THE EFFECT OF ELECTROLYTES ON THE MUSCLE OF THE FORE-GUT OF DYTISCUS MARGINALIS, WITH SPECIAL REFERENCE TO THE ACTION OF POTASSIUM

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1 3*5 THE EFFECT OF ELECTROLYTES ON THE MUSCLE OF THE FORE-GUT OF DYTISCUS MARGINALIS, WITH SPECIAL REFERENCE TO THE ACTION OF POTASSIUM BY A. D. HOBSON, MA., Lecturer in Experimental Zoology in the University of Edinburgh. (Received zstk February 1928.) (With One Plate and One Text-figure.) THE spontaneous rhythm of the isolated fore-gut of Dytiscus was first described by Ten Cate (1924). The interest of the preparation lies in the fact that our knowledge of the relation of insect muscle to electrolytes is extremely small and also that, with the exception of heart muscle, rhythmically contractile striated muscle is rare. The part of the alimentary canal used in the following experiments consisted of the oesophagus and the crop. Silk threads were tied round the pharynx and the posterior end of the crop, just in front of the proventriculus, and these were used for suspending the preparation. The crop was always allowed to empty itself before tying up the ends. The preparation was suspended in a modified uterus bath as described by Hogben and Hobson (1924), and was attached to a light recording lever. The great contractility of the wall of this part of the gut is due to the presence of circular and longitudinal muscles, especially well developed in the crop which is directly continuous with the oesophagus. The details of the arrangement of these muscles, which are all striated, have been described by Rungius (1911). Using as a medium Ringer solution at a concentration corresponding to 0*95 per cent. NaCl, i.e. having approximately the same osmotic pressure as that used by the present writer, Ten Cate (1924) obtained a powerful but extremely irregular rhythm resembling that shown by a smooth muscle preparation. Since he was chiefly interested in the action of adrenalin and other drugs on the muscle, he did not attempt to obtain a more satisfactory physiological medium. The present writer has employed a medium having the composition NaCl M : KC M : CaCl M. In a fluid of this composition the muscle maintains its activity almost unchanged for many hours. Magnesium was found to be an unnecessary constituent and was omitted for the sake of simplicity. The medium was made from M/6 solutions of NaCl and KC1 and M/g CaCl 2, these being approximately isotonic. In many of the experiments the salts used were those prepared by British Drug Houses Ltd., A.R. quality. In all cases the results have been checked with Kahlbaum's purest salts, and these were used exclusively in the experiments on the elimination of sodium from the medium.

2 386 A. D. HOBSON The experimental media employed were always, wherever possible, buffered with sodium bicarbonate. The amount was usually 1 drop of saturated solution per 200 c.c. of sodium or potassium chloride. The hydrogen ion concentration in all the experiments corresponded approximately toph 7-2. In those media in which the proportion of calcium was very high effective buffering was not possible and the hydrogen ion concentration was adjusted with Ca(OH) 2. In such cases the ph. was liable to alter rapidly so that it was necessary to change the fluid at short intervals. The solutions were thoroughly aerated before use. Aeration was not provided during the experiments but so long as the solutions were changed at fairly short intervals the absence of aeration was found to have no harmful effect. In some cases where a very light lever was being used and a feeble rhythm was maintained it was desirable to avoid even the slight disturbance caused by gentle aeration. The temperature was not specially controlled and varied in different experiments from i5 C. toi8 C. In individual experiments the variation was never more than 1-5 C. The rhythmic contractions usually begin as soon as the preparation is suspended in the bath. At first the muscle is in a state of more or less complete tonic contraction and a slow fall of tone takes place during the first ten or fifteen minutes until complete relaxation is attained. Sometimes the tonic contraction is so great that rhythmic contractions do not begin until about ten minutes after setting up the preparation. The strength of the contractions made by preparations from specimens equally large and healthy may differ enormously. The nature of the rhythm also varies widely. Usually, in the medium described above, the rhythm is irregular. It is, however, more regular than that found by Ten Cate (1924) from which it differs in showing a well-marked base line, that is to say, after each contraction the muscle relaxes to the same extent. This can be seen clearly in the graphs. Occasionally a preparation is obtained showing contractions almost as regular as, though slower than, those of a heart (Fig. 1). This type of rhythm can usually be obtained by increasing the concentration of potassium and calcium while keeping the ratio K/Ca constant. An instance of this is illustrated in PL VII, fig. 3. THE EFFECT OF VARIATIONS IN THE POTASSIUM AND CALCIUM CONCENTRATIONS IN THE PRESENCE OF SODIUM. In considering the effects of variations in the concentrations of the ions of the medium it must be borne in mind that, in the balanced mixture of salts taken as normal, relaxation seems to be very nearly complete. Consequently no marked loss of tone can be caused by changing directly from the normal medium to any other mixture. Tonic relaxation can only be clearly demonstrated when the tone of the muscle has been raised by previous treatment such as an increase in the potassium concentration. In general this preparation shows a strong resemblance to the crustacean hearts employed by Hogben (1925) and Wells (1928). The differences seem to be quantitative rather than qualitative. Both Hogben and Wells found that the optimum calcium concentration for the crustacean heart varies considerably and

3 Effect of Electrolytes on Muscle 387 that consequently the amount of Ca necessary to produce a given effect is also variable. The gut muscle of Dytiscus also shows differences in its quantitative relations not only to the calcium content but also to the potassium content of the surrounding medium. Increasing the potassium content of the normal medium causes a sharp rise in tone and greater amplitude and frequency. These effects may be very slight if the potassium concentration is only doubled, but if it is trebled or quadrupled the result becomes more and more striking. High concentrations of potassium cause a great increase in tone, which afterwards falls slowly, and immediate cessation of the con- Fig. 1. Muscle in NaCl M: KC M : CaClj M: ph 7-5. First signal, changed to NaCl 0164 M : CaCl, 0002 M : ph 7 5. Second signal, changed to NaCl 0*167 M : ph 7 5. tractions. Wells (1928) has called attention to a depressing effect of low concentration of potassium. This is not well marked in this preparation, probably because of its peculiarly complete relaxation. It can however be perceived if a potassium-free solution is changed for one containing 0003 Af K; there is depression of tone and a pause which may last for a minute or two before contractions begin again. The effect of potassium can also be seen when calcium is removed from the medium. If potassium is absent there is an abrupt rise of tone and a rapid succession of irregular contractions (Fig. 1, second signal) which gradually decrease in amplitude until the muscle is at rest in a condition of almost complete relaxation. If potassium is present the same succession of events occurs but the final arrest of the contractions takes place more quickly.

4 388 A. D. HOBSON If potassium is removed from the medium there is a rise in tone, usually very slight. The amplitude is reduced and the frequency may be increased. Fig. 1 illustrates these effects on a particularly powerful and regular preparation. The rise in tone is here so small as to be almost imperceptible. The relation of the muscle of the fore-gut of Dytiscus to the calcium concentration of the surrounding fluid seems to be essentially similar to that of the crustacean heart. A slight increase causes diminution of the amplitude and slowing of the contractions (PI. VII, fig. 2). If for the normal medium there is substituted a series of solutions in which the calcium content is gradually increased, a point can be found at which all contractions are stopped and the muscle is completely relaxed. If the calcium concentration is now further increased there is usually a drop in tone lasting for one or two minutes and then slow, tonic contraction takes place. The initial temporary lowering of tone is very characteristic and closely resembles that described by Wells as occurring under similar circumstances in the heart of Maia. It often occurs even if the increase in calcium concentration is insufficient to cause subsequent tonic contraction; in such cases the muscle quickly recovers its original tone. This can be seen in Fig. 2 and, to a more marked degree, in Fig. 4 (third signal). Only if the potassium concentration is above that of the normal medium or if the calcium content is deficient does an increase in the calcium cause a permanent lowering of tone. Calcium, therefore, produces different effects according to the concentration; slight excess causes arrest in relaxation; great excess causes tonic contraction. Another notable point is that recovery from the effects produced by excess of calcium is not immediate but takes several minutes at least. Wells has observed a similar phenomenon in the heart of Maia and has pointed out that it tallies with the view that calcium forms an insoluble calcium-lipoid compound on the surface of the cell. In his work on invertebrate muscle preparations Wells (1928) has advocated and brought considerable evidence in favour of the view that the tonic condition of these muscles is independent of the K/Ca ratio and dependent only on the absolute concentrations of potassium and calcium. While the results of this investigation of the muscle of the fore-gut of Dytiscus support this view in general, there is a certain amount of evidence which seems to oppose it. If the minimum concentration of calcium necessary to cause tonic contraction be determined for media containing varying amounts of potassium, it is found that as the potassium concentration rises so must also the calcium content be increased. In one experiment, for example, it was found that when the potassium concentration was M tonic contraction was induced by M calcium. Raising the potassium concentration to o-on M made it necessary to increase the calcium to M before tonic contraction took place. Similar results can be obtained if complete suppression of the contractions is taken as the point of comparison. While it is probable that the absolute concentrations of potassium and calcium are the most important factors, it does not seem as though the K/Ca ratio is entirely unimportant in determining tone.

5 Effect of Electrolytes on Muscle 389 THE REPLACEMENT OF SODIUM BY POTASSIUM. In the physiology of muscle, potassium plays so striking a role that there has been a tendency to attribute to this element specific properties distinguishing it from sodium. These specific properties appear to have their basis rather in quantitative than in qualitative differences. The essential similarity of potassium and sodium appears most obviously in the study of the tissues of marine invertebrates, especially in the case of those cells which are normally exposed to sea-water. As is now known, the surface membrane of such cells can in certain cases be maintained for long periods in normal condition in properly balanced mixtures of either sodium or potassium and calcium. Even with so specialised a tissue as muscle, the many points of resemblance between the effects of sodium and potassium are clear. These considerations led to the attempt to find a mixture containing only potassium and calcium in which the rhythmic contractions could be maintained. A preliminary experiment was performed with a very active preparation by testing the action of a series of mixtures in which the sodium concentration was gradually diminished while the ratio K/Ca was at first kept constant. The initial mixture was as usual NaCl M : KCl M : CaCl M. The sodium was gradually reduced until the mixture became NaCl : KCl M : CaCl M. Up to this point the only marked effect was a gradual decrease in the completeness of relaxation (Fig. 3). The degree of contraction remained unaltered, and, indeed, did not change throughout the experiment, and so the amplitude became gradually reduced. That this effect was not due to excess calcium was shown by the fact that it was accentuated by an increase in the potassium concentration, while the addition of calcium caused more complete relaxation (Fig. 4). As the sodium concentration became smaller in the later stages of the experiment, so was it necessary to increase the proportion of calcium to maintain a rhythm. A regular rhythm of contractions of very small amplitude persisted up to the point where a solution of composition NaCl M : KCl M: CaCl M was used. With further decrease in the sodium content no steady rhythm could be obtained whose amplitude was great enough to allow a satisfactory graphical record to be made. Very faint twitches could, however, be observed in the binary mixture KCl M : CaCl 2 0*077 M. As subsequent work showed, the failure to maintain a good rhythm in the final solution was due simply to exhaustion, the experiment having occupied over four hours. Different stages in the elimination of sodium in this experiment are illustrated in Figs. 3 and 4 taken from the same preparation. Fig. 4 shows the small amplitude and high tone of the muscle when the sodium is reduced to less than half the normal concentration. The decreased amplitude caused by raising the potassium concentration and the opposite effect of calcium can also be seen. Changing directly from the normal medium to one of the composition KCl M : CaCl M or preferably one with a somewhat smaller potassium content causes an immediate rise of the base line and temporary cessation of contractions.

6 390 A. D. HOBSON Contractions begin again in five minutes or less and a steady rhythm is maintained (Fig. 5). The amplitude is usually considerably less than the normal and the tone is always high. The limits between which a rhythm can be maintained in a potassium-calcium mixture are fairly clearly marked. No trace of contraction was ever obtained when the ratio K/Ca exceeded 1*3 or fell below 0-3. Between these limits the muscle maintains a rhythm which is fairly normal except that the tone is high and the amplitude small; increase in the potassium concentration causes rise in tone (Fig 6); increase in the calcium concentration causes decreased tone (Fig. 7). The optimum ratio varies considerably in different preparations but in most cases is probably about 0-4. The amplitude seems to fall off more rapidly when the potassium concentration is raised above the optimum than when the calcium is similarly increased. Often a good rhythm is maintained when the K/Ca ratio is 0-3 as is shown in Fig. 7. It is necessary to emphasise that the activity of the muscle in potassium-calcium mixtures is not of short duration. Experiments have been made in which the tissue has been kept in sodium-free media for z\ hr. Various changes in the K/Ca ratio were made in this period and at the end the muscle was just as active and reacted as readily to ionic changes as at the beginning of the experiment. Fig. 6 shows a portion of the record of this experiment, illustrating the characteristic effects produced by increasing the K/Ca ratio. Fig. 7 illustrates, from another experiment, the effect of decreasing the K/Ca ratio. In the first part shown the ratio K/Ca = 0-51 which was, for this preparation, too high. Reducing the ratio to 0-33 caused a good rhythm to begin immediately and there was a marked lowering of tone. Changing the medium to pure calcium chloride caused the contractions to disappear immediately; the tone decreased at first but the muscle then passed into slow tonic contraction. The effect of pure calcium chloride is completely reversible when the requisite amount of potassium is added. The time taken for recovery, which is never immediate, varies with the duration of the exposure to the calcium chloride solution. It may be stated generally that the behaviour of the muscle in solutions containing potassium and calcium chlorides only is in all essential features the same as in sodium-calcium or in sodium-potassium-calcium mixtures. The only important difference is that the tone is high and the amplitude is correspondingly reduced. The muscle is equally sensitive to ionic changes and these produce similar results which may be summarised as follows. A rise in the alkali-metal/ca ratio causes decreased power of relaxation and the amplitude is reduced; the frequency is increased slightly but is usually comparatively little affected. These effects can be removed almost instantly by returning to the optimum alkali-metal/ca ratio. Decrease in the alkali-metal/ca ratio causes nearly always an initial lowering of the tone; the amplitude of the contractions is reduced and the frequency is usually decreased. If a large excess of calcium is introduced into the medium, the contractions are arrested immediately and the initial lowering of tone is followed by tonic contraction (Fig. 7, second signal). These changes in the muscle are only comparatively slowly reversible.

7 Effect of Electrolytes on Muscle 391 DISCUSSION. The most interesting fact discovered during this investigation is that it is possible for striated muscle to maintain rhythmic activity in a solution containing potassium and calcium alone. Cousy and Noyons (1923) have concluded that sodium and potassium are similar in their action on the heart of the frog and, to a certain extent, mutually replaceable. Cousy (1923) found that the heart of the frog would continue to beat if the whole of the sodium was replaced by glucose with the exception to 0-02 per cent. NaHCO 3. No attempt was made to replace any of the sodium by potassium which was, in fact, reduced in concentration. Under these conditions the presence of o-oi CaCl 2 in the absence of potassium stopped the rhythm, o-oi per cent. KC1 allowed the rhythm to continue whether calcium was present or not. Activity could also be maintained for many hours by perfusion with a simple isotonic solution of glucose containing about 0-05 per cent. NaHCO 3. It is concluded from these results that only a small part of each of the salts present in Ringer is concerned in the actual balance of ions. Clark (1927), however, has suggested that these results are to be explained by traces of serum remaining in the intercellular spaces in the heart tissue. Clark (1926) has found in the frog's heart that the antagonism between potassium and calcium occurs only over a limited range of concentrations. Whereas M KC1 is completely antagonised by M CaCl 2, the effect of M KC1 is hardly altered by M CaCi 2 - The results obtained with the Dytiscns preparation show that this is not true of all muscle. The evidence points to amuchhigher tolerance to potassium in the external medium than has hitherto been recorded for muscle. Pantin (1926), working on Amoeba, has demonstrated that activity can be maintained for considerable periods in solutions in which potassium provided the only monovalent cation. R. S. Lillie (1906) and Gray (1920), working with the ciliated epithelium of the gills of Mytilus, found that potassium does not form a completely satisfactory substitute for sodium although movement may continue for some hours. The muscle of Dytiscus furnishes an example of a condition intermediate between that found in the marine Amoeba and that in the muscle cells of vertebrates. The muscles of marine invertebrates would probably provide material still more suitable for investigation along these lines. It is of interest to compare the optimum alkali-metal/ca ratios for Dytiscus muscle immersed in binary solutions with those found by Pantin (1926) for the marine Amoeba. For Dytiscus the optimum Na/Ca ratio is about 50. This agrees well with the variable value found by both Hogben (1925) and Wells (1928) for the crustacean heart but is higher than the value of 20 found by Pantin for Amoeba. It is, however, when the K/Ca ratios are compared that the most remarkable difference appears. For the muscle the optimum K/Ca ratio is probably about 0-4 while for Amoeba the value is about 40. The extraordinary low optimum K/Ca ratio might seem to suggest that the condition of the muscle and its relation to the surrounding medium is entirely abnormal in the potassium-calcium mixtures. The

8 392 A. D. HOBSON sensitivity to ionic changes is, however, as great as in sodium-calcium mixtures, and the normal character of the responses to such changes seems to preclude this view. It is, however, undesirable to discuss the precise significance of this low optimum K/Ca ratio until further evidence is available. Reference has already been made to the view recently put forward by Wells (1928) that the tonic condition of the muscles investigated by him is dependent on the absolute concentrations of potassium and calcium and is independent of the K/Ca ratio. While the results described in the earlier part of this paper are in general confirmatory of those obtained by Wells on the heart oimaia, it was pointed out that the concentration of calcium necessary to cause tonic contraction did not appear to be entirely independent of the potassium concentration. During the experiment on the elimination of sodium already described the tone became steadily increased with each reduction of the sodium concentration. That this increase in tone throughout was due to the increasing absolute concentration of potassium was proved by the fact that it was further increased by addition of potassium and that addition of calcium had the opposite effect. In the subsequent experiments with sodium-free media the tone was always high, due to the high potassium content. In all the potassium-calcium mixtures in which a rhythm could be maintained the potassium concentration was so high that, had sodium and calcium been present in the concentrations of the normal medium, the muscle would have undergone complete tonic concentration. The absence of this phenomenon in such potassiumcalcium mixtures shows that in this muscle, at least, the tone is dependent, not only on the absolute concentrations of potassium and calcium but also on the K/Ca ratio. SUMMARY. 1. The muscle of the isolated fore-gut of Dytiscus marginalis will maintain rhythmic contractions for long periods when suspended in a medium of the composition NaCl O-I6I M : KC M : CaCl M at a ph of about In the presence of normal concentrations of sodium, alterations in the potassium and calcium concentrations cause changes in the muscle essentially similar to those already described by Hogben and by Wells in the crustacean heart. 3. Rhythmic contractions can be maintained for extended periods in mixtures of potassium and calcium chlorides alone. 4. In potassium-calcium mixtures the tone is always high, but the contractions are normal in form and the muscle reacts in the normal manner to changes in the K/Ca ratio. The optimum K/Ca ratio is extraordinarily low, having a value of about The tone of the muscle is dependent, not only on the absolute concentration of potassium and calcium but also on the K/Ca ratio. This investigation was begun in the Department of Zoology, University College, London in 1925, and it has been continued in the Department of Zoology of the

9 EXPERIMENTAL BIOLOGY VOL. V, PLATE VII. HOBSON EFFECT OF ELECTROLYTES ON MUSCLE.

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11 Effect of Electrolytes on Muscle 393 University of Edinburgh and in the Marine Biological Laboratory, Plymouth. The author Is Indebted to the Royal Society and to the Earl of Moray Endowment of the University of Edinburgh for grants covering part of the cost of the Investigation and to the Committee of the DIxon Fund of the University of London for a grant for the purchase of a kymograph. Acknowledgment for the use of a table at Plymouth during part of the summer of 1927 Is made to the British Association. REFERENCES CLARK, A. J. (1926). Journ. Pharm. Exp. Therap. 29, 311. (1927). The Comparative Physiology of the Heart. Camb. Univ. Press. COUSY, R. (1923). Arch. Internat. Physiol. 21, 90. COUSY, R. and NOYONS, A. K. (1923). Arch. Internat. Physiol. 20, 1. GRAY, J. (1920). Quart. Journ. Micr. Set. 64, 345. HOGBEN, L. T. (192s). Quart. Journ. Exp. Physiol. 15, 263. HOGBEN, L. T. and HOBSON, A. D. (1924). This Journal, 1, 487. LILLIE, R. S. (1906). Amer. Journ. Physiol. 17, 89. PANTIN, C. F. A. (1926). This Journal, 3, 275. RUNGIUS, H. (1911). Zeitsckr.f. Wiss.Zool. 98, 179. TEN GATE, J. (1924). Arch. Neerland. Physiol. 9, 598. WELLS, G. P. (1928). This Journal, 5 S 258. EXPLANATION OF PLATE VII. Fig. 2. Muscle in NaCl O-I6IM:KC M: CaCl 2 o-oozmtpm 7-1. Signal, changed to NaCi M : KCi M : CaCI* M ipb Fig. 3 (a). Muscle in NaCl M : KCI M : CaCl M : J»H 7-2. Signal, changed to NaCI M: KCI M : CaCl M:pH 7-2. (&) Muscle in NaCl M : KCI o-oi2 M : CaCl M :ph 7-2. Signal, changed to NaCl M t KCI Af : CaCl 2 o-oiiaf:ph 7-1. Fig. 4. Muscle in NaCl M : KCI M : CaCl M :pm 7-2. First signal, changed to NaCl M: KCI M:CaCl M ipb Second signal, changed to NaCl M : KCI M : CaCl M : ph 7*2. Third signal, changed to NaCl M : KCI M : CaC! M : pb Fig. 5. Muscle in NaCl M : KCI M : CaCl M :ph 7-2. Signal, changed to KCI M : CaCl M :ph 7-3. Fig. 6. Muscle in KCI M : CaCl M :ph.t3* First signal, changed to KCI 0-042M : CaCl M : ph 7-1. Second signal, changed to KCI M : CaC! M : ^H 7-3. Fig. 7. Muscle in KCI M : CaCl? M : ph J-z. First signal, changed to KCI M : CaCl M :ph 7-2. Second signal, changed to CaCl g O-III M ipb. 7-4.