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Biological reactions of fluoroacetylcholine
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 62, 293-298 (1956)
Biological Reactions of Fluoroacetylcholine Alan H. Mehler and Y. T. Chang’ From the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, United States Public Health Service, Department of Health, Education, and Welfare, Bethesda, Maryland
Received October 31, 1955
Fluoroacetate has been recognized as a powerful poison for many animals for several years, and, as its sodium salt, is marketed as the rodentitide, 1080. The toxic action of this compound has recently been attributed to its effect on the tricarboxylic acid cycle (1, 2). Presumably through the intermediate formation of fluoroacetyl coenzyme A (CoA), an analog of citric acid is formed which is a potent inhibitor of the enzyme aconitase (3). The demonstration of enzyme inhibition caused by fluorocitrate, a derivative of fluoroacetate, has led to attempts to find other such derivatives. Although interference with citrate oxidation is an adequate mechanism for producing toxic symptoms, additional mechanisms may also occur. The various manifestations of fluoroacetate poisoning among different species usually appear to involve the heart or central nervous system (4). Therefore it was considered that fluoroacetylcholine might be involved as a toxic agent. Accordingly, this compound was synthesized and tested enzymatically and pharmacologically. MATERIALS
Fluoroacetylcholine was synthesized by adding two equivalents of fluoroacetylchloride to choline chloride and warming to dissolve. The product obtained by concentration was fluoroacetylcholine chloride, which was recrystallized from ethanol. The following analysis2 was obtained on the very hygroscopic crystals. r Fellow, Leonard Wood Memorial (American Leprosy Foundation). 2 C, H, N, and Cl analyses were performed in the analytical laboratory under the direction of Dr. W. C. Alford of this Institute. We are indebted to Dr. I. Zipkin of the National Institute of Dental Research for the fluorine analysis. 293
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ALAN
H. MEHLER
AND Y. T. CHANG
Anal. Calcd. for C~HIEOZNCIF: C, 42.12; H, 7.57; N, 7.01; Cl, 17.76; F, 9.52. Found: C, 41.06; H, 7.76; N, 6.72; Cl, 17.11; F, 9.16. Fluoroacetylcholine reacts with hydroxylamine under the conditions of Hestrin (5), but the color intensity of the iron complex of the hydroxamic acid is somewhat less than that given by an equivalent amount of acetylcholine. Serum cholinesterase (pseudocholinesterase) was obtained as a purified fraction of human serum supplied by the Cutter Laboratories, Berkeley, California. Guinea pig brain homogenates were used as a source of the so-called “true” cholinesterase. The brains were homogenized in a Waring blendor with 5 vol. of cold 1% potassium chloride, then filtered through cheesecloth. EXPERIMENTAL
Choline&eraseReactions The abilities of both true and pseudocholinesterases to hydrolyze fluoroacetylcholine were measured manometrically. The relative rates of hydrolysis of fluoroacetylcholine and acetylcholine are shown in Fig. 1. In the case of the serum enzyme, fluoroacetylcholine hydrolysis increases in rate with increasing substrate concentration. This enzyme appears to be saturated with acetylcholine until the concentration is reduced to less
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than lO+ M. The brain enzyme, on the other hand, hydrolyzes both esters at essentially the same rate throughout the concentration range shown in Fig. 1. At higher concentrations ( W2 M) , fluoroacetylcholine does not produce the inhibition found with acetylcholine.
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FIG. 1B IFIG. 1. Hydrolysis
of choline esters by guinea pig brain and serum cholinesterase. The reaction was followed by measuring CO* evolved from a COrbicarbonate buffer in conventional Warburg manometers. Each vessel contained 2.0 ml. 0.02 M NaHC03 saturated with 5% COZ-95% Nz , enzyme, 0.1 M substrate as indicated, and water to make a final volume of 4.0 ml. The substrate solution and an equal volume of buffer were placed in a side arm. The following substrates were used: a, 0.1 ml. fluoroacetylcholine; b, 0.1 ml. acetylcholine; c, 0.2 ml. fluoroacetylcholine; d, 0.2 ml. acetylcholine. In A, the enzyme was 1.8 ml. of guinea pig brain homogenate, and in B, 0.1 ml. of a 1:5 dilution of serum cholinest,erase was used. The gas phase was 5’% C02-950J0Ng and the temperature was 25”. Control experiments showed insignificant hydrolysis of both esters in the absence of enzyme.
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MEHLER
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FIG. 2. Effects of acetylcholine and fluoroacetylcholine on the frog heart. Acetylcholine (A) or fluoroacetylcholine (8’) dissolved in Ringer’s solution at the concentrations indicated was added to the preparation through a cannula extending into the ventricle through the aorta. At W, the ester was removed by several rinses with Ringer’s solution.
Pharmacological E$ects Salle (6) has recently reported some pharmacological effects of fluoroacetylcholine bromide, although no details were given about the synthesis or analysis of the material used. His studies indicate that fluoroacetylcholine has a toxicity comparable to fluoroacetate and that it has effects similar to acetylcholine on the arterial pressure of the dog and on guinea pig bronchial musculature and intestine, but that 10 or 20 times as much material is required. The kymograph tracing of the frog heart preparation (Fig. 2) shows that acetylcholine has a marked effect on the amplitude at a concentration of lo-’ M. At this concentration fluoroacetylcholine has no measurable effect. A small effect of fluoroacetylcholine was obtained with a concentration of 10e6 M, and at 10m4M it abolished the beat. There was no apparent effect of fluoroacetylcholine on the response to acetylcholine. All effects were completely reversed by washing the preparation with Ringer’s solution. Similar experiments were carried out with the frog rectus abdominus. The muscle was suspended between a fixed rod and a lever with a small load and immersed in a cylinder through which oxygen was bubbled. Five milliliters of appropriate concentrations of choline esters was added to 95 ml. of Ringer’s solution for each assay, and the cylinder was emptied and rinsed after each assay. With acetylcholine, the muscle shortened, giving a response roughly proportional to concentration in the range 10m6to 5 X 10m6.At these concentrations fluoroacetylcholine had no effect on the muscle length or on the response to acetylcholine. At
BIOLOGICAL REACTIONS OF FLUOROACETYLCHOLINE
10m4M, fluoroacetylcholine were reversed immediately
also caused a strong contraction. by washing.
297 All effects
DISCUSSION
The experiments in this paper together with those of Salle (6) demonstrate that fluoroacetylcholine is rapidly hydrolyzed by the enzymes tha,t hydrolyze acetylcholine and that the fluoro analog does not interfere with the physiological functions of acetylcholine. Fluoroacetylcholine has similar physiological effects, but approximately 100 times as much is required to duplicate the effects of acetylcholine. From the results of these experiments, a hypothesis can be proposed to explain the toxicity of Auoroacetate through effects on the acetylcholine system. If fluoroacetylcholine is made by the choline-acetylating system, a relatively inert molecule is formed in place of the physiologically active acetylcholine. This inert fluoroacetylcholine need not inhibit the formation of acetylcholine or the reaction with receptors; it merely decreases the effective concentration by being formed in place of the more active compound. In this negative fashion, functions requiring certain amounts of acetylcholine may be prevented. This hypothesis requires that fluoroacetyl CoA be formed and that it react with choline acetylase. The formation of fluoroacetyl CoA has been demonstrated by Brady (7)) who showed it to be active with various condensing enzymes. The reaction with choline has not been reported, but it seems probable that this proceeds as has been reported for the analogous acetylation of aromatic amines (7). The report of Dawson and Peters (8) that gross changes in phosphate metabolism are not found in the brains of fluoroacetate-poisoned animals supports the concept that a specific metabolic lesion occurs rather than a general interference with oxidation. Of course, this specificity might be caused by sensitivity of individual cells, as Dawson and Peters suggest, instead of an effect on systems other than the Krebs cycle, and further work will be required to determine whether the acetylcholine system is involved in fluoroacetate toxicity. Since serum cholinesterase hydrolyzes fluoroacetylcholine, parenterally administered fluoroacetylcholine is rapidly converted to fluoroacetate. Therefore it is to be expected that both fluoroacetate and fluoroacetylcholine will exhibit the same effects in intact animals, as indicated by the toxicity studies of Salle (6).
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ALAN H. MEHLER AND Y. T. CHANG SUMMARY
Fluoroacetylcholine chloride has been synthesized and crystallized. It is hydrolyzed by both brain and serum cholinesterase. It is much less active than acetylcholine in inhibiting the beat of the frog heart or stimulating the contraction of frog rectus abdominus muscle. REFERENCES 1. MARTIUS, C., Ann. 661, 227 (1949). 2. LIEBECQ, C., AND PETERS, R. A., Biochim. et Biophys. Acta 3, 215 (1949). 3. MORRISON, J. F., AND PETERS, R. A., Biochem. J. 66, xxxvi (1954). 4. CHENOWETH, M. B., Pharmocol. Revs. 1,383 (1949). 5. HESTRIN, S., J. Biol. Chem. 180, 249 (1949). 6. SALLE, J., Arch. intern. pharmacodynamie 91, 339 (1952). 7. BRADY, R. O., J. Biol. Chem. 217, 213 (1955). 8. DAWSON, R. M. C., AND PETERS, R. A., Biochim. et Biophys. Acta 16,254 (1955).
Biological Reactions of Fluoroacetylcholine Alan H. Mehler and Y. T. Chang’ From the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, United States Public Health Service, Department of Health, Education, and Welfare, Bethesda, Maryland
Received October 31, 1955
Fluoroacetate has been recognized as a powerful poison for many animals for several years, and, as its sodium salt, is marketed as the rodentitide, 1080. The toxic action of this compound has recently been attributed to its effect on the tricarboxylic acid cycle (1, 2). Presumably through the intermediate formation of fluoroacetyl coenzyme A (CoA), an analog of citric acid is formed which is a potent inhibitor of the enzyme aconitase (3). The demonstration of enzyme inhibition caused by fluorocitrate, a derivative of fluoroacetate, has led to attempts to find other such derivatives. Although interference with citrate oxidation is an adequate mechanism for producing toxic symptoms, additional mechanisms may also occur. The various manifestations of fluoroacetate poisoning among different species usually appear to involve the heart or central nervous system (4). Therefore it was considered that fluoroacetylcholine might be involved as a toxic agent. Accordingly, this compound was synthesized and tested enzymatically and pharmacologically. MATERIALS
Fluoroacetylcholine was synthesized by adding two equivalents of fluoroacetylchloride to choline chloride and warming to dissolve. The product obtained by concentration was fluoroacetylcholine chloride, which was recrystallized from ethanol. The following analysis2 was obtained on the very hygroscopic crystals. r Fellow, Leonard Wood Memorial (American Leprosy Foundation). 2 C, H, N, and Cl analyses were performed in the analytical laboratory under the direction of Dr. W. C. Alford of this Institute. We are indebted to Dr. I. Zipkin of the National Institute of Dental Research for the fluorine analysis. 293
294
ALAN
H. MEHLER
AND Y. T. CHANG
Anal. Calcd. for C~HIEOZNCIF: C, 42.12; H, 7.57; N, 7.01; Cl, 17.76; F, 9.52. Found: C, 41.06; H, 7.76; N, 6.72; Cl, 17.11; F, 9.16. Fluoroacetylcholine reacts with hydroxylamine under the conditions of Hestrin (5), but the color intensity of the iron complex of the hydroxamic acid is somewhat less than that given by an equivalent amount of acetylcholine. Serum cholinesterase (pseudocholinesterase) was obtained as a purified fraction of human serum supplied by the Cutter Laboratories, Berkeley, California. Guinea pig brain homogenates were used as a source of the so-called “true” cholinesterase. The brains were homogenized in a Waring blendor with 5 vol. of cold 1% potassium chloride, then filtered through cheesecloth. EXPERIMENTAL
Choline&eraseReactions The abilities of both true and pseudocholinesterases to hydrolyze fluoroacetylcholine were measured manometrically. The relative rates of hydrolysis of fluoroacetylcholine and acetylcholine are shown in Fig. 1. In the case of the serum enzyme, fluoroacetylcholine hydrolysis increases in rate with increasing substrate concentration. This enzyme appears to be saturated with acetylcholine until the concentration is reduced to less
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than lO+ M. The brain enzyme, on the other hand, hydrolyzes both esters at essentially the same rate throughout the concentration range shown in Fig. 1. At higher concentrations ( W2 M) , fluoroacetylcholine does not produce the inhibition found with acetylcholine.
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FIG. 1B IFIG. 1. Hydrolysis
of choline esters by guinea pig brain and serum cholinesterase. The reaction was followed by measuring CO* evolved from a COrbicarbonate buffer in conventional Warburg manometers. Each vessel contained 2.0 ml. 0.02 M NaHC03 saturated with 5% COZ-95% Nz , enzyme, 0.1 M substrate as indicated, and water to make a final volume of 4.0 ml. The substrate solution and an equal volume of buffer were placed in a side arm. The following substrates were used: a, 0.1 ml. fluoroacetylcholine; b, 0.1 ml. acetylcholine; c, 0.2 ml. fluoroacetylcholine; d, 0.2 ml. acetylcholine. In A, the enzyme was 1.8 ml. of guinea pig brain homogenate, and in B, 0.1 ml. of a 1:5 dilution of serum cholinest,erase was used. The gas phase was 5’% C02-950J0Ng and the temperature was 25”. Control experiments showed insignificant hydrolysis of both esters in the absence of enzyme.
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FIG. 2. Effects of acetylcholine and fluoroacetylcholine on the frog heart. Acetylcholine (A) or fluoroacetylcholine (8’) dissolved in Ringer’s solution at the concentrations indicated was added to the preparation through a cannula extending into the ventricle through the aorta. At W, the ester was removed by several rinses with Ringer’s solution.
Pharmacological E$ects Salle (6) has recently reported some pharmacological effects of fluoroacetylcholine bromide, although no details were given about the synthesis or analysis of the material used. His studies indicate that fluoroacetylcholine has a toxicity comparable to fluoroacetate and that it has effects similar to acetylcholine on the arterial pressure of the dog and on guinea pig bronchial musculature and intestine, but that 10 or 20 times as much material is required. The kymograph tracing of the frog heart preparation (Fig. 2) shows that acetylcholine has a marked effect on the amplitude at a concentration of lo-’ M. At this concentration fluoroacetylcholine has no measurable effect. A small effect of fluoroacetylcholine was obtained with a concentration of 10e6 M, and at 10m4M it abolished the beat. There was no apparent effect of fluoroacetylcholine on the response to acetylcholine. All effects were completely reversed by washing the preparation with Ringer’s solution. Similar experiments were carried out with the frog rectus abdominus. The muscle was suspended between a fixed rod and a lever with a small load and immersed in a cylinder through which oxygen was bubbled. Five milliliters of appropriate concentrations of choline esters was added to 95 ml. of Ringer’s solution for each assay, and the cylinder was emptied and rinsed after each assay. With acetylcholine, the muscle shortened, giving a response roughly proportional to concentration in the range 10m6to 5 X 10m6.At these concentrations fluoroacetylcholine had no effect on the muscle length or on the response to acetylcholine. At
BIOLOGICAL REACTIONS OF FLUOROACETYLCHOLINE
10m4M, fluoroacetylcholine were reversed immediately
also caused a strong contraction. by washing.
297 All effects
DISCUSSION
The experiments in this paper together with those of Salle (6) demonstrate that fluoroacetylcholine is rapidly hydrolyzed by the enzymes tha,t hydrolyze acetylcholine and that the fluoro analog does not interfere with the physiological functions of acetylcholine. Fluoroacetylcholine has similar physiological effects, but approximately 100 times as much is required to duplicate the effects of acetylcholine. From the results of these experiments, a hypothesis can be proposed to explain the toxicity of Auoroacetate through effects on the acetylcholine system. If fluoroacetylcholine is made by the choline-acetylating system, a relatively inert molecule is formed in place of the physiologically active acetylcholine. This inert fluoroacetylcholine need not inhibit the formation of acetylcholine or the reaction with receptors; it merely decreases the effective concentration by being formed in place of the more active compound. In this negative fashion, functions requiring certain amounts of acetylcholine may be prevented. This hypothesis requires that fluoroacetyl CoA be formed and that it react with choline acetylase. The formation of fluoroacetyl CoA has been demonstrated by Brady (7)) who showed it to be active with various condensing enzymes. The reaction with choline has not been reported, but it seems probable that this proceeds as has been reported for the analogous acetylation of aromatic amines (7). The report of Dawson and Peters (8) that gross changes in phosphate metabolism are not found in the brains of fluoroacetate-poisoned animals supports the concept that a specific metabolic lesion occurs rather than a general interference with oxidation. Of course, this specificity might be caused by sensitivity of individual cells, as Dawson and Peters suggest, instead of an effect on systems other than the Krebs cycle, and further work will be required to determine whether the acetylcholine system is involved in fluoroacetate toxicity. Since serum cholinesterase hydrolyzes fluoroacetylcholine, parenterally administered fluoroacetylcholine is rapidly converted to fluoroacetate. Therefore it is to be expected that both fluoroacetate and fluoroacetylcholine will exhibit the same effects in intact animals, as indicated by the toxicity studies of Salle (6).
298
ALAN H. MEHLER AND Y. T. CHANG SUMMARY
Fluoroacetylcholine chloride has been synthesized and crystallized. It is hydrolyzed by both brain and serum cholinesterase. It is much less active than acetylcholine in inhibiting the beat of the frog heart or stimulating the contraction of frog rectus abdominus muscle. REFERENCES 1. MARTIUS, C., Ann. 661, 227 (1949). 2. LIEBECQ, C., AND PETERS, R. A., Biochim. et Biophys. Acta 3, 215 (1949). 3. MORRISON, J. F., AND PETERS, R. A., Biochem. J. 66, xxxvi (1954). 4. CHENOWETH, M. B., Pharmocol. Revs. 1,383 (1949). 5. HESTRIN, S., J. Biol. Chem. 180, 249 (1949). 6. SALLE, J., Arch. intern. pharmacodynamie 91, 339 (1952). 7. BRADY, R. O., J. Biol. Chem. 217, 213 (1955). 8. DAWSON, R. M. C., AND PETERS, R. A., Biochim. et Biophys. Acta 16,254 (1955).