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Factors influencing the hemolysis of human erythrocytes by three snake venom cardiotoxins
9th World Congress
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muscle is decreased after pretreatment with 10 #M (67 #g) antx-a(s) as compared to controls. To determine if antx-a(s) has potassium and/or chloride channel activity, membrane potential in fiber bundles of the frog semitendinosus muscle were monitored. Membrane potential was monitored before and after antx-a(s) administration (1 - 10/~M) in normal amphibian Ringer's, high potassium and no potassium Ringer's. Chloride channel activity was determined in a similar manner using solutions of various chloride ion concentrations. Preand postsynaptic effects of antx-a(s) were analyzed using the sciatic nerve - sartorius muscle preparation. REFERENCES HYDE, E. G. and CARMICHAEL,W. W. (1988) Toxicologist g, 40. MAHMOOD, N. A. and CARMICHAEL, W. W. (1987) Toxicon 25, 1221.
Factors influencing the hemolysis of human erythrocytes by three snake venom cardiotoxins. MING-SHI JIANG,1 JEFFREY E. FLETCHER1 and LEONARD A. SMITH2 (Departments of 1Anesthesiology, Hahnemann University, Philadelphia, PA 19102-1192, U.S.A.; 2Department of Toxicology, Pathology Division, USAMRIID, Ft Detrick, Frederick, MD 21701, U.S.A.). THE EFFECTSof cell age, pH, divalent cation type and concentration, temperature and glucose on red blood cell (RBC) hemolysis induced by highly purified cardiotoxins (CTXs) from Naja naja atra, Naja naja kaouthia (each about 7000 mol.wt) and Bungarus fasciatus (16,300 mol.wt) venoms were examined. Using aged RBCs (Ca 2÷ 2 mH, pH 7.4, 37°C, 2 hr) and a 30 #M concentration of each toxin hemolysis levels (as % of hemolysis in distilled H20 ) were: N.n.k., 80%; N.n.a., 40%; Bf. 10%. These values were reduced by a factor of four when fresh RBCs were used. In Ca 2 ÷ or Sr 2 ÷ (2 mM) medium, increasing pH from 7 to 8 greatly increased levels of hemolysis by the three CTXs. In Ba 2 ÷ (2 mM) containing medium, the increased hemolysis at high pH was blocked for B.f. CTX, but not for N.n.k. or N.n.a. CTXs. Divalent cations at 0.5 - 2.0 mM enhanced (Ca 2÷) or slightly decreased (Sr 2÷, Ba 2÷) hemolysis due to N.n.k. and N.n.a. CTXs, and greatly decreased hemolysis at higher concentrations (4 - 40 mM). For the B.f CTX Ba 2 ÷ inhibited hemolysis at all concentrations, Sr 2÷ had little effect and high Ca 2÷ (>40 mM) enhanced hemolysis. Increasing temperature from 20° to 37°C doubled the hemolysis values for all three CTXs. Glucose increased hemolysis by the CTXs at low concentrations of divalent cations (2 mM) and decreased hemolysis at high concentrations (40 mM). The same binding site and mechanism may be involved in hemolysis by three CTXs. However, divalent cations appear to have effects on either binding or the mode of action that are dependent on the CTX studied. Supported in part by the U.S. Army Medical Research and Development Command, contract no. DAMD1787-C-7155.
Pathogenesis of hemorrhage and myonecrosis induced by copperhead (Agkistrodon contortrix sp.) venom. EDWARD K. JOHNSON and CHARLOTTE L. OWNBY (Department of Physiological Sciences, Oklahoma State University, Stillwater, OK 74078, U.S.A.). THE PATHOGENESIS of hemorrhage and myonecrosis induced by copperhead (A. contortrix sp.) venom was studied using light and electron microscopy. Mice were injected i.m. with a sublethal dose (5.0 mg/kg) of venom and muscle tissue taken at varying time intervals (5, 15 and 30 min; 1, 3, 6, 12 and 24 hr) after the injection. Tissue samples were fixed for 2 hr in 2% glutaraldehyde in cacodylate buffer, post-fixed for I hr in 1% osmium tetroxide, and embedded in Polybed 812 resin. For light microscopy, thick sections (1 gm) were obtained and stained with Mallory's trichromer dye. For electron microscopy, thin sections (70 - 90 nm) were obtained and stained with uranyl acetate and lead citrate. Gross observation of the tissue after injection of the venom revealed hemorrhage at all time intervals. Light microscopic examination of the muscle revealed extensive myonecrosis and capillaries in various stages of degeneration. Damaged capillaries and muscle cells were both present as early as 5 min after injection of the venom, however, very few intact capillaries were present at later time intervals. At the light microscope level necrotic muscle cells containing clumped myofilaments were the predominant pattern of myonecrosis although there were some qualitative differences in the lesions at different time periods. At the electron microscopic level these necrotic muscle cells contained disoriented sarcomeres and the sarcolemma was disrupted. Mitochondria in these cells appeared swollen with vesiculated cristae and flocculent densities.
Effects of Conus amidis toxin on the hematological variables in the estuarine fish, Mugil cephalus. R. KASINATHAN, V.S. CnANDRASEr,ARAN and J. TAGOP,E (Centre of Advanced Study in Marine Biology, Annamalai University, Parangipettai 608 502 Tamil Nadu, India). THE EFFECTSof toxin of the venomous cone Conus amadis on the hematological variables such as hematocrit, hemaglobin, red blood cell (RBC) count, blood serum protein, total free amino acid (FAA) and blood glucose
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muscle is decreased after pretreatment with 10 #M (67 #g) antx-a(s) as compared to controls. To determine if antx-a(s) has potassium and/or chloride channel activity, membrane potential in fiber bundles of the frog semitendinosus muscle were monitored. Membrane potential was monitored before and after antx-a(s) administration (1 - 10/~M) in normal amphibian Ringer's, high potassium and no potassium Ringer's. Chloride channel activity was determined in a similar manner using solutions of various chloride ion concentrations. Preand postsynaptic effects of antx-a(s) were analyzed using the sciatic nerve - sartorius muscle preparation. REFERENCES HYDE, E. G. and CARMICHAEL,W. W. (1988) Toxicologist g, 40. MAHMOOD, N. A. and CARMICHAEL, W. W. (1987) Toxicon 25, 1221.
Factors influencing the hemolysis of human erythrocytes by three snake venom cardiotoxins. MING-SHI JIANG,1 JEFFREY E. FLETCHER1 and LEONARD A. SMITH2 (Departments of 1Anesthesiology, Hahnemann University, Philadelphia, PA 19102-1192, U.S.A.; 2Department of Toxicology, Pathology Division, USAMRIID, Ft Detrick, Frederick, MD 21701, U.S.A.). THE EFFECTSof cell age, pH, divalent cation type and concentration, temperature and glucose on red blood cell (RBC) hemolysis induced by highly purified cardiotoxins (CTXs) from Naja naja atra, Naja naja kaouthia (each about 7000 mol.wt) and Bungarus fasciatus (16,300 mol.wt) venoms were examined. Using aged RBCs (Ca 2÷ 2 mH, pH 7.4, 37°C, 2 hr) and a 30 #M concentration of each toxin hemolysis levels (as % of hemolysis in distilled H20 ) were: N.n.k., 80%; N.n.a., 40%; Bf. 10%. These values were reduced by a factor of four when fresh RBCs were used. In Ca 2 ÷ or Sr 2 ÷ (2 mM) medium, increasing pH from 7 to 8 greatly increased levels of hemolysis by the three CTXs. In Ba 2 ÷ (2 mM) containing medium, the increased hemolysis at high pH was blocked for B.f. CTX, but not for N.n.k. or N.n.a. CTXs. Divalent cations at 0.5 - 2.0 mM enhanced (Ca 2÷) or slightly decreased (Sr 2÷, Ba 2÷) hemolysis due to N.n.k. and N.n.a. CTXs, and greatly decreased hemolysis at higher concentrations (4 - 40 mM). For the B.f CTX Ba 2 ÷ inhibited hemolysis at all concentrations, Sr 2÷ had little effect and high Ca 2÷ (>40 mM) enhanced hemolysis. Increasing temperature from 20° to 37°C doubled the hemolysis values for all three CTXs. Glucose increased hemolysis by the CTXs at low concentrations of divalent cations (2 mM) and decreased hemolysis at high concentrations (40 mM). The same binding site and mechanism may be involved in hemolysis by three CTXs. However, divalent cations appear to have effects on either binding or the mode of action that are dependent on the CTX studied. Supported in part by the U.S. Army Medical Research and Development Command, contract no. DAMD1787-C-7155.
Pathogenesis of hemorrhage and myonecrosis induced by copperhead (Agkistrodon contortrix sp.) venom. EDWARD K. JOHNSON and CHARLOTTE L. OWNBY (Department of Physiological Sciences, Oklahoma State University, Stillwater, OK 74078, U.S.A.). THE PATHOGENESIS of hemorrhage and myonecrosis induced by copperhead (A. contortrix sp.) venom was studied using light and electron microscopy. Mice were injected i.m. with a sublethal dose (5.0 mg/kg) of venom and muscle tissue taken at varying time intervals (5, 15 and 30 min; 1, 3, 6, 12 and 24 hr) after the injection. Tissue samples were fixed for 2 hr in 2% glutaraldehyde in cacodylate buffer, post-fixed for I hr in 1% osmium tetroxide, and embedded in Polybed 812 resin. For light microscopy, thick sections (1 gm) were obtained and stained with Mallory's trichromer dye. For electron microscopy, thin sections (70 - 90 nm) were obtained and stained with uranyl acetate and lead citrate. Gross observation of the tissue after injection of the venom revealed hemorrhage at all time intervals. Light microscopic examination of the muscle revealed extensive myonecrosis and capillaries in various stages of degeneration. Damaged capillaries and muscle cells were both present as early as 5 min after injection of the venom, however, very few intact capillaries were present at later time intervals. At the light microscope level necrotic muscle cells containing clumped myofilaments were the predominant pattern of myonecrosis although there were some qualitative differences in the lesions at different time periods. At the electron microscopic level these necrotic muscle cells contained disoriented sarcomeres and the sarcolemma was disrupted. Mitochondria in these cells appeared swollen with vesiculated cristae and flocculent densities.
Effects of Conus amidis toxin on the hematological variables in the estuarine fish, Mugil cephalus. R. KASINATHAN, V.S. CnANDRASEr,ARAN and J. TAGOP,E (Centre of Advanced Study in Marine Biology, Annamalai University, Parangipettai 608 502 Tamil Nadu, India). THE EFFECTSof toxin of the venomous cone Conus amadis on the hematological variables such as hematocrit, hemaglobin, red blood cell (RBC) count, blood serum protein, total free amino acid (FAA) and blood glucose