Samples were analyzed on a 10% SDS-PAGE. Lane 1, molecular weight markers (Mr  103); Lanes 2 and 3, crude cell extracts of the transformed cells, before and after induction with 1 mM IPTG, respectively. Lanes 4 and 5, recombinant ACL myotoxin puri®ed on a Ni + 2-anity column, under denaturing conditions.

some of these proteins have been tested for other biological activities. Despite their destructive action at the sarcolemma, the D49 PLA2 myotoxins, in general, are not hemolytic agents, suggesting that there is a speci®city in membrane targeting, or in the interaction with membrane components. It is remarkable that extensive phospholipid hydrolysis does not always lead to a signi®cant increase in the permeability of a membrane, or cause cell lysis. For example, a very high concentration of a histidine-48 modi®ed basic PLA2 from Naja nigricollis venom causes 77% hydrolysis of phosphatidylcholine, 24% of phosphatidylethanolamine and 38% of phosphatidylserine and yet only induces 6% hemolysis of guinea pig red blood cells (Condrea et al., 1981). In this same study, the native form of two other PLA2 enzymes caused 65% hydrolysis or greater of phosphatidylcholine, the major phospholipid of red blood cells, while only inducing 0.6% hemolysis, or less. These levels of hydrolysis were determined in the same cells used in the hemolysis studies. In addition, we have observed no e€ects of the Naja naja atra PLA2 on cell viability in the NB41A3 cell line incubated for one hr (378C) at concentrations of enzyme of up to 5 mM, yet this enzyme causes 60±80% hydrolysis of the phospholipids in these same cells at a much lower (100 nM) concentration under the same conditions (J.E.F., unpublished data). Dhillon et al. (1987) showed that the K49 PLA2 isolated from Agkistrodon piscivorus piscivorus venom had low hemolytic activity, and more recently, Angulo et al. (1997) showed that a myotoxic phospholipase A2 from the venom of Bothriechis (Bothrops) schlegelii from Costa Rica is also indirectly hemolytic. However, Kihara et al. (1992) found that two K49 PLA2 myotoxins from Trimeresurus ¯avoviridis venom

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lack hemolytic activity. BthTX-I exhibits a Ca2+-dependent hemolysis, which is in contrast to the Ca2+-independent e€ects of BthTX-I on contractile activity of skeletal muscle (Rodrigues-Simioni et al., 1983). Since contamination of a K49 fraction with a D49 PLA2 from the same venom may contribute to hemolytic activity, it is important to address this possibility, as discussed in Section 11. Some K49 phospholipase A2 myotoxins have been tested for anticoagulant activity. The K49 PLA2 toxin isolated from Agkistrodon piscivorus piscivorus venom had low anticoagulant activity (Dhillon et al., 1987). Also, a myotoxic phospholipase A2 from the venom of Bothriechis (Bothrops) schlegelii from Costa Rica had low anticoagulant activity on sheep platelet-poor plasma and was devoid of procoagulant activity (Angulo et al., 1997). Lomonte and GutieÂrrez (1989) reported that myotoxin II from Bothrops asper venom did not have anticoagulant properties in an assay using platelet-poor plasma from sheep, even when tested at 70 mg/ml. Two K49 PLA2 myotoxins from Bothrops moojeni venom did not have any detectable anticoagulant e€ect using sheep platelet-poor plasma (Lomonte et al., 1990). In contrast, Dõ az et al. (1995) reported that myotoxin IV, a K49 PLA2 toxin from Bothrops asper venom has weak anticoagulant activity on sheep platelet-poor plasma at concentrations of 40 mg/ml or higher. Although very few studies have been performed to determine the exact nature and mechanism of edema caused by these proteins, most of them are described as inducing some type of edema upon i.m. injection. Swelling usually begins approximately 10±15 min after injection and remains for at least the ®rst 3±6 h after injection. Lomonte and GutieÂrrez (1989) showed that myotoxin II from Bothrops asper venom induced edema in the mouse footpad assay with a minimum edema-forming dose of about 38 mg per animal. Liu et al. (1991) reported that a K49 PLA2 toxin from Trimeresurus mucrosquamatus venom caused a dose-dependent edema in the rat hind-paw test. Also, Lomonte et al. (1994a) described leakage of plasma from small venules and capillaries at about 2 min which became extensive by 4±5 min after exposure to myotoxin II from Bothrops asper venom. Angulo et al. (1997) showed that a myotoxic phospholipase A2 from the venom of Bothriechis (Bothrops) schlegelii from Costa Rica induced signi®cant edema in mice which was of rapid onset, continued for at least 6 h, then gradually resolved. It was not prevented by pretreatment of the animals with diphenhydramine or indomethacin. None of the K49 PLA2 myotoxins isolated so far appear to be highly lethal whereas this is one of the main activities of some of the D49 PLA2 toxins. The K49 PLA2 toxin from Agkistrodon piscivorus piscivorus venom had higher intravenous lethality in mice compared to that of the D49 toxin from the same venom, i.e. 5 versus 25 mg/kg, respectively (Dhillon et al., 1987), and HomsiBrandeburgo et al. (1988) determined an i.v. LD50 in mice for bothropstoxin I of 4.8 mg/kg. Angulo et al. (1997) found an LD50 of 2.5 mg/kg upon intravenous injection into mice for a PLA2 myotoxin from the venom of Bothriechis (Bothrops) schlegelii from Costa Rica.

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5.2. E€ects on muscle cells - In vivo The best documented biological activity of the K49 PLA2 toxins is their ability to induce necrosis of skeletal muscle cells in vivo. The pathogenesis of myonecrosis induced by the K49 PLA2 myotoxins has been investigated for only a few proteins, i.e. bothropstoxin from Bothrops jararacussu venom (HomsiBrandeburgo et al., 1988), ACL myotoxin from Agkistrodon contortrix laticinctus venom (Johnson and Ownby, 1993), myotoxin II from Bothrops asper venom (Lomonte et al., 1994a) and myotoxin I from Bothrops schlegelii venom (Angulo et al., 1997). Yet, there is good agreement about the overall process of muscle cell degeneration and regeneration induced by the K49 PLA2 myotoxins and this process is very similar to that induced by the D49 PLA2 myotoxins (see Fletcher et al., 1997; Gopalakrishnakone et al., 1997). These proteins act very rapidly, within 5 min after intramuscular or subcutaneous injection, and the time of onset does not appear to depend on dose of toxin used. In fact, these proteins cause similar myonecrosis at low (<10 mg) or high (>50 mg) doses (Lomonte et al., 1994a). In an e€ort to determine the initial site and type of action of one of these toxins, Johnson and Ownby (1993) investigated muscle tissue taken only 5 min after i.m. injection of ACL myotoxin. They described three di€erent types of early pathological changes in muscle cells, all of which can be observed in a single muscle cell in longitudinal section (Fig. 4A). In cross sections (Fig. 4B), some cells may appear to have only one of these types of pathological changes. The ®rst pathological alteration appears as vacuoles in the cytoplasm at the light microscopic level and as swollen sarcoplasmic reticulum at the electron microscopic level (Fig. 5A). GutieÂrrez et al. (1989) also described the presence of vacuolated cells after the injection of a K49 PLA2 myotoxin from Bothrops nummifer venom. Mitochondria in these regions of swollen sarcoplasmic reticulum also appear to be swollen with dilated intercristal spaces, but transverse tubules appear to be structurally normal. The second type of pathological change consists of delta lesions and hypercontracted myo®laments (Fig. 5B and C). These hypercontracted myo®laments appear as areas of densely clumped myo®brils at the light microscopic level. A third type of pathological change consists of regions of damaged muscle cells which contain disorganized myo®brils, abnormally far apart (Fig. 5D). The Z disks in these areas do not appear to be damaged. In some areas the myo®laments are highly contracted forming contraction bands, and in some areas the myo®laments are contracted so tightly that individual sarcomeres can not be distinguished. Areas of highly contracted myo®laments alternate with areas containing ¯occulent material and organelles such as swollen mitochondria, sarcoplasmic reticulum vesicles and pyknotic nuclei. The nature of the spatial relationship of these pathological changes to each other along the length of one muscle cell and their relationship to the site of rupture of the cell membrane (see Fig. 4A) led Johnson and Ownby (1993) to propose that the initial site of action of the toxin is the plasma membrane,

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Fig. 4. E€ects of the K49 myotoxin from Agkistrodon contortrix laticinctus venom (ACL myotoxin) on murine skeletal muscle. Light micrographs of skeletal muscle taken from a mouse 5 minutes after i.m. injection of 1.0 mg/g ACL myotoxin; tissue embedded in Polybed, stained with toluidine blue. (A) Longitudinal section. (B) Transverse section. M, muscle cells. Pathological changes in muscle cells are indicated as follows: (1) clear vacuoles, (2) delta lesions, (3) hypercontracted myo®brils and (4) disorganized myo®brils. See text for detailed explanation of pathological changes. Bars = 20 mm.

and that the initial action is lysis of the membrane allowing the in¯ux of ions such as sodium and calcium from the extracellular matrix. The idea is that two of the pathological changes observed by Johnson and Ownby (1993), i.e. swollen sarcoplasmic reticulum and disorganized myo®brils, respectively, could be explained by the in¯ux of Na + followed by an in¯ux of water. It is well established that an in¯ux of Na + and water lead to a dilatation of

Fig. 5. E€ects of the K49 myotoxin from Agkistrodon contortrix laticinctus venom (ACL myotoxin) on murine skeletal muscle. Electron micrographs showing the ultrastructural appearance of the pathologic changes illustrated in Fig. 4. Same experiment; tissue embedded in Polybed, stained with uranyl acetate and lead citrate. Portions of two muscle cells (M) are shown in each micrograph. (A) Arrows indicate dilatated sarcoplasmic reticulum, seen as clear vacuoles indicated by `1' in Fig. 4; (B) arrows mark edges of a delta lesion indicated in Fig. 4 as `2'; (C) arrows indicate hypercontracted myo®brils, indicated as `3' in Fig. 4 and (D) cell labelled DM contains disorganized myo®brils as do the cells indicated as `4' in Fig. 4; M, normal muscle cell. Bars = 1 mm.

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the endoplasmic reticulum as it attempts to take up the water from the cytoplasm of the cell (Ginn et al., 1968). The inability of the sarcoplasmic reticulum to take up all of the water might lead to swelling of the entire cell which could explain the increased distance between myo®brils resulting in a disorganized appearance. In the ACL myotoxin damaged muscle cell, the area containing dilatated sarcoplasmic reticulum is farthest from the plasma membrane lesion whereas the area of disorganized myo®brils is closer and adjacent to the area of clumped myo®brils. The third pathological change, i.e. hypercontraction and clumping of myo®brils, could be explained by increased levels of calcium in the cytoplasm. It is possible that the in¯ux of Na + , after lysis of the cell membrane, could induce a release of Ca2+ from intracellular pools, thus increasing the cyotosolic Ca2+ concentration, leading to contraction of myo®laments. A swollen and damaged sarcoplasmic reticulum might be unable to sequester the Ca2+, leaving the cytosolic levels high, leading to hypercontraction of myo®laments without relaxation. Support for this hypothesis was presented by Johnson and Ownby (1994) in a study of the role of extracellular ions in the pathogenesis of myonecrosis induced by ACL myotoxin. Muscle cells containing contracted and densely clumped myo®laments were observed after incubation in a solution containing 150 mM NaCl, approximately equal to that of the extracellular ¯uid of animal tissues. However, a higher number of muscle cells contained clumped myo®brils when the tissue was exposed to 300 mM NaCl, indicating that this pathological change might be concentration dependent. Incubation of the cells in 300 mM KCl did not produce this pathological change, whereas incubation in 150 mM LiCl did produce the same change. Thus, this pathological change is related to increased Na + (or Li + ) levels in the extracellular ¯uid, but not to increase K + or Cl ÿ levels. Also, increased Ca2+ (200 mM) did not produce this change even though this concentration is much higher than the 1±3 mM normally present in the extracellular ¯uid. However, all of the changes occurring in the muscle cell after the rupture of the plasma membrane could be the result of an increase in the amount of intracellular Ca2+ regardless of the initiating mechanism (Harris and Cullen, 1990; McArdle and Jackson, 1994). Later stages in the pathogenesis of myonecrosis induced by several K49 PLA2 myotoxins have also been fairly well-described. In general, the early changes which occur within the ®rst 5±30 min after injection include lysis of the plasma membrane, appearance of `delta lesions' (wedge-shaped clear areas within the muscle cell lacking organelles), and cells with hypercontracted clumped myo®brils. At later time periods (3±12 h), these dense, hypercontracted clumps of myo®brils have become less dense and the cells have the more amorphous and hyaline appearance of necrotic cells. By 24 h after injection of the toxin, the amorphous cells contain many phagocytic cells such as macrophages which are removing the cellular debris. Throughout this entire process, the basal lamina appears to remain structurally intact, and usually by 3±4 days after injection small myotubes are present. With all of

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the K49 PLA2 myotoxins evaluated so far, the regeneration of skeletal muscle cells is good. Homsi-Brandeburgo et al. (1988) described good regeneration of necrotic muscle cells even after the induction of extensive myonecrosis 10 min after injection of bothropstoxin I (10 and 20 mg) into the tibialis anterior muscle of mice. By 6 h there was a moderate polymorphonuclear neutrophil in®ltration among the necrotic ®bers. After 1 week, all of the necrotic debris had been removed and regeneration took place. After 2 weeks, the muscle had a normal appearance except for `permanent' centrally located nuclei, but there was no ®brosis. Essentially the same results were reported for several K49 PLA2 myotoxins from other Bothrops venoms (see review by GutieÂrrez and Lomonte, 1997). Brie¯y, myoblast fusion and the appearance of myotubes begins on about the fourth day after toxin-induced necrosis. By 1±2 weeks, numerous regenerating ®bers are present, containing centrally-located nuclei. The size of these regenerating muscle cells increases, and by about 4 weeks, the cells are as large as normal muscle cells, but still retain a centrally located nucleus. 5.3. Sensitivity of di€erent muscle cell types It is generally assumed that PLA2 myotoxins act preferentially on type I oxidative and oxidative-glycolytic muscle ®bers (Harris, 1991). Recently, Morini et al. (1998) investigated the sensitivity of fast-twitch, glycolytic (gastrocnemius) and slow-twitch, oxidative/oxidative-glycolytic (soleus) muscles to ACL myotoxin, a K49 PLA2. Subcutaneous injection of the myotoxin (5 mg/kg) over these muscles induced necrosis at 3 h in both the super®cial and deep regions of both muscles. Alternate serial frozen sections cut from the medial region of the muscles were stained with either toluidine blue, or for acid phosphatase, myo®brillar ATPase activity after alkali (pH 10.3) or acid preincubation (pH 4.3), succinate dehydrogenase or acetylcholinesterase. Both ®ber types I and II were injured in both muscles. In the gastrocnemius 21 days after toxin injection, the number of type IIC ®bers was signi®cantly increased and the number of type II ®bers signi®cantly decreased, suggesting a change in muscle ®ber type from type II to type IIC and ®nally to type I. Split ®bers and acetylcholinesterase activity in clusters of regenerating ®bers indicate that injury by ACL myotoxin produces axonal remodeling and muscle ®ber type change. These results are in contrast to those reported for notexin, a D49 PLA2 myotoxin (Harris et al., 1975). In fact, Harris (1991) states that it is a general property of all myotoxins that oxidative and oxidative-glycolytic muscle ®bers are more susceptible to damage than glycolytic muscle ®bers. The di€erence in these results could be due to a real di€erence in susceptibility to K49 PLA2 versus D49 PLA2 myotoxins or it could be due to a di€erence in dose of toxin used. Studies are now in progress to evaluate the di€erence in susceptibility of type I and type II ®bers to lower doses (0.5 and 0.05 mg/kg) of a K49 PLA2 myotoxin. It is possible that these toxins use two di€erent mechanisms

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to damage the muscle cell, i.e. a very speci®c, selective mechanism at low doses and a less selective mechanism at higher doses. 5.4. Physiological/biochemical e€ects: in vivo Contractures of skeletal muscle are a prolonged increase in muscle tension and usually result from a sustained elevation in intracellular Ca2+ levels. Most myotoxic agents induce contractures of skeletal muscle in vivo, although this is a relatively nonspeci®c indicator of myotoxicity (Harris, 1985, 1991; Mebs and Ownby, 1990; Ownby, 1990). Application of myotoxin II from Bothrops asper venom (Lomonte et al., 1994a) induced contractions within 10± 60 s which then cease after 1±2 min and were followed after 3±4 min by the development of transverse bands and loss of striations in focal areas. Angulo et al. (1997) reported essentially the same results for a K49 PLA2 myotoxin from the venom of Bothriechis schlegelii. Elevation of CK and/or lactate dehydrogenase (LDH) values, peaking in the ®rst few hours, suggest that muscle membrane breakdown has occurred and is a frequent ®nding following injection of a myotoxin (Lomonte and GutieÂrrez, 1989; Mebs and Ownby, 1990; Ownby, 1990; Dõ az et al., 1991, 1993, 1994; Kihara et al., 1992; Lomonte et al., 1992, 1994b; Melo et al., 1993; Angulo et al., 1997). More severe muscle damage is associated with the release of myoglobin, usually manifested as myoglobinuria (Mebs and Samejima, 1980; Harris, 1985, 1991; Mebs and Ownby, 1990; Gopalakrishnakone et al., 1997). The elevation of CK might be a useful indicator of the extent of myonecrosis in some cases in which the myotoxicity of an agent has already been established (Nakada et al., 1984); however, caution should be used in interpreting the results, since there is not always a good correlation between extent of myonecrosis and CK levels (Harris, 1985, 1991; Mebs and Ownby, 1990; Ownby, 1990). Histological evaluation of muscle tissue should always accompany CK data.

6. Overview of potential mechanisms of action on the muscle cell Most investigators agree that the plasma membrane is the initial site of action of these myotoxins. However, the nature of the initial insult to the muscle cell has been a bit more elusive. This is in part due to the extreme rapidity of action of these toxins. By routine microscopy it is dicult to observe the initial site or mechanism of contact between the toxin and the muscle cell membrane prior to rupture of the membrane. It is not clear whether the events leading to transient contractures are associated with long-term e€ects such as cell death. It is also unclear whether the changes in the muscle cell which lead to death are a result only of lysis of the plasma membrane or whether the toxin must exert an action within the cell, either by being internalized or through signal transduction.

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There are many aspects of skeletal muscle structure or function that could be disrupted by the K49 PLA2 myotoxins and lead to cell death. The K49 PLA2 myotoxins likely a€ect several of these systems, either directly or indirectly, by initiating a chain of events that leads to a loss of cellular homeostasis. It is most probable that the toxins ultimately irreversibly impair ion regulation. In the normal state the levels of various ions are maintained by adenosine triphosphate (ATP)-driven ion pumps that remove intracellular ions introduced by leakage or cell signaling. Inhibition of ion pumps by the toxin would lead to loss of ion regulation. An alternative mechanism is to overwhelm the capacities of the ion pumps, or exhaust supplies of ATP, thereby antagonizing the extrusion of ions from the cytoplasm. This latter mechanism could involve making the membrane `leaky', or increasing the current though extracellular or intracellular ion channels, thereby causing depletion of ATP stores by compensatory pumping and ®nally a total loss of ion regulation. Normally, intracellular levels of ions are altered by cell signaling mechanisms that open ion channels and allow Na + and Ca2+ ions to ¯ow inward, driven by their concentration gradients. The action potential, generated in the sarcolemma by opening voltage-gated Na + channels, is carried along the sarcolemma into the t-tubules to activate the excitation±contraction coupling mechanism, causing Ca2+ to be released from intracellular stores into the myoplasm. The action potential is terminated by inactivation of Na + channels and opening of K + channels. The disequilibrium in Na + and K + ions thus created across the sarcolemma is restored to normal by the Na + /K + -ATPase. The action potential propagated along the sarcolemma is carried down into the interior of the muscle by invaginations of the plasma membrane, termed t-tubules. T-tubules contain the dihydropyridine receptors, or sarcolemmal Ca2+ channels. In skeletal muscle the dihydropyridine receptors function as voltage sensors that open the ryanodine receptors. The skeletal muscle ryanodine receptor is embedded in the terminal cisternae region of the sarcoplasmic reticulum. Ca2+ is released into the myoplasm from the sarcoplasmic reticulum primarily through the ryanodine receptor (MacLennan and Phillips, 1992; McPherson and Campbell, 1993; Coronado et al., 1994; Melzer et al., 1995). Muscle contraction is terminated by the removal of Ca2+ from the myoplasm by the ATP-driven Ca2+ pump (MacLennan and Toyofuku, 1992). The longitudinal sarcoplasmic reticulum has a very dense concentration of Ca2+ pumps for maximum removal of Ca2+ from the myo®brils for relaxation of the muscle and reloading of the sarcoplasmic reticulum stores. It is not known whether these toxins are internalized. The toxin could act only at the plasma membrane level, either disrupting the structural integrity of the membrane by phospholipid hydrolysis or by disrupting the normal function of the membrane by altering its control of ion transport. Using immunohistochemistry at the light and electronmicroscopic levels, there is evidence for binding of myotoxin II from Bothrops asper venom to the sarcolemma, but no evidence for internalization of the toxin (GutieÂrrez and Lomonte, 1995). The toxins may remain at the level of the sarcolemma, yet still alter the intracellular lea¯et of the

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plasma membrane bilayer. The interaction of myotoxin II with the membrane is very weak when the temperature is lowered to 2±48C (Lomonte et al., 1994d), suggesting that internalization may be required. One interesting study on a D49 PLA2 toxin should be mentioned. Ollivier-Bousquet et al. (1991) showed that the basic PLA2 crotoxin (B subunit) from Crotalus durissus terri®cus venom is at least partially internalized inducing secretion in epithelial mammary cells. These results support the possibility that PLA2 toxins may act intracellularly. It remains to be con®rmed whether these e€ects on secretion or other e€ects of PLA2 toxins such as neurotoxicity, myotoxicity, etc. are achieved via intracellular action. In many cases a single mechanism appears to account for the action of a toxin. For example, in the case of tetrodotoxin, the only reported mechanism is blockade of sodium channels and this mechanism is believed to account for its action on all a€ected tissues. The myotoxic PLA2 toxins could act through more than one mechanism. At least two pharmacologically distinct mechanisms can be dissected, as described below. In addition, it appears that several di€erent systems can be a€ected by the toxins. The most likely explanation for altering several systems simultaneously is through a signaling mechanism with wide-spread consequences. The fatty acids released by PLA2 activity are known to alter the function of many di€erent proteins including ion channels and pumps. Therefore, these products of PLA2 activity may be involved in at least some aspects of myotoxicity. However, it is clear that there is speci®city to the action of these toxins and that the production of fatty acids alone cannot account for toxicity, as will be discussed below.

7. E€ects on skeletal muscle in vitro 7.1. Contractile properties Nerve terminal or acetylcholine receptor e€ects of a toxin can be isolated from direct action on the muscle by monitoring muscle twitch tension evoked by brief pulses of `indirect' nerve or `direct' muscle stimulation. The use of nerve±muscle preparations has been proposed as the basis for screening venoms for neurotoxic and myotoxic e€ects (Harvey et al., 1994). The directly elicited depolarization of the muscle opens voltage-gated Na + channels, causing propagation of an action potential that is independent of neurotransmission. Myotoxicity causes the loss of the direct and indirect twitch. Myotoxicity must be further assessed by light and electron microscopy to verify that necrosis has occurred and that the actions of the toxin are speci®c to skeletal muscle. For example, K49 PLA2 myotoxin from Bothrops jararacussu (BthTX-I) has no e€ect on nerve endings, as determined either by electrophysiological or ultrastructural analysis (Rodrigues-Simioni et al., 1983). BthTX-I has been demonstrated to block directly-elicited twitches of skeletal muscle irreversibly in parallel with a decrease in compound action potential

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amplitude (Rodrigues-Simioni et al., 1983, 1995; Heluany et al., 1992). This K49 PLA2 myotoxin can induce contractures in vitro in chick (Heluany et al., 1992) and frog (Rodrigues-Simioni et al., 1983) skeletal muscle and in mouse diaphragm (Hawgood and Smith, 1977; Heluany et al., 1992; Rodrigues-Simioni et al., 1995) preparations. Determining the e€ects of altering the divalent cation composition has been a common approach to explore the role of enzymatic activity in the action of the Ca2+-dependent D49 PLA2 toxins. However, this approach has provided interesting observations that are likely independent of e€ects on enzymatic activity. Substitution of Sr2+ for Ca2+ has very little e€ect on the magnitude of contracture to BthTX-I (Rodrigues-Simioni et al., 1995), despite the reduction of any enzymatic activity that should occur. However, this alteration of divalent cation composition greatly reduces the time to 50% block of the muscle twitch. Simply omitting Ca2+ from the bathing medium increased the magnitude and decreased the time to onset of contractures to BthTX-I in the chick biventer cervicis preparation (Heluany et al., 1992). A Ca2+-free medium does not alter the inhibition of frog cutaneous pectoris direct twitch by BthTX-I (RodriguesSimioni et al., 1983) or LDH release from frog toe muscles induced by Bothrops jararacussu venom (Melo and Suarez-Kurtz, 1988). Elevating the Ca2+ concentration in the medium to 10 mM antagonizes contractures, twitch depression and membrane depolarization induced by BthTX-I (Heluany et al., 1992). Unlike with other snake venom components, this antagonism is apparently not due to antagonism of binding (Heluany et al., 1992). Decreasing the temperature of the bath from 37 to 238C had no e€ect on the time to paralysis of the mouse phrenic nerve±diaphragm preparation (Rodrigues-Simioni et al., 1995). Overall, the e€ects of divalent cations are complex and some vary with the preparation examined. This complexity is partly due to the role these ions play in cell function, in addition to their e€ects on catalytic activity and binding of the toxin. 7.2. Membrane potential and Na + channels A K49 PLA2 myotoxin from Trimeresurus ¯avoviridis venom (Kihara et al., 1992) and BthTX-I (Rodrigues-Simioni et al., 1983; Heluany et al., 1992) depolarize skeletal muscle. Depolarization is Ca2+-dependent, but not required for myotoxicity, since some myotoxins, such as an D49 PLA2 myotoxin from Trimeresurus ¯avoviridis venom, do not cause depolarization (Kihara et al., 1992). Fibrillations (spontaneous muscle twitches, possibly fasciculations) induced by BthTX-I (Rodrigues-Simioni et al., 1983, 1995) are prevented by tetrodotoxin (Rodrigues-Simioni et al., 1983), suggesting the potential involvement of the Na + channel in at least one mode of action of K49 PLA2 myotoxins. In contrast, toxin-induced depolarization was una€ected by tetrodotoxin (Rodrigues-Simioni et al., 1983; Heluany et al., 1992). In further agreement with a role for Na + currents in at least one mechanism, the myotoxicities of the K49 ACL myotoxin

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(Johnson and Ownby, 1994; Selistre de Araujo et al., 1996a) and a K49 myotoxin from Bothrops nummifer venom (GutieÂrrez et al., 1986) have been shown to be dependent on Na + and not Ca2+ in the extracellular medium. Bupivacaine is a local anesthetic that acts on sodium channels and is myotoxic at higher than clinically e€ective concentrations (Harris et al., 1980). Therefore, the e€ects on Na + channels are not unique to PLA2 myotoxins, but appear to be a general characteristic of several types of myotoxic agents. Fatty acids are a major product of PLA2 activity that alter the function of a large number of ion currents, including Na + , Ca2+, Cl ÿ and K + (Wieland et al., 1992, 1996; Graber et al., 1994). The fatty acids produced by the catalytic activity of lipolytic enzymes may also generate a current independent of ion channels (Alix and Woodbury, 1997). It is dicult to determine which e€ects might be caused directly by the PLA2 myotoxin and which might be caused indirectly by liberated fatty acids. The Na + channel is an acylated protein (Bizzozero et al., 1994; Milligan et al., 1995; Wedegaertner et al., 1995) and this mechanism of covalent attachment of fatty acids to the channel could be crucial in toxin action. The possible role of lipolytic activity in the toxicity of PLA2 myotoxins is discussed in detail below. 7.3. Intracellular Ca2+ regulation Two K49 myotoxins, BthTX-I (Rodrigues-Simioni et al., 1995) and ACL myotoxin (Fletcher et al., 1996), stimulate the opening of the Ca2+-induced Ca2+ release channel, or ryanodine receptor, in heavy sarcoplasmic reticulum fractions. This action of the toxin is manifest as a net decrease in the rate at which the sarcoplasmic reticulum vesicles can sequester Ca2+ and is completely antagonized by ruthenium red, a blocker of ryanodine receptor. Furthermore, BthTX-I decreases the threshold of Ca2+-induced Ca2+ release through a mechanism independent of the ryanodine receptor (Rodrigues-Simioni et al., 1995). While BthTX-I does cause Ca2+ release from isolated sarcoplasmic reticulum preparations, the dantrolene-sensitive Ca2+ stores do not appear to be involved in myotoxicity in intact tissue, as judged by the blockade of the directlyelicited twitch (Rodrigues-Simioni et al., 1995). Therefore, the relationship of studies in isolated organelles to e€ects of the toxin on intact tissues is presently unclear.

8. Lysis of arti®cial membranes: model of interaction with sarcolemmal lipid The PLA2 myotoxins have been examined using lysis of liposomes as a model membrane system. Liposomes are arti®cial membrane vesicles composed of one or more phospholipids. K49 PLA2 myotoxins from several di€erent venoms, including Bothrops asper (myotoxin II; myotoxin IV), Bothrops moojeni (myotoxin II) and Agkistrodon piscivorus piscivorus, cause the release of peroxidase or

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¯uorescent markers from negatively charged, but not positively charged liposomes (Dõ az et al., 1991, 1993, 1994, 1995; Ru®ni et al., 1992; Shen and Cho, 1995). Small and large unilamellar liposomes were disrupted by myotoxin II from Bothrops asper venom and myotoxin II from Bothrops moojeni venom (Dõ az et al., 1991). The eight amino acids at the NH2-terminal appear to be involved in lysis of liposomes (Dõ az et al., 1994). The leakage of dyes from the liposomes was independent of Ca2+ in the bu€er for three di€erent myotoxins (Dõ az et al., 1991; Ru®ni et al., 1992), suggesting PLA2 activity plays no role in the liposome lysing activity of these toxins. This is in contrast to the Ca2+-dependent hemolysis induced by BthTX-I (Rodrigues-Simioni et al., 1983). Further evidence that PLA2 activity plays no role in lysis of liposomes by myotoxin II from Bothrops asper was the release of entrapped dye from liposomes comprised of nonhydrolyzable analogues of phospholipids (Pedersen et al., 1994). It is not surprising that the lysis of liposomes by the K49 myotoxins is Ca2+-independent, since they do not hydrolyze arti®cial membranes to any signi®cant extent (see below). For some D49 PLA2 myotoxins enzymatic activity is not essential for lysis of liposomes, albeit, it seems to enhance the lytic activity (Dõ az et al., 1991; BultroÂn et al., 1993). In agreement, a D49 PLA2 from Agkistrodon piscivorus piscivorus venom enhanced lysis induced by a K49 PLA2 from the same venom (Shen and Cho, 1995). There is not a perfect correlation between liposome disruption and in vivo myotoxicity, although PLA2 myotoxins (D49 and K49) may be unique relative to nomyotoxic PLA2 enzymes in that Ca2+ is not essential for lysis (Dõ az et al., 1991; Ru®ni et al., 1992).

9. Acute e€ects on cell cultures ACL myotoxin and BthTX-I cause lysis of cell cultures, as determined by trypan blue exclusion (Fletcher et al., 1996). The K49 PLA2 myotoxins also cause release of CK (BruseÂs et al., 1993; Lomonte et al., 1994d) and LDH (BruseÂs et al., 1993; Lomonte et al., 1994b) from a variety of cell cultures. Cytolysis is enhanced when phosphate-bu€ered saline is utilized in place of growth medium and abolished when the temperature is lowered to 2±48C (Lomonte et al., 1994d). Few studies have described in any detail the morphologic e€ects of K49 (or even D49) PLA2 myotoxins in cell culture (BruseÂs et al., 1993; Lomonte et al., 1994d). Myotoxin II, a K49 PLA2 myotoxin from Bothrops asper venom, caused cytoplasmic granulation and dissolution of the plasma membrane in L6 myoblasts (Lomonte et al., 1994d). The PLA2 myotoxins in fractions IV and V from Bothrops nummifer cause vacuolation of the sarcoplasmic reticulum and t-tubules and release of all CK activity from primary cultures of chick muscle and the C2C9 mouse muscle cell line (BruseÂs et al., 1993). However, the integrity of the plasma membrane was maintained, the myo®brils were relatively una€ected and no delta lesions were observed (BruseÂs et al., 1993). Thus, the e€ects of these PLA2

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myotoxins in nonenzymatic, than to those morphological myotoxins in myotoxicity.

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cell culture are more similar to the in vivo action of the noncytolytic peptide myotoxins such as myotoxin a and crotamine, of the PLA2 myotoxins. Therefore, it is essential to evaluate the changes and compare these to the documented actions of vivo when establishing a cell culture model of PLA2-induced

10. K49 PLA2 myotoxins and lipolysis in arti®cial and cell culture membranes The enzymatic activity of a D49 PLA2 molecule provides a number of potentially toxic mechanisms. First, the integrity of the membrane is disrupted by the hydrolysis of the phospholipids. Second, both fatty acids (Ordway et al., 1991; Graber et al., 1994) and lysophospholipids (Pestronk et al., 1982; Lundbaek and Andersen, 1994) are highly active and potentially destructive molecules. Some PLA2 enzymes are catalytically very active, yet they are far less toxic than other less enzymatically active PLA2s. Therefore, if enzymatic activity is important in myotoxicity, it is most likely a highly speci®c and/or localized event that would be masked by a high level of nonspeci®c hydrolysis. Rosenberg (1979, 1997) has repeatedly demonstrated that there is no relationship between enzymatic activity in isolated systems and that in systems in which toxicity is determined. However, most investigators continue to extrapolate enzymatic data derived from arti®cial substrates to toxicity studies in complex biological membranes and this has greatly complicated our understanding of this ®eld. The uses and limitations of omitting Ca2+ from the bathing medium and substituting Sr2+ for Ca2+, as well as other methods for pharmacologically examining the role of enzymatic activity in toxic mechanisms of these Ca2+dependent enzymes have been reviewed elsewhere (Fletcher et al., 1997; Rosenberg, 1997). Modi®cation of the histidine residue at position 48 with p-BPB abolishes the enzymatic activity of PLA2 enzymes (Volwerk et al., 1974; Kondo et al., 1978). BSA can antagonize PLA2 activity by removing fatty acids from the cell plasma membrane (Fletcher and Jiang, 1995). 10.1. Arti®cial versus cell culture substrates The types of substrates examined in arti®cial substrate systems do not match the complex array of substrates presented in a biological membrane. Also, the large number of peptides, proteins and cofactors that could be essential for the enzymatic activity in cell cultures is not presented in the arti®cial substrate system. This topic and associated methodologies has been reviewed elsewhere (Fletcher et al., 1997; Rosenberg, 1997). Enzymatic activity of highly puri®ed K49 PLA2 myotoxin fractions on arti®cial substrates is virtually undetectable even when high concentrations of toxin are employed (Homsi-Brandeburgo et al., 1988). When similar or even lower concentrations of K49 PLA2 myotoxins are examined on

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biological membranes, lipolytic activity can be very extensive (Rodrigues-Simioni et al., 1995; Fletcher et al., 1996, 1997). Whether this activity is due to the K49 PLA2 or activation of one or more tissue lipolytic enzymes is currently under investigation.

11. Role of lipolytic activity in myotoxicity 11.1. Studies using arti®cial substrates and tissues Several investigators have dismissed a role for catalytic activity in the toxicity of enzymatically active D49 PLA2 myotoxins, based on the lack of correlation of enzymatic activity with toxicity. The studies of Rosenberg and coworkers (Rosenberg, 1979, 1986, 1997; Rosenberg et al., 1989), determining enzymatic activity on biological substrates, have been the most convincing. This view gained strength when K49 PLA2 toxins were reported to be catalytically inactive. The mutant D49K of recombinant pancreatic PLA2 enzymes are devoid of [if derived from porcine pancreatic D49 PLA2 (Van den Bergh et al., 1988)], or possess very low levels of [if derived from bovine pancreatic D49 PLA2 (Li et al., 1994)], enzymatic activity on arti®cial substrates. Mutant D49K PLA2 enzymes also have little or no Ca2+ binding activity (Li et al., 1994). The residual PLA2 activity of the K49 mutant of bovine pancreatic enzyme is Ca2+-dependent (Li et al., 1994), suggesting the possibility of trace contamination with a D49 PLA2. Some caution should be used in extrapolation of the results of these site-directed mutagenesis studies to toxicity of the actual K49 PLA2 myotoxins for two reasons. First, the enzyme activity studies were conducted with arti®cial substrates and there is general agreement that K49 enzymes are inactive on these systems. Second, there are additional structural features possessed by the K49 PLA2 myotoxins not shared by the D49 PLA2 myotoxins, including a phosphorylation site (Selistre de Araujo et al., 1996b; Fletcher et al., 1997), that may in¯uence the toxic function and enzymatic activity of the molecule on cell membranes. For example, the K49 PLA2 from Agkistrodon piscivorus piscivorus venom can form complexes with phospholipid in the absence of Ca2+ (Maraganore and Heinrikson, 1986), which is a property not shared by the D49K mutants (Li et al., 1994). The site-directed mutagenesis studies suggest that contamination with D49 PLA2 accounts for trace enzymatic activity in impure K49 PLA2 myotoxin fractions when using arti®cial substrates and con®rm that high concentrations of K49 PLA2 (100±200 mg/ml) must be tested to rule out this possibility. Although the possible role of enzymatic activity in myotoxicity for D49 PLA2 myotoxins has not been fully resolved, at least some questions have been answered. Details of studies investigating the role of PLA2 activity in a variety of toxic mechanisms are reviewed elsewhere (Rosenberg, 1979, 1986, 1997; Rosenberg et al., 1989; Fletcher et al., 1997). It is clear that if phospholipid hydrolysis plays a role in myotoxicity, then it must be a highly speci®c and/or localized activity (e.g.,

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ref. Fletcher and Jiang, 1995). In agreement, a recent study of the K49 BtxTX-I has observed no correlation between the e€ects of various ionic conditions on the toxic action (Heluany et al., 1992; Rodrigues-Simioni et al., 1995) and on overall enzymatic activity determined on a biological substrate (skeletal muscle cultures) (Rodrigues-Simioni et al., 1995). Therefore, the bulk of the enzymatic activity appears to play no role in toxicity, at least in some cases. A study of a K49 PLA2 myotoxin (myotoxin II from Bothrops asper venom) found that modi®cation at histidine 48 with p-BPB caused a decrease in in vivo myotoxicity (Dõ az et al., 1993), which would suggest that PLA2 activity might play a role. Recently, similar results have been obtained for ACL myotoxin (Melo and Ownby, unpublished results). Eclipta prostrata extract inhibits myotoxicity of BthTX-I and PLA2 activity for several PLA2 myotoxins (Melo et al., 1994). Thus, there exists evidence for lipolysis induced by a K49 PLA2 myotoxin and arguments for and against a primary role for PLA2 activity in myotoxicity. 11.2. K49 PLA2 myotoxin-induced lipolysis in cell culture Recently, lipolytic activity has been observed in primary cultures of human skeletal muscle treated with two K49 PLA2 myotoxins (Rodrigues-Simioni et al., 1995; Fletcher et al., 1996). ACL myotoxin is cytolytic (by trypan blue exclusion) to human muscle cells within 60 min, causing 50% loss of cell viability at about 2.5 mM (Fletcher et al., 1996). ACL myotoxin (5.0 mM) was also found to decrease viability in a cell line (NB41A3) by about 50% (J.E.F. and C.L.O., unpublished observations) and this was associated with signi®cant levels of lipolysis, especially with elevated fatty acid production (Fletcher et al., 1996, 1997). The puri®ed ACL myotoxin (Selistre de Araujo et al., 1996a) and BthTX-I (Homsi-Brandeburgo et al., 1988) K49 PLA2 myotoxins were both found to be inactive on arti®cial substrates at 100±200 mg/ml, ruling out contamination with a D49 PLA2 as liberating fatty acids. Studies are ongoing to test whether the fatty acids are liberated by the K49 PLA2, or by tissue lipases activated by the K49 PLA2 myotoxin. The role of highly localized lipolytic activity in myotoxicity is also under investigation.

12. Chronic e€ects on cell cultures: methylprednisolone-sensitive lysis Higher concentrations of BthTX-I or ACL myotoxin are required to lyse cell cultures within 1 h than over a 24 h incubation (Fletcher et al., 1996). Electron microscopic examination of the NB41A3 cell line treated with ACL myotoxin (0.5 mM, 24 h) revealed ruptures in the plasma membrane and a swollen or dilated and empty endoplasmic reticulum (C.L.O. and J.E.F., unpublished observations). While we initially suspected that lysis by both low and high concentrations were mediated through similar mechanisms, we subsequently found that cytolysis of the NB41A3 cell line by low concentrations of ACL myotoxin was antagonized by

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methylprednisolone (MePDN; 50 mM), whereas that by high concentrations was only slightly antagonized by MePDN (Fig. 6A). If the concentration of ACL myotoxin was decreased to 1.0 mM for the 1 h incubation to achieve a greater percentage viability [61 22% (3); mean 2S.E.M. (n)] similar to that for the cells treated with a 0.5 mM concentration of ACL myotoxin for 24 h (Fig. 6A), then the addition of MePDN (50 mM) still did not signi®cantly elevate the percentage viability [67 2 2% (3)]. Therefore, there appears to be a rapid, MePDN-insensitive mechanism at high concentrations of ACL myotoxin and a slower, MePDN sensitive mechanism at lower concentrations of ACL myotoxin. Notice that the lower concentration of ACL myotoxin also exerts some of the MePDN-insensitive mechanism, as the viability does not return to 100% of the control (Fig. 6A). The ratio of triglyceride (TG) to free fatty acid (FFA) was about 39 in control NB41A3 cells, whether incubated for 1 or 24 h in parallel with toxin-treated preparations (Fig. 6B). If these cells are incubated with ACL myotoxin (5.0 mM) for a brief (1 h) period of time, the ratio (Fig. 6B) drops dramatically due to the production of free fatty acids from 0.03 2 0.06 (n = 3) in controls to 10.4 20.6% of total radiolabeled lipid (n = 3) in toxin-treated cultures. In contrast, when cells are incubated for 24 h at a 0.5 mM concentration of ACL myotoxin, the ratio of TG/FFA is approximately equal to control values (Fig. 6B). This suggests that the cellular enzymes are maintaining the levels of FFA produced by ACL myotoxin low by reacylating them to TG and phospholipid. In contrast, to ACL myotoxin, the Naja naja atra acidic D49 PLA2 (1 mM, 24 h) does not cause detectable morphological e€ects on the NB41A3 cell line (C.L.O. and J.E.F., unpublished observations), despite levels of FFA reaching 14.5 2 0.6% of total radiolabeled lipid (n = 4). The FFAs liberated by the Naja naja atra acidic D49 PLA2 are not readily reacylated to TG and FFA, as the ratio of TG/FFA is very low (Fig. 6B). These ®ndings do not support a role for high levels of PLA2 activity in cell lysis, as the high concentration of ACL myotoxin produced even less fatty acid than the noncytolytic Naja naja atra acidic D49 PLA2. However, the lower concentration of ACL myotoxin incubated for 24 h (the MePDN-sensitive component of lysis) may still be acting through a FFA-dependent mechanism as the FFA are apparently reaching the cell FFA processing enzymes and this area is currently under investigation.

13. A model for a potential role of fatty acid production in one mechanism of myotoxicity Recall that a FFA-mediated mechanism would be only one of at least two mechanisms of action for K49 PLA2 myotoxins. One model suggesting how such a mechanism could alter cellular function is explored below. The data of several groups have con®rmed that gross enzymatic activity of a PLA2 does not cause cell lysis (see above) and that large amounts of FFA can be extracted from cells treated with a noncytolytic PLA2 (Fig. 6B and associated text). In

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Fig. 6. E€ects of the K49 myotoxin from Agkistrodon contortrix laticinctus venom (ACL myotoxin, ACLMT) on the NB41A3 cell line. (A) Antagonism of ACLMT-induced cell lysis (trypan blue exclusion) by methylprednisolone (MePDN). NB41A3 cells were preincubated in the absence or presence of MePDN (50 mM) for 24 h and then separated into controls, ACLMT-treated, or ACLMT plus MePDN-treated and incubated an additional 1 or 24 h. Therefore, the total exposure time to MePDN was 25 or 48 h in all cells tested with this agent. Percent viability was determined relative to untreated control cells incubated for the same time periods (Fletcher et al., 1996). (B) E€ects of ACLMT and the Naja naja atra acidic D49 PLA2 (Nna PLA2) on the ratio of triglyceride/free fatty acid (TG/FFA). Cells were radiolabeled with [14 C] linoleic acid (10 mM) for three days before starting the exposure to MePDN. Following incubation cells were extracted, neutral lipids separated by TLC and the radioactivity in each lipid fraction determined using a radioactivity scanner (Fletcher et al., 1996). Data from the 1 and 24 h control incubations were not signi®cantly di€erent and were pooled to yield a single control group. Values are the mean2S.E.M. for the number of determinations indicated in parentheses over each bar.

Fig. 7. Di€erences between noncytolytic and cytolytic PLA2 enzymes in localization of phospholipid hydrolysis. (A) The noncytolytic mechanism can involve the production of large amounts of FFAs that remain within the plasma membrane, inaccessible to cellular FFA processing enzymes. These latter enzymes normally utilize dietary FFAs, transported across the plasma membrane by fatty acid transport proteins. These FFAs are used for energy (ATP generation by the mitochondria), synthesis of phospholipid and triglyceride and acylation of proteins, potentially involved in cell signaling. (B) The cytolytic mechanism is dependent on FFAs reaching the intracellular space so that the appropriate enzymes can process them. Whether these FFAs enter by the same fatty acid transport proteins used for dietary FFAs, or are produced by intracellular phospholipases, is unknown. The FFAs are activated to acylCoAs prior to further processing, which can include acylation of proteins, such as the Na + channel. The Na + channel is merely used in this model as an example of a protein that can be functionally altered by intracellular FFA and is likely only one of several proteins involved in the mechanisms of the PLA2 myotoxins.

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contrast, the FFAs produced by a cytolytic K49 PLA2 myotoxin over a 24 h period do not accumulate in a FFA pool, but are further metabolized to triglyceride and, possibly through other routes of metabolism (see above). Therefore, we hypothesize that a noncytolytic PLA2 (Fig. 7A) releases FFAs that remain associated with the cell membrane and are not made readily available to the FFA processing enzymes in the cell. Since only normal levels of dietary FFA are being utilized for ATP production, synthesis of phospholipid and neutral lipid and acylation of proteins, the amounts and types of fatty acids acylated to proteins within the cell can be well-controlled. These will consist primarily of saturated FFAs. In contrast, a cytolytic PLA2, such as a PLA2 myotoxin, produces mostly unsaturated FFAs that enter the cell and are utilized by the cellular FFA processing enzymes. Entry of the FFAs may be mediated by fatty acid transport proteins that are either the receptor for the PLA2, or are closely associated with PLA2 receptors. We hypothesize that these newly introduced, primarily unsaturated, FFAs, di€er from the saturated fatty acids normally acylated covalently to proteins and likely alter the function of some acylated proteins, causing leakage of ions into the cytoplasm (Fig. 7). For example, intracellularly injected FFAs cause normally latent Na + channels to become voltage-gated Na + channels (Wieland et al., 1996). Most PLA2 myotoxins likely induce both cytolytic and noncytolytic hydrolysis of phospholipids. While this model is easily applied directly to the D49 PLA2 myotoxins, it is important to remember that the FFA release by the K49 PLA2 myotoxins may actually be mediated by activation of tissue lipases through a signal transduction mechanism.

Acknowledgements The authors gratefully acknowledge the data generously provided by M.-S. Jiang (Allegheny University of the Health Sciences) and the expert technical assistance of Terry R. Colberg (Oklahoma State University).

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