CREATINE FORMATION DURING TONIC MUSCLE CONTRACTION. BY K. UYENO AND T. MITSUDA. (From the Physiological and Biochemical Laboratories, Cambridge.) Creatine of the amphibition muscles in the breeding season. KAHN(1) found less creatine in the flexor muscles of the arm and a part of the pectoral muscle than in the muscles of the leg in the male frog while clasping the female in the breeding season. He interpreted this fact as a decrease of creatine in muscles contracting tonically. Riesser() showed that this interpretation was not justified since in the frog the normal content of creatine is less in the arm than in the leg muscles, but he considered that creatine was not produced by the clasping contraction. The contraction was held by Kahn to be tonic on account of the absence of action currents. As our experiments with nicotine (3) suggested that creatine is formed during the tonic contraction of frog's muscle it seemed desirable to investigate the question on the clasping muscles. The fact shown by Rie sse r is inconclusive as he did not compare the creatine of the normal frogs with that of the clasping frogs in the same season. Our scheme was, therefore, to compare the creatine of the arm and leg muscles in the breeding season in frogs and toads which were clasping and those which were not. Muscles taken for analysis were the flexor muscles of the arm and the pectoral muscles on one hand and the whole muscle of the hind limb, except small muscles of the foot, on the other. The muscles of the two sides were taken. Creatine was estimated as creatinine by Folin's method. For the comparison we use the quotient creatine in clasping muscles 100 creatine in hind limb muscles X, since the creatine content of the normal frogs varies in fairly wide range so that it may sometimes cover the range of actual increase by tonic contraction'. Experiments were begun with toads. Males during clasping, most freshly caught, some from the laboratory stock, were observed for several 1 In accordance with custom we use the term "tonic contraction" for the persistent contractions dealt with in this paper, but we do so without prejudice as to the nature or frequency of the nerve impulses causing them.
314 K. UYENO AND T. MITSUDA. days in order to ensure that the contraction had been continuous. They were then killed and the muscles were analysed. Others, separated from the females or found single, were kept for several days isolated. Some clasped and some isolated females were also used. The results are shown in Table I. From this we see that the quotient in clasping males varied from 90 to 99 and averaged about 95 whilst that in non-clasping males varied from 81 to 88 and averaged about 84. Thus the creatine increased in the clasping muscles while the males were coupling and the amount TABLE I. Creatinine in p.c. in the muscles of the toad. 1 34 5 67 8 9 10 11 1 13 (a) (b) of the arm and Muscles of the pectoral muscle hind limb *7 *30 *34 '30 '4 *6 '30 *5 *48 *36 *40 *37 *37 *34 Males not coupling. *03 *30 '04 *43 *31 '85 *0 *49 *01 *44 '03-39 a X 100 90-1' 9'8 99-95'8 94'6 95-4 98'3 mean 95' ne Females not coupling. 14 '03 *35 15 '191 '14 Female during coupling. 16 '190 *5 88-3 84'0 81.1 81' 8'4 84'9,an 83-7 86-5 89'3 84'5 TABLE II. Creatinine in P.C. in the muscles of the frog. 1 3 4 5 6 7 8 9 (a) of the arm and pectoral muscle *335 *318-35 *301 (b) Muscles of the hind lim.349 '330 *38 *37 Males not coupling *99 '340 *75 *351 *30 *351 *8 *318 *77 *30 Females during coupling. 10 *90 *331 11 *88 *335 Female not coupling. 1 '75 *37 of the arm 13 *49 *314 14 '70 *343 15 *68 *333 16 17 18 19 Males not coupling. '40 '340 '3 '34 *3 *318-34 *35 ax100 b b 96'0 96'4 99' 9'1 mean 95-9 88-0 78-3 86-1 88-7 86'6 mean 85'5 I 87-7 86-1 84-1 79.4 78'8 80'6 nean 79'6 70'6 71'6 70' 7'1 nean 71'1
CREATINE AND MUSCLE TONE. 315 of increase was about 10 p.c. of the creatine content in the hind limb muscles. The creatine of the clasped and non-clasped females did not show much difference from that of non-clasping males. There was also, as was to be expected, no difference in creatine content in the hind limb muscles of the clasping and non-clasping males. The figures in the clasping muscles were remarkably constant except in one case, so that we could see at a glance the increase of creatine in the clasping muscles. Even in the exceptional case, i.e. in No. 10, in which the creatine content of the clasping muscles was abnormally great near to the maximum it was also great in the hind limb muscles and the quotient was as low as 81. This case (whether the abnormally high percentage was due to experimental error or to actual individual variation) shows the usefulness of the quotient in interpreting the results. Similar experiments were then made on the frog and the results are shown in Table II. Here again we see about 10 p.c. increase of creatine in the clasping males though the actual percentage was, as known already, much greater than that in the toad. When pectoral muscles were omitted and the flexor muscles of the arm only taken for analysis the quotient was much smaller, even smaller than that of the non-clasping males, but it was found that reckoning in the same manner the quotient in the nonclasping males was also less than that of the clasping males. This shows that creatine is increased in the flexor muscles of the arm as well as in the pectoral muscles though the normal content is greater in the latter. From these results we conclude that creatine is formed in the clasping muscles both in male toads and frogs during coupling in the breeding season. Decerebrate rigidity and creatine formation. Dusser de Barenne and Tervaert(4) have recently published experiments which controvert the statement of Pekelharing and Hoogenhuyze(5) that creatine is formed in decerebrate rigidity. Our experiments were originally designed to reinvestigate this question and also, on the suggestion of Professor Hop kins, to see if carnosine, another non-protein nitrogenous substance, changes in the same way as creatine. We found that carnosine did not change in amount, so that the following account is concerned solely with creatine. The experiments were made on cats and were carried out in three series. In the first we have compared the amount of cre'aine in the limb muscles of the two sides. In the second the motor nerves to some of the
316 K. UYENO AND T. MITSUDA. muscles were cut and after 4 hours, during which the cats were ansesthetised with urethane, the muscles were taken for analysis. In the last series the cat was decerebrated, the motor nerves to muscles being cut on one side before decerebration, and killed after four hours. In all cases creatine was estimated as creatinine by Folin's method. The results of the first series are shown in Table III. From this we see that the difference of the two sides is very slight though the absolute values differ in different muscles as well as in different cats. The percentage difference varies from 0 to 3-5. Roughly speaking there is a difference of about p.c. Hence if we find any difference greater than this we may probably attribute it to an altered condition of the muscles. In Table IV we show the results of the second series. The amount of creatine was always less on the side on which the motor nerves were cut. Except in two cases of the triceps muscle, in one of which we noticed a distinct stiffness of the muscle on the side with intact motor nerve, the percentage difference was about 3. The decrease of creatine in paralysed Creatinine (in p.c.) in decerebrate rigidity (cat). TABLE III. Comparison of the two sides in normal cats. Cat Right Diff. left P.c. diff. Triceps brachii 1-385 - 007 1-8 35-513 + -007 1-4 -450 + -004-9 6 *445 +-010-3 Vastocrureus 1-396 --004 1-0 5-465 --011-4 -41 --009-1 6-48 + -006 1-3 Gastrocnemius *435 0 0 34 *533 --008 1-5 *494 --01-4 5 *495 -*010 0 Semimembranosus 4-414 0 0 TABLE IV. Section of motor nerves on one side; cats killed after 4 hours. Nerve Diff. Cat intact nerve cut P.c. diff. Triceps brachii 1-656 --01 3- * -440 --063 14-3 3-46 --030 7-0 Vastocrureus 1-615 --018-9 -41 --009-1 3-44# --014 3- * The fore leg was stiff and kept thrust forward, cause unknown. TABLE V a. Decerebration, rigidity developed strongly, kept 4 hours. Triceps brachii Cat Non-rigid Diff. rigid 1-519 +-05 3 *481 + -040 *455 + -040 Vastocrureus 1-580 + -045 3* *455 +-035-446 +-059 Gastrocnemius 3-510 -51 +-033 +-00-341 +-015 P.c. diff. 10-8 8-3 8-8 7-8 7-7 13-6-5 3-8 4.4 4-8 Soleus 3-315 +*015 * Rigidity was little stronger in the hind leg than in the fore leg. TABLE V b. Decerebration with slight and non-persistent (Cat 1) or no rigidity (Cat ). Triceps brachii 1-396 -460 --011 +-03-6 4-8 Vastocrureus 1-41 -476 +-014 +-007 3-3 1-5
CREATINE AND MUSCLE.TONE. muscle may be, as suggested by various workers, referred either to the increased blood flow and consequent increased washing away of creatine or to a decreased formation of creatine in the paralysed muscles, or to both of them. This question we have not investigated. The results of the third series of the experiments are shown in Table V a. The percentage increase of creatine in the rigid muscles was 7'7 to 13- in the triceps and vastocrureus and much less (about half) in the gastrocnemius and soleus. Pekelharing and Hoogenhuyzewho abolished rigidity by cutting posterior roots-found 7-9 to 19-4 p.c. in the triceps and Dusser de Barenne and Tervaert,-also cutting posterior roots, 1*5 to 9*7 in triceps and 1-9 to 11 in gastrocnemius. We may point out that the difference of creatine was much less in the gastrocnemius, that this was corresponding to the less degree of rigidity, and that it was on the gastrocnemius that most of the experiments by Dusser de Barenne and Tervaert were made. The cases of decerebration in which rigidity was slight and not persistent or did not develop at all are shown in Table V b. In these cases there was no distinct increase of creatine. Our results we think show that decerebrate rigidity is accompanied by a definite increase in creatine formation. SUMMARY. 1. In male toads and frogs creatine was found to increase in the clasping muscles during coupling in the breeding season.. In decerebrate rigidity creatine increases in the rigid muscles. We wish to thank Prof. Langley and Prof. Hopkins for the facilities given us in their laboratories. 317 REFERENCES. (1) Kahn. Pfliiger's Arch. 177, p. 94. 1919. () Riesser. Ztschr. f. physiol. Chem. 0, p. 189. 19. (3) Mitsuda and Uyeno. This Journal, 57, p. 80. 193. (4) Dusser de Barenne and Tervaert. Pfluger's Arch. 195, p. 370. 19. (5) Pekelharing and Hoogenhuyze. Ztschr. f. physiol. Chem. 64, p. 6. 1910. PH. LVII. 1