From the Department of Physiology, University of Lund.

Effect of Acetylcholine and Adenosine Triphos- phste on Denervsted Muscle.

BY FRITZ BUCHTHAL and GEORG KAHLSON.

Received 2 February 1946.

In previous papers (BUCHTHAL et al. 1944 a b c) it could be demonstrated that minute amounts of adenosine triphosphate (ATP) release contraction in frog and mammalian striated muscle. It was suggested that ATP constitutes a further link in the chain of reactions initiated by the injection of acetylcholine into the artery of a muscle. This high energetic phosphate is even effec- tive after complete curarization. Apart from the release of con- traction by ATP another property of this substance was dis- closed, viz. its sensitizing effect on subsequent applications of acetylcholine. Thus, it seemed likely that in some way or other more intimate interactions take place in the nerve muscle system between acetylcholine and ATP. We thought it of interest t o investigate the effect of ATP on denervated muscle, as denerva- tion causes profound changes in the reaction to acetylcholine. It is generally accepted that normal skeletal muscle is relatively insensitive to intra-arterially injected acetylcholine, while de- nervated muscle after application of minute amounts of this substance exhibits a considerable increase in tension and dura- tion of contraction.

Method. The experiments were performed on the anterior t'bial muscles of

the cat under chloralose or decerebrate as described by BROWN (1938). The substances were applied by close arterial injection into the distal part of the anterior tibial artery, the proximal part being temporarily closed by traction on a ligature, when the substances are applied. The tension developed by tibial muscles on intra-arterial injection of ace- tylcholine may show rather iarge individual differences in different cats. It therefore seemed of special interest t o record alternately within

EFFECT OF ACETYLCHOLINE AND ADENOSINE TRIPHOSPHATE. 285

short intervals the tension developed by normal and denervated muscles, thus enabling a quantitative comparison under standardized conditions. Therefore, both tibia1 muscles were mounted in the Brown-Schuster myograph exerting tension on the same isometric lever.

Section of the right sciatic nerve was performed 6-85 days before the experiments in all 25 cats. The completeness of the denervation was controlled by electrical stimulation of the peroneal nerve. The reduction in weight of the denervated muscle amounted to 25-60 per cent as compared with the normal muscle, depending on the time allowed for degeneration.

All substances were applied iso-osmotically by substituting an equal amount of NaCl + water in the Tyrode solution by the staple solution of ATP. The pH of the injected solution was 7 . 3 and its temperature 37" C. The ATP was applied as sodium salt (for description of prepara- tion and analysis cf. BUCHTHAL et al. 1944 a). The substance was kindly provided by D r . A. DEUTSCH, research laboratory A. B. Leo, Halsing- borg.

Results. 1. Sensitivity to acetylcholine. Close arterial injection of minute amounts of acetylcholine into

denervated muscle releases a double mechanical response, con- sisting of a quick initial phase followed by a protracted develop- ment of tension. The quick phase is generally absent a t the second injection, but may exceptionally persist during the first three applications. This is in agreement with the observations of BROWN (1937). Prom the 6th day onwards after denervation no system- atic correlation exists between duration of tension development and time allowed for degeneration. However, in 3 of 25 experi- ments with a degeneration period of 46, 47 and 52 days respec- tively, an exceptional behaviour was observed, the muscle res- ponding to acetylcholine solely by contractions of normal type and duration. I n another series of experiments comprising 3 animals with a similar degeneration time of 46, 47 and 49 days resp. the denervated muscle responded with contractions of the protracted type.

The present technique allows a direct comparison of the sen- sitivity of the normal and denervated muscle to acetylcholine on one and the same animal. The difference in threshold was con- siderable, the denesvated muscle being 20-200 times more sensitive than the normal.

The blocking action of acetylcholine on subsequent injection of this substance is well known from normal muscle. I n de- nervated muscle it i s even present when injections are made with intervals of several minutes, denervated muscle becoming in-

286 FRITZ BUCBTHAL AND GEORQ KAHLSON.

Fig. 1. Cat under chloralose. Mechanical responses from right (r) and left (1) anterior tibia1 muscles to close arterial injection of acetylcholine. Right muscle denervated four weeks previously. Time marks 1 per second. To the right: direct electrical stimulation of the denervated muscle. Weights: 1 = 6.4 g; r = 3.0 g.

sensitive t o acetylcholine after 10-20 injections. The response to direct electric stimulation is retained (Fig. 1).

2. Effect of Na-adenosine triphosphate. A solution of AT9 injected into the artery in amounts causing

submaximal responses evokes contractions of different types in normal and denervated muscles. Compared with the response in normal muscle ATP contractions of denervated muscles are con- siderably prolonged, the difference in duration being of a similar ratio as that observed with acetylcholine (Fig. 2). As is the case for the latter, duration of contractions released by ATP, from the 6th day onwards, is independent of the time allowed for degeneration. In ATP contractions there is no quick phase even with the first injection. The peak of tension developed during contractions, with the same dose, was generally 30-50 per cent higher in denervated - though atrophied - muscle than in the corresponding normal muscle. A definite small amount of ATP, inactive in the normal, causes a pronounced contraction in the corresponding denervated muscle (Fig. 2). There is, however, no paralleIism in the sensitivity of normal and denervated muscle to acetylcholine and ATP. Thus, it was frequently observed that a muscle relatively insensitive to acetylcholine developed strong tension when ATP was injected or vice versa.

I n denervated muscle, contrary to findings in normal, previous injection of ATP does not enhance the tension developed by subsequent application of acetylcholine.

EFFECT OF .ACETYLCHOLINE AND ADENOSINE TRIPHOSPHATE. 281

In the course of this investigation evidence accumulated that under cer ta in condi t ions ATP was ineffective in elicitirbg mechanical responses i n denervated muscle. This was always the case when previously to the application of ATP acetylcholine had been injected into the muscle. The response to acetyl- choline and to *direct electrical stimulation is, however, retained. For one experiment of this type a supply of lithium ATP was available and i t was striking that Li-ATP was active even after pre- vious injection of acetyl- choline when the muscle was refractory to Na-ATP.

Fig. 2. Cat under chloralose. Mechanical re- sponses from right (r) and left (1) anterior tibia1 muscles to close arterial injection of acetylcholine and ATP. Right muscle denervated five weeks previously. Time marks 1 every ten seconds.

Weights: 1 = 7.0 g; r = 2.8 g.

This is in agreement with our observations on smooth muscle where Li-ATP proved effective in cases where the action of Na-ATP was strongly reduced (BUCHTHAL and KAHLSON 1944).

Discussion.

The aim of this investigation was a direct comparison of the reaction of normal and denervated muscle to injections of ace- tylcholine and ATP. Independent of the time of degeneration both substances release a contraction of long duration in'de- nervated muscle. It is well known that acetylcholine, ineffective in initiating contractions in curarized muscle, evokes a special type of response in denervated muscles. Since curarization, in normal muscle, does not alter the type of contraction, i t is obvious that denervation, apart from changes in the peripheral nerves and motor end plates causes changes in the reaction of the muscle substance itself t o chemical stimulation. It is on the other hand worth mentioning that the greater sensitivity of denervated muscle to chemical stimuli is abolished by curarine (BROWN 1937) and even after a degeneration period of almost 3 months, the motor end plate still is highly sensitive to acetylcholine.

The depressing effect of acetylcholine on subsequent applica- tion of ATP in denervated muscle indicates that acetylcholine interferes with the reaction of the muscle substance. As the re- sponse to acetylcholine is retained when the preparation has be-

288 FRITZ BUCHTHAL AND QEORO KAIILSON.

come refractory to ATP, we are forced to suppose that in denerv- ated muscle acetylcholine prevents the interaction of the intra- arterially applied ATP with the contractile substance. The mechan- ism of this inhibition remains obscure. It may, however, be noted in this connection that acetylcholine actually interferes with the enzymatic activity of myosin. Acetylcholine has a considerable inhibitory influence on the adenosinetriphosphatase. Furthermore, previous application of acetylcholine abolishes the changes in birefringence produced by ATP in normal frog muscle fibres. This, too, indicates that acetylcholine, apart from its effect on the motor end plate, in some way reacts on the contractile protein.

Summary.

Direct comparison of denervated with normal anterior tibia1 niuscles of the cat showed:

1. Sodium adenosine triphosphate initiates contractions in de- nervated muscle which last considerably longer than in normal muscle.

2 . Previous application of acetylcholine to denervated muscle abolishes its sensitivity to ATP, while the reaction of normal muscle to this substance is uninfluenced by previous injection of acetylcholine.

3. I n agreement with previous investigations we find denerv- ated muscle considerably more sensitive to acetylcholine (average 40-50 times) than normal muscle; the curare-like action observed on repeated applications of acetylcholine is highly pronounced in denervated muscle. Since the increased sensitivity still is present when degeneration has been allowed to proceed for 75-85 days, and since this effect can be abolished by curarization, it must be concluded that the motor end plate or part of it survives even a t this high degree of muscle atrophy.

References.

BROWN, G. L., J. Physiol. 1937. 89. 438. BROWN, G. L., Ibidem 1938. 92. 22 P. BUCHTHAL, F.: A. DEUTSCH, and G. G. KNAPPEIS, Acta Physiol. Scand.

BUCHTHAL, F., and B. FOLKOW, Ibidem 1944 b. 8. 312. BUCHTHAL, F., and G. KAHLSON, Ibidem 1944 c. 8. 317, 325.

1944 a. 8. 271.