The Dynamics of Living Matter

An observation mentioned already in a former lecture shows con- clusively that the mixture of 100 NaCl, 2 KC1, and 2 CaCl2 cannot be considered as a nutritive solution for animals, but must play a different role. Fundulus lives just as well in distilled water as in sea water. This fact proves that these animals do not depend for their nutrition t The fact that Locke also mentions sugar as one of the necessary constituents of his solution indicates that he considers the other constituents also as nutritive material. This would, however, be wrong. From my own experiments I do not think that the addition of sugar is of any value.

and development upon the salts dissolved in the sea water. If the young fish, however, are put into a pure solution of NaCl of the concen- dies in less than twelve hours. If CaCl2 is added, the animal does not live more than twenty-four hours. If it is desired to keep animals alive permanently, - - my experiments lasted for ten days, - - not only 2 CaCl2 but also 2 KC1 must be added to 100 NaCl. This is exactly the solution which is generally considered as a nutritive solution for animals.* I believe that these facts show that we must discriminate between nutritive and protective solutions. Ringer's solution, as well as the sea water, are primarily protective and not nutritive solutions. What is meant by this becomes clear if we remember what was said concern-

ing the antagonistic effects of salt. A - - solution of NaCl, as well as a '- solution of ZnSO4, alone are each poisonous for the eggs of Fundulus. If mixed, the solution becomes considerably less poisonous. It is prob- able that these two salts if together in solution materially diminish their rate of diffusion into the tissues. It follows from these experi- ments that the role of the Ca and Mg in the sea water, as well as in a Ringer's solution, consists partly in antagonizing the effects which would be produced by the NaCl were it alone in solution. The experiments with Fundulus suggest that in this case the presence of the Ca and Mg in the sea water diminishes the rapidity of diffusion of the NaCl into the tissues. It is possible that the Zn acts in some protective way in the case of Raulin's solution, although in regard to this it is not possible to make a definite statement.

But there remains the other fact that K is also needed. The life of Gammarus and many other marine animals is not essentially pro- longed by the addition of CaCl2 or MgCL, or both to the NaCl solution, but is materially prolonged by the addition of CaCl2 and KC1 to the NaCl solutions. Besides we cannot, in general, substitute any other bivalent metal (with the exception of Sr) for Ca; nor can we sub- stitute any other univalent cations for Na and K. This indicates that the metals Na, Ca, K, and Mg play a role in life phenomena other than that of serving for the synthesis of living matter, and also other than that of merely regulating the velocity of diffusion. We have already alluded to the possibility of their necessity for phenomena of secretion. We are inclined to believe that what is generally called irritability and contractility is due to the influence of these ions.

In 1899 I outlined a general theory of irritability which may be briefly summarized in the following sentences which I quote from a former paper: "The salts, or electrolytes in general, do not exist in living tissues as such exclusively, but are partly in combination with proteids (or fatty acids). The salts or electrolytes do not enter into this combination as a whole, but through their ions. The great importance of these ion-proteid compounds (or soaps) lies in the fact that, by the substitution of one ion for another, the physical properties of the proteid compounds change (e.g. their surface tension, their power to absorb water, or their viscosity or state of matter). We thus possess in these ion-proteid or soap compounds essential constituents of living matter, which can be modified at desire, and hence enable us to vary and control the life phenomena themselves." *

Life phenomena, and especially irritability, depend "on the pres- ence in the tissues of a number of various metal proteids, or soaps (Na, Ca, K, and Mg), in definite proportions." I first applied this conception to a phenomenon which had hitherto been observed only occasionally ; namely, rhythmical contraction of the muscles of the skeleton.f I found that such rhythmical contractions occur only in solutions of electrolytes, i.e. in compounds which are capable of ionization. In solutions of nonconductors (urea, various sugars, and glycerine), these rhythmical contractions are entirely or practically impossible. Only in certain, not in all, salt solutions are such rhythmical contractions possible. All the solutions of Na-salts are able to produce them, but in a 0.7 per cent NaCl solution, contrac- tions begin later, and are less powerful, than in an equimolccular NaBr solution. The experiments on the rhythmical contractions of the muscles of the skeleton led to some other data concerning the effects of those salts. Solutions of Na-salts produce rhythmical contractions only if the muscle cells contain Ca-ions in sufficient numbers. As soon as there is a lack of Ca-ions in the tissues, the Na-ions are no longer able to cause rhythmical contractions. On the other hand, if we add Ca-salts in sufficient quantity to the NaCl solution, it will no longer cause rhythmical contraction in a fresh muscle of the frog. It there-

The idea of the general existence of such ion-proteid compounds was developed inde- pendently by Pauli and myself in 1899. Loeb, Pfliigeijs Archiv, Vol. 75, p. 303, 1899 ; and Festschrift fur Pick, 1899 ; and W. Pauli, Wiener akaJemischer Anzeiger, October 12, 1899 ; and Ueber physikalisch-chemische Methoden und Probletne in der Medizin, Wien, 1900. fore looks as if the presence of a certain quantity of Na-ions caused contractions ; but if the quantity of the Na-ions becomes too great in proportion to the Ca-ions, the muscle loses its irritability. On the other hand, if there are too many Ca-ions present, the rhythmical con- tractions become also impossible. The quotient of the concentration

of the Na-ions over the concentration of the Ca-ions, — — a, becomes therefore of importance for phenomena of irritability. We shall see later that Mg acts very much like Ca in this respect. It is hardly necessary to mention that this suggested the possibility that muscular contraction, in general, is due to a substitution of Na for Ca, or vice -versa, in certain compounds (proteins or soaps) in the muscle. Every substance or agency will act as a stimulant which brings about such a change of the metals in these compounds in the muscle.

It may be added that all the salts of univalent metals act like the Na-salts, inasmuch as they cause rhythmical contractions when the muscle is put into them. All these salts, however, have secondary effects which usually prevent the contraction from lasting as long as in NaCl. In KC1 the muscle gives only a few twitches when thrown into the solution, and then stops. In LiCl the twitches may last over a day. As of the salts with a univalent metal, only the Na- and K-salts occur in the muscle, only the substitution of one of these salts need be considered for the theory of irritability.

Not only Ca- but also Sr- and Mg-salts are capable of antagonizing the stimulating effects of a pure NaCl solution when added to the same. As I stated six years ago, we owe it to the Ca- and Mg-salts in our blood that our skeletal muscles do not contract rhythmically like our heart. We may now give a provisional answer to the question why it is that the Na-salts, which are unnecessary in the nutritive solution of a plant, become of so great importance for the life of an animal. If our hypothesis be correct, the answer should be that all the muscular contractions are due to a substitution of Na-(or K) for Ca- or Mg-ions, or vice versa. In the plant, which has no muscles, there is no need for any NaCl. Likewise we can understand why CaCL, plays a lesser role in plants than in animals, as Raulin's investigations indicate.

In an - - solution of NaCl it requires, as a rule, a long time — an hour or more — before the contractions begin at ordinary room tem- perature; while in a more concentrated solution the contractions begin more rapidly. I concluded from this that a NaCl solution produces this effect only through the diffusion of NaCl into the muscle. As soon as the concentration of the NaCl or the Na-ions in the muscle has reached a certain value, the muscle fiber will begin to contract.

Normally the quotient — — in the muscle is too small to permit such contractions. The addition of a slight amount of NaHO accelerates the process, possibly by accelerating the diffusion of the salt into the muscle.* The fact that CaCL, inhibits the rhythmical contractions which are produced by the NaCl, suggested experiments on the effects of salts which precipitate Ca or diminish the concentration of free Ca-ions through the formation of salts with a low degree of dissociation. It was found that such salts, e.g. sodium-oxalate, -fluoride, -citrate, and -tartrate, act much more powerfully than NaCl or sodium-acetate or -succinate. If such salts be applied to the nerve they produce before the twitchings begin a condition of increased irritability, comparable to the catelectrotonic condition caused by the constant current at the region of the cathode. f

The addition of a slight amount of acid to a - NaCl solution shortens the latent period for the beginning of the rhythmical contractions. We shall see later on that the acid acts possibly like the above-mentioned salts; namely, by liberating the calcium from certain organic combi- nations in the muscle or nerve. Inasmuch as CO2 is produced in the muscle itself, this relation is of importance. These facts suggested the idea that the process of contraction is caused by an exchange of Na or K for Ca or Mg, or vice versa, in cer- tain compounds in the muscle (or nerve). This change must be accom- panied by a change in some physical property of the compound, e.g. the surface tension, the state of aggregation, viscosity, absorbing power for water, etc. The change in such a physical property may deter- mine or facilitate the process of contraction..

I applied the facts found in the muscle to the study of rhythmical contractions in a more favorable object; namely, the swimming bell of the Medusa. The jellyfish is comparable to a free-swimming heart which beats rhythmically, with this difference, however, that the jellyfish does not beat incessantly, like the heart, but intermittently, with long pauses between series of contractions. It is known that the central nervous system of the Hydromedusa is situated in a ring near the edge of the Medusa. Romanes had already observed that if the

edge of a Hydromedusa be cut off from the center, the former continues to beat while the latter stops beating. This fact was utilized as an argument to prove that the contractions in a Medusa originate normally from the nerves in the margin. This may be so, but it seemed to me that the center of a Medusa (deprived of its nerve ring) might also be able to beat if it were not prevented from so doing by the constitution of the sea water. I found, indeed, that the isolated center of Gonio- nemus, a Hydromedusa, common at Woods Hole, is able to beat rhyth- mically in a pure solution of NaCL* The center beats in such a solu- tion very rapidly, and the more rapidly the higher, within certain limits, the concentration of the NaCl solution. The addition of a small quan- tity of CaCl2 or MgCl2 retards or inhibits the contractions caused in the NaCl solution. If a salt which precipitates Ca or diminishes the concentration of its ions is added in excess to sea water (e.g. sodium oxalate, fluoride, citrate), the center can be caused to beat in sea water also.

It is thus obvious that the case of the center of the Medusa seems very analogous to that of the muscle. Just as the latter is prevented from twitching in the blood on account of the presence of CaCl2 and MgCl2, so the isolated center of Gonionemus is prevented from beat- ing in sea water on account of the presence of CaCl2 and MgCl2. . After these data had been obtained I asked Dr. Lingle to deter- mine whether similar laws hold for the heartbeat. It was known that if the sinus venosus of a frog's heart be severed from the heart, the former goes on beating as before ; while the rest of the heart, especially the isolated ventricle, stops beating in blood. This observation is comparable to the one made by Romanes on jellyfish; and we may carry the analogy a step farther, by comparing the center of the Medusa to the ventricle ; the edge to the sinus venosus of a frog's heart. Lingle worked on the heart of a tortoise.f He found that the ventricle is only able to beat after it has been put for about half an hour into a pure solution of NaCl. When the ventricle remains permanently in the sodium chloride solution, the heartbeats will stop after a certain time, as Lingle believes, on account of the diffusion of too much NaCl into the heart muscle. If the strip of the ventricle is put into a moist chamber after the beats are once started in a NaCl solution, they may continue for a number of days, until the process of putrefaction puts an end to the contraction. No other substance can take the place of Na. Li, which acted so well in the case of the isolated frog's muscle, can be only partially substituted for NiCl. One of the most remarkable

facts which Dr. Lingle found is that even the heart stimulants, such as caffeine, cannot cause the strip to beat, except in the presence of NaCl. These experiments give the impression that the ventricle of the tor- toise does not beat in the blood, because of the fact that the NaCl in the blood is prevented from entering into the heart cells or from acting upon them through the presence of another salt; namely, CaCl2, or MgCl2, or both. These experiments show that in order to start the heartbeat, a pure NaCl solution, or a pure solution of an Na-salt, is required; but if the heart remains permanently in a pure NaCl solution, it stops beating. The pure solution of NaCl acts like a poison. If, however, a small amount of CaCl2 be added to the NaCl solution after the heartbeats have once started, the beats can go on for a long time. They can also continue in serum after they are once started in a pure NaCl solution. The addition of Ca therefore acts antagonistically to the injurious action of the pure NaCl solution. Ca cannot start rhythmical con- tractions in the ventricle, but it is necessary to sustain the rhythmical action once started by NaCl. It is possible again that the pure NaCl solution becomes toxic through the fact that in such a solution too many Na-ions take the place of Ca, in the ion-colloids, and that this is prevented by the presence of a trace of Ca. It is possible that the Ca prevents or retards the diffusion of Na into the muscle. In a pure NaCl solution, however, the rhythmical action of the heart strip can also be sustained for a long time without the addition of Ca, if pure O is allowed to bubble through the NaCl solution, or if a trace of H2O2 is added to the solution. I consider these observations of the greatest importance, inasmuch as they show that the processes of oxidation going on in the muscle give the latter equal protection against the NaCl poisoning, just as the addition of CaCl2 to the NaCl solution. Could it be possible that the increase in oxidations leads to the setting free of Ca inside the muscle, and that the Ca acts in this case?

When I entered upon the investigation of the effect of salts on the rhythmical contractions of the muscle, I had in mind the solution of the problem of electrical stimulation. Previous experiments on the effects of the galvanic current had led me to the idea that the polar effects of the current are due to the ions which are blocked in their prog- ress by the semipermeable membranes in these organs; and my idea was that experiments with salts would show which ions are responsible for the effects of a galvanic current. If the hypothesis that ions are responsible for the stimulating effects of a current were correct, it was to be expected that in solutions of nonconductors no twitchings would occur. One of the first facts that I ascertained was that this idea was

generally correct. In solutions of dextrose, cane sugar, milk sugar, and glycerine, no twitchings of the common muscles occurred, no matter how concentrated the solution. In solutions of urea the same was true, in general, but occasionally transitory contractions were observed. In alcohol I also observed occasionally a slight twitching, but it is possible that in these latter cases the twitchings were caused by an indirect effect of urea and alcohol upon reactions or changes inside the muscle fibers. On the whole, I am under the impression that the muscle cannot be, or only exceptionally, and to a slight extent, caused to contract by solutions of nonconductors. I found that the same is true for the rhythmical contractions of the center of the jelly- fish (Gonionemus). Dr. Lingle tried the same experiments on the strips from the ventricle of the tortoise, with the same result. In pure solutions of dextrose, cane sugar, and glycerine, no beats originated, even if the strip remained in these solutions for a day; but when the ventricle was afterward put into a pure NaCl solution the contrac- tions began, showing that these solutions had only prevented the contractions without permanently injuring the heart. Lingle also de- termined what the minimum concentration of NaCl was that was able

to start heartbeats. He found that in a mixture of 98 c.c. - cane sugar All these experiments seemed to support or, at least, not to contra- dict the idea set forth in my first paper on this subject, that the rhythmical contractions depend upon the exchange of Na (or K) and Ca (or Mg) in certain compounds, possibly proteids or soaps, in the muscle. Such an exchange might alter the physical properties, e.g. the surface tension, or viscosity, etc., of the substance; and a sudden change in one of the properties might result in a change of form such as underlies contrac- tion. I figured to myself that the change starting rhythmical contrac- tions was a sudden change in surface tension, e.g. a phenomenon of spreading, and gave expression to this possibility in my book on Brain Physiology. A series of new observations confirms me in the idea that we are dealing here with phenomena which must occur at the surface of the elements.

In 1901 I described a form of irritability in the muscle which, to my knowledge, had never been noticed before, and which is produced by such salts as precipitate Ca or diminish the concentration of free Ca-ions, such as citrates, oxalates, fluorides, carbonates, phosphates, etc., and especially the sodium salts of these acids.* The experiment is as follows: If the gastrocnemius of a frog is put into a — solution of sodium citrate and left there for two or three minutes, it will go into powerful tetanic contractions or rather cramplike clonic contractions when taken out of the solution; while these contractions stop at once when the muscle is put back immediately. This can be repeated at desire, the muscle always going into contractions when exposed to the air, and relaxing again when put back into the solution. Zoethout found that this reaction can be produced quicker and with greater certainty when a slight amount of a K-salt is added to the solution. I believe that the K antagonizes the tendency to rhythmical contractions which the muscle possesses in a sodium-citrate solution. It seems to be necessary that

this tendency to rhythmical contractions be overcome in order to obtain the phenomenon which we are now discussing, and which I called in a pre- liminary way the contact re- action of muscle, inasmuch as it can be produced by changing the nature of the medium which surrounds the muscle. The apparatus used for the demon- stration of this experiment is shown in Fig. 15. When only part of the muscle is lifted out of the citrate solution, only those fibers go into tonic contraction which are in contact with the air; while those fibers which remain in the solution do not contract. The reaction, therefore, is a purely local one in each in- dividual muscle cell. This re- action not only occurs when the muscle is brought from the solution into contact with air, but also when it is brought into contact with CO2, oil, toluol, sugar, or glycerine solutions. All these solutions are nonconductors, and

I at first believed that I was dealing here only with a break shock caused by the muscle's own current; but this was contradicted by various facts: first, that only those individual fibers contracted which were lifted out of the solution, while the others remained relaxed; second, that the latent period for the contraction after the muscle has left the solution is too long; namely, as much as a second or more. The most convincing proofs against such an assumption are, however, the following facts. The irritability of the muscle for the contact reaction does not reach its maximum at once, but only after a certain time. When the sensitiveness of the muscle has reached its height, a glycerine or a sugar solution can be substituted for the citrate solution. Whenever the muscle is at that time taken out from the glycerine or sugar solution and brought into contact with the air, the contraction occurs; while it ceases when the muscle is put back into the sugar solution. After a short time, however, the muscle loses its contact irritability in the sugar or glycerine solution. These experiments, however, certainly prove that the contact reaction is not a break shock caused by the resting current of the muscle itself when it is lifted out of the citrate solution.

It is a very interesting and theoretically important fact that the muscle loses this peculiar form of irritability very soon when it remains in contact with air, oil, sugar solution, glycerine, or salt solutions, different from those that produce this specific irritability. In LiCl or NaCl solutions the contact irritability is lost as fast, if not faster, than in a sugar or glycerine solution. We can reestablish the irritability, however, by putting the muscle back into the sodium citrate solution for some time. This fact, together with those mentioned before, suggests the following as the most probable explanation of the peculiar phenomena of contraction with which we are dealing in this case. The solutions which produce the contact irritability possess anions which are liable to form insoluble calcium compounds. Whatever the effects of these anions may be, the fact that in less than a minute the contact effects are noticeable, indicates that only the surface layer of the muscle, or the surface layer of each individual fiber, is altered. It is impossible for the anions to migrate deeper into the muscle in so short a time. In the surface layer of the muscle or the individual fibers, we have there- fore temporarily a diminution of Ca-ions. We have then a muscle whose surface layer differs from that of an ordinary excised muscle. If this layer is once established, the muscle contracts at any change from the citrate, carbonate, fluoride, etc., solutions to air, CO2, oil, 2 n sugar solution, glycerine, chloroform, or toluol. If the muscle be left in these media, or put into a NaCl or a CaCl2 solution, it loses this contact

irritability. This loss of contact irritability of the muscle in air, oil, etc., may be due to the migration of Ca-ions from the interior of the fiber or muscle to the surface, thus reestablishing approximately the original normal surface condition. If we then put the muscle back for a short time into a sodium citrate or sodium fluoride, etc., solution, a diminu- tion of Ca-ions will again occur in the surface layers, and the contact irritability will be reestablished. As we should expect, the length of time that the muscle remains in the solution is as important as the concentration of the solution. If we dip a muscle for a few seconds only into a sodium citrate solution (i-g. molecule in 10 1.) the contact irritability cannot be produced, as there is not time for a large enough number of citrate-ions to enter the muscle.

Dr. Zoethout carried these investigations still farther. He found that if a muscle be put into a pure -- solution of any potassium salt, e.g. KC1, KNO3, KI, K2SO4, K-oxalate, etc., the tone of a muscle increases, i.e. the muscle shortens while it is in the solution. If, however, the muscle is put into a pure — CaCl2 or NaCl solution, it again relaxes.* o The minimum concentration of KC1 for bringing about this increase in tone of the gastrocnemius of a frog was i c.c. — KC1 + 9 c.c. H2O or

— glycerine. If, however, a potassium salt was chosen whose anion is liable to decalcify the muscle, the minimum concentration could be Zoethout interprets this as showing that the Ca-ions of the muscle itself are liable to antagonize the shortening action of the K-ions. This interpretation he supported by a number of experiments. "It occurred to me that since potassium increases the tone of the muscle and calcium inhibits this action of the potassium, it might be possible that the pre- cipitation of the calcium salts causes contact irritability, becauses it destroys the normal equilibrium between these two salts in the muscle." " Upon testing this view we found it to be correct. The contact reaction produced by sodium citrate is increased if we previously, or simul- taneously, introduce K-ions into the muscle." f Zoethout's conclusions are as follows: "If the calcium salts in the muscle are decreased, the efficiency of the K-ions to increase the tone of the muscle is increased. If the K-ions in the muscle are increased, the efficiency of such salts as Na-oxalate and Na-citrate to cause contact irritability is increased.

The contact irritability depends (as Loeb suggested) on the disturbance of the normal ratio of salts in the muscle. Perhaps it is the disturbed ratio between the potassium and calcium salts which makes the contact reaction possible." Of course the experiment of Zoethout does not explain why the contact reaction only occurs when the muscle is taken out of the citrate solution. I am inclined to believe that not only the K but also the Na-salts are concerned in this reaction ; but it is certain that Zoethout's observa- tions establish the fact that the antagonism between K and Ca-salts is to be considered in the theory of animal irritability and stimulation. The statement contained in my older publications, namely, that possibly the substitution of Na for Ca, or vice versa, caused the twitch- ing of the muscle must be modified so as to include also the substitution of K for Ca, or vice versa. Perhaps it may be said that the substitution of any univalent cation for Ca in the muscle, or vice versa, causes a twitching. As of the univalent metals, however, only Na and K occur in the tissues, they are the principal ones to be considered. It seems from these observations and others, all of which cannot be considered in this short sketch, as if indeed the substitution of Na or K for Ca-salts, or vice versa, is the essential feature of the twitching or of muscular stimulation. It would be of the utmost importance to determine which of the two possible changes was the real cause. We know that in general a substitution of Ca for K or Na in colloids favors the formation of more solid or insoluble compounds, e.g. in the case of soaps. In the case of the coagulation of blood or milk, it is also obvious that Ca in moderate quantities favors coagulation.

Ringer had already observed that barium salts have a stimulating effect upon muscle, and I have been able to confirm this observation. A pure solution of any soluble barium salts gives rise to powerful rhyth- mical contractions of the muscle ; and the threshold for this stimulation is much lower for Ba than for the corresponding Na-salts. In a NaCl solution the contractions last longer than in a BaCl2 solution, on account of the greater toxicity of the BaCl2. I expected from this that a pure solution of CaCl2 or SrCL, might act similarly to BaCl2, but this does not seem to be the case for the muscle. Two years ago, however, I found some new facts concerning the influence of salts upon rhythmical contractions of the center of a Californian jellyfish, Polyorchis, which meet this expectation.* If the margin containing the central nervous system of this Medusa be cut off from the center of the swimming bell, the center no longer contracts spontaneously in sea water; nor if it be put into a pure NaCl solution of about the concentration of the

sea water do contractions begin. It differs in this regard very markedly from Gonionemus, whose center begins to beat at once in a pure NaCl solution. After a certain time, however, generally a number of hours, rhythmical contractions will begin in the isolated center of Polyorchis in a pure NaCl solution, and may last with long interruptions for two or three days. When they have ceased in a pure NaCl solution, a few single contractions can at any time be produced by touching the sub-um- brella with a drop of a solution of a potassium salt; but the addition of a potassium salt to the NaCl solution, although it promptly calls forth a single contraction or a short series of contractions, does not maintain the rhythm.