Sensitivity of cultured embryonic heart cells to cardiotoxin obtained from Naja naja siamensis venom.

Clumps of embryonic chick heart cells in culture were exposed to cardiotoxin prepared by Sephadex CM-50 fractionation from snake (Naja naja siamensis) venom. Cardiotoxin irreversibly depolarized these cells. More of the cells cultured from 4-day embryos, than from 12–13-day embryos, continued to beat spontaneously after 30 min exposure to the toxin at a concentration of 15–20 μg per ml.     

Monoclonal antibodies against Vero cells that protect against diphtheria toxin

Mice were immunized with a cell line (Vero) that possesses a high number of membrane receptors for diphtheria toxin. Spleen cells from these mice were fused with SP2/0-Ag14 cells and two cell lines (1A2 and 2D2) isolated by screening for the ability of their secreted antibodies to inhibit binding of radiolabeled diphtheria toxin to Vero cells. These antibodies protected Vero cells from the inhibition of protein synthesis mediated by diphtheria toxin. The antibodies were purified, iodinated, and their binding characteristics investigated. At 4 degrees C, the association of 1A2 and 2D2 with Vero cells was saturable (KD approximately 10(-8) M) and indicated about 10(6) binding sites/cell. Diphtheria toxin did not inhibit the binding of either radiolabeled antibody. Monoclonal antibody 1A2 completely inhibited 125I-2D2 binding and vice versa. Trypsin or phospholipase C treatment of Vero cells had no effect on the ability of the monoclonal antibodies to bind to the cells. These findings suggest that: (1) the two monoclonal antibodies recognize the same or closely related epitopes and (2) the antibodies bind a domain distinct from the toxin binding site or to a subcomponent of the diphtheria toxin receptor that is present at many other cell surface sites. These antibodies offer a powerful tool to study the structure, processing and mode of action of diphtheria toxin receptors.     

The chemistry and biological effects of cardiotoxin from the Chinese cobra (N. naja Linn.) on hormonal responses in isolated cell systems.

A cardiotoxin (Toxin III) was purified from Chinese cobra (N. naja Linn.) venom. Toxin III showed only one band on acidic polyacrylamide gel electrophoresis, had an N-terminal leucine, a molecular weight of 7250 and an isoelectric point around pH 11·1. The amino acid composition of Toxin III was essentially the same or a close analogue of the cardiotoxin isolated from Formosan cobra (N. naja atra) (Lo et al., J. Chin. chem. Soc.13, 25, 1966).Cardiotoxin produced a biphasic effect on hormonal-responses in isolated cell systems. At higher concentrations, it inhibited ACTH-stimulated lipolysis in isolated fat cells and steroidogenesis in isolated adrenal cells. Inhibition became apparent at 3 μg per ml and attained 100 per cent at 100 μg per ml. At lower dosages (less than 3 μg per ml), cardiotoxin produced a stimulatory effect on lipolysis and steroidogenesis. This stimulatory effect was additive with a suboptimal dose of the hormone and disappeared when hormonal stimulation approached maximum. The inhibitory and stimulatory effects of cardiotoxin were accompanied, respectively, by a decrease and increase in the total cyclic AMP level. Cardiotoxin inhibition could not be reversed by increasing the ACTH concentration in isolated adrenal cells or isolated fat cells. Cardiotoxin effects could not be reversed by extensive washing of the isolated adrenal cells but could be partially reversed by cardiotoxin antiserum in isolated fat cells. It is postulated that cardiotoxin might act at the plasma membrane level, either binding irreversibly on the membrane or perturbing the plasma membrane conformation leading to an irreversible change in the activity of the membrane-bound adenyl cyclase.     

Not as simple as just punching a hole.

Like a variety of other pathogenic bacteria, Aeromonas hydrophila secretes a pore-forming toxin that contribute to its virulence. The last decade has not only increased our knowledge about the structure of this toxin, called aerolysin, but has also shed light on how it interacts with its target cell and how the cell reacts to this stress. Whereas pore-forming toxins are generally thought to lead to brutal death by osmotic lysis of the cell, based on what is observed for erythrocytes, recent studies have started to reveal far more complicated pathways leading to death of nucleated mammalian cells.     

7-(D,L-alpha-fluoro-2-thienylacetamido)cephalosporanic acid (L 14655), a new semisynthetic cephalosporin. Synthesis and preliminary biological evaluation

We describe the synthesis and some preliminary antimicrobial and enzymatic properties of 7-(D,L-alpha-fluoro-2-thienylacetamido)cephalosporanic acid (L 14655), a fluorinated derivative of cephalothin. L 14655 had antimicrobial activity similar to that of cephalothin. Cell-free studies demonstrated that L 14655 was very stable to hydrolysis by type Ia beta-lactamase (325 times more than cephalothin) but not to TEM enzyme. In addition, L 14655 was 480 times more active than cephalothin in preventing the hydrolysis of nitrocephin by type Ia enzyme. Preliminary studies in intact beta-lactamase-producing cells did not however produce evidence of enhanced activity of L 14655 relative to cephalothin.     

Acute Inhalation Toxicity of T-2 Mycotoxin in the Rat and Guinea Pig

Acute Inhalation Toxicity of T-2 Mycotoxin in the Rat and GuineaPig. CREASIA, D. A., THURMAN, J. D., WANNEMACHER, R. W., JR.,AND BUNNER, D. L. (1990). Fundam. Appl. Toxicol 14, 54–59.In this study, concentration-response parameters were determinedfor rats and guinea pigs systematically exposed to an aerosolof T-2 toxin. The LC50 for a 10-min exposure to T-2 toxin aerosolwas 0.02 mg T-2/liter air for rats and 0.21 mg T-2/liter airfor guinea pigs. Data from total T-2 deposition in rats andguinea pigs exposed to their respective LC50 aerosol concentrationgave an LD50 of 0.05 mg T-2/kg body weight for the rat and 0.4mg T-2/kg body weight for the guinea pig. These data show thatinhaled T-2 toxin is approximately 20 times more toxic to therat (0.05 mg T-2/kg body wt inhaled vs 1.0 mg T-2/kg body wtip) and at least twice as toxic to the guinea pig (0.4 mg T-2/kgbody wt inhaled vs 1-2 mg T-2/kg body wt ip) than ip administeredT-2 toxin. Histopathologic examination of major organs in boththe rat and guinea pig after respiratory exposure to T-2 toxinindicated that lesions were similar to those described aftersystemic administration of the toxin. Gross and microscopicalterations of respiratory tract tissue after T-2 aerosol exposurewere minimal and could not account for the increase in toxicity.     

Kinetics of hemolysis induced by a toxin from Bacillus thuringiensis israelensis

S. A. Weinstein, A. W. Bernheimer and J. D. Oppenheim. Kinetics of hemolysis induced by a toxin from Bacillus thuringiensis israelensis. Toxicon26, 1177–1185, 1988.—The kinetics of hemolysis resulting from the action on rabbit erythrocytes of a highly purified cytolytic toxin (26,000 mol.wt) isolated from a spore-crystal mixture of Bacillus thuringiensis israelensis was studied. Course of hemolysis, as determined by release of hemoglobin, yielded sigmoid curves whose maximum slopes were taken as a measure of the rate of lysis. Hemolysis occurred without an induction period, and the rate of lysis was a linear function of toxin concentration. Rate of hemolysis as a function of temperature yielded an Arrhenius constant of 9300 calories per mole. The toxin was active between pH 4.5 and 8.0. Lysis was strongly inhibited by Cu²⁺, Fe²⁺ and Zn²⁺ in concentrations as low as 0.025 M. Phosphatidylserine, phosphatidylinositol, phosphatidylcholine and sphingomyelin inhibited lysis, whereas phosphatidylethanolamine, cerebroside, cholesterol and major integral erythrocyte membrane proteins caused little or no inhibition. Inhibition of lysis by sucrose indicates that hemolysis is of the colloid-osmotic type.     

Mechanisms of cardiotoxin lll-induced apoptosis in human colorectal cancer colo205 cells

Cardiotoxin III (CTX III) is a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom. This is the first report on the mechanism of the anticancer effect of CTX III in human colorectal cancer Colo205 cells. 2. Cardiotoxin III-induced Colo205 cell apoptosis was confirmed by DNA fragmentation (DNA ladder and sub-G1 formation) with an IC(50) of 4 mg/mL at 48 h. 3. Further mechanistic analysis demonstrate that CTX III induced the loss of mitochondrial membrane potential (Dym), cytochrome c release from mitochondria into the cytosol and activation of capase-9, caspase 3, as well as markedly enhancing the expression of Bax, but not Bcl-2, protein in the cells. Moreover, the CTX III-induced apoptosis was significantly blocked by the broad-spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. 4. However, CTX III did not generate the formation of reactive oxygen species and anti-oxidants, including N-acetylcysteine, and catalase could not block CTX III-induced apoptosis in the Colo205 cells. 5. Taken together, these results suggest that CTX III may induce apoptosis through a mitochondrial- and caspase-dependent mechanism and alteration of Bax/Bcl-2 ratio in human colorectal Colo205 cancer cells.     

Involvement of both endoplasmic reticulum- and mitochondria-dependent pathways in cardiotoxin III-induced apoptosis in HL-60 cells

Cardiotoxin (CTX) III, a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom, has been reported to have anticancer activity. In the present study, we investigated the mechanisms underlying the anticancer activity of CTX III in human leukaemia (HL-60 cells). Cardiotoxin III activated the endoplasmic reticulum (ER) pathway of apoptosis in HL-60 cells, as indicated by increased levels of calcium and glucose-related protein 78 (Grp78), and triggered the subsequent activation of micro-calpain and caspase 12. In addition, CTX III initiated the mitochondrial apoptotic pathway in HL-60 cells, as evidenced by an increased Bax/Bcl-2 ratio, the release of cytochrome c and activation of caspase 9. In the presence of 50 micromol/L Z-ATAD-FMK (a caspase 12 inhibitor) and 100 micromol/L Z-LEHD-FMK (a caspase 9 inhibitor), the CTX III-mediated activation of caspase 9 and caspase 3 was significantly reduced. There was no significant effect of the caspase 12 inhibitor Z-ATAD-FMK on mitochondrial cytochrome c release. Cardiotoxin III-mediated activation of caspase 12 was not abrogated in the presence of the caspase 9 inhibitor Z-LEHD-FMK, indicating that caspase 12 activation was not downstream of caspase 9. These results indicate that CTX III induces cell apoptosis via both ER stress and a mitochondrial death pathway.     

Tetanus toxin: Biochemical and pharmacological comparison between its protoxin and some isotoxins obtained by limited proteolysis

Summary Single-chain tetanus toxin (toxin S) was prepared from short-term cultures by lysis under protection with protease inhibitors, precipitation with 40% ammonium sulfate, gel filtration, and chromatography on DEAE ion exchanger. Its limited proteolysis by trypsin, post-arginine cleaving enzyme from mouse submaxillary gland and clostripain led to bichainal derivatives (BT, BA, BCl) consisting of a heavy chain and a larger version of the light chain. The latter was then converted by trypsin into a small version which comigrated with the light chain of bichainal extracellular toxin (BE). The light chain produced by chymotrypsin (BC) and elastase (BEI) was of intermediate size. The nick region serves as substrate for all esteroproteases investigated and comprises between one and two kDa. Limited proteolysis increased the hydrophilicity (BT > BE > S) in hydrophobic interaction HPLC, and anionic behaviour (BC > BE > BT > S) in DEAE ion exchanger HPLC.The bichainal toxins assessed (BC, BE or BT) were about two times more toxic than toxin S (LD₅₀, mouse s. c. 2 ng/kg vs. 4 ng/kg). They were five to twelve times more potent than toxin S in three in vitro assays measuring the prevention of neurotransmitter release, i. c. on the phrenic nerve-hemidiaphragm preparation of the mouse (acetylcholine, with toxin BE and BT), on primary brain cell cultures from the mouse ([³H]noradrenaline, with toxin BE and BT), and on brain homogenate from rats ([³H]noradrenaline, with toxin BA, BC, BE and BT).Thus single-chain toxin is a less potent precursor of, or protoxin for, various bichainal isotoxins. The term tetanus toxin covers a family of agents that differ by their posttranslational processing.Abbreviations Toxin S single-chain tetanus toxin - toxin B bichainal tetanus toxins, prepared by treatment of toxin S with trypsin (BT), chymotrypsin (BC), post-arginine cleaving enzyme (BA), clostripain (BCI), or elatase (BEI) - toxin BE bichainal extracellular tetanus toxin Send offprint requests to E. Habermann at the above address     

T-2 toxin induces apoptosis via the Bax-dependent caspase-3 activation in mouse primary Leydig cells

To explore the toxic effect of T-2 toxin on mouse Leydig cells and its underlying molecular mechanisms, we isolated Leydig cells from mature mice, set-up Leydig cells culture, treated cells with T-2 toxin, evaluated cell proliferation, detected the caspase-3 activity, mitochondrial activity and apoptosis rate, and measured the mRNA levels of Bcl-2, Bax, PARP and caspase-3. T-2 toxin inhibited cell proliferation at concentrations higher than 10^?9 M or time more than 12?h, T-2 toxin also decreased Bcl-2 expression at the mRNA levels and mitochondrial activity at concentrations higher than 10^?9 M. While, T-2 toxin increased the mRNA expressions of Bax and PARP at concentrations higher than 10^?8 M and 10^?9 M, respectively, triggered mitochondria-mediated apoptosis, activated downstream caspase-3, and then increased caspase-3 at the activity and mRNA levels at concentrations higher than 10^?9 M. These data showed that T-2 toxin appears to activate specific intracellular death-related pathways leading to Bax-dependent caspase-3 activation and the induction of apoptosis in Leydig cells.     

Cardiotoxin III induces apoptosis in K562 cells through a mitochondrial-mediated pathway

1. Cardiotoxin (CTX) III is a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom. This is the first report on the mechanism of the anticancer effect of CTX III on human leukaemia K562 cells. 2. Cardiotoxin III was found to inhibit the growth of K562 cells in a time- and dose-dependent manner, with an IC(50) value of 1.7 mug/mL, and displayed several features of apoptosis, including apoptotic body formation, an increase in the sub-G(1) population, DNA fragmentation and poly (ADP-ribose) polymerase (PARP) cleavage. 3. Investigation of the mechanism of CTX III-induced apoptosis revealed that treatment of K562 cells with CTX III resulted in the loss of mitochondrial membrane potential, cytochrome c release from mitochondria into the cytosol and activation of caspase-9 and caspase-3 and the subsequent cleavage of the caspase-3 substrate PARP; however, CTX III did not generate reactive oxygen species (ROS). 4. Taken together, the results indicate that CTX III induces apoptosis in K562 cells through an ROS-independent mitochondrial dysfunction pathway.     

Brown spider dermonecrotic toxin directly induces nephrotoxicity

Brown spider (Loxosceles genus) venom can induce dermonecrotic lesions at the bite site and systemic manifestations including fever, vomiting, convulsions, disseminated intravascular coagulation, hemolytic anemia and acute renal failure. The venom is composed of a mixture of proteins with several molecules biochemically and biologically well characterized. The mechanism by which the venom induces renal damage is unknown. By using mice exposed to Loxosceles intermedia recombinant dermonecrotic toxin (LiRecDT), we showed direct induction of renal injuries. Microscopic analysis of renal biopsies from dermonecrotic toxin-treated mice showed histological alterations including glomerular edema and tubular necrosis. Hyalinization of tubules with deposition of proteinaceous material in the tubule lumen, tubule epithelial cell vacuoles, tubular edema and epithelial cell lysis was also observed. Leukocytic infiltration was neither observed in the glomerulus nor the tubules. Renal vessels showed no sign of inflammatory response. Additionally, biochemical analyses showed such toxin-induced changes in renal function as urine alkalinization, hematuria and azotemia with elevation of blood urea nitrogen levels. Immunofluorescence with dermonecrotic toxin antibodies and confocal microscopy analysis showed deposition and direct binding of this toxin to renal intrinsic structures. By immunoblotting with a hyperimmune dermonecrotic toxin antiserum on renal lysates from toxin-treated mice, we detected a positive signal at the region of 33-35 kDa, which strengthens the idea that renal failure is directly induced by dermonecrotic toxin. Immunofluorescence reaction with dermonecrotic toxin antibodies revealed deposition and binding of this toxin directly in MDCK epithelial cells in culture. Similarly, dermonecrotic toxin treatment caused morphological alterations of MDCK cells including cytoplasmic vacuoles, blebs, evoked impaired spreading and detached cells from each other and from culture substratum. In addition, dermonecrotic toxin treatment of MDCK cells changed their viability evaluated by XTT and Neutral-Red Uptake methodologies. The present results point to brown spider dermonecrotic toxin cytotoxicity upon renal structures in vivo and renal cells in vitro and provide experimental evidence that this brown spider toxin is directly involved in nephrotoxicity evoked during Loxosceles spider venom accidents.     

Nodularin concentrations in Lakes Alexandrina and Albert,South Australia,during a bloom of the cyanobacterium (blue-green alga) Nodularia spumigena and degradation of the toxin

Nodularin together with cell numbers of Nodularia spumigena were monitored during a toxic bloom of this cyanobacterium during the summer/autumn of 1994–1995 in Lakes Alexandrina and Albert, South Australia. There was a good correlation between cell numbers and toxin concentration over the course of the bloom at a number of locations. The correlation was poorer toward the end of the bloom, and may have reflected cell lysis and release of toxin to the surrounding water where it was rapidly degraded. The results suggested that the suitability of the lake water for drinking could be better determined by monitoring toxin content rather than cell numbers. Degradation of nodularin was studied and found to be more rapid in lake water that was supporting a bloom compared with water collected that had not experienced a bloom for several months. Half-lives were 24 and 54 h, respectively. There was no lag phase in water that was supporting a bloom, suggesting a relatively high population of microorganisms capable of degrading the toxin to be present. Toxin released to the lake water from lysing cells would therefore disappear very quickly. Toxin degradation was inhibited in the presence of copper ions. Copper sulphate treatment of blooms may therefore be doubly disadvantageous. Not only are cells lysed by copper sulphate addition, thereby releasing toxin to the surrounding water, but the natural microbial population capable of degrading the toxin may be destroyed, or the enzymes capable of toxin degradation inhibited. © 1997 John Wiley & Sons, Inc. Environ Toxicol Water Qual 12 : 273–282, 1997     

Toxicity of the cyanobacterium Nodularia spumigena Mertens

The bloom forming cyanobacterium (blue-green alga) Nodularia spumigena produced a peptide hepatotoxin with an LD50 of 70 micrograms/kg i.p. in mice. The livers of lethally poisoned mice were haemorrhagic and enlarged, the weight doubling to about 10% of total body weight. Histologically there was centrilobular to midzonal disruption and lysis of hepatocytes resulting in haemorrhage and formation of blood lakes. Death occurred approximately 1 hr after i.p. injection. By 30 min significant increases had occurred in the plasma levels of lactate dehydrogenase, aspartate aminotransferase, alanine aminotransferase and glucose paralleling degeneration and necrosis of centrilobular hepatocytes. In vitro the toxin caused rapid dose-dependent deformation of freshly isolated rat hepatocytes, which was accompanied by the activation of phosphorylase a; 125 ng/ml of toxin being sufficient to cause these changes in 10(6) cells. This work demonstrates that, both in vivo and in vitro, Nodularia toxin shares many similarities in its action to the well characterized peptide toxins of another cyanobacterium, Microcystis aeruginosa.     

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.     

Production of diarrhetic shellfish poisoning toxins and pectenotoxins at depths within and below the euphotic zone

During a 10 day survey in the CelticSea near the Irish South-West coast (July 2007), Dinophysis acuta was observed in large numbers. The deployment of a profiler allowed for the identification of a D. acuta thin layer that reached 1910 cells/L. The aim of the study was to investigate if the bloom that occurred in low light environment was viable, dividing, actively producing toxins and if the toxin profile changed over a short term period. Several large concentrates of phytoplankton samples were obtained over a 14 h period, from evening to morning, by pumping Dinophysis from specific depths. In addition, D. acuta was collected in complete darkness at 81 m depth by concentrating 120 L of water. The cells were extracted and their toxin profiles were established by liquid chromatography – mass spectrometry (LC–MS). Passive samplers were deployed in a nearby location for 6 days at 30, 50, 70 and 110 m depth, and the toxin profiles were determined by LC–MS as above. The toxin profiles obtained in phytoplankton samples and in the SPATT were compared and correlated well. Sample concentrates and SPATT results suggested that toxic D. acuta occurred and produced similar toxin profiles at all water depths, including below the euphotic zone.     

Diphtheria fusion protein therapy of chemoresistant malignancies

Patients with widespread cancer respond initially to combination chemotherapy, immunotherapy, and/or radiotherapy, but most relapse with chemoresistant disease. Novel methods of killing resistant neoplastic stem cells are needed. One such approach is therapy with targeted toxins composed of tumor cell selective ligands covalently linked to group I peptide toxins (group II and III peptide toxins act on the cell surface). The targeted toxin is delivered to the cell by a tumor selective ligand. Once bound, the ligand-receptor complex is internalized. The catalytic domain escapes to the cytosol. The toxin then enzymatically modifies a critical cell function (protein synthesis, p21 Rho activity, protein kinase signaling, cyclic AMP signaling or others). The irreversibly damaged cells fails to divide and, eventually, undergoes lysis or programmed cell death. Targeted peptide toxins used to date in the treatment of chemotherapy refractory cancers include ricin toxin, Pseudomonas exotoxin, pokeweed antiviral protein, saporin, gelonin and diphtheria toxin. In this review, we have focused on the applications of genetically engineered diphtheria toxin for cancer therapy.     

Role of cardiotoxin and phospholipase A in the blockade of nerve conduction and depolarization of skeletal muscle induced by cobra venom

1. The effects of phospholipase A (PhA), cardiotoxin (CTX) and neurotoxin (cobrotoxin) isolated from Formosan cobra (Naja naja atra) venom on conduction of the rat phrenic nerve and membrane potential of the rat diaphragm were studied.2. Phospholipase A, lysolecithin and cobrotoxin were without effect on the axonal conduction. Cardiotoxin was the only active agent in cobra venom, but it was less potent than the crude venom.3. The blocking action of cardiotoxin was markedly accelerated by the simultaneous administration of phospholipase A. However, the minimum effective concentration of cardiotoxin (100 μg/ml), was not decreased by phospholipase A. Pretreatment of the nerve with phospholipase A, followed by washout, did not alter the activity of cardiotoxin.4. Cardiotoxin (3 μg/ml) completely depolarized the membrane of superficial muscle fibres within 60 min, being 3 times more potent than the crude venom. Phospholipase A, on the other hand, needed a dose 30 times higher and a prolonged period of incubation to induce depolarization of similar extent. Cobrotoxin was without effect on membrane potentials.5. CaCl₂ (10 mM) effectively antagonized the nerve blocking as well as the depolarizing effect of the crude venom, cardiotoxin or cardiotoxin plus phospholipase A. By contrast, the slow depolarizing effect of phospholipase A was enhanced by high concentrations of calcium.6. Cardiotoxic fractions of Indian cobra venom affected both nerve conduction and diaphragm membrane potential in exactly the same way as cardiotoxin. Toxin A of the same venom was without effect.7. It is concluded that the active agent in cobra venoms either on axonal conduction or on muscle membrane is cardiotoxin. The synergistic effect of phospholipase A on cardiotoxin appears to be due to acceleration rather than potentiation of its action. The mechanism of action of cardiotoxin and its synergism by phospholipase A are discussed.     

The effects of opiates on calcium accumulation on rat peritoneal mast cells

Opiate agonists, morphine, levorphanol and beta-endorphin increased calcium accumulation in rat peritoneal mast cells. This effect was dose dependent and beta-endorphin was 10 times more potent than morphine. The stimulation was stereospecific and inhibited by naloxone. The site of the opiate action appears to be on the outer surface of the plasma membrane since lysis of the mast cell did not alter the response to morphine. Tolerance to the opiate effect was not seen after chronic morphine administration. Morphine did not stimulate histamine release even at relatively high doses in vivo or high concentrations in vitro. It is reasoned that the enhancing effects on external calcium accumulation may reduce the critical cytosol calcium level for effecting histamine release.