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Release of eicosanoids induced by Crotalus durissus terrificus venom from guinea-pig isolated lungs
600
Third Pan-American S y m p o s i u m - - A b s t r a c t s
Exploration of the molecular structure and cellular distribution of ion channels' by neurotoxins. KIMON J. ANGELIDES (Department of Molecular Physiology and Biophysics, and Neuroscience, One Baylor Plaza, Baylor College of Medicine, Houston, TX 77030, U.S.A.). THE ISOLATION,chemical characterization, and use of neurotoxins in cell biology has increased significantly in the past decade. To dissect the molecular structure and cellular dynamics of ion channels in nerve, we have synthesized biologically active fluorescent channel-specific neurotoxin derivatives. Because the regulation of ion channel distribution is critical in determining the firing activity and integrative properties of neurons, we have used these neurotoxins together with a digital fluorescence imaging and photobleach recovery microscopy to m a p ion channel distribution and mobility on mature and developing nerve cells. On mature neurons Na ÷ and Ca 2÷ channels are segregated to morphologically distinct regions; labeling with fluorescent neurotoxins specific for Na ÷ channels show that the hillock is stained intensely, and the cell body is sparse and diffuse. Several foci of high Na + channel density are seen on the axon. In contrast, Ca 2+ channels, labeled with fluorescent ~o-conotoxin, are segregated to dendrites of the same neuron. When the lateral mobility of these channels is examined by fluorescence photobleach recovery, Na ÷ channels on the cell body are freely mobile while Na ÷ channels located at the hillock and along the axon are immobile. Ca 2+ channels on cell bodies are immobile, but Ca 2+ channels are mobile within dendritic spines. In addition, each channel is targeted to these regions early in development. Our imaging and photobleach studies with these toxins suggest that the distribution and mobility of Na ÷ and Ca2÷ channels have a great deal of molecular specificity. For Na ÷ channels their distribution is controlled through associations with the cytoskeleton, which on axons correlates with the expression of ankyrin and spectrin. While similar interactions m a y be responsible for immobilizing Ca 2+ channels on the cell body, the recruitment of mobile Ca 2÷ channels on dendrites confers additional plasticity to the neuron and greatly expand the neuron's ability to orchestrate and regulate Ca 2+ signaling. Neurotoxins, indeed, have provided exquisite probes to view the cellular dynamics of neuronal ion channels.
Biochemical characterization of variant Chinese-hamster ovary cell lines resistant to the microbial metabolite A23187. CINDY K. ANGERHOFERand W. THOMAS SHIER (Dept of Medicinal Chemistry, University of Minnesota, Minneapolis, M N 55455, U.S.A.). A23187, a cytotoxic metabolite secreted by Streptomyces chartreusis, has been shown to act as an ionophore able to shuttle divalent cations, including Ca 2+, across biological membranes. Chinese-hamster ovary (CHO) cells are killed by A23187 in the presence o f extracellular Ca 2÷ using m e m b r a n e permeabilization to Trypan blue dye as the criterion for cell death. Two variant C H O lines were selected in our laboratory which exhibit 10-fold increased resistance to killing by A23187 plus Ca 2+. The resistant lines showed cross-resistance to the other calcium ionophores lasolocid and ionomycin as well as to amphotericin B. Cytochalasin B (200/aM) largely reversed resistance o f the variants, while a panel of other metabolic inhibitors and Ca 2÷ antagonists had no effect on cell killing profiles. Total phospholipase activity was significantly less in the resistant variants, but preincubation with arachidonate metabolism inhibitors had no effect on A23187-stimulated cell killing. Ca2+/Mg 2÷ ATPase activities were determined to be lower in the variants than in the wild type, suggesting that increased p u m p i n g of Ca 2+ out of the cells is not the mechanism of resistance. SDS-polyacrylamide gel electrophoresis of cell homogenate and plasma m e m b r a n e preparations indicated a 5 x increase in a 57 k D membrane protein in the variants relative to the wild type cells. A 180 k D protein was also sporadically observed in variant membranes. No obvious differences were noted in protein phosphorylation patterns when wild type or variant lines were preloaded with 32PO4, with or without A23187 treatment. Photoafl]nity labeling revealed a 116 k D ATP-binding protein exclusively in wild type membranes; 2 - 4 x increased ATP-binding by a 190kD protein in variants relative to wild type, and a 4 x increase in GTP-binding by a 75 k D homogenate protein of a variant. These proteins m a y act to p u m p A23187 molecules out of variant cells. Electroblotting of electrophoresed proteins followed by 45Ca2+ overlay of the m e m b r a n e s detected a 2-3 x increase in Ca 2+ binding by a 17 k D membrane-associated protein in variants. Resistance to A23187 plus Ca -,÷ appears to be achieved by modest changes in activity of a variety of mechanisms for defending against elevated intracellular Ca 2+.
Release of eicosanoids induced by Crotalus durissus terrificus venom from guinea-pig isolated lungs. EDSON ANTUNES, JOLIA PRAt)O-FRANCESCHI and GILBERTO DE NUCCl (Dept of Pharmacology, F C M / U N I C A M P , Campinas-SP., Brazil). SNAKE venoms release histamine and SRS from guinea-pig isolated lungs (FELDBERGand KELLAWAY,1938). Convulxin, a high mol. wt toxin purified from C.d. terrificus venom (PRADO-FRANCESCHI and VITAL BRAZIL, 1981), also release T X A a from rabbit platelets (VARGAETIGe t al., 1980) . We report here that C.d. terrificus venom release TXA~ and PG12 from isolated lungs of guinea-pigs and this release is greater when the venom is infused via the trachea. The guinea-pigs (male, 300-350 g) were anaesthetized with pentobarbitone. After mid toracotomy, the pulmonary artery was cannulated and perfused for 5 min with beparinized Krebs' bicarbonate
Third Pan-American Symposium--Abstracts
601
solution. The trachea was cannulated with the lungs removed and placed in a heated chamber. The lungs were perfused via pulmonary artery or via trachea. C.d. terrificus venom (10 #g/ml) was infused through the lungs for 3 min and the samples collected for l0 rain. Prostacyclin and thromboxane release were monitored by radioimmunoassay of their breakdown products, 6-oxo-PGF~ alpha and TXB 2, respectively (SALMON, 1978). When infused via the pulmonary artery, the venom released 9.9+0.5 and 4.2+0.4ng/ml of TXB 2 and 6-oxo-PGF~ alpha respectively (n = 4). When infused via the trachea, the release of eicosanoids induced by the venom was greater (I 9.2 + 5.2 ng/ml of TXB2; n = 4). These results indicate that lungs perfused via the trachea provide a more sensitive model for studying the ability of snake venoms to release eicosanoids. REFERENCES FELDnFatG, W. and KELLWAY,C. H. (1938) J. Physiol. 94, 187. PRAvo-FRANce~m, J. and VITAL BRAZIL,O. (1981) Toxicon 19, 875. SALMON,J. A. (1978) Prostaglandins 15, 387. VAgOAFTIO,B. B., PRADO-FRAsCESCH1,J., CmGNARD, M., LEFORT,J. and MARLAS,G. (1980) Eur. J. Pharmacol 68, 451. Comparison o f the phospholipase A2, blood-clotting, caseinolytic esterolytic and hemorrhagic activities o f some Bothrops snake venoms. ALBETIZAL. DE ,M~AOJO,Jos~ L. DONATO, RONILSONA. MOp.£NO and J(JLIA PRADOFRANCF_.SCm(Dept. of Pharmacology, FCM/UNICAMP, P.O. Box 6111, 13082-Campinas-SP., Brazil).
DRIED crude venoms of eight Bothrop species (Bothrops alternatus, B. erythromelas, B insularis, B. jararaca, B. jararacussu, B. lanceolatus, B. moojeni and B. neuwiediO were assayed for phospholipase A2, blood-clotting, caseinolytic, esterolytic and hemorrhagic activities. Phospholipase activity measured using Triton X-100 solubilized lechithin showed that B. jararacussu venom had the highest activity (90/zmole/min/mg) and B. alternatus the lowest (23 #mole/min/mg). In blood-clotting and caseinoioytic activities, B. erythromelas venom was the most active (Minimal Coagulant Dose = 1.5 #g and caseinolytic activity 2.94 U/rag for caseinolytic activity), B. lanceolatu~ was the only venom devoid of clotting activity, while B. jararacussu had poor caseinolytic activity (0.73 U/mg). For B. insularis venom the highest activity was the esterolytic (p-toluenesulfonyl-L-arginine methyl ester, 1.83 U/mg) which was practically absent in the B. erythromelas sample (0.40 U/mg). In the hemorrhagic assay, B. jararaca venom was the most potent (Minimal Hemorrhagic Dose = 0.35 #g) and B. insularis venom the least potent (MHD = 20#g). The present results are a preliminary part of a project aimed at studying the pharmacological aspects of Bothrops snake venoms. Canatoxin induces in vivo neutrophil migration. CHRISTINA BAn~A-F1DALC,O,mCI~LIA R. S. CARLINI,2 JORGE A. GtnMAm~S,2 CARLOSA. FLOP.m,3 F~NAt,a~O Q. CUNrIA3 and SERGIOH. F~ut~UtA3. (~Dept. Pharmacology/IB/ Universidade do Estado do Rio de Janeiro, 2Dept. Biochemistry, ICB, Universidade Federal do Rio de Janeiro, and 3Dept. Pharmacology, Fac. Medicina Ribeirao Preto, Univ. Sao Paulo, Brazil).
CANATOXaS(CNTX), a toxic protein purified from Canavalia ensiformis seeds has been shown to induce in vitro neurotransmitter secretion in synaptosomes and platelets (CARL1NIet al., 1985; BA~A-FIDALGOet al., 1988) and hormone release in vivo (RIBEIRO DASILVAet al., 1988). Those effects seem to be related to a lipoxygenasedependent pathway. Leukotriene B4, a product of 5-1ipoxygenase, is well known as chemotactic for neutrophil in vitro and in vivo. Besides that, SOUZAet al., (1988) described that neutrophil migration induced by different inflammatory stimuli was dependent on the resident macrophages population. Thus, the aim of the present work was to investigate the potential ability of CNTX to induce neutrophil migration in vivo. Four hours after injection into rat peritoneal cavities, CNTX (250rig) induced a significant neutrophil migration (control : 7,6 + 3 x I0 vs CNTX : 3,4 + 5,2 x 10 cells/ml). When macrophage number was increased in 3% thioglycollate pretreated rats (10ml/i.p.), CNTX induced a significantly greater neutrophil migration into the cavities. On the other hand, in rats with reduced resident macrophages population (saline-washed peritoneal cavities) CNTX-induced migratory effect was greatly diminished. Nentrophil migration was also observed into rat skin air-pouches injected with CNTX. Pretreatment of the rats with dexamethasone (0.5/zg/kg) inhibited bot)t effects. These data suggest that CNTX-induced neutrophil migration would be dependent on the resident macrophage population. The mechanism by which resident macrophages can control this migration and the involvement of arachidonate pathways on this effect of CNTX are now being studied. Acknowledgement - - Supported by FINEP, CNPq & IFS (Sweden). REFERENCES BAgJA-FIDALGOet al. (1988) Braz. J. Med. Biol. Res. 21, 549. CAgLIm et al. (1985) Brit. J. Pharrnacol. 84, 551. RiaEmo DASILVAet al. (1988) Braz. J. Med. Biol. Res. 22, 303. SOUZA et al. (1988) Agent & Actions 24, 377.
Third Pan-American S y m p o s i u m - - A b s t r a c t s
Exploration of the molecular structure and cellular distribution of ion channels' by neurotoxins. KIMON J. ANGELIDES (Department of Molecular Physiology and Biophysics, and Neuroscience, One Baylor Plaza, Baylor College of Medicine, Houston, TX 77030, U.S.A.). THE ISOLATION,chemical characterization, and use of neurotoxins in cell biology has increased significantly in the past decade. To dissect the molecular structure and cellular dynamics of ion channels in nerve, we have synthesized biologically active fluorescent channel-specific neurotoxin derivatives. Because the regulation of ion channel distribution is critical in determining the firing activity and integrative properties of neurons, we have used these neurotoxins together with a digital fluorescence imaging and photobleach recovery microscopy to m a p ion channel distribution and mobility on mature and developing nerve cells. On mature neurons Na ÷ and Ca 2÷ channels are segregated to morphologically distinct regions; labeling with fluorescent neurotoxins specific for Na ÷ channels show that the hillock is stained intensely, and the cell body is sparse and diffuse. Several foci of high Na + channel density are seen on the axon. In contrast, Ca 2+ channels, labeled with fluorescent ~o-conotoxin, are segregated to dendrites of the same neuron. When the lateral mobility of these channels is examined by fluorescence photobleach recovery, Na ÷ channels on the cell body are freely mobile while Na ÷ channels located at the hillock and along the axon are immobile. Ca 2+ channels on cell bodies are immobile, but Ca 2+ channels are mobile within dendritic spines. In addition, each channel is targeted to these regions early in development. Our imaging and photobleach studies with these toxins suggest that the distribution and mobility of Na ÷ and Ca2÷ channels have a great deal of molecular specificity. For Na ÷ channels their distribution is controlled through associations with the cytoskeleton, which on axons correlates with the expression of ankyrin and spectrin. While similar interactions m a y be responsible for immobilizing Ca 2+ channels on the cell body, the recruitment of mobile Ca 2÷ channels on dendrites confers additional plasticity to the neuron and greatly expand the neuron's ability to orchestrate and regulate Ca 2+ signaling. Neurotoxins, indeed, have provided exquisite probes to view the cellular dynamics of neuronal ion channels.
Biochemical characterization of variant Chinese-hamster ovary cell lines resistant to the microbial metabolite A23187. CINDY K. ANGERHOFERand W. THOMAS SHIER (Dept of Medicinal Chemistry, University of Minnesota, Minneapolis, M N 55455, U.S.A.). A23187, a cytotoxic metabolite secreted by Streptomyces chartreusis, has been shown to act as an ionophore able to shuttle divalent cations, including Ca 2+, across biological membranes. Chinese-hamster ovary (CHO) cells are killed by A23187 in the presence o f extracellular Ca 2÷ using m e m b r a n e permeabilization to Trypan blue dye as the criterion for cell death. Two variant C H O lines were selected in our laboratory which exhibit 10-fold increased resistance to killing by A23187 plus Ca 2+. The resistant lines showed cross-resistance to the other calcium ionophores lasolocid and ionomycin as well as to amphotericin B. Cytochalasin B (200/aM) largely reversed resistance o f the variants, while a panel of other metabolic inhibitors and Ca 2÷ antagonists had no effect on cell killing profiles. Total phospholipase activity was significantly less in the resistant variants, but preincubation with arachidonate metabolism inhibitors had no effect on A23187-stimulated cell killing. Ca2+/Mg 2÷ ATPase activities were determined to be lower in the variants than in the wild type, suggesting that increased p u m p i n g of Ca 2+ out of the cells is not the mechanism of resistance. SDS-polyacrylamide gel electrophoresis of cell homogenate and plasma m e m b r a n e preparations indicated a 5 x increase in a 57 k D membrane protein in the variants relative to the wild type cells. A 180 k D protein was also sporadically observed in variant membranes. No obvious differences were noted in protein phosphorylation patterns when wild type or variant lines were preloaded with 32PO4, with or without A23187 treatment. Photoafl]nity labeling revealed a 116 k D ATP-binding protein exclusively in wild type membranes; 2 - 4 x increased ATP-binding by a 190kD protein in variants relative to wild type, and a 4 x increase in GTP-binding by a 75 k D homogenate protein of a variant. These proteins m a y act to p u m p A23187 molecules out of variant cells. Electroblotting of electrophoresed proteins followed by 45Ca2+ overlay of the m e m b r a n e s detected a 2-3 x increase in Ca 2+ binding by a 17 k D membrane-associated protein in variants. Resistance to A23187 plus Ca -,÷ appears to be achieved by modest changes in activity of a variety of mechanisms for defending against elevated intracellular Ca 2+.
Release of eicosanoids induced by Crotalus durissus terrificus venom from guinea-pig isolated lungs. EDSON ANTUNES, JOLIA PRAt)O-FRANCESCHI and GILBERTO DE NUCCl (Dept of Pharmacology, F C M / U N I C A M P , Campinas-SP., Brazil). SNAKE venoms release histamine and SRS from guinea-pig isolated lungs (FELDBERGand KELLAWAY,1938). Convulxin, a high mol. wt toxin purified from C.d. terrificus venom (PRADO-FRANCESCHI and VITAL BRAZIL, 1981), also release T X A a from rabbit platelets (VARGAETIGe t al., 1980) . We report here that C.d. terrificus venom release TXA~ and PG12 from isolated lungs of guinea-pigs and this release is greater when the venom is infused via the trachea. The guinea-pigs (male, 300-350 g) were anaesthetized with pentobarbitone. After mid toracotomy, the pulmonary artery was cannulated and perfused for 5 min with beparinized Krebs' bicarbonate
Third Pan-American Symposium--Abstracts
601
solution. The trachea was cannulated with the lungs removed and placed in a heated chamber. The lungs were perfused via pulmonary artery or via trachea. C.d. terrificus venom (10 #g/ml) was infused through the lungs for 3 min and the samples collected for l0 rain. Prostacyclin and thromboxane release were monitored by radioimmunoassay of their breakdown products, 6-oxo-PGF~ alpha and TXB 2, respectively (SALMON, 1978). When infused via the pulmonary artery, the venom released 9.9+0.5 and 4.2+0.4ng/ml of TXB 2 and 6-oxo-PGF~ alpha respectively (n = 4). When infused via the trachea, the release of eicosanoids induced by the venom was greater (I 9.2 + 5.2 ng/ml of TXB2; n = 4). These results indicate that lungs perfused via the trachea provide a more sensitive model for studying the ability of snake venoms to release eicosanoids. REFERENCES FELDnFatG, W. and KELLWAY,C. H. (1938) J. Physiol. 94, 187. PRAvo-FRANce~m, J. and VITAL BRAZIL,O. (1981) Toxicon 19, 875. SALMON,J. A. (1978) Prostaglandins 15, 387. VAgOAFTIO,B. B., PRADO-FRAsCESCH1,J., CmGNARD, M., LEFORT,J. and MARLAS,G. (1980) Eur. J. Pharmacol 68, 451. Comparison o f the phospholipase A2, blood-clotting, caseinolytic esterolytic and hemorrhagic activities o f some Bothrops snake venoms. ALBETIZAL. DE ,M~AOJO,Jos~ L. DONATO, RONILSONA. MOp.£NO and J(JLIA PRADOFRANCF_.SCm(Dept. of Pharmacology, FCM/UNICAMP, P.O. Box 6111, 13082-Campinas-SP., Brazil).
DRIED crude venoms of eight Bothrop species (Bothrops alternatus, B. erythromelas, B insularis, B. jararaca, B. jararacussu, B. lanceolatus, B. moojeni and B. neuwiediO were assayed for phospholipase A2, blood-clotting, caseinolytic, esterolytic and hemorrhagic activities. Phospholipase activity measured using Triton X-100 solubilized lechithin showed that B. jararacussu venom had the highest activity (90/zmole/min/mg) and B. alternatus the lowest (23 #mole/min/mg). In blood-clotting and caseinoioytic activities, B. erythromelas venom was the most active (Minimal Coagulant Dose = 1.5 #g and caseinolytic activity 2.94 U/rag for caseinolytic activity), B. lanceolatu~ was the only venom devoid of clotting activity, while B. jararacussu had poor caseinolytic activity (0.73 U/mg). For B. insularis venom the highest activity was the esterolytic (p-toluenesulfonyl-L-arginine methyl ester, 1.83 U/mg) which was practically absent in the B. erythromelas sample (0.40 U/mg). In the hemorrhagic assay, B. jararaca venom was the most potent (Minimal Hemorrhagic Dose = 0.35 #g) and B. insularis venom the least potent (MHD = 20#g). The present results are a preliminary part of a project aimed at studying the pharmacological aspects of Bothrops snake venoms. Canatoxin induces in vivo neutrophil migration. CHRISTINA BAn~A-F1DALC,O,mCI~LIA R. S. CARLINI,2 JORGE A. GtnMAm~S,2 CARLOSA. FLOP.m,3 F~NAt,a~O Q. CUNrIA3 and SERGIOH. F~ut~UtA3. (~Dept. Pharmacology/IB/ Universidade do Estado do Rio de Janeiro, 2Dept. Biochemistry, ICB, Universidade Federal do Rio de Janeiro, and 3Dept. Pharmacology, Fac. Medicina Ribeirao Preto, Univ. Sao Paulo, Brazil).
CANATOXaS(CNTX), a toxic protein purified from Canavalia ensiformis seeds has been shown to induce in vitro neurotransmitter secretion in synaptosomes and platelets (CARL1NIet al., 1985; BA~A-FIDALGOet al., 1988) and hormone release in vivo (RIBEIRO DASILVAet al., 1988). Those effects seem to be related to a lipoxygenasedependent pathway. Leukotriene B4, a product of 5-1ipoxygenase, is well known as chemotactic for neutrophil in vitro and in vivo. Besides that, SOUZAet al., (1988) described that neutrophil migration induced by different inflammatory stimuli was dependent on the resident macrophages population. Thus, the aim of the present work was to investigate the potential ability of CNTX to induce neutrophil migration in vivo. Four hours after injection into rat peritoneal cavities, CNTX (250rig) induced a significant neutrophil migration (control : 7,6 + 3 x I0 vs CNTX : 3,4 + 5,2 x 10 cells/ml). When macrophage number was increased in 3% thioglycollate pretreated rats (10ml/i.p.), CNTX induced a significantly greater neutrophil migration into the cavities. On the other hand, in rats with reduced resident macrophages population (saline-washed peritoneal cavities) CNTX-induced migratory effect was greatly diminished. Nentrophil migration was also observed into rat skin air-pouches injected with CNTX. Pretreatment of the rats with dexamethasone (0.5/zg/kg) inhibited bot)t effects. These data suggest that CNTX-induced neutrophil migration would be dependent on the resident macrophage population. The mechanism by which resident macrophages can control this migration and the involvement of arachidonate pathways on this effect of CNTX are now being studied. Acknowledgement - - Supported by FINEP, CNPq & IFS (Sweden). REFERENCES BAgJA-FIDALGOet al. (1988) Braz. J. Med. Biol. Res. 21, 549. CAgLIm et al. (1985) Brit. J. Pharrnacol. 84, 551. RiaEmo DASILVAet al. (1988) Braz. J. Med. Biol. Res. 22, 303. SOUZA et al. (1988) Agent & Actions 24, 377.