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Effect of a snake venom cardiotoxin (CTX Bungarus fasciatus) on epithelial Ca2+-activated K+ currents
9th World Congress
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It is well established that cytotoxic activity is related to hemorrhagic and proteolytic factors in Crotalidae and Viperidae venoms, although the results of this study clearly indicated that some non-hemorrhagic and nonproteolytic fractions are cytotoxic, as well. The sensitivity of hybridoma systems would be valuable in further studies of cytotoxic effects.
Effect of presynaptic phospholipase A 2 (PLA2) toxins, fl-bungarotoxin (fl-BuTX) and notexin ( N T X ) , and nonspecific P L A 2 enzymes, N. n. atra and N. nigricollis, on rat brain synaptosomal membrane integrity, phospholipid hydrolysis and fluidity. ANNAmTA GHASSEMIand PmLlP ROSENaERG(Section of Pharmacology and Toxicology, School of Pharmacy, University of Connecticut, Storrs, CT 06268, U.S.A.).
PRIOR TO studying the effects of PLA 2 toxins and enzymes on ionic and neurotransmitter fluxes and asymmetry of synaptic plasma membrane (SPM), it was necessary to determine their effects on membrane integrity. Integrity of synaptosomes isolated from cerebral cortex, corpus striatum and hippocampus was evaluated by measuring lactate dehydrogenase leakage following 15 - 60 min (37°C) exposure to 5 and 50 mM toxins and enzymes. Disruption of SPM in response to the PLA2 toxins and enzymes was concentration and time but not tissue dependent. In each tissue, the order of potency was: (50 nM) N. n. atra >> N. nigricollis >i N T X >1 fl-BuTX; (5 nM) N. n. atra > NTX /> fl-BuTX = N. nigricollis. Ten mM EDTA + Ca 2+ in the incubation medium reduced the effects of PLA 2 preparations, while BSA (1 mg/ml) inhibited the effect of N. n. atra PLA 2 but potentiated the effect of fl-BuTX. Using the steady state fluorescence polarization probe, DPH, PLA2 toxins and enzymes decreased synaptosomal and SPM fluidity, with fl-BuTX causing the least and PLA 2 enzymes causing the greatest effects. Changes in membrane asymmetry and pharmacological effects can be studied on intact synaptosomes provided that short incubation times and low concentrations of PLA2 toxins and enzymes are used. There is no positive correlation between the presynaptic specificity of action and the potency on SPM integrity and fluidity. PLA2 activity might be required for the non-specific membrane disruption discussed. The disruptive effects of N. n. atra PLA 2 but not fl-BuTX may be due to liberation of free fatty acids. Supported by U.S.P.H.S. Grant ROINS 14521 to P.R. and by Boehringer-Ingelheim Pharm. Co. Intergradation o f venom A and venom B populations o f the Mojave rattlesnake (Crotalus scutulatus scutulatus) in Arizona. JAMESL. GLENN and RICHARD C. Sa~AIGHT (Venom Research Laboratory, Veterans Administration Medical Center, Salt Lake City, UT 84148, U.S.A.).
THE VENOMSof 15 Crotalus scutulatus scutulatus from regions betwern the venom A and venom B populations in Arizona were examined for the presence of the phospholipase (PLA2) neurotoxin (Mojave toxin) by immunochemical assay, mouse i.p. LDso, proteolytic activity, esterase activity and hemorrhagic activity (mice) as previously described (GLENN et al., 1983). Seven venoms were intergradal in composition, containing both the Mojave toxin of venom A and the proteolytic and hemorrhagic activities of venom B. The i.p. LDso values of the A + B venoms were 0.8-2.6mg/kg, compared to 0.2-0.5mg/kg for venom A individuals and 2.1 - 5.3 mg/kg for the venom B individuals. These data indicate that an intergrade zone exists between the two venom types which arcs around the northwest, west and southern regions of the venom B population. Within these regions, three major venom types can occur in Crotalus s. scutulatus, i.e. venom A, venom B and the combined activities of venom A + B. REFERENCE GLENN, J. L., STRAIGHT, R. C., WOLFE, M. C. and HARDY, D. L. (1983) Toxicon 21, 119. Effect o f a snake venom cardiotoxin ( C T X Bungarus fasciatus) on epithelial Ca 2 +-activated K + currents. QI H. GONG,1 STEVENJ. WIELAND,2 JEFFREYE. FLETCHER,l and MING S. JIANGl (Departments of 1Anesthesiology and 2Anatomy, Hahnemann University, Philadelphia, PA 19102, U.S.A.).
THE ACTION of a 16,300 mol.wt CTX from B. fasciatus venom on membrane electrical properties of a human respiratory epithelial cell line was studied by using tight-seal whole-cell recording methods. Baseline voltageactivated currents were measured by voltage-clamping the cells in the absence of CTX. These cells exhibited a voltage and Ca2+-activated K + current; the threshold for voltage activation of the K + current was greatly reduced by increasing free Ca z + in the recording pipette from nominally 0 to 2 x 10 -6 M. The activation was decreased by replacing external Ca 2+ with 5 x I 0-3 M Mg 2+ . Current was blocked by replacing pipette K + with Cs +, or by 2x 10-aM Ba 2+ in the bath. B. fasciatus CTX (0.5 - 5 x 10 -6 M), for 5 - 30 min in the bath, did not alter membrane leakage current, as judged by the maintenance of low pre-treatment values over the range of - 1 4 0 mV to - 4 0 inV. However, the threshold for voltage activation of the K + current was reduced even at the lowest concentrations tested. In contrast to the results with epithelial cells, CTX exposure inhibited the activation of voltage-activated K +
48
9th World Congress
currents in human lymphocytes; these currents have been shown by others to be inhibited by increased intracellular Ca 2 ÷. Thus, the action of this CTX appears to either mimic, or to cause an intracellular increase in free Ca 2 +.
Functional anatomy of the venom apparatus of spitting cobras. P. GOPALAKRISHNAKONE1 and E.
KOCHVA 2
(1Department of Anatomy, National University of Singapore; 2Department of Zoology, Tel Aviv University, Israel). SERIAL sections of the venom glands of the black spitting cobra Naja naja sputatrix were studied by light microscopy. A detailed study of transmission electron microscopy from various parts of the gland such as the accessory gland, duct, fang sheath, main gland, and associated muscles were done. Scanning electron microscopy of the fangs with special attention to the orifices were done on Naja naja sputatrix, Naja nigricollis, Hemachatus haemachatus, and Naja melanoleuca. A summary of the observations will be presented.
Morphology of the venom glands of fourteen species of sea snakes from the South China Sea and the Straits of Malacca. P. GOPALAKRISHNAKONE1 and E. KOCHVA 2 (1Department of Anatomy, National University of Singapore; 2Department of Zoology, Tel Aviv University, Israel). SERIAL sections of the venom gland of the following sea snakes Acalyptophus peronii, Aipysurus eydouxi, Astrotia stokes, Enhydrina schistosa, Hydrophis cyanocinctus, Hydrophisfasciatus, Hydrophis klossi, Hydrophis ornatus, Hydrophis inornatus, Hydrophis torquatus, Lapemis hardwicki, Laticauda colubrina, Thalassophina viperina and Pelamisplaturus, were examined under the light microscope. A detailed transmission electron microscopic study of the venom glands of Lapemis hardwicki, Hydrophis cyanocinctus and Laticauda colubrina were done. In addition, scanning electron microscopic examination of the fangs of some species were done. A summary of the findings and a comparison between these observations and terrestrial elapids will also be presented.
Glutamate receptor antagonists from spider venom. EUGENE V. GRISHIN (Shemyakin Institute of Bioorganic Chemistry, U.S.S.R. Academy of Sciences, 117871 Moscow, U.S.S.R.). HOMOLOGOUS low molecular weight compounds blocking postsynaptic glutamate receptors were isolated from the Argiope lobata spider venom by ion exchange as well as reverse phase high performance liquid chromatographies. Structures of nine different blocking components were determined by NMR and mass spectroscopy. All the isolated glutamate receptor antagonists can be divided into three structural groups: argiopin (mol.wt 636), argiopinins (759, 744, 659, 630, 658) and pseudoargiopinins (743, 728, 373). The main principles of the structural organization of the novel class of glutamate receptor antagonists were revealed. REFERENCES GRISHIN, E. V., VOLKOVA,T. M., ARSENIEV, k. S., RESHETOVA, O. S., ONOPRIENKO, V. V., MAGAZANIK, L. G., ANTONOV, S. M. and FEDOROVA, I. M. (1986) Bioorg. Khimia. 12, 1121 (in Russian). MAGAZANIK, L. G., ANTONOV, S. M., FEOOROVA, I. M., VOLKOVA, T. i . and GRISHIN, E. V. (1986) Biol. Membr. 3, 1204 (in Russian).
Toxic proteins behaving as hemilectins. JORGE A. GUIMAR.~tES and Cl~LIA R. CARLINI (Department of Biochemistry, ICB, Universidade Federal do Rio de Janeiro, CP 68041, Rio de Janeiro, R.J., Brasil). THE PRESENCE of lectins and toxic proteins in plants has been known for several decades. The lectins, carbohydrate-specific binding proteins, have been thoroughly investigated and shown to be present in several hundreds in seed extracts. The cell recognition and agglutination properties of the lectins are due to the presence in these proteins of at least two specific binding sites for a carbohydrate moiety. In contrast, few of the toxic proteins which are usually associated with the lectins in the same seed have been purified. Among them, the first one to be isolated was ricin found in Ricinus communis (castor beans), together with a galactose-specific lectin. Other of these plant toxic proteins already purified include abrin (Abrus precatorius), moddecin (Adenia diggitata), mormodin (Mormodica charantia) and canatoxin (Canavalia ensiformis). It has been shown that these toxic proteins presents only one binding site for a hexose and sometimes a more complex carbohydrate. These toxins are still able to bind glycoproteins or glycolipids in the cell surface but do not cause cell agglutination. Thus, these toxic proteins behave as monovalent lectins or hemilectins (CARLINI e l al., 1988). Proteins behaving as hemilectins usually have two polypeptide chains or protein domains. One of them, designated 'haptomer', is able to recognize the cell surface as a monovalent lectin. The other chain or domain, the 'effectomer', displays toxicity of different types: inhibition of protein synthesis, ADP ribosylation, interference with neurotransmitter
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It is well established that cytotoxic activity is related to hemorrhagic and proteolytic factors in Crotalidae and Viperidae venoms, although the results of this study clearly indicated that some non-hemorrhagic and nonproteolytic fractions are cytotoxic, as well. The sensitivity of hybridoma systems would be valuable in further studies of cytotoxic effects.
Effect of presynaptic phospholipase A 2 (PLA2) toxins, fl-bungarotoxin (fl-BuTX) and notexin ( N T X ) , and nonspecific P L A 2 enzymes, N. n. atra and N. nigricollis, on rat brain synaptosomal membrane integrity, phospholipid hydrolysis and fluidity. ANNAmTA GHASSEMIand PmLlP ROSENaERG(Section of Pharmacology and Toxicology, School of Pharmacy, University of Connecticut, Storrs, CT 06268, U.S.A.).
PRIOR TO studying the effects of PLA 2 toxins and enzymes on ionic and neurotransmitter fluxes and asymmetry of synaptic plasma membrane (SPM), it was necessary to determine their effects on membrane integrity. Integrity of synaptosomes isolated from cerebral cortex, corpus striatum and hippocampus was evaluated by measuring lactate dehydrogenase leakage following 15 - 60 min (37°C) exposure to 5 and 50 mM toxins and enzymes. Disruption of SPM in response to the PLA2 toxins and enzymes was concentration and time but not tissue dependent. In each tissue, the order of potency was: (50 nM) N. n. atra >> N. nigricollis >i N T X >1 fl-BuTX; (5 nM) N. n. atra > NTX /> fl-BuTX = N. nigricollis. Ten mM EDTA + Ca 2+ in the incubation medium reduced the effects of PLA 2 preparations, while BSA (1 mg/ml) inhibited the effect of N. n. atra PLA 2 but potentiated the effect of fl-BuTX. Using the steady state fluorescence polarization probe, DPH, PLA2 toxins and enzymes decreased synaptosomal and SPM fluidity, with fl-BuTX causing the least and PLA 2 enzymes causing the greatest effects. Changes in membrane asymmetry and pharmacological effects can be studied on intact synaptosomes provided that short incubation times and low concentrations of PLA2 toxins and enzymes are used. There is no positive correlation between the presynaptic specificity of action and the potency on SPM integrity and fluidity. PLA2 activity might be required for the non-specific membrane disruption discussed. The disruptive effects of N. n. atra PLA 2 but not fl-BuTX may be due to liberation of free fatty acids. Supported by U.S.P.H.S. Grant ROINS 14521 to P.R. and by Boehringer-Ingelheim Pharm. Co. Intergradation o f venom A and venom B populations o f the Mojave rattlesnake (Crotalus scutulatus scutulatus) in Arizona. JAMESL. GLENN and RICHARD C. Sa~AIGHT (Venom Research Laboratory, Veterans Administration Medical Center, Salt Lake City, UT 84148, U.S.A.).
THE VENOMSof 15 Crotalus scutulatus scutulatus from regions betwern the venom A and venom B populations in Arizona were examined for the presence of the phospholipase (PLA2) neurotoxin (Mojave toxin) by immunochemical assay, mouse i.p. LDso, proteolytic activity, esterase activity and hemorrhagic activity (mice) as previously described (GLENN et al., 1983). Seven venoms were intergradal in composition, containing both the Mojave toxin of venom A and the proteolytic and hemorrhagic activities of venom B. The i.p. LDso values of the A + B venoms were 0.8-2.6mg/kg, compared to 0.2-0.5mg/kg for venom A individuals and 2.1 - 5.3 mg/kg for the venom B individuals. These data indicate that an intergrade zone exists between the two venom types which arcs around the northwest, west and southern regions of the venom B population. Within these regions, three major venom types can occur in Crotalus s. scutulatus, i.e. venom A, venom B and the combined activities of venom A + B. REFERENCE GLENN, J. L., STRAIGHT, R. C., WOLFE, M. C. and HARDY, D. L. (1983) Toxicon 21, 119. Effect o f a snake venom cardiotoxin ( C T X Bungarus fasciatus) on epithelial Ca 2 +-activated K + currents. QI H. GONG,1 STEVENJ. WIELAND,2 JEFFREYE. FLETCHER,l and MING S. JIANGl (Departments of 1Anesthesiology and 2Anatomy, Hahnemann University, Philadelphia, PA 19102, U.S.A.).
THE ACTION of a 16,300 mol.wt CTX from B. fasciatus venom on membrane electrical properties of a human respiratory epithelial cell line was studied by using tight-seal whole-cell recording methods. Baseline voltageactivated currents were measured by voltage-clamping the cells in the absence of CTX. These cells exhibited a voltage and Ca2+-activated K + current; the threshold for voltage activation of the K + current was greatly reduced by increasing free Ca z + in the recording pipette from nominally 0 to 2 x 10 -6 M. The activation was decreased by replacing external Ca 2+ with 5 x I 0-3 M Mg 2+ . Current was blocked by replacing pipette K + with Cs +, or by 2x 10-aM Ba 2+ in the bath. B. fasciatus CTX (0.5 - 5 x 10 -6 M), for 5 - 30 min in the bath, did not alter membrane leakage current, as judged by the maintenance of low pre-treatment values over the range of - 1 4 0 mV to - 4 0 inV. However, the threshold for voltage activation of the K + current was reduced even at the lowest concentrations tested. In contrast to the results with epithelial cells, CTX exposure inhibited the activation of voltage-activated K +
48
9th World Congress
currents in human lymphocytes; these currents have been shown by others to be inhibited by increased intracellular Ca 2 ÷. Thus, the action of this CTX appears to either mimic, or to cause an intracellular increase in free Ca 2 +.
Functional anatomy of the venom apparatus of spitting cobras. P. GOPALAKRISHNAKONE1 and E.
KOCHVA 2
(1Department of Anatomy, National University of Singapore; 2Department of Zoology, Tel Aviv University, Israel). SERIAL sections of the venom glands of the black spitting cobra Naja naja sputatrix were studied by light microscopy. A detailed study of transmission electron microscopy from various parts of the gland such as the accessory gland, duct, fang sheath, main gland, and associated muscles were done. Scanning electron microscopy of the fangs with special attention to the orifices were done on Naja naja sputatrix, Naja nigricollis, Hemachatus haemachatus, and Naja melanoleuca. A summary of the observations will be presented.
Morphology of the venom glands of fourteen species of sea snakes from the South China Sea and the Straits of Malacca. P. GOPALAKRISHNAKONE1 and E. KOCHVA 2 (1Department of Anatomy, National University of Singapore; 2Department of Zoology, Tel Aviv University, Israel). SERIAL sections of the venom gland of the following sea snakes Acalyptophus peronii, Aipysurus eydouxi, Astrotia stokes, Enhydrina schistosa, Hydrophis cyanocinctus, Hydrophisfasciatus, Hydrophis klossi, Hydrophis ornatus, Hydrophis inornatus, Hydrophis torquatus, Lapemis hardwicki, Laticauda colubrina, Thalassophina viperina and Pelamisplaturus, were examined under the light microscope. A detailed transmission electron microscopic study of the venom glands of Lapemis hardwicki, Hydrophis cyanocinctus and Laticauda colubrina were done. In addition, scanning electron microscopic examination of the fangs of some species were done. A summary of the findings and a comparison between these observations and terrestrial elapids will also be presented.
Glutamate receptor antagonists from spider venom. EUGENE V. GRISHIN (Shemyakin Institute of Bioorganic Chemistry, U.S.S.R. Academy of Sciences, 117871 Moscow, U.S.S.R.). HOMOLOGOUS low molecular weight compounds blocking postsynaptic glutamate receptors were isolated from the Argiope lobata spider venom by ion exchange as well as reverse phase high performance liquid chromatographies. Structures of nine different blocking components were determined by NMR and mass spectroscopy. All the isolated glutamate receptor antagonists can be divided into three structural groups: argiopin (mol.wt 636), argiopinins (759, 744, 659, 630, 658) and pseudoargiopinins (743, 728, 373). The main principles of the structural organization of the novel class of glutamate receptor antagonists were revealed. REFERENCES GRISHIN, E. V., VOLKOVA,T. M., ARSENIEV, k. S., RESHETOVA, O. S., ONOPRIENKO, V. V., MAGAZANIK, L. G., ANTONOV, S. M. and FEDOROVA, I. M. (1986) Bioorg. Khimia. 12, 1121 (in Russian). MAGAZANIK, L. G., ANTONOV, S. M., FEOOROVA, I. M., VOLKOVA, T. i . and GRISHIN, E. V. (1986) Biol. Membr. 3, 1204 (in Russian).
Toxic proteins behaving as hemilectins. JORGE A. GUIMAR.~tES and Cl~LIA R. CARLINI (Department of Biochemistry, ICB, Universidade Federal do Rio de Janeiro, CP 68041, Rio de Janeiro, R.J., Brasil). THE PRESENCE of lectins and toxic proteins in plants has been known for several decades. The lectins, carbohydrate-specific binding proteins, have been thoroughly investigated and shown to be present in several hundreds in seed extracts. The cell recognition and agglutination properties of the lectins are due to the presence in these proteins of at least two specific binding sites for a carbohydrate moiety. In contrast, few of the toxic proteins which are usually associated with the lectins in the same seed have been purified. Among them, the first one to be isolated was ricin found in Ricinus communis (castor beans), together with a galactose-specific lectin. Other of these plant toxic proteins already purified include abrin (Abrus precatorius), moddecin (Adenia diggitata), mormodin (Mormodica charantia) and canatoxin (Canavalia ensiformis). It has been shown that these toxic proteins presents only one binding site for a hexose and sometimes a more complex carbohydrate. These toxins are still able to bind glycoproteins or glycolipids in the cell surface but do not cause cell agglutination. Thus, these toxic proteins behave as monovalent lectins or hemilectins (CARLINI e l al., 1988). Proteins behaving as hemilectins usually have two polypeptide chains or protein domains. One of them, designated 'haptomer', is able to recognize the cell surface as a monovalent lectin. The other chain or domain, the 'effectomer', displays toxicity of different types: inhibition of protein synthesis, ADP ribosylation, interference with neurotransmitter