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Antagonists of reactive oxygen species fail to antagonize the effect of β-bungarotoxin on the rat phrenic nerve-diaphragm preparation
0041-0101(94)00169-3
Toxicon. Vol. 33, No. 2, pp. 121-123. 199.5. Elsevier Science Ltd. Printed in Great Britain
LETTER TO THE EDITOR ANTAGONISTS OF REACTIVE OXYGEN SPECIES FAIL TO ANTAGONIZE THE EFFECT OF /3-BUNGAROTOXIN ON THE RAT PHRENIC NERVE-DIAPHRAGM PREPARATION
Presynaptic neurotoxins from snake venoms inhibit twitch tension in mammalian neuroskeletomuscular preparations by blocking the release of acetylchloline from presynaptic terminals through an unknown molecular mechanism (Chang, 1985). These same toxins have phospholipase A2 activity that releases fatty acids from membranes (Noremberg and Parsons, 1986; Yates and Rosenberg, 1991), and this activity has been implicated in the action of the toxins. Arachidonic acid is the predominant fatty acid in the 2 position of phospholipids of mammalian membranes, and it has been shown that metabolism of arachidonic acid and other unsaturated fatty acids can generate reactive oxygen species such as superoxide anion, hydrogen peroxide and hydroxyl radical (Chan et al., 1988; Needleman et al., 1986). In this regard, Crosland (1993) reported that unsaturated fatty acids inhibited twitch tension in the rat phrenic nerve-diaphragm preparation and that the order of potency of the fatty acids paralleled their order of potency in producing superoxide anion in cultured astrocytes (Chan et al., 1988). Superoxide dismutase eliminated the inhibition of twitch tension caused by arachidonic acid, further implicating superoxide anion (and possibly other reactive oxygen species) in the inhibitory action of the unsaturated fatty acids. These observations suggest that snake presynaptic neurotoxins may release arachidonic acid and other unsaturated fatty acids which in turn generate reactive oxygen species that contribute to the inhibition of twitch tension characteristic of the toxins. I tested this hypothesis by determining whether antagonists of reactive oxygen species would eliminate the reduction in twitch tension caused by /I-bungarotoxin, the most studied of the snake venom presynaptic toxins. Chemicals were purchased from the indicated manufacturers: catalase, deferoxamine, superoxide dismutase, vitamin E (Sigma Chemical Co., St Louis, MO, U.S.A.); [Cu(II)], (3,5-diisopropylsalicylate),, 1,2-dimethyl-3-hydroxy-pyridone, 1,3-dimethyl-2-thiourea, N-(2-mercaptopropionyl)-glycine (Aldrich Chemical Co., Milwaukee, WI, U.S.A.); fl-bungarotoxin (Miami Serpentarium Laboratories, Salt Lake City, UT, U.S.A.). Male, Sprague-Dawley rats (80-120 g; Harlan Sprague-Dawley, Inc., Frederick, MD, U.S.A.) were housed four per cage, maintained on a 12 hr light-dark (18:0046:00) cycle, and allowed free access to food and water. Their care and use were in compliance with the Animal Welfare Act (U.S.A.) and the National Institutes of Health’s Guide for the Care 121
122
Letter
to the Editor
and Use of Laboratory Animals (U.S.A.). Rats were anaesthetized with gaseous carbon dioxide and decapitated. Dissection of phrenic nerve-hemidiaphragms and measurement of twitch tension were performed according to Kitchen (1984) after Biilbring (1946) with phrenic nerve electrodes, transducers, and paper chart recorder from Harvard Apparatus (South Natick, MA, U.S.A.) and four channel stimulator from Grass Instruments (Quincy, MA, U.S.A.). I performed experiments in 100ml Teflon@ beakers (Scientific Products, McGaw Park, IL, U.S.A.). The organ bath solution (60 ml) was 1.8 mM CaCl,, 11 mM glucose, 5.0 mM KCI, 0.50 mM MgSO,, 24 mM NaHCO,, 137 mM NaCl, and 1.OmM NaH,PO, (pH 7.3). The solution was maintained at 37°C and oxygenated with 95% 0,/5% CO,. All water-insoluble compounds were dissolved in dimethyl sulfoxide. The resulting concentration of dimethyl sulfoxide (no more than 0.10% v/v) had no effect on twitch tension over the time periods studied (data not shown). All stimulation was indirect using the following parameters: 7.6 V amplitude, 250 n input impedance, 0.25 msec pulse duration, and 1 Hz. All stimulation was maximal and continuous during the experiments. Detecting low levels of reactive oxygen species is difficult, and would be especially so in the presynaptic terminals of the phrenic nerve-diaphragm preparation in view of the fact that their small relative mass makes it impossible to detect any /?-bungarotoxinstimulated phospholipid hydrolysis at the presynaptic terminals of this preparation (Ghassemi et al., 1988). I therefore tested the efficacy of several disparate antagonists of reactive oxygen species to attenuate the effect of j?-bungarotoxin on twitch tension. Superoxide dismutase, catalase, and deferoxamine are membrane-impermeant compounds that act extracellularly. Superoxide dismutase converts superoxide ion into hydrogen peroxide and molecular oxygen, while catalase converts hydrogen peroxide into water and molecular oxygen. Deferoxamine chelates iron, which prevents the formation of hydroxyl radical. The remainder of the antagonists are membrane permeant and can act intracellularly. [Copper(II)],(3,5-diisopropylsalicylate), is a superoxide dismutase mimetic. 1,2Dimethyl-3-hydroxy-pyridone is an iron chelator. 1,3-Dimethyl-2-thiourea scavenges (i.e. reacts with and inactivates) hydroxyl radical and hydrogen peroxide. N-(&mercaptopropionyl)-glycine scavenges superoxide anion and hydroxyl radical. Vitamin E is a general antioxidant. Phrenic nervediaphragm preparations were preincubated in the presence of antagonist. After 10 min, 23 nM B-bungarotoxin was added. I used the time to 50% reduction in twitch tension after the addition of /?-bungarotoxin to compare the effects of the antagonists. The results were (mean min + S.E.M.): no antagonist, 43 f 1; 250 U/ml superoxide dismutase + 640 U/ml catalase + 442 PM deferoxamine, 38 f 0.5; 2.0 p M [copper(H)], (3,5-diisopropylsalicylate), , 34 f 3; 50 PM 1,2-dimethyl-3-hydroxy37+ 1; 1.OmM pyridone, 40 +_0.3; 7.5 mM 1,3-dimethyl-2-thiourea, N-(2mercaptopropionyl)-glycine, 39 + 2; 200 p M vitamin E, 40 + 2. At these known effective concentrations (Lesnefsky, 1992), none of the antagonists attenuated the effect of /?-bungarotoxin. The results clearly demonstrate that none of the antagonists of reactive oxygen species that I tested was able to antagonize /3-bungarotoxin’s reduction in twitch tension at the rat phrenic nerve-diaphragm preparation. These antagonists are effective against several reactive oxygen species (Lesnefsky, 1992) and are both membrane impermeant and membrane permeant. If one can generalize from the effects of the antagonists and toxin studied here, it does not appear that reactive oxygen species are involved in the action of snake presynaptic phospholipase A, toxins.
Letter to the Editor
123
REFERENCES Bulbring, E. (1946) Observations on the isolated phrenic nerve diaphragm preparation of the rat. Br. J. Phnrmac. 1, 38-61. Chan, P. H., Chen, S. F. and Yu, A. C. H. (1988) Induction of intracellular superoxide radical formation by arachidonic acid and by polyunsaturated fatty acids in primary astrocytic cultures. J. Neurochem. 50, 1185-I 193. Chang, C. C. (1985) Neurotoxins with phospholipase A, activity in snake venoms. Proc. natn. Sci. Count. B. ROC 9, 126142. Crosland, R. D. (1993) Effect of arachidonic acid on twitch tension of the rat phrenic nerve-diaphragm. J. Pharmac. exp. Ther. 264, 1311-1314.
Ghassemi, A., Dhillon, D. S. and Rosenberg, P. (1988) j?-Bungarotoxin-induced phospholipid hydrolysis in rat brain synaptosomes: effect of replacement of calcium by strontium. Toxicon 26, 5099514. Kitchen, I. (1984) Textbook of in vitro Practical Pharmacology. Oxford: Plenum Press. Lesnefsky, E. J. (1992) Reduction of infarct size by cell-permeable oxygen metabolite scavengers. Free Rad. Biol. Med. 12, 429-446.
Needleman, P., Turk, J., Jakschik, B. A., Morriso, A. R. and Lefkowith, J. B. (1986) Arachidonic acid metabolism. A. Rev. Biochem. 55, 69-102. Noremberg, K. and Parsons, S. M. (1986) Selectivity and regulation in the phospholipase AI-mediated attack on cholinergic synaptic vesicles by /I-bungarotoxin. J. Neurochem. 47, 1312-1317. Yates, S. L. and Rosenberg, P. (1991) Comparative effects of phospholipase A, neurotoxins and enzymes on membrane potential and Na+/K+ ATPase activitv of rat brain svnodosomes. Toxic. aool. Pharmac. 109. 207-218. _ RICHARD D. CROSLAND Toxinology Division United States Army Medical Research Institute of Infectious Diseases Frederick MD 21702-501 I U.S.A.
Toxicon. Vol. 33, No. 2, pp. 121-123. 199.5. Elsevier Science Ltd. Printed in Great Britain
LETTER TO THE EDITOR ANTAGONISTS OF REACTIVE OXYGEN SPECIES FAIL TO ANTAGONIZE THE EFFECT OF /3-BUNGAROTOXIN ON THE RAT PHRENIC NERVE-DIAPHRAGM PREPARATION
Presynaptic neurotoxins from snake venoms inhibit twitch tension in mammalian neuroskeletomuscular preparations by blocking the release of acetylchloline from presynaptic terminals through an unknown molecular mechanism (Chang, 1985). These same toxins have phospholipase A2 activity that releases fatty acids from membranes (Noremberg and Parsons, 1986; Yates and Rosenberg, 1991), and this activity has been implicated in the action of the toxins. Arachidonic acid is the predominant fatty acid in the 2 position of phospholipids of mammalian membranes, and it has been shown that metabolism of arachidonic acid and other unsaturated fatty acids can generate reactive oxygen species such as superoxide anion, hydrogen peroxide and hydroxyl radical (Chan et al., 1988; Needleman et al., 1986). In this regard, Crosland (1993) reported that unsaturated fatty acids inhibited twitch tension in the rat phrenic nerve-diaphragm preparation and that the order of potency of the fatty acids paralleled their order of potency in producing superoxide anion in cultured astrocytes (Chan et al., 1988). Superoxide dismutase eliminated the inhibition of twitch tension caused by arachidonic acid, further implicating superoxide anion (and possibly other reactive oxygen species) in the inhibitory action of the unsaturated fatty acids. These observations suggest that snake presynaptic neurotoxins may release arachidonic acid and other unsaturated fatty acids which in turn generate reactive oxygen species that contribute to the inhibition of twitch tension characteristic of the toxins. I tested this hypothesis by determining whether antagonists of reactive oxygen species would eliminate the reduction in twitch tension caused by /I-bungarotoxin, the most studied of the snake venom presynaptic toxins. Chemicals were purchased from the indicated manufacturers: catalase, deferoxamine, superoxide dismutase, vitamin E (Sigma Chemical Co., St Louis, MO, U.S.A.); [Cu(II)], (3,5-diisopropylsalicylate),, 1,2-dimethyl-3-hydroxy-pyridone, 1,3-dimethyl-2-thiourea, N-(2-mercaptopropionyl)-glycine (Aldrich Chemical Co., Milwaukee, WI, U.S.A.); fl-bungarotoxin (Miami Serpentarium Laboratories, Salt Lake City, UT, U.S.A.). Male, Sprague-Dawley rats (80-120 g; Harlan Sprague-Dawley, Inc., Frederick, MD, U.S.A.) were housed four per cage, maintained on a 12 hr light-dark (18:0046:00) cycle, and allowed free access to food and water. Their care and use were in compliance with the Animal Welfare Act (U.S.A.) and the National Institutes of Health’s Guide for the Care 121
122
Letter
to the Editor
and Use of Laboratory Animals (U.S.A.). Rats were anaesthetized with gaseous carbon dioxide and decapitated. Dissection of phrenic nerve-hemidiaphragms and measurement of twitch tension were performed according to Kitchen (1984) after Biilbring (1946) with phrenic nerve electrodes, transducers, and paper chart recorder from Harvard Apparatus (South Natick, MA, U.S.A.) and four channel stimulator from Grass Instruments (Quincy, MA, U.S.A.). I performed experiments in 100ml Teflon@ beakers (Scientific Products, McGaw Park, IL, U.S.A.). The organ bath solution (60 ml) was 1.8 mM CaCl,, 11 mM glucose, 5.0 mM KCI, 0.50 mM MgSO,, 24 mM NaHCO,, 137 mM NaCl, and 1.OmM NaH,PO, (pH 7.3). The solution was maintained at 37°C and oxygenated with 95% 0,/5% CO,. All water-insoluble compounds were dissolved in dimethyl sulfoxide. The resulting concentration of dimethyl sulfoxide (no more than 0.10% v/v) had no effect on twitch tension over the time periods studied (data not shown). All stimulation was indirect using the following parameters: 7.6 V amplitude, 250 n input impedance, 0.25 msec pulse duration, and 1 Hz. All stimulation was maximal and continuous during the experiments. Detecting low levels of reactive oxygen species is difficult, and would be especially so in the presynaptic terminals of the phrenic nerve-diaphragm preparation in view of the fact that their small relative mass makes it impossible to detect any /?-bungarotoxinstimulated phospholipid hydrolysis at the presynaptic terminals of this preparation (Ghassemi et al., 1988). I therefore tested the efficacy of several disparate antagonists of reactive oxygen species to attenuate the effect of j?-bungarotoxin on twitch tension. Superoxide dismutase, catalase, and deferoxamine are membrane-impermeant compounds that act extracellularly. Superoxide dismutase converts superoxide ion into hydrogen peroxide and molecular oxygen, while catalase converts hydrogen peroxide into water and molecular oxygen. Deferoxamine chelates iron, which prevents the formation of hydroxyl radical. The remainder of the antagonists are membrane permeant and can act intracellularly. [Copper(II)],(3,5-diisopropylsalicylate), is a superoxide dismutase mimetic. 1,2Dimethyl-3-hydroxy-pyridone is an iron chelator. 1,3-Dimethyl-2-thiourea scavenges (i.e. reacts with and inactivates) hydroxyl radical and hydrogen peroxide. N-(&mercaptopropionyl)-glycine scavenges superoxide anion and hydroxyl radical. Vitamin E is a general antioxidant. Phrenic nervediaphragm preparations were preincubated in the presence of antagonist. After 10 min, 23 nM B-bungarotoxin was added. I used the time to 50% reduction in twitch tension after the addition of /?-bungarotoxin to compare the effects of the antagonists. The results were (mean min + S.E.M.): no antagonist, 43 f 1; 250 U/ml superoxide dismutase + 640 U/ml catalase + 442 PM deferoxamine, 38 f 0.5; 2.0 p M [copper(H)], (3,5-diisopropylsalicylate), , 34 f 3; 50 PM 1,2-dimethyl-3-hydroxy37+ 1; 1.OmM pyridone, 40 +_0.3; 7.5 mM 1,3-dimethyl-2-thiourea, N-(2mercaptopropionyl)-glycine, 39 + 2; 200 p M vitamin E, 40 + 2. At these known effective concentrations (Lesnefsky, 1992), none of the antagonists attenuated the effect of /?-bungarotoxin. The results clearly demonstrate that none of the antagonists of reactive oxygen species that I tested was able to antagonize /3-bungarotoxin’s reduction in twitch tension at the rat phrenic nerve-diaphragm preparation. These antagonists are effective against several reactive oxygen species (Lesnefsky, 1992) and are both membrane impermeant and membrane permeant. If one can generalize from the effects of the antagonists and toxin studied here, it does not appear that reactive oxygen species are involved in the action of snake presynaptic phospholipase A, toxins.
Letter to the Editor
123
REFERENCES Bulbring, E. (1946) Observations on the isolated phrenic nerve diaphragm preparation of the rat. Br. J. Phnrmac. 1, 38-61. Chan, P. H., Chen, S. F. and Yu, A. C. H. (1988) Induction of intracellular superoxide radical formation by arachidonic acid and by polyunsaturated fatty acids in primary astrocytic cultures. J. Neurochem. 50, 1185-I 193. Chang, C. C. (1985) Neurotoxins with phospholipase A, activity in snake venoms. Proc. natn. Sci. Count. B. ROC 9, 126142. Crosland, R. D. (1993) Effect of arachidonic acid on twitch tension of the rat phrenic nerve-diaphragm. J. Pharmac. exp. Ther. 264, 1311-1314.
Ghassemi, A., Dhillon, D. S. and Rosenberg, P. (1988) j?-Bungarotoxin-induced phospholipid hydrolysis in rat brain synaptosomes: effect of replacement of calcium by strontium. Toxicon 26, 5099514. Kitchen, I. (1984) Textbook of in vitro Practical Pharmacology. Oxford: Plenum Press. Lesnefsky, E. J. (1992) Reduction of infarct size by cell-permeable oxygen metabolite scavengers. Free Rad. Biol. Med. 12, 429-446.
Needleman, P., Turk, J., Jakschik, B. A., Morriso, A. R. and Lefkowith, J. B. (1986) Arachidonic acid metabolism. A. Rev. Biochem. 55, 69-102. Noremberg, K. and Parsons, S. M. (1986) Selectivity and regulation in the phospholipase AI-mediated attack on cholinergic synaptic vesicles by /I-bungarotoxin. J. Neurochem. 47, 1312-1317. Yates, S. L. and Rosenberg, P. (1991) Comparative effects of phospholipase A, neurotoxins and enzymes on membrane potential and Na+/K+ ATPase activitv of rat brain svnodosomes. Toxic. aool. Pharmac. 109. 207-218. _ RICHARD D. CROSLAND Toxinology Division United States Army Medical Research Institute of Infectious Diseases Frederick MD 21702-501 I U.S.A.