From the Department of Physiology, University of Lund.

Close Arterial Injection of Adenosine Triphos- phrtte and Inorganic Triphosphate

into Frog Muscle BY

FRITZ BUCHTHAL’ and BJORN FOLKOW.

Rcccived 8 July 1944.

I n the preceding conimunication (BUCHTHAL e t al. 1944) the striking effect of micro-application of adenosine triphosphate (ATP) and related compounds has been described, ATP releasing itiechanical responses, changes in birefringence and action poten- tials. Apart from a paper b y ABDON (1942), who applied ATP intra-arterially to the gastrocnemius of the frog, no other in- vestigation concerning the effect of this substance on striated muscle seems to exist. I n contrast t o the observations hy RTTCH- THAI, e t al., AmoN could not observe any stiniulating action of ATP and found only inhibiting effects on the release of contrac- tion by acetylcholine. We therefore thought it of interest t o investigate anew the effect of intra-arterial application of ATP to frog muscle.

Method.

The method used for intra-arterial injectioii was abont the w i l t

as tha t used by BROWN (1937) for the investigation of the pffect of acetylcholine on frog musqle. All branches of the sciatic artery except those supplying the gastrocnemius musle were ligatpd As the frogs available (Rana esculenta and Rana temporaria) were only * j 3 the size of those used by BROWX, a fine glass cannula with an opening of 110 I ( was inserted in the sciatic artery instead of a hypodermic cannula, the gastrocnemius muscle bring flxed in a horizontal position. The nright of the muscles used wab 300 to 400 nig.

1 Working on a. fellow.ship fiom the Hockefeller E’omrlrrfeoii.

CLOSE ARTERIAL INJECTION OF ADENOSINE TRIPHOSPEIATE. 313 ATP and inorganic triphosphate were applied in an iso-osmotic con-

dition and with a pH of 7 . 3 . The preparation of the substances which were kindly provided by Dr. A. I~EVTSCH (Research laboiatory, A. B. Leo, Halsingborg), is described in the preceding paper. The staple solution of ATP contained 4.35 mp,ml and that of inorganic sodium tri-poly-phosphate 2. B rng/ml. 811 arterial injections were made in a volume of 0 . 0 3 ml Ringer. The conipo~ition of the Ringer's solution was as follows: 6.5 g NaC1, 0 . 2 g RC1. 0 . 1 4 g anhydrous CaCl, and 0 . 2 g glucose, distilled water to 1 litrt.. By a suitable amount of NaHCO, and by passing a gas mixture of 1 per cent CO, and 99 per cent 0, through the solution its pH was adjusted to 7 . 3 . The experiments were performed at 18-19' C. The mechanical responses were either re- gistered isometrically or isotonicslly aud in some experiments action potentials were simultaneously recorded by leading off with dg-AgC1 electrodes to an A.-C. aniplificlr and electrostatic oscillograph.

Results.

Intra-arterial injection of A T P into the sciatic artery supply- ing the gastrocnemius of t h r frog sets up a tetanus-like con- traction, the threshold amount being approximatelv PO ,ug in 0.03 ml Ringer (0 .04 x 10-6 mol). The tension produced by 40 ,up ATP (0.08 x 10-6 niol) corresponds on an average to tha t released by 1 pg acetylcholine. After injection of ATP tension rises quickly and is accompanied by an outburst of asynchronous action potentials. The contraction niay be niaintained over a considerable time, and the per sibting tension. too, is accompanied by electrical activity (Fig. 1 B). Tension increases with increasing concentration of ATP.

When acetylcholine is applied after previous injection of ATP, the sensitivity of the muscle to acetylcholine is essentially in- creased (Fig. 1 C). The effect of acetylcholine after previous application of ATP is 4-10 times that hefore the use of it (Figs. 1 and 2).

Inorganic tripolyphosphatc, likewise initiates tetanic contrac- tion. The mechanical response indlcates here a materially more asynchronous activity of the tllfferent fibres than is the case after application of ATP or acetylcholine. The abrupt rise in tension following injection o f tripliosphate is supeiposecl by a diffuse fibrillary activity (Fig. 3 B). l h e threshold amount is somewhat higher than for -1TP. 50-60 r ( g triphosphate (0.1- 0.13 X mol) releasing a inechanical response with the same tension as that initiated bv 1 ccg acetylcholine.

314 FRITZ BUCATHAL AND W6RN FOLKOM'.

Figure 1. Action potentials (upper curves) and mechanical tension (lower curres) of the frog gastrocnemius after close arterial injection of:

A. 6 rig acetylcholine in 0.03 ml Ringer. B. 217 ,ug sodium adenosine triphosphate (0.44 X 10-8 mol) in 0.03 ml Ringer C. O.G Ng acetylcholine in 0.03 m1 Ringer after previous application of ade-

nosine triphosphate (B).

A 0 c 0 Figure 2. Mechanical tension of the frog gastrocnemius muscle after arterial in-

jection of: A. 5 ,pg acetylcholine in 0.03 ml Ringer. B. 20 ,pg sodium adenosine triphosphate (0.04 x mol, applied 5 times

C. 5 pg acetylcholine in 0.03 rnl Ringer after prereding injection of adenosine

D. 5 pg acetylcholine 4.5 min after C. Time marks: 1 sec.

in 0.03 ml Ringer).

triphosphate (B).

CLOSE ARTERIAL INJECTION OF ADENOSINE TRIPHOSPHATE. 315

I n a series of experiments made a t another time of the year the sensitivity of the muscle to acetylcholine and triphosphate was essentially higher (c. 20 times), the threshold for acetyl- choline being 0.025 pg.

Application of acetylcholine after previous treatment of the preparation with triphosphate - as after ATP - releases a considerably higher tension than before application of the phos-

A B C Figure 3. Intra-arterial injection of:

A. 0.6 pg acetylcholine in 0.03 ml Ringer. B. 58 pg (0.13 x 10-8 mol) sodium tripolyphosphate in 0.03 ml Ringer. C. 0.6 pg acetylcholine in 0.03 ml Ringer after preceding application of sodium

Time marks: 1 8ec. tripolyphosphate (B).

phate (Fig. 3 C). Furthermore, the responses produced by tri- phosphate are in their turn enhanced by previous application of ATP.

It can be seen from these experiments that close arterial in- jection of ATP and inorganic triphosphate in relatively low con- centrations releases a tetanus-like contraction in the gastrocne- mius muscle of the frog, and they furnish further evidence that ATP is an important agent in the initiation of contraction. When the reaction to acetylcholine is used as a basis for com- parison, frog muscle is about 3-4 times more sensitive to ATP than mammalian muscle (BUCHTHAL and KAHLSON 1944). The negative results obtained by ABDON (1942) with injection of adeno- sine triphosphate by means of a similar technique to that used here are difficult to explain. The doses of ATP apparently cor- respond to those used in our experiments. A factor involved may, however, be the acetylcholine contracture produced before ATP is applied by adding 200-300 mg acetylcholine to the surrounding bath.

316 FRITZ BUCHTHAL AND BJSRN FOLKOW.

Summary.

1. I n the gastrocnemius of the frog close arterial injectmionof small amounts of adenosine triphosphate and inorganic triphos- phate evoke tetanus-like contractions. The mechanical response produced by 40 pug ATP (0.08 x mol) or 50-60 !(g triphos- phate (0.10-0.13 x 10-6 mol) corresponds to that released by 1 pg acetylcholine.

2. The sensitivity of the preparation to acetylcholine and in- organic triphosphate is greatly increased by previous application of adenosine triphosphate.

References.

ABDOK-, K. O., Om Kreatirifosforsyrens och Adeuosintrifosforsyrens Betydelse for de parasympaticomiinetiska Farniakas Verkari, Dis- sert. Lund. 1942.

BROWS, G. L., J. Physiol. 1937, 89. 438. BUCHTHAL, F., A. DEUTSCH, and G. G. KNAPPEIS, Acta Physiol. Scand.

BUCHTHAL, F., 8. DEUTSCH, and (;. c:. I i N A P P E I S , YtItllI't? 1944. 153.

BUCHTHAL, I?., and G. KAHLSOK, Ibidem 1944. 154. 178.

1944.

774.