slowing of the muscle. Bronk [1933] has given a striking

Transcription

1 106 6I2.74I.I2 THE EFFECT OF ACTIVITY ON THE FORM OF THE MUSCLE TWITCH. BY J. L. PARKINSON. (From the Department of Physiology and Biochemistry, University College, London.) IT has been found by various workers [Hartree and Hill, 1921; Bronk, 1930; Bozler, 1931; Feng, 1931] that in isolated muscle an increase occurs, during a prolonged contraction, in the economy with which the contraction is maintained. BozIer found in the retractor of the pharynx of the snail that with one shock every 3 sec. an incomplete tetanus was at first obtained, gradually developing into a smooth contraction. The rate of heat production gradually diminished while the tension remained constant. Feng [1931] has related this increase of economy to a progressive slowing of the muscle. Bronk [1933] has given a striking demonstration of the same phenomenon in crab's muscle. Hill [1931], during the course of experiments with the frog's gastrocnemius, observed during a series of twitches a very rapid "spreadingout" of the contraction. It was more noticeable in large Hungarian R. esculenta than in Dutch R. esculenta or English R. temporaria. In his paper (Fig. 7, p. 293) are shown series of single twitches at 3 and 2j sec. intervals. The " spreading-out " of the contraction is immediately obvious, the sixth twitch of a series lasting almost twice as long as the first twitch. In tetani the summation was at first incomplete, but became complete as stimulation continued. It was also found that the slowing occurred earlier in the later series of twitches and that recovery was almost complete in 30 sec. This recovery was so quick that it had obviously nothing to do with the ordinary oxidative recovery process, especially since it occurred equally in large gastrocnemii so thick that oxygen could not possibly have penetrated into the interior. The experiments described in this paper represent an attempt to investigate further this "spreading-out" of the contraction in twitches. Since the rapid recovery observed could not be associated with oxidation, the question naturally arose: could it be due to the delayed lactic acid

2 FORM OF MUSCLE TWITCH. 107 formation which is known to be associated with the restoration of phosphagen [see e.g. Lundsgaard, 1930]? The question could be answered by seeing whether the " spreading-out " occurred, and whether, if it did, it was followed by equally rapid recovery, in a series of twitches in muscles poisoned with iodoacetate. It was desirable also to know whether it came on as quickly in muscles which were entirely rested and normally supplied with blood through the circulation. A. EXPERIMENTS ON ISOLATED NORMAL GASTROCNEMII. Preparations from large Hungarian R. esculenta were used in the first experiments. The muscle was left attached to part of the femur, which was held in an ebonite clamp. This was fitted to a frame of the type described by Hill [1931]. One end of a thin, straight wire was tied to the tendon and the other end attached to an isometric lever. The nerve was stimulated through a pair of silver electrodes: the stimuli used were provided by condenser discharges, single shocks being given by hand at the required intervals with a Morse key. The muscle was surrounded with Ringer's solution (buffered to ph 7-2 by phosphate solution 10 mg. P per 100 g.) for about 30 min. before an experiment, the solution being stirred by oxygen or nitrogen. In the earlier experiments the following procedure was adopted. A series of ten stimuli was given at intervals of j to 2 sec. and one stimulus each at 30, 45 and 60 sec. after the last of the series. The object of the three later shocks was to determine the interval necessary for the recovery or partial recovery of the muscle. The mechanical response was recorded on a rapidly moving smoked drum driven by a constant speed motor. Experiments performed in this way confirmed the observations of Hill [1931]. The first twitch of a series was invariably less prolonged than the succeeding ones, "spreading-out" of the contraction being obvious in the third or fourth twitch. Recovery was generally complete in 60 sec., or at least as complete as it would be finally. Successive series showed increasing "spread." The effects were observed in nitrogen as well as in oxygen. B. EXPERIMENTS ON ISOLATED POISONED GASTROCNEMII. In order to determine whether lactic acid formation was connected with this phenomenon experiments were made on muscles poisoned with mono-iodoacetic acid (I.A.A.). Since it was impossible to poison a gastrocnemius effectively by soaking in Ringer's solution containing the acid, it was necessary to inject the poison into the frog. The following

3 108 J. L. PARKINSON. Fig. 1. First two rows: first series of ten twitches at 1 sec. intervals of fresh isolated gastroonemius of Hungarian B. e8cul*nta. The tenth twitch lasted about twice as long as the first. (The duration of each twitch is measured at 40 p.c. of its height.) Note that the response increased slightly in size during the series. Third row: single twitch taken 60 sec. after the last of the series. "Recovery" was almost complete, both in width and height. Fig. 2. Series of twitches, similar to Fig. 1, by the opposite gastrocnemius of the same frog, but poisoned with iodoacetate. The "spreading-out" was almost identical with that observed with the normal muscle, and "recovery" was again complete in 60 sec. The adequacy of the poisoning is shown by the typical I.A.A. contracture setting in after sixty to eighty twitches (drum slowed).

4 FORM OF MUSCLE TWITCH. 109 method was adopted. The frog's brain was pithed and the skin cut at the back of the leg, avoiding damage to the dorsal lymph sac, the sciatic nerves were exposed and carefully separated from adjoining arteries. Fine silk threads were then tied round the nerves about 2 cm. from the junction with the muscles and the nerves cut through above this point. Care was taken to avoid loss of blood. The skin was replaced and the frog injected in the dorsal lymph sac with iodoacetic acid (in the form of the sodium salt), 2 c.c. of a 2 p.c. solution per 50 g. of frog being given. When the forelimbs became rigid (in i to 1i hours) the frog was laid on ice and the muscles with nerves dissected and placed in ice-cold Ringer. Generally speaking, the whole frog, with the exception of the gastrocnemii, was rigid before dissection was complete. The muscles were mounted in frames and placed in Ringer's solution at temperatures varying from 7.50 C. to 180 C. The Ringer's solution was stirred by nitrogen. After 15 min. the solution was withdrawn and the experiments made in nitrogen. The same "spreading-out" was observed in these poisoned muscles, though only three or four series of ten twitches were possible before contracture began. The contracture, which was large in every case, proved that the poisoning was adequate. It seemed desirable for comparison to use a poisoned and a normal muscle from the same frog. The brain having been destroyed and the nerves cut, the frog was left for 2 hours with the circulation intact to recover from previous activity. (Recovery by soaking in oxygenated Ringer's solution would take too long in the case of a muscle as thick as the gastrocnemius of a Hungarian frog.) A ligature was then tied round the upper part of one thigh and this leg cut off. The frog was then injected with I.A.A. The muscle from the normal leg was used for experiment immediately and the poisoned muscle used when the frog had become rigid. The "spreading-out" of the contraction appeared to be the same in both normal and poisoned muscles, and " recovery " also occurred in both. These results, as well as those described in the following sections, were found in a large series of experiments without exception. C. NORMAL AND POISONED GASTROCNEMII IN SITU. In these experiments the conditions for normal and poisoned muscles were identical. The brain was destroyed; the nerves were tied and cut as usual and the skin around each Achilles tendon was removed. A thread

5 110 J. L. PARKINSON. was tied round the tendon and the latter cut just below it, avoiding injury to the blood vessels. The frog was then pinned down on a board, the thread from one tendon connected to one arm of a crank, and the other arm of the crank (at 900) attached by a thin wire to an isometric lever above. The crank was light, frictionless, and adjustable in a horizontal direction: the isometric lever was attached to a heavy stand adjustable vertically. The frog was left in the position described for 2 hours. Since the circulation was not hindered in any way, both muscles recovered in this time from activity during dissection. When required for stimulation the nerves were raised by threads and laid across a pair of silver electrodes fixed to the board; an initial tension of about 10 g. was placed on the muscles by raising the isometric lever. When the experiment on the normal leg was complete the tendon of the other leg was attached to the crank, the frog was injected with the appropriate amount of I.A.A. and allowed to remain until the forelimbs became rigid. The muscle was then stimulated and responses and contracture recorded. It was noticed in these experiments that the "spreading-out" of the contraction began much later than in the isolated muscles. In the latter, 50 to 100 p.c. "spread" was observed in the tenth twitch, but fifty to seventy twitches were necessary to produce the same "spreading-out" in muscles in situ. In the tenth twitch no "spread" was noticeable. A highly rested condition is clearly unfavourable to the appearance of the phenomenon in question. D. THE EFFECT OF CIRCULATION IN NORMAL GASTROCNEMII. In order to determine whether the circulation continuing during stimulation had any effect on retarding the " spread " in muscles in situ, the following experiment was made. The frog was prepared as described above, and a record was obtained from a normal muscle with circulation intact. Immediately before stimulation of the other muscle a ligature was tightly tied round the upper part of the leg in order to stop the circulation. The muscles exhibited almost identical responses. The normal muscle recovered in about 20 min. from the fatigue produced by 200 twitches, while, as expected, the muscle without circulation showed no recovery. Apparently the absence of circulation during.such rapid stimulation does not affect the "spreading-out."

6 FORM OF MUSCLE TWITCH. ill E. EXPERIMENTS ON ISOLATED SARTORIUS MUSCLES. Isolated sartorius muscles from Hungarian R. esculenta exhibit the same "spreading-out" of the contraction as the gastrocnemii. The muscles were soaked in oxygenated Ringer's solution for at least 2 hours to avoid reversible inexcitability [Duliere and Horton, 1929]. Nitrogen was then passed through the Ringer for 30 min., the Ringer was withdrawn and stimulation (direct) carried out in nitrogen. Poisoning with I.A.A. by soaking in Ringer's solution containing 1 part in 12,500 of that substance had no effect on the "spreading-out." The muscles exhibited a typical poison contracture after about 100 twitches. The effect of varying the ph of the Ringer surrounding the muscle was examined. After 2 hours in oxygenated Ringer the muscle was stimulated in nitrogen and the responses recorded. The Ringer was then replaced and oxygen passed through it for 30 min. The oxygen was then replaced by a mixture of 5 p.c. C02 and 95 p.c. nitrogen, which was allowed to bubble through the Ringer for about 1 hour. The Ringer was then removed and the muscle stimulated in the atmosphere of C02 in nitrogen. The "spreading-out" obtained after this treatment was identical with that obtained in oxygen. The Ringer's solution at the end of the experiment had a ph of 5-6. An experiment was made on the sartorius muscle first under normal conditions and then after soaking for 1j hours in calcium-free Ringer's solution. The "spreading-out " and recovery remained unaltered. A pair of sartorius muscles from a curarized frog also showed the same phenomenon. Sartorius muscles from Dutch R. esculenta and English R. temporaria behaved in a similar manner to those of Hungarian frogs but to a less degree. F. TEMPERATURE AND FATIGUE. Alteration of the temperature appeared to have no effect on the "spreading-out" of the contraction; the "spread" was noticeable as early as the tenth twitch and more noticeable in later twitches. No fatigue was seen even after 50 or 100 twitches-indeed the response was generally greater than at the beginning. DISCUSSION. Contrary to expectation, the recovery which sets in rapidly after a few twitches and causes the return of the normal form of contraction within a minute has nothing to do with the delayed lactic acid formation

7 112 J. L. PARKINSON. by which phosphagen is restored. Quantitatively the " spreading-out " and recovery are very striking phenomena, at least in Hungarian frogs, and at present one can suggest no chemical reaction corresponding to either of them. Complete previous oxidative recovery is obviously not favourable to the rapid onset of the "spreading-out," but it does not prevent it, only makes it necessary to give a larger number of twitches. It is not possible at present to do more than describe the phenomenon. Its relation to chemical processes occurring in muscle is not obvious. SUMMARY. During a series of muscle twitches at intervals of a second or two a considerable "spreading-out" of the contraction occurs. "Recovery," which is independent of the presence of oxygen, is complete within a minute. The " spreading-out " is not connected with lactic acid formation, nor is the " recovery " associated with phosphagen restoration, since both occur unchanged in muscles adequately poisoned with iodoacetate. A completely resting condition (absence of stimulation with previous intact circulation) delays the "spreading-out," but its onset is not quickened by circulatory stoppage during the series. Sartorii showed the same phenomenon. "Fatigue " is not the cause of it, since usually the response increased during a series. Temperature and ph have no effect on it. Its relation to known chemical changes in muscle is not obvious. REFERENCES. Bozler, E. (1931). J. Phy8iol. 72, 24 P. Bronk, D. W. (1930). Ibid. 69, 306. Bronk, D. W. (1933). J. cell. comp. Phy8iol. 2, 285. Duliere, W. and Horton, H. V. (1929). J. Phy8iol. 67, 152. Feng, T. P. (1931). Proc. Roy. Soc. B, 108, 522. Hartree, W. and Hill, A. V. (1921). J. Physiol. 55, 133. Hill, A. V. (1931). Proc. Roy. Soc. B, 109, 267. Lundsgaard, E. (1930). Biochem. Z. 227, 51.