Venom PLA Research Articles

Toxic effects of human pancreatic and snake and bee venom phospholipases A 2 on MCF-7 cells in culture

The toxicity of phospholipase A 2 (PLA 2) has been suggested to be involved in the pathology of a number of severe diseases including septic shock and acute pancreatitis. However, testing the toxicity of these substances is difficult in vivo. In the present study we compared the toxicity of PLA 2s from three snake venoms, bee venom and human pancreas on MCF-7 cells grown in culture. Tetrazolium microculture assays were developed to test the cytostatic and cytotoxic effects of PLA 2 on MCF-7 cells. These tests are based on the ability of viable cells to reduce a tetrazolium-based compound MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] to a blue formazan product. Leakage of lactate dehydrogenase (LD) from the cells into the culture medium was also measured. There were marked differences in the toxicity of the PLA 2s tested. Cobra ( Naja mosambique mosambique) venom PLA 2 was toxic to the cells at a concentration of 4.5 U/ml. Light microscopic changes were seen in the injured cells after 3 hr treatment. Sixty-seven per cent of cells were dead after 24hr treatment. Treatment for 4 hr caused irreversible changes in the cells. Leakage of LD was noted from 4 hr onwards. Other snake ( Crotalus adamanteus and Laticauda semifasciata) venom PLA 2s, even after continuous exposure to 4.5 U/ml caused only slight decreases in values obtained in the MTT test. No morphologic changes suggesting a cytotoxic effect were seen. PLA 2 from bee ( Apis mellifera) venom had no toxic effect, either. Continuous exposure of cells to human pancreatic PLA 2 caused a 15% decrease in the MTT-test. The treated cells appeared morphologically normal and after discontinuation of the treatment their growth rate was equal to the control cells.

Effect of aristolochic acid on arachidonic acid cascade and in vivo models of inflammation

Recently, it has been reported that aristolochic acid inhibits phospholipase A2 (PLA2in vitro and also decrease either oedema induced by snake venom and human synovial fluid PLA2. The aim of this research was to study the antiiflammatory activity of aristolochic acid and to investigate the effect of the alkaloid on the enzymes involved in the release of eicosanoids. Our results demonstrate that aristolochic acid is able to inhibit inflammation induced by immunological, immune complexes, and nonimmunological agents such as carrageenan or croton oil. We suggest that one mechanism of the antiinflammatory activity of alkaloid may be directly blocking PLA2 catalyzed release of arachidonic acid. Moreover, this study demonstrates that aristolochic acid could also inhibit other steps involved in eicosanoids release such as cyclooxygenase and lipooxygenase pathways.

Human recombinant non pancreatic secreted platelet phospholipase A2 has anticoagulant activity in vitro on human plasma

Extracellular phospholipase A2 is secreted from the platelets upon activation by a stimulus such as thrombin. The secreted enzyme has been recently cloned and the recombinant protein produced. Snake venom PLA2 effect on platelet and coagulation has been extensively studied (for review see 3,4) and it has been proposed that the anticoagulant phospholipases may inhibit coagulation by competing with clotting proteins for the lipid surface. Structure function relationship for PLA2s with anticoagulant activity indicates that the activity is conferred by positively charged aminoacids between residues 54 and 77. The corresponding segment of human recombinant secreted platelet PLA2 (r-hnps-PLA2) possesses five positively charged aminoacids in this region being at the identical positions to those of PLA2s with known anticoagulant activities. Here using human and rat plasma we have demonstrated for the first time that the recombinant human extracellular secreted platelet PLA2 increases activated partial thromboplastin time but not prothrombin time.

Recombinant human type II phospholipase A 2 lacks edema producing activity in rat

The rat paw edema-inducing, acute inflammatory activity of four snake venom phospholipase A 2s (PLA 2) ( Naja naja, Naja mocambique mocambique, Crotalus atrox and recombinant Naja naja naja) and of recombinant human type II PLA 2 (rh-PLA 2) found in rheumatoid synovial fluid, were compared after a bolus subplantar injection. The snake venom-derived PLA 2s, including the recombinant Naja naja naja, were potent inducers of paw edema. On the other hand, when given in similar amounts (protein and/or enzymatic activity), the rh-PLA 2 did not produce paw edema. Furthermore, the addition of Naja naja PLA 2, blood plasma from rats with adjuvant arthritis or the E. coli-based enzymatic incubation mixture used to measure PLA 2 activity to the injection mixture containing rh-PLA 2, did not result in paw edema-inducing activity. These results suggest that the lack of paw edema-inducing activity of rh-PLA 2, may be due to significant structural differences between snake venom PLA 2s and human PLA 2 which allow snake venom PLA 2s, but not the human II PLA 2, to express inflammatory activity as measured by paw edema in rat induced by a bolus injection.

Effect of diacylglycerols on the activity of cobra venom, bee venom, and pig pancreatic phospholipases A2

The effects of a series of diacylglycerols (DAGs) with varying acyl chain lengths and degree of unsaturation on the activity of cobra venom, bee venom, and pig pancreatic phospholipases A2 (PL-A2S) were studied using two lipid substrates: dipalmitoylphosphatidylcholine (DPPC) or bovine liver phosphatidylcholine (BL-PC). The activities of the phospholipases critically depended on the chain length and degree of unsaturation of the added DAGs and on the chemical composition of the substrate. The effects of DAGs on cobra or bee venom PL-A2S were similar, but significantly different from the pig pancreatic PL-A2. The data, taken together with our previous NMR studies on physicochemical effects of these DAGs on lipid bilayer structure [De Boeck, H., & Zidovetzki, R. (1989) Biochemistry 28, 7439; (1992) Biochemistry 31, 623], allowed detailed correlation of the type of a bilayer perturbation induced by DAG with the activation or inhibition of the phospholipase on the same system. In general, the activation of the phospholipases correlated with the DAG-induced defects of the lipid bilayer structure. The results, however, argue against general designation of DAGs as "activators" or "inhibitors" of PL-A2S. Thus, for example, diolein activated phospholipases with the BL-PC lipid substrate, but inhibited them with the DPPC substrate. Dihexanoylglycerol and dioctanoylglycerol inhibited pig pancreatic PL-A2 with both lipid substrates and inhibited cobra or been venom PL-A2 with the DPPC substrate, but activated the latter two enzymes with the BL-PC substrate. Longer-chain DAGs (C greater than 12), which induce lateral phase separation of the bilayers into the regions of different fluidities, activated all PL-A2S with both lipid substrates.(ABSTRACT TRUNCATED AT 250 WORDS)

Antiflammins: Anti-inflammatory activity and effect on human phospholipase A 2

Two anti-inflammatory peptides (antiflammins) corresponding to a high amino acid similarity region between lipocortin I and uteroglobin were tested for their ability to inhibit purified human synovial fluid phospholipase A 2 (HSF-PLA 2). No inhibitory activity was observed, even at such high concentrations of peptides as 50 μM. When antiflammins were preincubated with the enzyme and/or the substrate, no HSF-PLA 2 inhibition was detected. In vivo anti-inflammatory activity of these peptides was evaluated in several experimental models of inflammation induced by carrageenan, croton-oil, oxazolone and Naja naja naja venom phospholipase A 2 (PLA 2). In contrast to the in vitro results, anti-inflammatory activity was observed in all tests, except when inflammation was induced by snake venom PLA 2. Taken together, our results do not support the hypothesis that the in vivo anti-inflammatory effect of antiflammins is directly related to inhibition of PLA 2 activity.

Phospholipase A 2 inhibition by alkylbenzoylacrylic acids

3-(4-Alkylbenzoyl)acrylic acids (ABAAs) were synthesized by acylation of alkylbenzenes with maleic anhydride and then screened in vitro for inhibition of phospholipase A 2 (PLA 2) from snake venom and from porcine pancreas. The inhibitory potency of ABAAs increased with the length of the alkyl residues resulting in IC 50 values of between 10 −7 and 10 −4 mol/L. The most potent inhibitors of the snake venom PLA 2 were the 4-( n)-hexadecyl and octadecyl (OBAA) derivatives. Kinetic experiments referred to a time-dependent inhibitory reaction. Irreversibility was examined by dilution and dialysis. A molar ratio of inactivation of OBAA of nearly 20 was estimated. Double reciprocal replots of the apparent inactivation constants to the concentration of OBAA gave a (pseudo) first order rate constant of inactivation of 2.3 min −1. For the dissociation constant of the enzyme-inhibitor intermediate, a value of 6 × 10 −6mol/L was obtained. On the other hand, the PLA 2 from porcine pancreas seemed hardly to be inhibited by ABAAs. The present data are discussed in relation to the proposed model for PLA 2 inactivation by manoalide. In human PMNs leukotriene B 4 and 5-HETE production was essentially reduced. In human platelets the thrombin-induced TxA 2 production was reduced. Since these effects disappeared after addition of arachidonic acid, these findings refer to a PLA 2 inhibition. The immunologically induced bronchospasm in guinea pigs was significantly and dose-dependently inhibited by OBAA. This indicates that ABAAs might be useful in treating allergic diseases, such as asthma, eczema, allergic shock and others.

A prediction of the tertiary structure of human phospholipase A 2 from synovial fluid and a model of substrate binding

The tertiary structure of human Phospholipase A 2 (PLA 2) from synovial fluid has been predicted by modelling the sequence onto the structure of the homologous snake venom PLA 2 structure ( Crotalus atrox). A model of substrate binding to the protein has been on the predicted structure of the apoenzyme. These modelled structures are discussed.

Characterization of a high affinity binding site for pancreatic-type phospholipase A2 in the rat. Its cellular and tissue distribution.

We showed in an earlier study (Arita, H., Hanasaki, K., Nakano, T., Oka, S., Teraoka, H., and Matsumoto, K. (1991) J. Biol. Chem. 266, 19139-19141) that there is a high affinity and specific binding site for mammalian group I phospholipase A2 (PLA2-I) in Swiss 3T3 fibroblast cells. Analysis of the cellular distribution in rat using 125I-PLA2-I as a radioligand indicated the presence of this site in various cells, including vascular smooth muscle cells (VSMC), vascular endothelial cells, synovial cells, chondrocytes, and gastric mucosal cells. Scatchard analysis of rat VSMC revealed the existence of a single class of binding site with a Kd value of 1.60 nM and a Bmax value of 51.0 fmol/10(6) cells. The mammalian mature type of PLA2s-I derived from several animal species specifically recognized the same site in cells and stimulated DNA synthesis, whereas its inactive zymogen, mammalian group II PLA2s, and snake and bee venom PLA2s showed much lesser activities. 125I-PLA2-I bound to VSMC was rapidly internalized and subsequently released from the cells as trichloroacetic acid-soluble radioactivity. Down-regulation of the PLA2-I site was observed in the treatment of VSMC with cAMP-elevating agents, as well as glucocorticoids. Affinity labeling experiments indicated that PLA2-I binds to a single polypeptide with a mass of approximately 200,000 daltons. These results suggest a novel PLA2-I action on the cellular function via its specific binding site.

Identification of different receptor types for toxic phospholipases A2 in rabbit skeletal muscle

Oxyuranus scutellatus scutellatus toxins I (OS 1) and 2 (OS 2) are two phospholipase A 2s (PLA 2) isolated from the venom of the Australian Taipan snake. Their iodinated derivatives have been used to characterize PLA 2 binding sites on rabbit skeletal muscle. Competition and cross-linking experiments indicate that 125I-labelled OS 2 binding sites in rabbit skeletal muscle in vivo are distributed into two classes of receptors. One class binds OS 2 and OS 1 and is insensitive to the bee venom PLA 2. It is composed of a 180 kDa binding protein. This class of PLA 2 receptor is expressed at a high level in rabbit myotube membranes. The other class of PLA 2 receptor identified with 125I-OS 2 also binds with high affinity the bee venom PLA 2 but not OS 1 and is composed of major polypeptides of 34.48 and 82 kDa. This second class of receptor is similar to the one found in brain membranes. The density of the two classes of receptors varies during muscle development.

Two-step inactivation of bee venom phospholipase A 2 by scalaradial

Scalaradial (SLD), a marine natural product isolated from the sponge ( Cacospongia sp., possesses anti-inflammatory properties in vivo and in vitro (Pharmacologist 32: 168, 1990). In this study we characterize its effects against bee venom phospholipase A 2 (PLA 2; EC 3.1.1.4). SLD is a potent inactivator of bee venom PLA 2 with an ic 50 value of 0.07 μM. Inactivation of bee venom PLA 2 occurred in a time-dependent, irreversible manner. The rate of inactivation followed first-order reaction kinetics and was dependent on the concentration of SLD. Kinetic analysis suggested a two-step mechanism of inactivation: an initial apparent noncovalent binding ( K i = 4.5 × 10 −5 M) followed by covalent modification. The rate of inactivation was reduced markedly in the presence of excess phosphatidylcholine, suggesting that modification of the enzyme occurs at or near the substrate binding site.

Evidence that a 16-kilodalton integral membrane protein antigen from Schistosoma japonicum adult worms is a type A2 phospholipase

Type A2 phospholipase (PLA2) activity has been observed in integral membrane protein extracts of Schistosoma japonicum. Antiserum raised against bee venom PLA2 recognized a single 16-kDa band in the parasite extracts; it also localized to antigen in the gut lining of fixed adult schistosomes as shown by immunofluorescence techniques. Evidence was obtained that the molecule was expressed at low levels in comparison with other integral membrane proteins and was weakly immunogenic in rabbits. Two oligonucleotide probes were constructed on the basis of highly conserved regions between the nucleotide sequences of rat, bovine, rattlesnake, and bee venom PLA2; these probes were used to isolate S. japonicum genomic DNA phage clones. A 1.8-kb FnuD2 fragment was shown by Southern blot analysis to strongly hybridize with the 5' 32P-labeled PLA2 oligonucleotides in both S. japonicum genomic DNA and DNA from one of the phage clones. The nucleotide and predicted amino acid sequences of this fragment revealed homology with the C terminus of PLA2s from different species.

Effects of a cardiotoxin from Naja naja kaouthia venom on skeletal muscle: Involvement of calcium-induced calcium release, sodium ion currents and phospholipases A 2 and C

Snake venom cardiotoxin (CTX) fractions induce contractures of skeletal muscle and hemolysis of red blood cells. The fractions also contain trace amounts of venom-derived phospholipase A 2 (PLA 2) contamination and activate tissue phospholipase C (PLC) activity. The present study examines the mechanisms of action of a CTX fraction from Naja naja kaouthia venom in skeletal muscle. Sphingosine competitively antagonized CTX-induced red blood cell hemolysis, but not skeletal muscle contractures. CTX rapidly lowered the threshold for Ca 2+-induced Ca 2+ release in heavy sarcoplasmic reticulum fractions, as monitored with arsenazo III. There was also a slower time-dependent reduction of Na + currents, as assessed by whole cell patch-clamp techniques. The CTX fractions elevated levels of free fatty acids and diacylglycerol for 2 hr in primary cultures of human skeletal muscle by a combined action of venom-derived PLA 2 contamination in the fraction and activation of endogenous PLC activity. The activation of tissue PLC activity could be readily distinguished from the contribution of the venom PLA 2 by p-bromophenacyl bromide treatment of CTX fractions. The mechanism of action involved in contractures of skeletal muscle appears to be related to the immediate and specific effect of CTX (Ca 2+ release by the sarcoplasmic reticulum), while the mechanisms involved in hemolysis of red blood cells and decreased Na + currents in skeletal muscle most likely relate to long-term effects on lipid metabolism.

Contribution of bee venom phospholipase A 2 contamination in melittin fractions to presumed activation of tissue phospholipase A 2

Melittin from bee venom has been suggested to activate tissue phospholipase A 2 (PLA 2) activity, and subsequently has been used as a specific PLA 2 probe. The melittin in most cases was obtained commercially and used without further purification or treatment. To test the hypothesis that commercially obtained melittin specifically activates tissue PLA 2, we radiolabeled the lipids of immortalized epithelial cells by incubating the cells for 22 hr with 14C-linoleic acid. The cells were then incubated with 2 μM melittin, 2 nM bee venom PLA 2, 2 μM melittin treated with p-bromophenacyl bromide ( p-BPB) or PLA 2 plus p-BPB-treated melittin. Lipids were extracted and separated by thin-layer chromatography. The radioactivity in each lipid fraction was then quantitated. The melittin-stimulated PLA 2 activity observed in cells was primarily associated with phosphatidylcholine. Fatty acid release was decreased by 75% when the melittin fraction was pretreated with p-BPB to reduce contaminating venom PLA 2 activity. Adding PLA 2 to the p-BPB-treated melittin at an amount about equal to the original contamination (0.1%) resulted in the same PLA 2 activity in cells as observed with the untreated melittin fraction. These findings suggest that bee venom PLA 2 contamination, even at very low levels, can account for approximately 75% of the PLA 2 activity in cells treated with commercial melittin fractions.

Pharmacologic modulation of D-49 phospholipase A2-induced paw edema in the mouse.

Paw edema was produced in CD-1 mice by the injection of 0.3 micrograms of snake venom PLA2 (A.p. piscivorus D-49) into the hind paw. Edema peaked at 10 min, remained elevated until 60 min, and then declined slowly. The PLA2 inhibitors, luffariellolide and aristolochic acid, reduced the edema but only when coinjected with the PLA2. The histamine/serotonin antagonists were the most effective drug class against PLA2-induced paw edema. The PAF antagonists, CV-6202 (iv) and kadsurenone (coinjected) reduced the PLA2-induced edema, whereas high doses of the corticosteroids, dexamethasone and hydrocortisone, were also effective. NSAIDs only partially inhibited the paw edema. The LO/CO inhibitors yielded varying activities, with only BW755C and NDGA inhibiting the edema. These results suggest that PLA2 induces paw edema in the mouse via the action of several classes of inflammatory mediators.

A model to explain the pharmacological effects of snake venom phospholipases A 2

R. M. Kini and H. J. Evans. A model to explain the pharmacological effects of snake venom phospholipases A 2. Toxicon 27, 613–635, 1989.—Snake venom phospholipase A 2 enzymes induce a wide variety of pathological symptoms in animals, despite sharing a common catalytic activity and similar structural features with nontoxic mammalian pancreatic enzymes. A hypothetical model is described to explain how specific pharmacological effects, such as presynaptic neurotoxicity, cardiotoxicity, myotoxicity, anticoagulant and platelet effects are exhibited by venom PLA 2 enzymes. The model is an effort to elucidate many controversial and contradictory observations which have previously been difficult to interpret. The essential feature of the model is the targeting of venom PLA 2 enzymes to the specific tissue or cell due to their affinity towards specific proteins, rather than lipid domains. After the initial binding, PLA 2 enzymes induce various pharmacological effects by mechanisms which are either dependent or independent of their enzymatic activity. The model and its predicted target proteins thus provide a new focus for toxin research

Involvement of phospholipase A 2 in the regulation of [ 3H]hemicholinium-3 binding

We have examined the effects of exogenous phospholipase A 2 (PLA 2) on the sodium- dependent high-affinity choline uptake mechanism as assessed by the specific binding of [ 3H]hemi- cholinium-3 ([ 3H]HCh-3). Incubation of striatal synaptic membranes with bee venom PLA 2 resulted in a concentration-dependent increase in the specific binding of [ 3H]HCh-3. The effect of PLA 2 on [ 3H]HCh-3 binding was inhibited by quinacrine, a PLA 2 inhibitor, and by removal of calcium. Scatchard analysis revealed that the observed changes in binding reflected a 2-fold increase in both the capacity and affinity of [ 3H]HCh-3 for its binding site. Choline and tN-butylcholine inhibited the specific binding of [ 3H]HCh-3 in both control and PLA 2-treated membranes with similar potency. When a low concentration of PLA 2 was incubated with the striatal synaptosomes, a small but significant increase in high-affinity [ 3H]choline uptake was observed. However, higher concentrations of PLA 2, which further increased the specific binding of [ 3H]HCh-3, caused a reduction of [ 3H]choline uptake, apparently due to disruption of synaptosomal integrity by PLA 2. Finally, potassium depolarization- and PLA 2-induced increases in specific [ 3H]HCh-3 binding were not additive. These results suggest a possible role for endogenous PLA 2 in the calcium-dependent regulation of sodium-dependent high-affinity choline uptake.

Inactivation of phospholipase A 2 by manoalide : Localization of the manoalide binding site on bee venom phospholipase A 2

The marine natural product manoalide (MLD), a potent inhibitor of phospholipases, completely inactivates bee venom phospholipase A 2 (PLA 2) by an irreversible mechanism. It has been proposed [K.B. Glaser and R.S. Jacobs, Biochem. Pharmac. 36, 2079 (1987)] that the reaction of MLD with PLA 2 may involve the selective reactivity of MLD to a peptide sequence, possibly a Lys-X-X-Lys peptide. Localization of the MLD binding site on bee venom PLA 2 demonstrated that upon MLD modification of bee venom PLA 2 the only change in amino acid content was an apparent loss of Lys, corresponding to approximately three of the eleven Lys residues present. Selective chemical modification of Lys residues with [ 14C]maleic anhydride demonstrated that all eleven Lys residues on bee venom PLA 2 were accessible to this reagent (11.6 mol maleyl group incorporated/mol of PLA 2). Pretreatment of PLA 2 with MLD(<0.7% residual activity) resulted in a molar ratio of 8.7, also consistent with the loss of three Lys residues upon modification by MLD. Reverse phase high performance liquid chromatography (RP-HPLC) of the cyanogen bromide (CNBr) digestion product of MLD-treated PLA 2 produced three peaks ( A 280). The second peak showed the most intense absorbance at 434 nm. This material corresponded to residues 81–128, as determined by gas-phase microsequence analysis. Sequencing failure was observed at Lys-88 in the MLD-treated fragment. The control carboxy-methylated-PLA 2 fragment corresponding to residues 81–128 sequenced beyond Lys-88 without significant change in the expected yield. These data suggest that Lys-88 may correspond to one of the three MLD-modified Lys residues. The minor absorbance at 434 nm of the CNBr fragments containing residues 42–80 and 1–36 as compared to the fragment of residues 81–128 suggests that the major MLD binding fragment resides in residues 81–128.

Comparison of enzymatic and pharmacological activities of lysine-49 and aspartate-49 phospholipases A 2 from Agkistrodon piscivorus piscivorus snake venom

The basic Lys-49 phospholipase A 2 (PLA 2) from Agkistrodon pisciuorus piscivorus venom is homologous to the basic Asp-49 PLA 2 from the same venom as well as other snake venom PLA 2 enzymes. It differs, however, in several respects, most important being replacement of the previously invariant Asp-49 at the calcium binding site by Lys, resulting in a reversed order of addition of calcium and phospholipid, phospholipid binding first. Although the preferences for phospholipid substrates of the two enzymes are identical, the apparent V max of the Lys-49 PLA 2 was only 1.4 to 3% that of the Asp-49 enzyme. Similarly, the Lys-49 PLA 2, compared to the Asp-49 PLA 2 had < 3% of the intraventricular lethal potency and 4% of the anticoagulant activity. The intravenous lethal potency of the Lys-49 enzyme was 20% that of the Asp-49 PLA 2 and both had little direct hemolytic activity. In contrast, both enzymes were approximately equipotent on the phrenic nerve-diaphragm preparation and on the isolated ventricle strip of the heart. On the cardiac and neuromuscular preparations, the effects of the Asp-49 PLA 2 were accompanied by hydrolysis of phosphatidylcholine and phosphatidylethanolamine, whereas no phospholipid hydrolysis was observed with the Lys-49 PLA 2. Evaluation of the present results, along with earlier findings using Asp-49 PLA 2 enzymes from Naja nigricollis, Hemachatus haemachatus and Naja naja atra venoms, allows us to conclude that: (1) The A. p. piscivorus Asp-49 PLA 2 enzyme resembles the Asp-49 enzymes fron N. n. atra and H. haemachatus. In contrast, the A. p. piscivorus Lys-49 PLA 2 has much lower enzymatic and anticoagulant activities than the Asp-49 enzymes, but equal cardiotoxic and junctional effects. (2) In contrast to some previous suggestions, basic PLA 2 enzymes are not necessarily more toxic than neutral or acidic enzymes. (3) Pharmacological effects upon the heart and phrenic nerve-diaphragm preparation correlate neither with in vitro measurements of PLA 2 activity nor with actual levels of phospholipid hydrolysis in the heart or diaphragm. This suggests that PLA 2 enzymes exert effects independent of phospholipid hydrolysis.

Contracture induction by snake venom cardiotoxin in skeletal muscle from humans and rats

J. E. Fletcher and F. H. Lizzo. Contracture induction by snake venom cardiotoxin in skeletal muscle from humans and rats. Toxicon 25, 1003 – 1010, 1987. — Contracture responses to cardiotoxin (CTX) from Naja naja kaouthia venom were investigated in rat and human skeletal muscle of similar fiber type distribution to determine species differences in mechanism of action. Rat diaphragm strips and human vastus lateralis preparations were directly stimulated in a tissue bath. The calcium dependence of toxin action, synergism between CTX and phospholipase A 2 (PLA 2) activity and roles of Na + + K +-ATPase activity and the sarcoplasmic reticulum Ca 2+ stores in contracture induction were examined. The threshold of contracture to CTX was decreased in human and rat muscle when Sr 2+ was substituted for Ca 2+ in the bathing medium. In rat, but not in human muscle the threshold of contracture to CTX was decreased in a medium in which Ca 2+ had been omitted. The decrease in contracture threshold may relate to toxin binding. The maximum height of contracture for preparations from humans, but not for those from rats was considerably depressed in a medium in which Ca 2+ had been omitted. Exogenously added bee venom PLA 2 acts synergistically with CTX in skeletal muscle in a manner similar to that in erythrocytes. Ouabain (100 μM) did not elicit contractures in any of the media tested nor affect CTX-induced contractures in Sr 2+-containing medium. Dantrolene antagonized CTX-induced contractures, suggesting a role for Ca 2+ derived from the sarcoplasmic reticulum in CTX action. The species difference in CTX action may reflect differences in the relative contribution of Ca 2+ from the sarcolemma and sarcoplasmic reticulum to the contracture.