(Received 6 June 1951)

Transcription

1 237 J. Physiol. (95') I15, EXPERIMENTS ON THE ADENOSINETRIPHOSPHATE CONTRACTION OF THE FROG'S RECTUS MUSCLE BY A. B. L. BEZNAK From the Departmen of Physiology, University of Birminngham* (Received 6 June 1951) The adenosinetriphosphate (ATP) hypothesis of muscular contraction has been developed in its present form by Szent-Gy6rgyi and co-workers (Szent- Gyorgyi, 1947, with earlier refs.; 1950) on the basis of biochemical experiments, and the effects of ATP on skeletal muscle of frogs and cats have been regarded as 'physiological' evidence for this theory. Buchthal and co-workers (Buchthal, Deutach & Knappeis, 1944, 1946; Buchthal & Folkow, 1944; Buchthal & Kahlson, 1944) had found that ATP applied externally to a muscle causes contraction. The effect was obtained after denervation as well as after curare. The conditions, however, for obtaining these ATP contractions appear not to be clear. On the frog's gastrocnemius muscle, Abdon (1942) was unable to obtain contractions when ATP was injected arterially. Torda & Wolff (1946), on the other hand, obtained contractions of the frog's rectus abdominis muscle suspended in Ringer's solution containing ATP 1 x 10-3 (about 0.002M) or more. Eserine did not influence the contractions, which were thought to be due to an action of ATP on the contractile protein. Emmelin & Feldberg (1948) studied arterial injections of ATP on the cat's tibialis anticus muscle. With large doses 'weak contractions were obtained sometimes, but by no means always'. In this present paper a comparison is made of the ATP contractions of the frog rectus muscle with those produced by acetylcholine. In addition, evidence will be presented to show that it is not the ATP itself which produces the contraction, but that it releases from the muscle preparation acetylcholine or an acetylcholine-like substance which then in its turn elicits the contraction. METHODS The experiments were made throughout the year on frog recti of Hungarian and English R. e8culenta of both sexes. Twin strip preparations were prepared, one from the left and one from the right * The experiments here recorded were begun in 1944 in the Physiological Institute of the University of Budapest, and partly in collaboration with Dr Zs. GApIar-R&dy. When they were interrupted there, I had the privilege of continuing and completing the work at the Department of Physiology of the University of Birmingham.

2 238 A. B. L. BEZNATK muscle. The strips, which were mm. long, 3-4 mm. wide, and weighed mg., were suspended in separate containers and their contractions recorded isotonically with frontal writing levers. The magnification was fivefold to sixfold. The composition of the bath fluid was: 6-5 g. NaCl; 0O28 g. KCI; 0-12 g. CaCI2; 0-2 g. NaHCO3; 001 g. NaH3PO4; distilled water, 1 1. The ph of the solution was 6-9. The strips were suspended in 9 ml. of the solution, and the substances to be tested were added in 1 ml. samples to the bath. At the beginning of each experiment the tension against which the strips had to contract was adjusted so as to produce 4-10 mm. shortening in response to a concentration of acetylcholine (usually 01 Ag./ml.) which caused 20-50% of maximal shortening. Three different preparations of ATP were used. One was supplied by Professor Szent-Gyorgyi and Dr Banga (Budapest), the second by Professor Buchthal (Copenhagen), and the third by Mr James Morgan (Cambridge). I should like to make grateful acknowledgement for these gifts. The ATP was converted from its Ca or Ba salt into its Na salt with sodium oxalate or sulphate respectively. All solutions and apparatus used for preparing the sodium salt were ice cooled. Acetylcholine was used as bromide or chloride (British Drugs House or Roche Products), physostigmine as salicylate. The figures in the text always refer to the salt and not to the base. In a few experiments the acetylcholine content of the strips of frog recti was determined. For this purpose the muscle was extracted with 5% trichloroacetic acid, which was removed by ether and the solution aerated to remove all traces of ether. After neutralization, the solution was assayed on the eserinized frog rectus muscle (Chang & Gaddum, 1933; Beznak, 1934). In several experiments the ATP was removed from the bath fluid and the latter was assayed for the presence of active substances, particularly for acetylcholine. For this purpose, the ATP in the fluid was removed by precipitation with barium according to the method described by Harpur & Quastel (1949). Their findings could be confirmed, that this procedure does not entail loss, or appreciable loss, of acetylcholine. The procedure of removing the ATP caused dilution of the bath fluid by about 50%. Therefore the final solution had to be readjusted by the addition of sodium, potassium and calcium chloride. It was shown that traces of barium or sulphate which might possibly remain in the solution did not interfere with the assay on the frog rectus. In concentrations of 10 mg./100 ml. they had no effect on the frog rectus; these concentrations are much higher than those that could possibly have been present in the bath fluid. RESULTS The effect of ATP was examined on 59 frog recti; 24 were insensitive to concentrations up to 1 mm. and 35 responded with contraction, but the sensitivity of these to ATP varied greatly. The ATP contractions differed usually in three characteristic ways from the responses to acetylcholine. (1) Whereas with acetylcholine, graded responses are regularly obtained with increasing doses, this is the exception with ATP. In the majority of the recti examined the contraction obtained with the threshold dose is the same as that obtained with a 10 to 100,000-fold increased dose. For instance, in the experiment of Fig. 1, doses between and 7800,ug. ATP produce practically the same degree of shortening; the response is thus of the 'all or none' nature. On such recti increasing amounts of ATP successively added to the bath without washing the muscle between the ATP additions have no effect, and the steady, slow contraction produced by a threshold dose continues at the same rate even when the dose is increased a hundredfold or more. This is illustrated in the experiment of Fig. 2, in which the effects of increasing amounts of ATP and of acetylcholine are compared with each other.

3 ATP ON FROG RECTUS 239 (2) The maximal shortening produced by ATP is smaller than that produced by acetylcholine. A maximal acetylcholine contraction produces a shortening of the muscle of about 30 %, i.e. about 12 mm.; whereas it is rarely possible ; 2 i3 n 4 5n ix. 44, Fig. 1. Contractions of frog rectus muscle in 10 ml. bath to 1 pg./ml. acetylcholine (at 1 and 8j and to varying doses of ATP (at 2-7). The figures on top of the ATP contractions represent the amounts in pg./bath (10 ml.). ^239 p.m p.tml p.m Fig. 2. Contractions of twin frog recti to increasing doses of acetylcholine (upper tracing) or ATP (lower tracing) applied without renewal of the bath fluid. The figures on the bottom of the tracing refer to the time. W=renewal of bath fluid. to produce shortening by ATP of more than 15%, i.e. 6 mm. Fig. 3 gives as ordinates maximal ATP contractions in 35 frog recti; in most, maximal shortening is produced by the threshold concentration of ATP; in those in which graded responses were obtained, the ATP concentration in the abscissae refers to that which causes maximal contraction.

4 240 A. B. L. BEZN4K (3) In the majority of the recti, contractions produced by ATP proceed 4-10 times more slowly than those produced by acetylcholine. In the experiment of Fig. 2, the acetylcholine contraction takes place in 50 sec., the ATP contraction in 500 sec. Fig. 1, on the other hand, is from a rectus muscle in which- the contraction to ATP proceeds as quickly as that to acetylcholine. mm Shortening of recti in ATP 0O e 3 0 * p ~~~~~~~~~ * I 00I to. I I I DAM. AM. 1M. m M. mm. mm. M. Fig. 3. Maximal shortening of 35 frog recti to ATP expressed in mm. The abscisse give the doses of ATP used in the different experiments. In about two-thirds of the muscles the shortening was less than 2 mm., as indicated by the horizontal line. For details see text. The type of the ATP response can sometimes change in the course of an experiment. There is no strict relation between speed of contraction and the finding of an 'all or none' response, but the most frequent combination is an 'all or none' response which proceeds slowly. There is no relation between the sensitivity of a preparation to ATP and to acetylcholine, as seen from the results of Table 1, in which the sensitivity to acetylcholine is represented by the dose of acetylcholine which produces 50%

5 ATP ON FROG RECTUS 241 of the maximal contraction. Some of the 21 muscles which are most sensitive to ATP are those least sensitive to acetylcholine. TABLE 1. Sensitivities of frog recti to acetylcholine and ATP Number of recti responding to varying jum. acetylcholine concentrations of ATP (mm.) producing 50% contraction * * Total The sensitivity of a preparation is usually not affected by the time the muscle has been suspended in the bath, but in three experiments in which the time required for setting up the preparation and applying the ATP was reduced 2 27 P.rm p.m p.m Fig. 4. Continuation of experiment of Fig. 2, 2i hr. later. Responses of the eserinized preparations to acetylcholine, ATP, and 'ATP bath'. Eserine given at 1.27 p.m. till the end of the experiment. Time, 10 sec. For details see text. to a few minutes, an initial sensitivity to ATP disappeared within 30 min. No effect was found on the sensitivity of a preparation to ATP by altering the composition of Ca, Mg, K and Na ions of the bath fluid within physiological limits or by adding blood to the bath. Preceding ATP or acetylcholine contractions also did not alter the sensitivity to ATP. Eserine has a pronounced effect on the ATP contraction. When a muscle has been bathed in eserine 1 x 10-5 for about an hour, it becomes more sensitive to ATP: the contractions become several times stronger and, in addition, proceed more quickly. This is illustrated by the tracings of an experiment reproduced in Figs. 2 and 4. Fig. 2 gives the contraction to 2-66,ug. ATP before, and Fig. 4 after the eserine treatment. The results of five experi-

6 242 A. B. L. BEZNiK ments are given in Table 2; in three of them eserine has increased the ATP contractions 2X6 to 21-5 times; but in two eserine has no effect. In one of these the muscle has been insensitive to ATP and remained so after the eserine treatment. This is not always so. Some muscles which do not respond to ATP in the absence of eserine do so after the eserine treatment. TABLE 2. Effect of eserine 1 x 10-5 on ATP contractions Contractions in mm. ATP,_ A, concentration Without With % (Ag./ml.) eserine eserine increase D-Tubocurarine abolishes the ATP contractions. For instance, in one experiment on a non-eserinized preparation, 0x2 mg. D-tubocurarine abolished the effect of both 266 jug. ATP and 0 1 pg. acetylcholine. On eserinized preparations larger doses of D-tubocurarine (up to 5 mg.) are required to abolish ATP as well as acetylcholine contractions. The release of an acetylcholtine-ltike substance by ATP During the ATP contraction, a substance appears in the bath fluid which has the properties of acetylcholine and is probably responsible, or responsible to a great extent, for the ATP contractions of the frog rectus muscle. To demonstrate the release of this substance, the ATP contraction was elicited in eserinized preparations. In the experiment of Fig. 4, the twin recti had been soaked for 1 hr. in Ringer's solution containing eserine 1 x 10-5 when ATP was added to the one (lower tracing) and acetylcholine to the other (upper tracing). The acetylcholine contraction reached its maximum in about 3 min., the ATP contraction in about 10 min., when the bath was pipetted off and stored for re-testing. Both muscles were washed three times with eserinized Ringer's solution and allowed to relax. The stored bath fluid (referred to in the following as 'ATP bath') was then added to the upper muscle and, within about 10 sec., a contraction occurred which was stronger and proceeded more quickly than the previous ATP and even the acetylcholine contraction. It was so unlike a usual ATP contraction that it appeared unlikely to be the result of the presence of ATP. This was proved by the fact that the 'ATP bath' retained its contracting property after the ATP had been removed according to the procedure of Harpur & Quastel. This is illustrated in Fig. 4 on the lower tracing, which shows the strong quick contraction of the muscle on the addition of 'ATP bath' from which the ATP had been removed. Further experiments are recorded in Table 3, in which the size of contractions

7 ATP ON FROG RECTUS 243 obtained by 'ATP bath' before and after removal of the ATP is given in mm. These contractions are compared with those produced by acetylcholine. Columns 4 and 5 give the acetylcholine concentrations in ug./ml. which were found to produce the same contraction as 'ATP bath'. In the last six experiments the assay was made on two frog recti; on one 'ATP bath' was assayed against acetylcholine before, on the other after removal of the ATP. It will be seen that in most experiments the muscle stimulating effect of 'ATP bath', when compared with the effect of acetylcholine, has not decreased, or decreased only slightly after the ATP removal. When a muscle is very insensitive to acetylcholine, 'ATP bath' may be ineffective, but when it is tested after ATP removal on another muscle sensitive to acetylcholine, a strong contraction occurs. Such a result is obtained in Exp. 11 of Table 3. TABLE 3. Contractions of frog rectus to 'ATP bath' (a) before and (b) after removal of ATP; eserine concentration of bath between 0-5 and 2 x 10-6 Size of contraction Acetylcholine (tig./ml.) in mm. producing the same produced by contraction as ATP concentra- A - Pecovery tion (ug./ml.) (a) (b) (a) (b) % 460 1;2 1; ;11 3; ; The contractions obtained with 'ATP bath' before and after removal of the ATP might be explained as follows: the contraction before removal of the ATP is due to ATP itself; the contraction after removal, to the fact that in the process of ATP removal the 'ATP bath' acquires new stimulating properties. This objection is met by the result of the following experiment. Eserinized Ringer's solutions, to which either ATP or ATP with acetylcholine is added, are subjected to the treatment of Harpur & Quastel for removal of ATP. This results in a loss of about 20% of the added acetylcholine, but the samples to which no acetylcholine has been added cause either no contractions or very minute, slow ones, entirely different from the strong contractions produced with 'ATP bath' from which ATP had been removed. Negative results are also obtained when the Harpur-Quastel treatment is applied to eserinized Ringer's solution which has been in contact with a rectus muscle for several

8 A. B. L. BEZNAK 244 minutes but without ATP, which is only added to the fluid after it has been pipetted off. Therefore, the contractions of ATP-free 'ATP bath' can only be due to a substance which was released into the bath fluid during the action of ATP on the muscle. The fact that ATP contractions were potentiated by eserine and abolished by D-tubocurarine suggested that the substance appearing in the bath fluid during an ATP contraction might be acetylcholine itself, and that the stimulating effect of ATP on the frog muscle was brought about indirectly by this mechanism. The conclusion that the released substance is probably identical with acetylcholine is strengthened by the following findings. (1) The presence of eserine in the bath during the ATP contraction facilitates the demonstration of the stimulating effect obtained with 'ATP bath' after removal of ATP. 40Z. Fig. 5. Continuation of experiment of Figs. 2 and 4, about Ij hr. after Fig. 4. Effect of D-tubocurarine (T-C) on contraction produced by ATP-free 'ATP bath' (lower tracing). The upper tracing illustrates the spontaneous relaxation after washing out the 'ATP bath' (W.Es.-R). (Time, 10 sec.) (2) A muscle contracted by ATP-free 'ATP bath' relaxes quickly when the Ringer's solution is renewed (see Fig. 5). The relaxation proceeds as quickly as the relaxation which follows an acetylcholine contraction after washing out the acetylcholine. (3) Eserine augments, but D-tubocurarine and atropine abolish or reduce the contractions produced by 'ATP bath'. These experiments were performed with 'ATP bath' before and after removal of ATP. For instance, Fig. 5 shows the effect of D-tubocurarine, and Fig. 6 of atropine, on the contractions produced by 'ATP bath' after ATP removal. The concentration of atropine used in this experiment was that shown by de Elio (1948) to be characteristic for antagonizing an acetylcholine effect on the frog rectus. (4) The ATP-free 'ATP bath' loses its stimulating effect after it has been boiled for a minute in alkali. The appearance of acetylcholine or of an acetylcholine-like substance in the

9 245 ATP ON FROG RECTUS bath fluid by the action of ATP on the muscle is not asociated with a corresponding diminution in the acetylcholine content of the muscle. This is shown as follows. Twin recti from a frog are suspended in separate baths. To one ATP is added, to the other acetylcholine in an amount sufficient to contract the muscle as strongly as its twin. At the height of the contractions, the muscles are removed, blotted between filter paper, extracted with trichloroacetic acid and assayed for acetylcholine. Furthermore, the acetylcholine Fig. 6. Effect of atropine on the contractions of eserinized twin frog recti to ATP-free 'ATP bath' (upper tracing) and to acetylcholine (lower tracing). At 5.28 p.m. 700jug. atropine. (Time, 10 sec.) TABLE 4. Effect of ATP contraction on acetylcholine content of frog rectus muscle yg. acetylcholine (a) in 'ATP bath'; (b) in muscle extracted during ATP contractions; (c) in twin muscle extracted during acetylcholine contraction. In all but the first and the last experiment, the values for acetylcholine are the mean of two determinations on twin preparations. (a) (b) (a + b) (c) *4 0* * * X58 1* content of the 'ATP bath' is assayed after removal of its ATP. The results of seven such experiments are given in Table 4; in four of these the muscle treated with ATP contains either as much as, or more, acetylcholine than its control twin; in the remaining three it contains less. But even in two of these the acetylcholine of the muscle plus that of 'ATP bath' is higher than that of the control muscle. Table 4 illustrates, further, that 'ATP bath' usually

10 A. B. L. BBzNAK 246 contains several times more acetylcholine than the muscle. From these results it must be concluded either that the acetylcholine is released from the muscle by the ATP and at once replaced by synthesis, or that acetylcholine is synthesized when ATP is brought in contact with the muscle, and that this excess acetylcholine is then at once released. DISCUSSION If a frog rectus muscle is made to contract by the administration of ATP, it has been found that the eserinized Ringer's solution in which it is immersed becomes itself capable of stimulating muscles; this is due to the presence of acetylcholine or an acetylcholine-like substance. The question is raised, therefore, whether ATP causes the muscle to contract as a result of its leading to the release, or formation, of acetylcholine. Several of the findings presented in this paper are in favour of such an interpretation of the ATP contraction: for instance, the observation that the ATP contraction is augmented by eserine but abolished by D-tubocurarine and atropine under the same conditions as an acetylcholine contraction. Although no complete pharmacological identification was made of the muscle stimulating substance appearing in the bath under the action of ATP on the muscle, all tests so far applied are in agreement with the conclusion that the substance is acetylcholine itself. Other possible substances to be considered are phosphoryl or organic choline esters, pyrophosphorylaneurine and inorganic pyrophosphate. All phosphoryl esters, including those of choline and aneurine, can be excluded because they are probably removed by the Harpur-Quastel treatment. In addition, phosphorylcholine is 1000 times less active than acetylcholine (Beznak & Chain, 1936) and pyrophosphorylaneurine contracts the frog rectus only in concentrations of 1 x 10-2 (Torda & Wolff, 1944). Such high concentrations could not have been liberated from the muscle, especially not in those experiments in which the ATP concentrations used for producing contraction were low, since the ATP would be the phosphate donor. The possibility of pyrophosphate being the stimulating substance had to be considered because it causes contraction in a concentration of about 15 mm. (Buchthal et al. 1944) and could be split off from ATP by an apyrase system which is present in frog muscle (Steinbach, 1949). The slowness of the ATP contraction, in contrast to the quick contraction produced by 'ATP bath', could then be explained by a gradual splitting off of this small and quickly moving molecule of pyrophosphate. The effects of eserine, curare and atropine on the contractions produced by ATP-freed 'ATP bath', however, would be difficult to explain if the substance were inorganic pyrophosphate. The effects of these drugs, on the other hand, would be accounted for if the substance were acetylcholine. The fact that the substance is sensitive to alkali also speaks for acetylcholine and against pyrophosphate.

11 ATP ON FROG RECTUS 247 The results of the present experiment do not indicate the site of the ATP action, whether the acetylcholine is derived from the motor nerve endings or from the muscle fibres. As has been mentioned before, the ATP may either act by releasing the acetylcholine which is then replaced by synthesis, or by stimulating synthesis with subsequent release. If the action of ATP is on the release it could be due to withdrawal of calcium: if on the synthesis, to its known catalytic action on the choline acetylase system. Both mechanisms may be responsible for the appearance of acetylcholine in the bath fluid. The conclusion that the ATP contractions of the frog recti are either wholly or mainly indirect actions due to the release by the ATP of a substance which is probably acetylcholine, does not allow us to assume a similar mode of action of ATP in mammalian muscle, or even in frog muscles other than the rectus. We have to realize that the muscle fibres of the frog rectus have special properties; they are endowed with the ability to respond to acetylcholine with localized contracture instead of propagated contractions. According to Hunt & Kuffler (1950) most of the muscle fibres of the frog rectus are innervated by the small diameter motor nerve fibres. Release of acetylcholine by ATP may only play a minor part in the contractions produced by ATP on muscles which consist predominantly of muscle fibres innervated by the large diameter nerve fibres and endowed with the property to respond to acetylcholine with propagated contractions. This would, for instance, explain the finding of Buchthal et at. (1944) mentioned in the introduction that curare does not antagonize the ATP contractions obtained on such muscles. SUMMARY 1. The effect of ATP on the frog rectus muscle is examined. Some muscles do not contract to ATP, some to large doses only, but others to small doses. 2. The ATP contractions differ from the acetylcholine contractions mainly in the following: (a) Graded reponses are regularly obtained with increasing doses of acetylcholine; this is the exception with ATP. The shortening obtained with the threshold dose of ATP is usually the maximal effect. (b) This ATP contraction is smaller than the maximal acetylcholine contraction. (e) Usually the ATP contraction proceeds more slowly than an acetylcholine contraction. 3. During the contraction produced by ATP on the frog rectus muscle a substance appears in the bath fluid which has acetylcholine-like properties and is probably acetylcholine itself. 4. It is concluded that the ATP contractions of the frog recvuw muscle are not direct effects of ATP on this muscle but are secondary effects of the released acetylcholine. 5. The question whether this mode of action of ATP necessarily applies to the ATP contractions of other striated muscles, particularly of mammals, is discussed.

12 248 A. B. L. BEZNAK It is a pleasure to thank Professor H. P. Gilding for his perfect hospitality, as well as for his kind interest in this work. I am also grateful to Dr M. Beznalk for her help in some of the experiments. During part of the work I have enjoyed the assistance of Mr G. H. Taylor. Finally, my grateful thanks to Dr W. Feldberg, without whose patient help in composition, this script would not have seen the light of day. REFERENCES Abdon, N. 0. (1942). Dissertation, University of Lund. Cited by Buchthal & Folkow in Acta phy8iol. Scand. 1944, 8, 312. Beznak, A. B. L. (1934). J. Phy8iol. 82, 129. Beznak, A. B. L. & Chain, E. (1936). Quart, J. exp. Phy8iol. 26, 201. Buchthal, F., Deutsch, A. & Knappeis, G. G. (1944). Acta physiol. Scand. 8, 271. Buchthal, F., Deutsch, A. & Knappeis, G. G. (1946). Acta physio. Scand. It, 325. Buchthal, F. & Folkow, B. (1944). Acta phy8iol. Scand. 8, 312. Buchthal, F. & Kahlson, C. (1944). Acta physio. Scand. 8, 317. Chang, H. C. & Gaddum, J. H. (1933). J. Physiol. 79, 255. de Elio (1948). Brit. J. Pharmacol. 3, 108. Emmelin, N. & Feldberg, W. (1948). Brit. J. Pharmacol. 3, 237. Harpur, R. P. & Quastel, J. H. (1949). Nature, Lond., 164, 779. Hunt, C. C. & Kuffler, S. W. (1950). Pharmaco. Rev. 2, 96. Steinbach, H. B. (1949). Arch. Biochem. 22, 328. Szent-Gyorgyi, A. (1947). Chemistry of Muscular Contraction. New York: Academic Press. Szent-Gy6rgyi, A. (1950). Abstr. XVIII int. phyil. Congr. Torda, C. & Wolff, H. G. (1944). Proc. Soc. exp. Biol., N.Y., 56, 89. Torda, C. & Wolff, H. G. (1946). Amer. J. Phy8l. 145, 419.