Modeling analysis of the global and microscopic distribution of immunoglobulin G, F(ab')2, and Fab in tumors.

In order to understand the pharmacology of monoclonal antibodies and their conjugates, one must consider both global and microscopic aspects of antibody distribution. Here we present an analysis of antibody distribution in tumors based on the following factors: (a) molecular weight and valence of the antibody; (b) global pharmacokinetic profile following i.v. bolus injection; (c) penetration through the vascular wall; (d) diffusive and convective transport through interstitial space in the tumor; (e) antigen-antibody interaction; (f) antibody metabolism. Partial differential equations were developed to incorporate these factors and then solved numerically using parameter values from animal experiments, from clinical protocols at our institution, from studies of antibody binding characteristics in vitro, and from the literature. Salient findings from this model are that (a) antigen-antibody interaction in the tumor can retard antibody percolation away from blood capillaries, thus constituting a "binding site barrier"; (b) high antibody affinity tends to decrease antibody penetration and result in a more heterogeneous distribution; (c) high molecular weight [IgG greater than F(ab')2 greater than Fab] slows percolation and results in less uniform spatial distribution; (d) the average antibody concentration in the tumor does not increase linearly with antibody dose; (e) raising the rate of antibody metabolism results in low concentration and poor percolation; (f) perhaps most interesting, there is predicted to be a range of antibody dose and affinity within which the specificity ratio and average concentration could be kept high while limiting the heterogeneity of distribution. PERC, the computer program package developed for these analyses, provides a convenient and flexible way to assess the impact of global and microscopic parameters on the distribution of immunoglobulin in tumors. For calculations presented here, the input data were obtained from experimental sources, and qualitative features of the output proved consistent with the few interpretable observations available. However, detailed validation would require much more data than are currently at hand. The mathematical findings should therefore be considered as aids to concept development and as a set of null hypotheses with which to guide experimentation. Experiments and simulations will continue in tandem. It should be noted that the PERC package (and also the general principles delineated here) can be applied as well to biological ligands other than antibodies.

Phenytoin increases specific triacylglycerol fatty esters in skeletal muscle from horses with hyperkalemic periodic paralysis.

Previous studies have demonstrated that phenytoin decreases the levels of triacylglycerols in several tissues other than skeletal muscle. Since phenytoin is clinically effective in several skeletal muscle disorders, triacylglycerol metabolism in skeletal muscle from four normal Quarter horses and four Quarter horses with hyperkalemic periodic paralysis was examined. The horses with hyperkalemic periodic paralysis had low levels of 18:3 in the phospholipids, low levels of 16:0, 16:1 and 18:3 in the free fatty acids and low levels of 20:4 in triacylglycerols. Triacylglycerol levels were increased in skeletal muscle from seven (three controls, four hyperkalemic periodic paralysis) of the eight horses on treatment with oral phenytoin for one week. Instead of an increase in all fatty ester types only 16:0, 16:1, 18:1 and 18:2 were significantly increased. Total lipid phosphorus and the distribution of phospholipid fatty esters and free fatty acids were not significantly altered by phenytoin treatment in most cases. An alteration in triacylglycerol metabolism by phenytoin was also observed in primary cultures of normal equine skeletal muscle radiolabeled with 18:1, but not in those radiolabeled with 18:2. These findings suggest that phenytoin does not just increase the levels of triacylglycerol in skeletal muscle, but alters the utilization and incorporation of fatty esters. These findings suggest a potential involvement of triacylglycerol metabolism in the clinical efficacy of phenytoin in hyperkalemic periodic paralysis.

The binding of palmitoyl-CoA to bovine serum albumin.

Bovine serum albumin (BSA) is routinely utilized in vitro to prevent the adverse detergent effects of long-chain acyl-CoA esters (i.e., palmitoyl-CoA) in enzyme assays. Determination of substrate saturation kinetics in the presence of albumin would only be valid if the relationship between bound and free substrate concentrations was known. To elucidate the relationship between bound and free palmitoyl-CoA concentrations in the presence of BSA, several different techniques including equilibrium dialysis, equilibrium partitioning, fluorescence polarization and direct fluorescence enhancement were investigated. Direct fluorescence enhancement using a custom synthesized fluorescent probe, 16-(9-anthroyloxy)palmitoyl-CoA (AP-CoA), was the best approach to this question. Measurement of the relationship between mol of palmitoyl-CoA bound per mol of BSA (nu) versus -log[free palmitoyl-CoA] revealed that the binding of palmitoyl-CoA to BSA, like palmitate was nonlinear, suggesting the presence of more than one class of acyl-CoA binding sites. Computer analyses of the binding data gave a best fit to the 2,4 two-class Scatchard model, suggesting the presence of two high-affinity primary binding sites (k1 = (1.55 +/- 0.46) x 10(-6) M-1) and four lower affinity secondary binding sites (k2 = (1.90 +/- 0.09) x 10(-8) M-1). Further analyses using the six parameter stoichiometric (stepwise) ligand binding model supports the existence of six binding sites with the higher affinities associated with the binding of the first mole of palmitoyl-CoA and weaker binding occurring after the first two sites are occupied. The association constants from this model of multiple binding diminish sequentially (i.e., K1 greater than K2 greater than K3 greater than...greater than or equal to K6), suggesting that each mol of long-chain acyl-CoA binds to BSA with decreasing affinities.

A modeling analysis of monoclonal antibody percolation through tumors: a binding-site barrier.

For successful use of radiolabeled monoclonal antibodies (MAbs) for diagnosis and therapy, it is helpful to understand both global and microscopic aspects of antibody biodistribution. In this study, antibody distribution in a tumor is simulated by splicing together information on global pharmacokinetics: transport across the capillary wall, diffusive penetration through the tumor interstitial space, and antigen-antibody interaction. The geometry simulated corresponds to spherical nodules of densely packed tumor cells. This modeling analysis demonstrates that: 1) antigen-antibody binding in tumors can retard antibody percolation; 2) high antibody affinity at a given dose tends to decrease antibody percolation because there are fewer free antibody molecules. The result is a more heterogeneous distribution; 3) the average antibody concentration in the tumor does not increase linearly with affinity; and 4) increasing antibody dose leads to better percolation and more uniform distribution. This mathematical model and the general principles developed here can be applied as well to other biologic ligands.

Antibodies having markedly different effects on enzymatic activity and induction of acetylcholine release by two presynaptically-acting phospholipase A2 neurotoxins.

The enzymatic and acetylcholine-releasing activities of two presynaptically-acting phospholipase A2 neurotoxins (pseudexin B and scutoxin) were studied in a synaptosomal fraction. Scutoxin (100 nM) induced greater [14C]acetylcholine release than did pseudexin B (100 nM). Both toxins caused fatty acid production in the synaptosomal fraction, although pseudexin B was more active than scutoxin. One monoclonal antibody raised against pseudexin B (#4) had no effect on the enzymatic activity of either pseudexin B or scutoxin. Two other monoclonal antibodies (#3 and #7), also raised against pseudexin B, antagonized the enzymatic activity of pseudexin B and scutoxin. Monoclonal antibody #3 was more effective than #7 in reducing the amount of acetylcholine released by the toxins, whereas #7 was more effective than #3 in reducing fatty acid production. Although antibody #3 caused complete inhibition of phospholipase A2 activity of pseudexin B on purified substrates, it only reduced phospholipase A2 activity by 35% in synaptosomes. These findings support the hypothesis that gross phospholipase A2 activity does not play a role in stimulation of acetylcholine release by the presynaptically-acting phospholipase A2 neurotoxins.

Possible mechanisms of action of cobra snake venom cardiotoxins and bee venom melittin.

Cobra snake venom cardiotoxins and bee venom melittin share a number of pharmacological properties in intact tissues including hemolysis, cytolysis, contractures of muscle, membrane depolarization and activation of tissue phospholipase C and, to a far lesser extent, an arachidonic acid-associated phospholipase A2. The toxins have also been demonstrated to open the Ca2+ release channel (ryanodine receptor) and alter the activity of the Ca(2+)+Mg(2+)-ATPase in isolated sarcoplasmic reticulum preparations derived from cardiac or skeletal muscle. However, a relationship of these actions in isolated organelles to contracture induction has not yet been established. The toxins also bind to and, in some cases, alter the function of a number of other proteins in disrupted tissues. The most difficult tasks in understanding the mechanism of action of these toxins have been dissociating the primary from secondary effects and distinguishing between effects that only occur in disrupted tissues and those that occur in intact tissue. The use of cardiotoxin and melittin fractions contaminated with trace ('undetectable') amounts of venom-derived phospholipases A2 has continued to be common practice, despite the problems associated with the synergism between the toxins and enzymes and the availability of methods to overcome this problem. With adequate precautions taken with regard to methodology and interpretation of results, the cobra venom cardiotoxins and bee venom melittin may prove to be useful probes of a number of cell processes, including lipid metabolism and Ca2+ regulation in skeletal and cardiac muscle.

Presynaptically acting snake venom phospholipase A2 enzymes attack unique substrates.

Synaptosomes were incubated with bovine serum albumin (BSA) to examine whether the presynaptic action of snake venom phospholipase A2 (PLA2) toxins is due either to the release of fatty acids resistant to extraction by BSA or to the liberation of a specific fatty acid type. In the presence of BSA (0.5% or 1.0%) two PLA2 enzymes from Naja naja atra and Naja naja kaouthia snake venoms that do not have a predominant presynaptic action at the neuromuscular junction (PS-) did not stimulate acetylcholine (ACh) release from synaptosomes. In contrast, two PLA2 enzymes (beta-bungarotoxin, scutoxin) that do have a predominant presynaptic action at the neuromuscular junction (PS+) did stimulate ACh release. BSA did not antagonize PS- enzymes by more efficiently extracting the fatty acids produced by these enzymes relative to PS+ enzymes. While absolute amounts of total and unsaturated fatty acid produced overlapped for the PS- and PS+ enzymes, the two PS+ enzymes produced a significantly greater absolute amount and relative percentage of palmitic acid (16:0) than did either of the PS- enzymes. However, the levels of free palmitic acid remaining in the synaptosomes where they would exert effects on ACh release were similar for the N. n. kaouthia PLA2 (PS-) and beta-bungarotoxin (PS+). Therefore, the total (supernatant plus synaptosomal) amount of palmitic acid produced per se did not account for stimulation of ACh release, since the greater amounts produced by the PS+ enzymes were removed from the synaptosomes by BSA. The production of higher levels of palmitic acid suggests either that PS+ enzymes gain access to sites containing phospholipid substrates unavailable to the PS- enzymes, or that they have a different substrate preference. These findings suggest new possibilities for the mechanism of PS+PLA2 action, including site-directed enzymatic activity and protein acylation.

Effects of beta-bungarotoxin and Naja naja atra snake venom phospholipase A2 on acetylcholine release and choline uptake in synaptosomes.

Beta-bungarotoxin is a potent presynaptically acting snake venom toxin that exhibits phospholipase A2 activity. We compared the effects of beta-bungarotoxin and a less toxic snake venom phospholipase A2 on synaptosomal 3H-acetylcholine release and 3H-choline uptake. The purpose of these experiments was to study the mode by which beta-bungarotoxin inhibits 3H-acetylcholine release in this preparation. Under non-depolarizing conditions, both beta-bungarotoxin and Naja naja atra phospholipase A2 stimulated 3H-acetylcholine release from a synaptosomal fraction preloaded with 3H-choline. Beta-bungarotoxin was more potent, but less efficacious, than N. naja atra phospholipase A2. In contrast, both toxins inhibited 3H-acetylcholine release from the synaptosomal fraction incubated with 3H-choline after toxin exposure. In agreement with the results obtained by monitoring acetylcholine release, beta-bungarotoxin and N. naja atra phospholipase A2 appeared to block 3H-choline uptake into the synaptosomal fraction non-competitively. Although the toxins may cause the release of unlabeled choline from synaptosomes, the block of labeled choline uptake could not be explained by decreased specific activity of 3H-choline in the bathing medium. Therefore, beta-bungarotoxin and N. naja atra phospholipase A2 block 3H-acetylcholine release from synaptosomes indirectly by inhibiting the uptake of 3H-choline necessary for 3H-acetylcholine synthesis. In comparing these results using 3H-choline to those in the literature obtained with deuterated choline, there appears to be a difference in apparent toxin action that relates to the type of label (3H or 2H) attached to choline.

LYS49 phospholipase A2 myotoxins lyse cell cultures by two distinct mechanisms.

Three Lys49 phospholipase A2 (PLA2) myotoxins from Agkistrodon contortrix laticinctus (ACLMT), Bothrops jararacussu (bothropstoxin-I) and Bothrops asper (myotoxin II) snake venoms are enzymatically inactive on artificial substrates, yet addition of these toxins to cell cultures causes the release of fatty acids derived from the hydrolysis of membrane phospholipids. Bothropstoxin-I treated with p-bromophenacyl bromide is no longer enzymatically active on cell cultures, suggesting the toxin, not tissue PLA2, may hydrolyze the phospholipids. The NB41A3 cell line is sensitive to lysis by ACLMT by two separate mechanisms. The first mechanism is predominant at lower concentrations of ACLMT (0.1-0.5 microM) and over long incubation periods (24 h) with toxin. This mechanism is antagonized by methylprednisolone (MePDN). The second is predominant at higher concentrations of toxin (1-5 microM) incubated over a short period (1 h) and is not antagonized by MePDN. There is no correlation between enzymatic activity and toxicity at the higher concentrations (5 microM; 1 h) when the enzymatic activity of ACLMT is compared with a noncytolytic PLA2 from Naja naja atra venom (1 microM). However, over a 24 h period, triglyceride formation relative to fatty acids remaining free is about 10-fold greater for ACLMT (ratio about 40:1) than for the PLA2 from Naja naja atra venom (ratio about 4:1), suggesting the two enzymes act on substrates associated with different cellular compartments under this condition. Therefore, two mechanisms of Lys49 PLA2-induced myonecrosis exist and these are dependent on toxin concentration. The MePDN-sensitive mechanism associated with triglyceride accumulation correlates with myotoxicity.

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

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 A2 (PLA2) activity and roles of Na+ + K+-ATPase activity and the sarcoplasmic reticulum Ca2+ stores in contracture induction were examined. The threshold of contracture to CTX was decreased in human and rat muscle when Sr2+ was substituted for Ca2+ in the bathing medium. In rat, but not in human muscle the threshold of contracture to CTX was decreased in a medium in which Ca2+ 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 Ca2+ had been omitted. Exogenously added bee venom PLA2 acts synergistically with CTX in skeletal muscle in a manner similar to that in erythrocytes. Ouabain (100 microM) did not elicit contractures in any of the media tested nor affect CTX-induced contractures in Sr2+-containing medium. Dantrolene antagonized CTX-induced contractures, suggesting a role for Ca2+ derived from the sarcoplasmic reticulum in CTX action. The species difference in CTX action may reflect differences in the relative contribution of Ca2+ from the sarcolemma and sarcoplasmic reticulum to the contracture.

Species difference in modulation of calcium release by Naja naja kaouthia snake venom cardiotoxin in terminal cisternae from human and equine skeletal muscle.

The modulation of Ca2+ release by a cardiotoxin (CTX) from Naja naja kaouthia snake venom was examined in terminal cisternae-containing fractions from equine and human skeletal muscle. Pretreatment with CTX (10 microM) decreased by 27% (human muscle), or had no effect on (equine muscle), the threshold of Ca(2+)-induced Ca2+ release. If terminal cisternae fractions were first preloaded with Ca2+ to greater than 65% of the threshold of Ca(2+)-induced Ca2+ release and then CTX added, an immediate and sustained release of Ca2+ occurred in preparations from both species. Addition of CTX after a Ca2+ preload of less than 60% of the threshold of Ca(2+)-induced Ca2+ release did not elicit Ca2+ release in preparations from either species. Ruthenium red (10 microM) antagonized CTX-induced Ca2+ release, whereas dantrolene (10 microM) did not. These findings suggest that the effects of CTX on the Ca2+ release channel are dependent on Ca2+ preload and that CTX may be an important probe of the Ca(2+)-modulated Ca2+ release process and in understanding regulation of Ca2+ release in skeletal muscle from different species.

Myotoxin a reduces the threshold for calcium-induced calcium release in skeletal muscle.

Myotoxin a, isolated from the venom of the prairie rattlesnake Crotalus viridis viridis, induces necrosis in skeletal muscle. In isolated organelles, it has been reported that myotoxin a reduces Ca2+ uptake into the sarcoplasmic reticulum. The effects of the toxin on Ca2+ regulation were examined in heavy sarcoplasmic reticulum fractions from human and equine skeletal muscle. Ca2+ uptake and release (the threshold of Ca(2+)-induced Ca2+ release) were examined by dual wavelength spectrophotometry. The toxin lowered the threshold of Ca(2+)-induced Ca2+ release in a dose-dependent manner (1-10 microM) and this effect was antagonized by ruthenium red, a Ca2+ release channel blocker. Ca2+ uptake into equine heavy sarcoplasmic reticulum was not decreased by myotoxin a (10 microM) when Ca2+ release was blocked by ruthenium red. [3H]Ryanodine binding to equine heavy sarcoplasmic reticulum was converted from a relatively low affinity state to a higher affinity state by myotoxin a. These results suggest that the dominant effect of myotoxin a is to increase the Ca2+ sensitivity for the opening of the calcium release channel (ryanodine receptor). Myotoxin a may prove to be a useful tool to probe the modulation of calcium release in sarcoplasmic reticulum fractions.

Contribution of bee venom phospholipase A2 contamination in melittin fractions to presumed activation of tissue phospholipase A2.

Melittin from bee venom has been suggested to activate tissue phospholipase A2 (PLA2) activity, and subsequently has been used as a specific PLA2 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 PLA2, 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 microM melittin, 2nM bee venom PLA2, 2 microM melittin treated with p-bromophenacyl bromide (p-BPB) or PLA2 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 PLA2 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 PLA2 activity. Adding PLA2 to the p-BPB-treated melittin at an amount about equal to the original contamination (0.1%) resulted in the same PLA2 activity in cell as observed with the untreated melittin fraction. These findings suggest that bee venom PLA2 contamination, even at very low levels, can account for approximately 75% of the PLA2 activity in cells treated with commercial melittin fractions.

Cardiotoxin 1 from cobra (Naja naja atra) venom causes necrosis of skeletal muscle in vivo.

Cardiotoxin 1 from cobra (Naja naja atra) venom was tested for its ability to cause necrosis of skeletal muscle cells after i.m. injection into mice. Light and electron microscopic examination of tissue indicated that the toxin caused necrosis of skeletal muscle as early as 30 min after injection. The plasma membranes of affected cells were ruptured in the area of delta lesions, and the myofibrils were condensed into dense clumps alternating with clear areas containing elements of the sarcotubular system and damaged mitochondria. By 24 hr the affected cells appeared as empty 'bags' containing only remnants of myofibrils and swollen mitochondria. To eliminate the possibility that the necrosis was due to contaminating phospholipase A2 (PLA2) activity of the sample, the sample was treated with p-bromophenacyl bromide (p-BPB), a known inhibitor of PLA2 activity. The p-BPB-treated preparation caused myonecrosis in vivo in mice, and the treatment caused a significant decrease in the release of fatty acids and no detectable lysophospholipid in human muscle cell cultures treated in vitro with the preparation, indicating the lack of PLA2 activity. Additionally, purified PLA2 from the same venom failed to cause myonecrosis in vivo at doses equal to or ten times the estimated contaminating concentration. Thus, it is concluded that cardiotoxin 1 from Naja naja atra venom causes necrosis of skeletal muscle cells in vivo upon i.m. injection.

Bovine serum albumin does not completely block synaptosomal cholinergic activities of presynaptically acting snake venom phospholipase A2 enzymes.

Bovine serum albumin (BSA), which binds fatty acids, was used to test the contribution of free fatty acid to the presynaptic toxicity of phospholipase A2 (PLA2) enzymes. The effects of BSA on inhibition of [14C]choline uptake and stimulation of [14C]acetylcholine (ACh) release in synaptosomes by PLA2 enzymes that do not have a predominant presynaptic action at the neuromuscular junction (PS-) were compared with those on the cholinergic actions of PLA2 enzymes that do have a predominant presynaptic action at the neuromuscular junction (PS+). The inhibition of choline uptake by the Naja naja atra PLA2, a PS- PLA2, was completely antagonized by BSA (0.5%); whereas that by beta-bungarotoxin, a PS+ PLA2, was unaffected by BSA. The inhibition of choline uptake by two other PS+ PLA2 toxins (scutoxin and pseudexin) was partially antagonized by BSA. The effects of the PLA2 enzymes were antagonized in the same manner by BSA whether on Na(+)-dependent or on Na(+)-independent choline uptake. Likewise, the stimulation of ACh release by two PS- PLA2 enzymes (from Naja naja atra and Naja naja kaouthia snake venoms) was completely blocked by BSA; whereas that by beta-bungarotoxin was unaffected and that by scutoxin and pseudexin was only partially antagonized by BSA. The results suggest that the PS- PLA2 enzymes are completely dependent on fatty acid production for their cholinergic toxicity and that BSA can be used to investigate further the neurotoxic mechanisms of PS+ PLA2 enzymes in synaptosomes.

Factors influencing the hemolysis of human erythrocytes by cardiotoxins from Naja naja kaouthia and Naja naja atra venoms and a phospholipase A2 with cardiotoxin-like activities from Bungarus fasciatus venom.

The effects of red blood cell age and incubation conditions (temperature, divalent cation type and concentration, pH and glucose) on hemolysis induced by cardiotoxin fractions from Naja naja atra and Naja naja kaouthia venoms, a phospholipase A2 with cardiotoxin-like activities from Bungarus fasciatus venom and bee venom phospholipase A2 were examined. Hemolysis by the snake venom toxins was dependent on red blood cell age (aged more susceptible than fresh) and the temperature of incubation (37 degrees C greater than 20 degrees C). Divalent cations at 0.5-2.0 mM enhanced (Ca2+) or slightly decreased (Sr2+, Ba2+) hemolysis due to N. n. kaouthia and N. n. atra toxins, and greatly decreased (Ca2+, Sr2+, Ba2+) hemolysis by these toxins at higher concentrations (5-40 mM). For the B. fasciatus phospholipase A2, Ba2+ and Sr2+ could not fully support hemolysis in any concentration while both low (less than 0.5 mM) and high (greater than 40 mM) Ca2+ enhanced hemolysis. Bee venom phospholipase A2 only induced hemolysis (greater than 10% at greater than 40 mM) at high concentrations of Ca2+. Increasing the pH from 7.5 to 8.5 greatly increased the levels of hemolysis by the snake venom toxins and enzyme. Glucose (5.3 mM) increased hemolysis by the snake venom components at low concentrations of divalent cations (2 mM) and slightly decreased hemolysis at high concentrations (40 mM). Treatment with p-bromophenacyl bromide abolished phospholipase A2 activity of bee venom and B. fasciatus phospholipases, but did not affect hemolytic potency of N. n. kaouthia or B. fasciatus toxins. A similar mechanism, which is independent of phospholipase A2 activity, may be involved in hemolysis by the N. n. kaouthia and N. n. atra cardiotoxins. The B. fasciatus cardiotoxin-like phospholipase A2 appears to have two mechanisms of hemolysis; the first is similar to that of the two typical cardiotoxins and the second appears dependent on phospholipase A2 activity and is only evident at high Ca2+ concentrations.

Effects of divalent cations on snake venom cardiotoxin-induced hemolysis and 3H-deoxyglucose-6-phosphate release from human red blood cells.

At a low concentration of Naja naja kaouthia cardiotoxin (3 microM) Ca2+, Sr2+ and Ba2+ (2 mM), had little to no effect on 3H-deoxyglucose-6-phosphate (3H-dGlu-6-p) or hemoglobin release. At higher concentrations of N. n. kaouthia cardiotoxin (greater than or equal to 10 microM), Ca2+ (2 mM), but not Sr2+ or Ba2+, significantly enhanced 3H-dGlu-6-p and hemoglobin release. Mn2+ (2 mM) almost completely inhibited 3H-dGlu-6-p release and hemolysis at both the 3 microM and 10 microM concentrations of cardiotoxin. At a fixed concentration of N. n. kaouthia cardiotoxin (3 microM). Ca2+ at low concentrations (0.5 mM) enhanced 3H-dGlu-6-p and hemoglobin release, but at higher concentrations caused a dose-dependent inhibition of cardiotoxin action. The cardiotoxin from N. n. kaouthia venom (3 microM) induced 3H-dGlu-6-p release and hemolysis release with similar time courses and to similar extents. 3H-dGlu-6-p release induced by cardiotoxin was greatly enhanced as the pH of the medium was increased from 7.0 to 8.5. Similarities between 3H-dGlu-6-p and hemoglobin release do not support opening of pores in the plasmalemma of all red blood cells as the mode of action of cardiotoxins, but suggests that complete lysis of a subpopulation of cells occurs. Cardiotoxins have two components of lysis, only one of which is Ca2+-dependent. The Ca2+-dependent lysis is only evident at higher cardiotoxin concentrations and is likely due to trace phospholipase A2 contamination in the toxin fraction. Mn2+ is an effective antagonist of cardiotoxin action.

Lysine 49 phospholipase A2 proteins.

The structures of several K49 PLA2 proteins have been determined and these differ as a group in several regions from the closely related D49 PLA2 enzymes. One outstanding difference is the presence of a high number of positively charged residues in the C-terminal 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 fiber types possibly more sensitive than others. Whereas it is clear that the K49 PLA2 myotoxins lyse the plasma membrane of the affected 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 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 artificial 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.

Interactions in red blood cells between fatty acids and either snake venom cardiotoxin or halothane.

Phospholipase A2 (PLA2) activity enhances snake venom cardiotoxin (CTX)-induced and general anesthetic (halothane)-induced hemolysis of red blood cells. In the case of halothane-induced hemolysis, this effect appears to be related primarily to free fatty acids. In the present study, the interaction between CTXs and halothane and the effects of different free fatty acids on cardiotoxin and halothane-induced hemolysis were examined. The hemolytic actions of halothane and a CTX from Naja naja kaouthia venom were examined in erythrocytes with different phospholipid and free fatty acid composition from five species. The extent of hemolysis by CTX or halothane was dependent upon the species examined and appeared to be inversely related to the amount of saturated free fatty acid in the membrane. The order of susceptibility of red blood cells from five species to hemolysis was similar for halothane- and N. n. kaouthia CTX-induced hemolysis, but very different for osmotic fragility. The slope of the hemolysis dose-response curve was considerably steeper for halothane than for CTX. Hemolysis due to N. n. kaouthia CTX was greatly increased by halothane in erythrocytes from humans and horses and to a lesser extent in erythrocytes from sheep, goats and cows. Hemolysis induced by halothane and the N. n. kaouthia or Naja naja atra CTXs was enhanced by unsaturated fatty acids. In contrast, hemolysis induced by halothane was decreased and that caused by the two CTXs was relatively unaffected by saturated fatty acids. Halothane and CTXs differ in their exact mechanisms, but appear to act upon similar fatty acid-sensitive processes.