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THE EFFECTS OF TEMPERATURE ON THE OSMOTIC PROPER- TIES OF MUSCLE. By D. H. DE SOUZA. (From the Physiological Laboratory, University of Sheffield.) (With six diagrams in the text.

1 THE EFFECTS OF TEMPERATURE ON THE OSMOTIC PROPER- TIES OF MUSCLE. By D. H. DE SOUZA. (From the Physiological Laboratory, University of Sheffield.) (With six diagrams in the text.) (Received for publication 22nd January 1909.) INTRODUCTORY. OF late years several attempts have been made to show that a muscle immersed in saline solutions behaves like a solution of salts enclosed in a semi-permeable membrane-i.e. that it withdraws fluid from solutions of lower concentration and gives up fluid to solutions of higher concentration, without, at the same time, parting with or receiving any addition to its salts. While it is generally agreed that an exchange of fluid does take place, yet most observers have found that this exchange is not in strict accordance with the laws of osmotic pressure. The discrepancy has been considered by some to be due to the state of the fluid originally in the muscle, by others to be due to the membrane. Overton,1 who has worked exhaustively at the subject, looked upon the limiting surface of. the muscle fibre itself as the only truly semipermeable membrane in the muscle complex; but although he was satisfied with the existence of such a membrane, yet he found it impossible to accept the view that it contained a simple solution of salts, seeing that there was lack of agreement with the laws of osmotic pressure when the behaviour of the muscle in salt solutions was studied. From a careful consideration of his experiments he came to the conclusion that all the water in the muscle did not act like a simple solvent for the salts. Some of it did so act, and this portion took part in the ordinary osmotic exchange; but in the muscle there was always present a certain amount of water of practically constant volume, which was inert from an osmotic point of view (" Quellungswasser.") This he found a satisfactory explanation of the discrepancies from the law, obtained in his experiments. Further observations have shown that even the semi-permeability of the membrane must be questioned. Fletcher2 has pointed out that some results are readily explained by "leakiness" of the membrane, so that an exchange of salts by diffusion goes on at the same time as an exchange of 1 Pfluiger's Archiv, xcii. p. 115, Journ. of Physiol., xxx. p. 414, VOL. II., NO

2 220 de Souza fluid by osmosis. It will be seen subsequently that some of the experiments in this paper seem to bear out Fletcher's contention. But another important fact has to be taken into account when considering the behaviour of the membrane and its contained fluid, viz. the frequent changes in concentration which this solution must undergo. There is no doubt that the quantity of substances in simple solution in it must depend upon the physiological condition of the muscle. It is known that a muscle which has been active for some time will take up fluid from a salt solution of low concentration more rapidly than one which has been previously at rest. This has been taken to mean that during activity chemical changes occur, and the substances produced raise the osmotic pressure in the interior of the muscle. If this be the case, any factor causing an increase in chemical change should cause a rise in osmotic pressure inside the muscle. Miss Cooke ' considered alterations of temperature capable of producing such changes. Working with the frog's gastrocnemius, she found that rise in temperature caused rise in osmotic pressure inside the muscle-that a solution isotonic for a muscle at one temperature parted with water to the muscle, i.e. was hypotonic for it, at a higher temperature. This she explained as being due to the presence within the muscle of dissociation products, the formation of which was accelerated by rise in temperature. Before accepting this explanation, however, it is well to remember that the substances produced are very diffusible, and that, if the membrane is partially permeable, as experiments seem to show, they may pass through it almost as rapidly as they are formed, and so affect the osmotic pressure either not at all, or only temporarily and to a slight extent. Now Macdonald2 has drawn attention to the importance of physical factors in producing such effects as those under consideration. He3 looks upon the fluid inside the muscle as a colloidal solution containing electrolytes, some of which are in simple solution, while others form adsorption complexes with the colloidal particles When a large colloidal particle is, for any reason, split up into smaller ones, there is a greater extent of surface available for adsorption, and fewer electrolytes are left in solution. On the other hand, the formation of large particles from smaller ones, "desolution," increases the number of electrolytes in solution. When the muscle is in an excited but not contracted state, these two conditions occur in neighbouring regions of the muscle, so that there are alternating segments of high and low osmotic pressure, leading to the passage of water from the region of low to that of high osmotic pressure and the consequent contraction of the muscle.4 It would seem then that any factor converting the large colloidal particles into smaller ones may cause a fall in osmotic pressure inside the muscle. while any factor producing the opposite change in the particles may cause a rise in osmotic pressure. It is probable that Journ. of Physiol., xxiii. p. 141, Science Progress, No. 7, p. 482, Proc. Roy. Soc., lxxvi. B, p. 322, Proc. Physiol. Soc., May 16, 1908.

The Effects of Temperature on the Osmotic Properties of Muscle 221 such an alteration in the internal solution may be brought about by physical means, and it is therefore necessary for a thorough

3 The Effects of Temperature on the Osmotic Properties of Muscle 221 such an alteration in the internal solution may be brought about by physical means, and it is therefore necessary for a thorough consideration of this matter to have an accurate knowledge of the effects of physical changes on the osmotic properties of muscle. Turning first to the action of temperature, one finds that Miss Cooke's work was, as she admits, incomplete, and that it has not since been extended. I have, therefore, at Professor Macdonald's suggestion, reinvestigated that part of the subject. EXPERIMENTAL METHODS. The experiments were carried out in the autumn. Both the sartorius and gastrocnemius of the frog were used. In order to avoid injury to the muscle the device employed by Overton, and subsequently by Fletcher, was adopted. A piece of fine silk thread was tied to the tendon of insertion of the sartorius, and half a centimetre of it was left attached to the muscle for purposes of manipulation. For the same reason a small portion of tendon was left attached to the gastrocnemius. A 0 7 per cent. solution of sodium chloride was taken as isotonic for the muscle. This was nearly always so for the sartorius, but was sometimes hypotonic for the gastroenemius, especially with big frogs. The muscle was put into 250 c.c. of this solution. The experiment was performed in the following way:-each one of a pair of muscles was put into a 0 7 per cent solution of sodium chloride and left at an initial temperature for from 1 to 1t hours. Each was then rapidly dried, weighed, and put into fresh 0 7 per cent. sodium chloride solution, the one at initial temperature, the other at some different temperature. They were taken out at intervals, dried, weighed, and put back. To avoid error the muscles were manipulated in the same order. The surface was rapidly dried between filter papers, the same number of manipulations being used in all cases. The weighings were carried out rapidly on a "Curie" balance, which comes quickly to rest, and in which all differences of weight less than a decigram are read off, by means of a magnifying glass, from a scale graduated to half-milligrams, attached to the beam. This balance has been tested and found accurate for differences of a milligram. In some experiments the initial temperature was the lower one, in others the higher. The ranges of temperature actually employed were from room temperature (140 C. to 180 C.) to 25 C., in one case to 300 C.: from 250 C. to room temperature, from room temperature to 00 C., and from 00 C. to room temperature. RESULTS WITH ISOTONIC SOLUTIONS. With the sartorius very constant results were obtained. A solution isotonic for that muscle at room temperature was isotonic for it at other temperatures. In other words, the increase in osmotic pressure in the

4 222 de Souza interior of the muscle produced by rise in temperature was the same as the increase in osmotic pressure produced in the surrounding solution. This is shown in fig. 1. In this case the muscles were kept at an initial temperature of 170 C. for 1 hour before the first weighings. The upper curve shows the variations in weight of the muscle at 170 C., and the lower one those of the muscle at 250 C. The time in hours is measured along the abscissa, and the weight in milligrams along the ordinate. The curves show that both muscles were in isotonic solution. Such parallel curves were obtained with the sartorius for all ranges of temperature, and the gastrocnemius gave similar results in the majority of cases. In two experiments, however, results of the type seen in fig. 2 were obtained. The muscles had been kept in 07 per cent. sodium chloride solution at 140 C. for 1 hour, after which they were transferred to solutions of the same " hours FiG. 1.-Clhanges in weight of a pair of sartorii in 017 per cent. NaCl solution, the one at 17 C., the other at 25 C. Both had been kept previously at an initial temperature of 17 C. for 1 hour. strength at 14 C. and 30 C. respectively. The lower curve shows the variations in weight of the warmer muscle. There was a continuous gain in weight for both muscles, but the gain was more rapid for the muscle at the higher temperature. In addition there was a marked difference between the initial weights of the muscles-30 in this experiment. This led to the suspicion that a knowledge of the previous history of the muscle might furnish an interpretation of the result. It was suggested, for example, that the smaller muscle had previously lost fluid, and consequently took up niore fluid than the larger to equalize the weights again. The second experiment of this type, however, negatived this supposition, for in that experiment the gain in weight was in favour of the larger muscle, which increased in weight from to 484 migms. in 5 hours, while the smaller increased from 400 mgins. to 418 migmns. in the same time. There is also the possibility that previously the one muscle mnay have been more active than the other. The presence of the products of activity would *cause a rise in osmotic pressure in the previously active muscle, and a gain in weight as compared with the other muscle, when put into the salt

5 The Effects of Temperature on the Osmotic Properties of Muscle 223 solution. If this were the complete explanation, the fact that the previously active muscle was, in both experiments, the one at the higher temperature would have to be regarded as a coincidence. Again, it was thought that the difference in the behaviour of the two muscles might be due to injury inflicted during preparation. There is no doubt that injury to a muscle causes a rise in osmotic pressure inside the muscle. Fig. 3 illustrates this. The curves show the variations in weight of a pair of gastrocnemii in 07 per cent. sodium chloride solution at 150 C. The muscle from whose weights the curve N was plotted was prepared carefully so as to avoid injury. The other muscle had a portion of its sheath cut away during preparation A! 1-0_00 ~~~~ hours hours FIG. 2.-Changes in weight of a pair of gastro- FIG. 3.-Changes in weight of a pair of gastrocnemii in 0 7 per cent. NaCl solution, the cnemii in 0 7 per cent. NaCl solution at one at 140 C., the other at 300C. Both 15 C. The one (I) had a portion of its had been kept previously at an initial sheath cut away, the other (N) was untemperature of 140 C. for 1 hour. injured. The solution remained isotonic for the first muscle, but the injured muscle gained in weight, so that the injury must have produced a rise in osmotic pressure inside the muscle. After half an hour, however, the gain in weight ceased, the weight remaining fairly constant for the rest of the experiment, showing that the muscle was then in isotonic solution. A similar result is obtained if, instead of removing the sheath, the fibres of the muscle be cut across some distance from the place of insertion. It is easy to injure the muscle in this way during preparation, and so to introduce a source of error into the experiment. A comparison of figs. 2 and 3 shows at once that the result depicted in fig. 2 was not due to this cause. The gain in weight was continuous, and the solution did not become isotonic for the muscle during the four hours of the experiment. But in the two experiments of which fig. 2 is a type, there is another factor to be considered. In both cases there was a gain in weight in the

6 224 de Souza two muscles. The surrounding solution was therefore hypotonic for the muscles. The question arises then whether a solution known to be hypotonic for a muscle will cause a greater increase in the weight of the muscle at a higher temperature than at a lower one. RESULTS WITH HYPOTONIC SOLUTIONS. The effect of temperature on the gastrocnemius muscle in a hypotonic solution was accordingly tested, with the result shown in fig. 4. In the experiment both gastrocnemii were put into 07 per cent. sodium chloride solution at 160 C. and left for 1j hours. They were then taken out and weighed, and put into 05 per cent. sodium chloride, the one at 160 C., the other at 250 C. The lower curve shows the subsequent variations in weight of the first muscle, the upper curve those of the second muscle. In order to keep the curves sufficiently close to each other for comparison along their whole extent, the ordinates in this and the following figure were drawn to a scale one-fifth the size of that used in the other figures. In the experiments with hypotonic solutions of which this is a. sample, the muscle at the higher temperature always experienced for several hours a greater increase in weight than the muscle at the lower temperature. This affords a sufficient explanation of results of the type depicted in fig. 2, in which the 07 per cent. sodium chloride solution was slightly hypotonic for the muscles. The subsequent loss in weight, shown in fig. 4, will be referred to later. Such results led one to consider why, in the case of hypotonic solutions, difference in temperature should cause difference in rate of fluid intake. To begin with, the osmotic pressure inside the muscle is greater than that of the surrounding fluid, and the difference must be exaggerated by rise in temperature. This exaggeration, however, is not enough numerically to account for the greater increase in weight in the one muscle as compared with the other in these experiments. It seemed probable that diffusion might be an important factor. The partial permeability of muscle has been pointed out by Fletcher. Owing to this property of the muscle, diffusion may take place between the fluids within and without the musclefibres. But the influence of diffusion may be exerted in another way. The differences in behaviour between the sartorius and gastrocnemius muscles in saline solutions suggest that the thickness of the latter muscle must be taken into consideration. A fibre in the middle of the muscle will take up fluid some time after the fibres at the periphery, and this time must depend upon the rate of diffusion in the fluid surrounding the muscle fibres. The rate of diffusion would be increased by rise in temperature, so that fluid would more readily penetrate the muscle. Hence the muscle at the higher temperature should gain weight more rapidly than the one at the lower temperature, and should reach its maximum first; and this is what actually happens.

7 The Effects of Temperature on the Osmotic Properties of Muscle 225 Now Fletcher has shown that a resting gastrocnemius in hypotonic solution first gains and then loses weight owing to its partial permeability Z co1 00 nz CO kc0 X h Since rate of diffusion is increased by rise in temperature, this loss of weight should be greater for the muscle at the higher temperature. That such is indeed the case will be seen from fig. 4. At first both muscles gained weight, the one at 25 C. more rapidly than the one at 16 C., and

226 de Souza they were still gaining at the end of 7 hours. The next day (23 to 29 hours) they were both losing weight, the muscle at 250 C. more rapidly than that at 160 C.

8 226 de Souza they were still gaining at the end of 7 hours. The next day (23 to 29 hours) they were both losing weight, the muscle at 250 C. more rapidly than that at 160 C., so that soon after 27 hours the curves crossed. RESULTS WITH HYPERTONIC SOLUTIONS. It was of interest to compare with this the result when a hypertonic solution was used (fig. 5). The experiment was carried out in the same way, but a 09 per cent. sodium chloride solution was used instead of the 05 per cent. solution. The two muscles first lost weight, the- curves keeping fairly parallel, then gained weight. The muscle at the higher temperature reached its minimum first, in this experiment at the end of 3 hours, as compared with 4 hours for the other muscle. In more concentrated solutions this difference is more marked. Thus in 1 per cent. solution the muscle at the higher A En...-. I. - - t _ a'7~~~/'t ad -f-h hours FIG. 5.-Changes in weight of a pair of gastrocnemii in 0 9 per cent. NaCl solution, the one at 17' C., the other at 25' C. Both had been kept previously in 0 7 per cent. NaCl solution at 17' C. for 1 hour. temperature reached its minimum in 2 hours, while the other muscle was still losing weight after 4 hours. The subsequent gain in weight was more rapid in the case of the warmer muscle, as is evidenced by the crossing of the curves in fig. 5. It will be noticed that both muscles took in fluid to such an extent that their weights exceeded the original weights. This remarkable fact came out also in Overton's experiments, and implies the liberation of molecules inside the muscle. Such molecules may be liberated from adsorption complexes through the agency of the strong salt solution, or may be the result of chemical activity due to the constant stimulus produced by the withdrawal of water by the salt solution. Sartorius in hypotonic and hypertonic solutions.-ithas already been mentioned that there are differences in the behaviour of the sartorius and gastrocnemius in saline solutions, and it has been suggested above that these differences may depend upon the thickness of -the muscle. In the sartorius fluid should be able to penetrate to the centre of the muscle more rapidly, so that in a hypotonic solution the maximum weight should be reached earlier than that of the gastrocnemius. Fletcher showed that this was the case, and my results confirm his. The effect of temperature on the process is shown in fig. 6.

9 The Effects of Temperature on the Osmotic Properties of Muscle 227 Both muscles were in a 0-5 per cent. sodium chloride solution, the one at 15' C., the other at 250 C. Both had been kept previously in 07 per cent. sodium chloride solution for one hour. The lower curve shows the variations in weight of the warmer muscle. The maximum of the curve for the muscle at 250 C. is lower than that of the curve for the muscle at 150 C. This is always so, and is no doubt due to the more rapid diffusion out of the muscle caused by rise in temperature. As was to be expected, when an intake of fluid by osmosis is going on at the same time as a diffusion of salts out of the muscle the relative rate of increase in weight of the two muscles is very variable. The gain is sometimes more rapid for the cooler muscle, as shown in fig. 6; at others for the -warmer muscle at first, then for the T hours FIG. 6.-Changes in weight of a pair of sartorii.in 0 5 per cent. NaCl solution, the one at 15'C., the other at 250 C. Both had been kept previously in 017 per cent. NaCl solution at 15 C. for 1 hour. cooler muscle; and so the ascending portions of the curves cross. The curves usually reach a maximum about the same time, but the cooler muscle always has the higher maximum. With hypertonic solutions no constant results could be obtained for the sartorius. This was probably due to chemical changes produced inside the muscle, for the strong solution stimulated the muscle, causing irregular contractions. SUMMARY. 1. A solution of sodium chloride isotonic for a muscle at one temperature is isotonic for that muscle at other temperatures, provided coagulation does not occur. 2. Apparent exceptions to this rule are due to inijury to the muscle or to hypotonicity of the solution. 3. Both the gain and loss in weight of a muscle in hypotonic or hypertonic solution are increased in rapidity by rise in temperature.

slowing of the muscle. Bronk [1933] has given a striking

slowing of the muscle. Bronk [1933] has given a striking 106 6I2.74I.I2 THE EFFECT OF ACTIVITY ON THE FORM OF THE MUSCLE TWITCH. BY J. L. PARKINSON. (From the Department of Physiology and Biochemistry, University College, London.) IT has been found by various