238. Picrotoxin: A Potentiator of Muscle Contraction

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1 No. 101 Proc. Japan Acad., 46 (1970) Picrotoxin: A Potentiator of Muscle Contraction By Kimihisa TAKEDA and Yutaka OOMURA Department of Physiology, Faculty of Medicine Kanazawa University, Kanazawa (Comm. by Masahiro OKADA, M. J. A., Dec. 12, 1970) We have shown that picrotoxin, known as a neurally acting convulsant, selectively inhibits conductance increases in the sarcotubular system (STS, internal membrane system) 1) of frog muscle fibers.2~,3) The aim of this investigation was to determine whether the inhibition of the conductance exerts any effects on excitationcontraction coupling. The results demonstrate the direct action of picrotoxin on the muscle fibers to augment twitch tension. The experiments were performed on the sartorius muscle of Rana catesbiana at room temperature (21-23 C). Two microelectrodes, one for intracellular stimulation and the other for potential recording, were inserted into a surface fiber of the isolated small bundle (for tension recording), or of the whole muscle, immersed in normal Ringer's solution with or without 10-3 g/ml of picrotoxin. The isometric tension produced by single fiber twitches was measured by means of a mechano-electronic transducer 5734A (Toshiba) attached to the bundle of fibers. In order to measure the mechanical threshold, 10J6 g/ml of tetrodotoxin was added to the Ringer's solution and 200 msec depolarization steps were applied using the constant current or point-voltage clamp technique. In the latter experiment, membrane potentials were clamped to -100 mv except for the duration of the depolarization steps. Least visible local contraction under a binocular microscope was the criterion to determine the threshold. Fig. 1 shows an example of picrotoxin-induced twitch potentiation in a fiber. The upper trace in the records shows a conducting action potential produced by direct intracellular stimulation. The lower trace shows the simultaneously recorded isometric twitch tension. Fig. 1 Al is a control record before application of picrotoxin. The action potentials and twitch tensions in three control records were almost identical, though the resting potential was reduced by a few millivolts (not illustrated). On the addition of picrotoxin, the twitch tension in the fiber was increased about three times after 5 minutes (A2), and about five times after 10 minutes (A3). Small increases were noticed in the time to peak and duration of the twitches (Fig. 1 B). The potential recording electrode was withdrawn by

2 1052 K. TAKEDA and Y. OOMURA Vol. 46, Fig. 1. Picrotoxin-induced twitch potentiation in a frog muscle fiber. Records before (A1), and 5 minutes (A2) and 10 minutes (A3) after 10.3 g/ml of picrotoxin are superimposed in B so as to make the rising phase of action potentials coincide. Upper trace, intracellular action potential produced by direct intracellular stimulation with a 5 msec pulse: the horizontal line registers external potential. Lower trace, simultaneously recorded isometric twitch tension of the fiber due to the conducted action potential. In B, solid lines, A1: broken lines, A2: dotted lines, A3: numbered arrows indicate peak of action potential in Al-A3: the oblique arrow shows a dip at the end of the spike potential: the short horizontal line in potential record indicates -50 mv. The spike potential in Al-A3 was retouched. The external potential in Al was drawn. the augmented twitches (Au, A3). Local damage produced by electrode withdrawal, however, was not taken as serious for the following reasons. (i) The recorded tensions were produced almost entirely by the conduction of action potentials along the fiber length, since large local depolarizations alone, which resulted in visible local contractions, did not show appreciable changes in the tension records. (ii) Although the resting potential of the fiber was reduced to -88 mv (A3) from --94 mv (A1), these values were far below the contraction threshold and might not affect contraction. The peak values of the action potentials were +36 mv in Al and +24 mv in A3. The changes in the falling phase of the action potentials after picrotoxin were conspicuous. The rate of repolarization was diminished and the dip at the end of the spike potential,4~ which was noticed

3 No. 10] Picrotoxin : Potentiator of Contraction 1053 Fig. 2. Picrotoxin-induced twitch potentiation as in Fig. 1. Note that an expanded sweep was used for potential recording. Artifacts in the potential record are due to a 5 msec stimulating pulse which is registered early in the tension record. Records from newly penetrated fibers before (A), and 15 minutes (B) and 90 minutes (C) after picrotoxin are superimposed in D. In D, solid lines, A: broken lines, B: dotted lines, C : an early portion of. the upper dotted line was omitted (indicated by solid line). The external potential was drawn in A and B. in the control record (oblique arrow), was then absent showing a smooth transition to the early of terpotential (Fig. 1 B). The amplitude of the early afterpotential was comparable to that obtained before picrotoxin. Fig. 2 shows these effects on an expanded time scale. Fig. 2 A is a control record before picrotoxin. Fig. 2 B was taken 15 minutes after the addition of picrotoxin from a newly penetrated adjacent fiber in the same preparation. The rising phase and peak of the action potentials in these records were identical (Fig. 2 D). The falling phase was prolonged after picrotoxin, but not more than twice the duration obtained without picrotoxin. The record in Fig. 2 C was taken from another preparation 90 minutes after picrotoxin. The potentiation in twitch tension with a plateau phase was tremendous (267 mg, more than ten times control values), but the twitch duration did not alter greatly. The duration of the action potential in this fiber was about the same as that in Fig. 2 B and its amplitude was even reduced (Fig. 2 D). A little more depolarization was brought about at the beginning of the action potential, but this local depolarization might not contribute much to the recorded tension, as previously described. All three fibers in Fig. 2

4 1054 K. TAKEDA and Y. OoMURA [Vol. 46, had resting potentials of -81 mv. The picrotoxin-induced twitch potentiation was reversible. It required more than 10 minutes to restore control tensions after the removal of picrotoxin. Fibers after long exposure to picrotoxin (20 minutes or more) occasionally displayed repetitive twitches, which sometimes lasted for several seconds, in response to single intracellular stimulation with a 5 msec pulse. Nevertheless, addition of 10,3 g/ml of picrotoxin by itself caused neither spontaneous twitches nor contractures. When whole muscle fibers in a small bundle were stimulated externally with a pair of electrodes, picrotoxin-induced twitch potentiation was not so large as in a single fiber. Tetanus tension thus observed was not altered by picrotoxin, although its fusion frequency was reduced. It was noticed that the falling phase of tetanus tension was slowed down after long exposure to picrotoxin. The mechanical thresholds measured in single fibers in the presence of picrotoxin were --51±1.66 mv (mean±s.d., 5 fibers) by the constant current method and -51.5±1.78 mv (10 fibers) by the voltage clamp method. Similar mean values (50.4 mv and 50.5 mv) were obtained in the fibers without picrotoxin.1 Thus 10-3 g/ml of picrotoxin did not alter the mechanical threshold. Present results demonstrate the direct action of picrotoxin (10-3 g/ml) on the muscle fibers as a potentiator of twitch contraction. Picrotoxin might satisfy the criteria for the type B potentiator5~ in that it has no effects on the mechanical threshold but decelerates the falling phase of the action potential. The enormous (over ten times) picrotoxin-induced twitch potentiation, however, with relatively minor (less than twice) prolongation in the action potential (Fig. 2C) makes it difficult to ascribe the potentiation to a simple extension in the mechanically effective period. A more fundamental process must be affected by picrotoxin. Unlike caffeine, picrotoxin at the concentration used neither lowered the mechanical threshold nor induced contractures, but slowed down the falling phase of tetanus tension. On the other hand, picrotoxin, while affecting the resting conductance only slightly,6~ inhibits both the hyperpolarization-activated early conductance increase in the STS which causes creep in normal Ringer's solution2~ and the regenerative depolarizing response in the STS observed in F-rich solution.3} It is quite possible that the picrotoxin-inhibited conductance in the STS is specifically related to muscle relaxation, and that its inhibition produces the twitch potentiation. It is tempting to speculate that the picrotoxin-inhibited conductance component is located in the membrane of the sarcoplasmic reticulum and is related to the accumulation of activator Ca,11

5 No. 10] Picrotoxin : Potentiator of Contraction 1055 Although the Ca-releasing period and, therefore, the contraction period, may be primarily determined by the duration of an action potential,' inhibition of the Ca accumulation may potentiate and prolong twitches, and, further, may somehow induce repetitive twitches in response to single stimulation. It is noted that an increase in the recorded tension in the present study may involve both an actual increase in tension and a prolongation of the contraction period in each longitudinal contractile unit, since a considerable time (approximately msec) was expended for the conduction of action potentials along the fiber. Finally, the direct twitch potentiating action of picrotoxin on muscle fibers and occasional induction of repetitive contractions in response to single direct stimulation should be taken into consideration to interpret the convulsant action of picrotoxin at high concentrations. This work was supported in part by grants from the Ministry of Education and by NIH grant (NB ). Crystalline tetrodotoxin used was supplied by the Sankyo Co., Ltd. References 1) 2) 3) 4) 5) 6) Sandow, A. (1970) : Ann. Rev. Physiol., 32, 87. Takeda, K., and Oomura, Y. (1969) : Proc. Japan Acad., 45, (1970) : Proc. Japan Acad., 46, Persson, A. (1963) : Acta Physiol, Scand., 58 (SuppL 205), 3. Sandow, A. (1965) : Pharmacol. Rev., 17, 265. Takeda, K., and Oomura, Y. (1968) : Proc. Japan Acad., 44, 1072.