extraordinary evanescence of the effect, when the substance is injected

Transcription

1 :612.I28 THE ACTION OF BLOOD ON ACETYLCHOLINE. BY K. MATTHES. (From the National Institute for Medical Research, Hampstead.) THE intense activity of acetylcholine has been well known since its first description by Hunt and Taveau [1906]. Dale [1914] emphasized the extraordinary evanescence of the effect, when the substance is injected into the blood stream, and suggested that it was probably due to hydrolysis into choline and acetic acid by an esterase in the blood. Loewi and his co-workers [1926], in studying the properties of the substance liberated in the frog's heart by stimulation of the vagus, found that this had the properties of a choline ester, and demonstrated the presence in the heart of an esterase, which destroyed the vagus substance or acetylcholine. In either case the destructive action was inhibited by eserine or ergotamine. Galehr and Plattner [1928] made a detailed study of the effect upon acetylcholine of mammalian blood in vitro. They found that it was, indeed, hydrolysed with extraordinary rapidity. The hydrolytic agent, however, possessed properties which they regarded as incompatible with its being an esteraso. Thus the activity of the blood serum resisted heating to 580 C. and exposure to ultra-violet light. Since they further observed that the destructive activity was concentrated in the corpuscles, so that a suspension of these acted more rapidly than the serum, they concluded that the destructive activity of blood on acetylcholine was due to a surface catalysis, and not to the action of a true ferment. After my own investigations, here recorded, had been largely completed, I was informed, through the courtesy of Prof. 0. Loewi, of an investigation which had been made independently in his laboratory by Dr Engelhardt, and which has since been published [Engelhardt and Lo ewi, 1930], covering to some extent the same ground as my own. Engelhardt and Loewi were able to show that the resistance of the hydrolytic agent to heat and to irradiation is due to the presence of protective substances, the esterase from frog's heart being similarly protected when added to mammalian serum. They also observed the remarkably potent inhibition of the hydrolytic action produced by adding eserine to the blood or serum, even in very low concentrations,

2 ACTION OF BLOOD ON ACETYLCHOLINE. which is the principal subject of this paper. They concluded, as I have done, that the destructive agent for acetylcholine in blood is an esterase. METHODS. To obtain a quantitative estimate of the rate of destruction of acetylcholine by blood or tissue extracts the action must be allowed to proceed for a definite time, and then brought to an end. This has been done by the method used by Galehr and Plattner [1928]: 1 c.c. of blood or organ extract is shaken up with 2 c.c. of Tyrode solution and 1 c.c. of 1:10,000 acetylcholine, and destruction is allowed to proceed for a definite time at room temperature. It was unnecessary to control the temperature, since the observations were in all cases comparative. The destructive agent is then precipitated by adding 1 c.c. of 20 p.c. trichloracetic acid. The solution is filtered and the filtrate diluted with ten times its volume of Tyrode's solution. This final solution is very acid (ph 2-3), and no appreciable alteration in its acetylcholine content takes place for several days. It has been tested by a method used by Dale [1914]. The action of the solution is compared with that of a standard acetylcholine solution on a loop of rabbit's jejunum suspended in warm, oxygenated Tyrode's solution. Suitable effects are seen on this preparation when c.c. of a 10-6 acetylcholine solution is added to a 15 c.c. bath, giving a final concentration of the order of As noted by Dale and Dudley [1929], acetylcholine can be measured by this test, with doses in the sensitive range, to an accuracy of 10 p.c., and with a favourable preparation to 5 p.c. THE MEASUREMENT OF DESTRUCTIVE ACTIVITY. It was found by Glalehr and Plattner [1928] that thedestruction of acetylcholine in the blood of some animals (dogs, rabbits, cats) resembled a monomolecular reaction, in that the rate of destruction at any time given was proportional to the concentration of acetylcholine present. Expressed mathematically, if x and xo are concentrations of acetylcholine at times t and to, dx/dt =-kx or r Iogl XOx 339 where r = k logloe. In other animals, such as pigs, cattle, horses and men, where acetylcholine was especially quickly destroyed, the exact value of r was found by P1 attn e r to become smaller with decreasing time. In our experiments with horse blood, however, unless times shorter than 20 seconds

3 340 K. MATTHES. were used, the value of r obtained was found to be constant over a wide range of times. In particular, it was always found to be constant if the rate of destruction was reduced by diluting the blood or by the action of some inhibiting agent. In the case of many enzymes it is found that the value of r so obtained is directly proportional to the quantity of enzyme present. Experiments have been carried out to test whether this is true of the agent destroying acetylcholine. The results of one such experiment are shown in Table I. A certain quantity of acetylcholine was treated, under otherwise identical conditions, with a series of different dilutions of the same sample of horse blood in Tyrode solution. The time allowed for action was increased in inverse proportion to the concentration of blood. Suspension of the corpuscles was maintained by means of a mechanical shaker. From Table I it will be seen that, if the observed value of r is used as an estimate of the quantity of enzyme present, the error is not greater than 14 p.c. TABLE I. Showing percentage destruction of acetylcholine by different concentrations of horse blood. Concentration of blood after Time of Deaddition of action on struction Rate of Rate acetylcholine acetylcholine (p.c.) destruction calculated m. S : : : : : : : ACTION OF ESERINE ON THE DESTRUCTIVE AGENT. The rate of destruction of acetylcholine by blood as measured in these experiments is diminished by the previous addition of eserine to the blood, even in extremely low concentrations. For example, the rate was usually halved by such addition of one part of eserine in 30 millions. Site and time of eserine action. Various experiments have been carried out to exclude the possibility that this apparent effect was due to some of the eserine surviving the precipitation with trichloracetic acid, and itself contributing to the action on the plain muscle. This it could do, either by its own stimulating action on the intestinal preparation, or by sensitizing the latter to the action of acetylcholine. Control experiments

4 ACTION OF BLOOD ON ACETYLCHOLINE. showed that such effects played no part. When eserine was added to the bath in sufficient concentration to cause contraction of the intestine, the effect of a dose of acetylcholine was simply superposed, the combined effect being merely additive. When the concentration of eserine was insufficient to produce a contraction by itself (less than 10-8 added to the bath) the action of acetylcholine was unaffected. It was found that when the concentration of eserine in the blood was 1/300,000, or less, the quantity surviving the treatment with trichloracetic acid was insufficient to affect the intestine in any way. It is, therefore, clear that the action of the eserine in the experiments with blood must have been to protect the acetylcholine from destruction by the blood, and not an action on the intestinal preparation. In order to get the full effect of the eserine it was necessary for it to act on the blood for some time before the acetylcholine was added. Some reaction appeared to occur between the eserine and the blood which was so slow that, with low concentrations of eserine, it was incomplete in fifteen minutes. The results shown in Table II illustrate these facts. TABLE II. Showing the percentage destruction of acetylcholine by blood treated for different lengths of time with eserine. Duration of interaction of blood and eserine Duration of before the addition of acetylcholine action of KA _ blood on 0 15 sec. 5 mn. 15 mi. 30 mn. 1 hr Concen- acetyltration of choline Percentage destruction of acetylcholine eserine (min.), A ^ These results show that the eserine acts by inhibiting the destructive agent. Its action does not depend on a combination with acetylcholine. Reversibility ofaction ofeserine. This effect of eserine on the destructive agent is completely reversible since, if the eserine is removed by dialysis, the blood destroys acetylcholine as rapidly as if no eserine had been added to it. In one experiment eserine was added to blood so as to make a concentration of A portion of the mixture was kept as a control, and two portions of 10 c.c. each were dialysed for different times by placing them in suitably folded pieces of cellophane. Under these conditions the volumes of the mixtures were increased to about 15 c.c. Equivalent quantities of the control, of the two dialysed portions and of the original blood, dialysed in the same way, were made up to the same

5 342 K. MATTHES. volumes, and tested for destructive action on acetylcholine. The results are given in Table III. TABLE III. Showing the percentage destruction of acetylcholine by mixtures of blood and eserine before and after dialysis. Duration of action on acetylcholine 2in. 5 min. 15 mm. Percentage destruction of acetylcholine Blood and eserine before dialysis Blood and eserine after dialysis for 5 hr Blood and eserine after dialysis for 24 hr Blood without eserine after dialysis for 24 hr Relation of inhibition to concentration of eserine. Experiments have been carried out to determine quantitatively the relation between the concentration of eserine and the degree of inhibition which it produces. Different quantities of eserine were added to portions of blood and the mixtures allowed to stand for one hour. The rate at which the mixture destroyed acetylcholine was then determined in each case, and r was calculated. The results of a typical experiment are shown in Table IV. The meaning of the last column is explained below. TABLE IV. Showing the inhibition of the destructive agent produced by different concentrations of eserine. Time, in Concen- seconds, of tration action on Destruction Rate of of eserine acetylcholine (p.c.) destruction K 109 c r O * O Since it has been found that the value of r gives an estimate of the amount of free agent present, the results given above represent the dissociation curve of the compound that is apparently formed by its combination with eserine. If the amount of free agent depends on the attainment of equilibrium between two simple opposing reactions, both

6 ACTION OF BLOOD ON ACETYLCHOLINE. 343 governed by the same law, the relationship between r and c' (where c' is the final concentration of free eserine) should be given by the equation where K is a constant, and ro is the value of r when no eserine is present. K in Table IV is a figure calculated for each concentration, on the assumption that the final concentration of free eserine is equal to the concentration, c, of the total eserine present. This assumption is certainly justifiable in the case of high concentrations, but in the case of low concentration c' might be expected to become appreciably less than c, and the calculated value of K to increase. It will be seen that the value of K is practically constant for concentrations of eserine of 10-7 or higher. With fall of the concentration to 10-8 K decreases, and then increases again, with further fall of the concentration. Similar changes in the value of K have occurred in every experiment except one, in which K remained constant to c = 1-7,5. These changes of K are considerably larger than could be accounted for by the error on the determination. There is no obvious explanation for the low values shown at concentrations of 10-7'5 and Whatever the cause, it would be expected still to act at concentration 10-8'5, and the fact that the value of K is there again higher may, perhaps, be due to the error introduced by assuming that c' = c in that low range. It may be noted that the deviation of the dissociation curve given by these results from the hyperbola, which is expected on the simplest assumption, occurs in the opposite direction to that seen in the dissociation curve of oxyheemoglobin in acid solutions. Action on other enzymes. Eserine is not a general ferment poison. I could not detect any change in the action of trypsin, takadiastase, or rennin in the presence of 1 : 10,000 eserine. Its effects on lipase action are being further studied by Ste dma n (personal communication). THE ACTION OF THE ENZYME ON OTHER SUBSTRATES. A specimen of pyruvylcholine was obtained through the kindness of Dr H. W. Dudley. As shown by Le Heux [1921], esterification with pyruvic acid intensifies the activity of choline in a manner similar to acetylation, but to a smaller degree. In stimulating the isolated rabbit intestine I found the pyruvyl ester to have about 22-8 p.c. of the activity of acetylcholine. Its activity is thus about 230 times that of choline, and its hydrolysis can be followed by the biological test in the same way as

7 344 K. MATTHES. that of acetylcholine. It was found in one experiment that, under conditions similar to those used for acetylcholine, pyruvylcholine was destroyed to the extent of 90 p.c. by blood in 5 minutes. When 1 part of eserine had been previously added to 106 of blood no destruction could be detected after that time. The quantity of pyruvylcholine available was small, and only one such experiment was made. AcTION OF OTHER SUBSTANCES. The action of miotine. Miotine is a base prepared by Stedman [1929]. It represents a part of the eserine molecule, and has been shown to have an approximately equal miotic activity on the pupil. Its other pharmacological properties have been studied by White (personal communication), and shown to be practically indistinguishable from those of eserine. Its relation to eserine is shown by the structural formulse: Miotine CH3NHOCO C6H4 CHCH3N(CH3)2HC1. Eserine CH3NHOCO C0H3 C7H14N2. Dr Stedman has kindly provided a sample of this substance, and its effect on the destruction of acetylcholine by blood has been compared with that of eserine. It has been found that equimolecular concentrations of the two substances produce indistinguishable effects both qualitatively and quantitatively. This conclusion is illustrated in Table V. TABLE V. Percentage destruction of acetylcholine by blood in the presence of eserine and miotine. Molar concentrations Duration of action on acetylcholine 30 sec. 1 mi. 2 min. 5 min. Percentage destruction Eserine, 2/ Miotine, 2/ Eserine, 2/ Miotine, 2/ No alkaloid Effects of other alkaloids, etc. Alkaloids belonging to other groups appear to have little effect on the enzyme. The effect of ergotoxine ethanesulfonate and ergotamine tartrate have also been studied, since Loewi [1926] states that ergotamine inhibits the acetylcholinase in heart extracts. These alkaloids in high concentrations also inhibit the destruction of acetylcholine by blood. The results of an experiment on this point are shown in Table VI.

8 ACTION OF BLOOD ON ACETYLCHOLINE. 345 TABLE VI. Percentage destruction of acetylcholine by blood in the presence of ergotoxine and ergotamine, allowed to act on blood for 1 hour before addition of acetylcholine. Concentration of alkaloids Duration of action on acetylcholine 30 sec. 1 min. 2 min. 5 min. 15mi. Percentage destruction Ergotoxine, 1/ Ergotamine, 1/ Ergotoxine, 1/ Ergotamine, 1/ No alkaloid The restraining action is perceptible in all cases, except with the weaker dilution of ergotamine, but is much weaker than that of eserine. The effects of quinine and atoxyl have been studied, because of their known inhibitory action on serum lipase and other lipases, shown first by Rona and his co-workers [1920, 1921]. Quinine has, in comparatively strong concentrations, a definite inhibitory effect on the destruction of acetylcholine by blood. Thus, the rate of destruction was reduced by quinine hydrochloride, in concentrations of 1: 100,000, 1: 10,000 and 1: 1000, to 85 p.c., 50 p.c., and 10 p.c. respectively of its original value. The destructive agent proved, on the other hand, to be very resistant to atoxyl; even a 1: 1000 solution of atoxyl did not alter the rate of destruction, and a 1 p.c. solution had only a slight inhibitory effect, the rate being reduced to 75 p.c. of its original value. Effects of inorganic salts. The effects of various inorganic salts on the destruction of acetylcholine by blood are in some ways similar to their effects on known lipase actions. When the 2 c.c. of Tyrode's solution was replaced by isotonic solutions of NaF, NaCl, NaBr, NaI and Na2SO4 in diluting the blood, it was found that fluoride inhibited the destruction, so that 40 p.c. of the acetylcholine was still undestroyed after one hour. In the presence of the other salts, as in the presence of Tyrode's solution, no acetylcholine could be detected after 2 minutes. In another experiment it was found that isotonic solutions of MgCl2, CaCl2, and BaCl2 diminished the rate of destruction by 13, 37 5, and 62 p.c. respectively, as compared with Tyrode's solution. When these salt solutions were diluted ten times with NaCl, they had no effect. In isotonic solutions of pure NaCl and KCI the destruction proceeded at the same rate as in the presence of Tyrode's solution. The fluorides were thus the only salts studied which had any distinct effect even in diluted solutions. This action is still shown, when the

9 346 K. MATTHES. concentration of fluoride is only 1: It is not due to the precipitation of calcium, since oxalate has no such effect. The relation between the concentration of the fluoride and the degree of inhibition produced has been studied. The result of one experiment is shown in Table VII. TABLE VII. The action of different concentrations of fluoride on the destruction of acetyl holine. Duration of Concentration of action on Destruction Rate of fluoride acetylcholine (p.c.) destruction 0 40 sec molar ,, 1 min ,, ,, ,, * ,, ,, While these results illustrate the general nature of the relation between the concentration of fluoride and its action, it is not possible to make exact calculations from them, because it is not known what quantity of the fluoride was precipitated by the calcium in the blood. DIsCUSSION. When I began my experiments, the only details available concerning the destruction of acetylcholine by blood were those furnished by Plattner and his colleagues, who had concluded that the effect was not due to enzyme action, but to a non-specific surface catalysis. Engelhardt and Loewi, as already mentioned, have since disposed of some of their more important arguments. Plattner's observation, which I have confirmed, that the destructive agent is more abundant in the corpuscles than in the serum, and his demonstration of the increase of the activity of the corpuscles with swelling, both appear to be entirely compatible with the action of an enzyme which is largely concentrated on the surface of the corpuscles. The alteration of its action by capillaryactive substances may also be compared with the effect of such substances on the action of invertin [Meyerhof, 1914]. Plattner [1930] has quite recently published observations on the destruction of acetylcholine by various organ extracts, accepting without question the view that this is due to an esterase, and it is no longer possible to find any adequate reason for regarding the destruction by blood as due to an action of a different kind. My own results, which at various points

10 ACTION OF BLOOD ON ACETYLCHOLINE. 347 confirm those of Plattner and his co-workers and of Engelhardt and Loewi, and are entirely compatible with the view that this action of blood is due to an esterase, may be briefly stated as follows: 1. As observed by IPlattner with some kinds of blood, I have regularly found that the time relations of the destruction of acetylcholine by horse blood, when tested in such dilutions as allow the measurement to be made with sufficient accuracy, are those characteristic of a monomolecular reaction. 2. The rate of destruction of acetylcholine by horse blood varies in strict proportion to the concentration of the destructive agent, which in this respect behaves like many enzymes. 3. Eserine has a highly specific inhibiting effect on the destruction of acetylcholine by blood, the rate being usually halved by adding eserine in the proportion of 1 to 30 millions. This effect, also observed by Engelhardt and Loewi, resembles the effect of eserine on the destruction of acetylcholine by a heart esterase, observed by Loewi. Recently Plattner has observed that eserine inhibits the destructive action of tissue extracts, which he accepts as that of an esterase. 4. Eserine must be left in contact with blood for a definite time before acetylcholine is added, for the full development of the inhibitory effect. This time increases with the dilution. 5. The relation between eserine and the blood esterase is a labile one, the full activity being restored when eserine is removed by dialysis. 6. Pyruvylcholine is destroyed by blood like acetylcholine, and the action is similarly inhibited by eserine. 7. Miotine, a cleavage product of eserine having a practically identical physiological activity, has also an identical inhibitory effect on the destruction of acetylcholine by blood. 8. Ergotoxine and ergotamine have some inhibitory effect on the destruction of acetylcholine by blood, but only if they are present in relatively high concentrations. Quinine has a similar effect in high concentrations, but practically none could be demonstrated for atoxyl; in these respects again, the blood esterase behaves like some of the tissue esterases studied by Plattner. 9. Of neutral salts only the fluorides, which are known to inhibit esterases, have a strong inhibitory effect on destruction of acetylcholine by blood. Salts of magnesium, calcium and barium have a slight inhibitory action. It seems clear that the rapid destruction of acetylcholine by blood is only a special instance of its liability to the action of tissue esterases

11 348 K. MATTHES. in general. This property has an obvious and important bearing on the course of its physiological actions, when it is injected in various ways into the living animal. Especially significant is the inhibitory effect of eserine (or miotine) on its destruction, in connection with its known action in intensifying and prolonging the actions not only of acetylcholine, but of parasympathetic nerves. SUMMARY. The destruction of acetylcholine by blood is due to the action of an esterase, the action of which is specifically inhibited by eserine, even in extreme dilutions, and also by fluorides. It is a pleasure to acknowledge my indebtedness to the Medical Research Council for the opportunity of carrying out this investigation in the National Institute for Medical Research in Dr Dale's department, and to Mr J. H. Gad d u m of that department for valuable help in making the experiments and presenting the results. REFERENCES. Dale, H. H. (1914). J. Pharmacol. 6, 147. Dale, H. H. and Dudley, H. W. (1929). J. Phy8iol. 68, 97. Engelhardt, E. and Loewi, 0. (1930). Arch. exp. Path. Pharmak. 150, 1. Galehr, 0. and Plattner, F. (1928). PJfueer8 Arch. 218, 488, 506. Hunt, R. and Taveau, R. de M. (1906). Brit. Med. J. 2, Le Heux, J. W. (1921). Pflueger Arch. 190, 280. Loewi, 0. and Navratil, E. (1926). Pftueger Arch. 214, 678, 689. Meyerhof, 0. (1914). Pfluegera Arch. 157, 251. Plattner, F. and Hintner, H. (1930). Pflueger8 Arch. 225, 19. Rona, P. and Bach, E. (1920). Biochem. Z. 111, 166. Rona, P. and Reinecke, D. (1921). Biochem. Z. 118, 213. Stedman, E. (1929). Amer. J. of Phy8iol. 90, 528.