The Dynamics of Living Matter

A sure means of producing rhythmical contractions of the isolated center of Polyorchis at once in a pure NaCl solution is the addition of a certain amount of a salt which precipitates Ca, or diminishes the concentration of the Ca-ions, e.g. Na-citrate, -tartrate, -oxalate, etc. I generally used the citrate as it seems to be the least harmful. If 10 c.c. of a m sodium citrate solution be added to a 100 c.c. f n NaCl solution, rhythmical contractions of the isolated center begin usually at once, and may last an hour or more. A second means of calling forth rhythmical contractions in a pure solution of NaCl at once is the addi- tion of a trace of an acid, e.g. HC1. CO2 acts in the same way, and I have wondered whether this acid which is formed regularly in the body does not thus play an important role in rhythmical contractions in general. The addition of acids may even cause the center to beat in

sea water. About i.s to 2 c.c--HCl to 100 c.c. of sea water is required for this purpose. Alkalis have the opposite effect. The action of the acid may be the same as that of the oxalate and citrate; namely, to set free Ca, which is in organic combinations in the cells, or at the surface of the cells, and make thus a substitution of Na or K for Ca, or vice versa, in these organic compounds, possible. It may be mentioned here that the oxalates, citrates, and similar salts, and the acids, are believed to play such a role in the process of the coagulation of milk.* One of the promptest means of producing rhythmical contractions in the isolated center of Polyorchis is putting it into a pure solution of CaCl2, BaCl2, or SrCl2. Instead of dissolving the CaCl2 in distilled

water, it may be dissolved in a f m or — sugar solution in order to have a solution which is more nearly isosmotic with sea water. If an isolated center is put into a solution of 10 c.c. f m CaCl2 + 50 c.c. f m cane sugar * A. S. Loevenhart, Hoppe- Stylets Zeitschr. fiir physiologische Chemie, Vol. 41, p. 177, or into 10 c.c. f m CaCl2 + 50 c.c. distilled- water, the center usually begins to beat rhythmically. The rhythmical contractions may last for three hours or more. Such rhythmical contractions can even be caused in a center which has been washed and kept for three hours in a pure solution of cane sugar, to make sure that the sea water at the surface of the muscle cells has been entirely removed. In this case, however, the contractions do not last as long, inasmuch as a solution of cane sugar as well as a pure CaCl2 solution are injurious to the muscle.

BaCl2 is much more effective than CaCl2, as it requires a much lower concentration of BaCl2 than of CaCl2 to produce rhythmical contractions in an isolated center of Polyorchis. Even in a solution of \ c.c. f m BaCl2 + 50 c.c. | m cane sugar, rhythmical contractions were produced which lasted about nine minutes. SrCL. is much less toxic than BaCl2 and it acts more like CaCl2. If it be true that the exchange of Ca for Na or K, or vice versa, in certain organic combinations be the cause of these rhythmical contrac- tions, we are apparently confronted with conflicting facts ; namely, that Ca-salts, as well as salts which precipitate Ca, produce rhyth- mical contractions; but the facts are no more in conflict in this case than in the similar case of the coagulation of milk, where both a previous treatment of the milk vvith decalcifying salts or acid, as well as the addition of a soluble calcium salt, favor coagulation. In this case the probable explanation, according to Loevenhart, is that a soluble calcium salt is necessary for the coagulation. This calcium salt may be added from without, or may be obtained from the milk itself, by freeing it from a combination in which it is held there. The same may be true for the rhythmical contractions in the center of Polyorchis. In order that a contraction may occur, the formation of a certain calcium compound (soap or a calcium proteid ?) is required. This condition may be satis- fied by the diffusion of calcium into the cells from without, or by cal- cium being freed from certain other compounds within the muscle cells themselves (by acid, certain salts, like the oxalates or citrates or, as we shall see later, by the action of enzymes). The process of contraction is due to the substitution of Na or K for the Ca, or vice versa. Ba and Sr act like Ca.

Why is it that the isolated center of Polyorchis does not contract rhythmically in normal sea water? If CaCL, acts as a stimulus, it is not probable that it is the CaCl2 of the sea water which inhibits its contraction. It might be possible that the action of the CaCl2 in the sea water is antagonized by the NaCl. There seems to be indeed a certain antagonism, inasmuch as it is easier to produce rhythmical contractions in a pure solution of CaCl2 or in a solution of CaCl2 in cane

sugar, than in a mixture of CaCl2 and NaCl solutions ;* but the antagonism is not complete, and I have actually obtained contractions in a mixture of NaCl + CaCL, in a center of Polyorchis. It can be shown that the Mg-salts contained in the sea, water inhibit the muscular contractions. If we start rhythmical contractions in a mixture of 50 c.c. f m NaCl + 10 c.c. m Na-citrate, and then put the center into a pure NaCl solution, or a solution of 50 c.c. NaCl + i or 2 c.c. CaCl2, the center continues to contract. If, however, the center be put into a solution of 50 c.c. f m NaCl + 5 c.c. | m MgCL, the contractions are inhibited. I believe that it is generally more due to the MgCL, than to the CaCl2 that an isolated center of a Medusa does not beat in normal sea water.

The facts thus far described give no clear answer to the question whether the substitution of NaCl or KC1 for CaCl2 or the reverse process causes the contraction. Possibly the following observation may throw light on this problem : If an isolated center is put into 50 c.c. f m cane sugar + 10 c.c. f m CaCl2 contractions will begin, which continue when the center is put into 50 c.c. § m cane sugar -f 10 c.c. m sodium citrate; but if the center is put from the latter into the former solution, it stops beating at least for some time. Various modifications of this experiment give similar results : they seem to speak in favor of the idea that the substitution of Na or K for Ca causes a contraction ; but the evidence is by no means unequivocal.

The question may be asked how it happens that in a pure solution of NaCl the beats do not start at once, but only after a number of hours. Is it necessary that NaCl enter the cells of the Medusa, and that this process requires time? Were this the reason, we should expect that within certain limits an increase in the concentration of the NaCl solu- tion should accelerate the beginning of the contractions. I have not been able to find that this is true. The contractions began about equally late in a |- m and a -|, |-, or | m NaCl solution. I am more inclined to believe that the Ca must enter into an appropriate com- bination in the muscles, and, for that purpose, must be freed from another combination which is responsible for the delay in the beginning of the spontaneous contractions of the center of Polyorchis in a pure NaCl solution. The beats of the center begin in a pure NaCl solution when the muscle cells of the center have had time to free an abundant amount of calcium from an organic combination contained in them. This might be done by a hydrolytic enzyme directly or indirectly, in the latter case through the influence of an acid, e.g. CO2, in the muscle cells. This supposition has its analogue in the action of rennet in the

* Tt is possible that this antagonism is due to the fact that the NaCl and CaCl2 retard each other's diffusion into the cells. coagulation of milk, where this enzyme seems solely concerned in ren- dering available Ca, which naturally is held in organic combination in the milk in such a form as to be of no use for the process of coagulation.* A word may be said in regard to the difference in the behavior of the isolated center of Gonionemus and Polyorchis toward a pure solution of NaCl. The former begins to beat in such a solution almost instantly, the other as a rule only after a long interval. The difference may con- sist simply in the fact that the cells of Gonionemus have from the start enough Ca in an available form, while this is not the case in Polyorchis. In the case of the former, the beats can begin immediately in a pure NaCl solution, while in the latter this is not possible.

The question may now be asked, What keeps the normal heart beat or the normal contractions of the jellyfish going? I believe the conditions are the same as those discussed in muscles, the strips of ven- tricle, or the center of the Medusa, with this difference only, that the salts or ions, which according to our hypothesis are needed for that purpose, are all supplied from within. I am inclined to believe that the constant chemical changes, such as oxidations, or the production of CO2, or other processes, lead also to an effect which in the isolated center can be brought about by certain salts, such as citrates or oxalates ; namely, the setting free of Ca so that it may form dissociable colloidal compounds and be then either replaced by Na or K, or vice versa. It is possible that the CO2 formed in the muscle may aid in this process.

The margin of a jellyfish which contains the central nervous system contracts for a time at least, rhythmically in any solution, and certainly in solutions of NaCl, KC1, and CaCl2. I was surprised to find that the addition of MgCl2 to any of these solutions makes the contractions of the margin more normal, as I believe, in an indirect way. For successful rhythmical contractions it is necessary that a real relaxation follows the contraction. I noticed that the edge of a Polyorchis has a tendency to remain permanently contracted in a mixture of 100 NaCl, 2 KC1, 2 CaCla, and this tendency finally interfered with the contractions. This effect is due to the Ca. I found, however, that upon the addition of MgCl2 this tendency to a continued contraction lessened and the Medusa showed a more normal type of contractions.

If it be true that the process of stimulation consists in an exchange of Na- and K-ions for Ca-ions (or Mg-ions?), or vice versa, in the tissues, and that normal irritability depends upon the presence of these ions in definite proportion in the tissues, it is to be expected that a change in these proportions would alter the irritability and give the tissues properties which they do not possess normally. I have already mentioned such an instance in the case of contact irritability which the muscle assumes when put into a solution of a decalcifying salt. As an example of abnormal sensitiveness on the part of sensory nerve endings, I may mention an observation on the nerve endings of the skin of a decapitated frog. If such a frog be suspended vertically over a dish containing dilute acid or alkali, so that the feet come in contact with the acid, they will be withdrawn. If, however, the feet be dipped into a dish of pure water, this will not occur; but if the feet of a frog are put for half a minute or a minute into a solution of A1C13 or Na-citrate, and are then put back into pure water, the feet are withdrawn immediately, in a most violent way,* which might suggest to an anthropomorphically inclined observer the idea that the contact with water caused the decapitated frog the most excruciating pain. This experiment is the more surprising as the contact with the sodium-citrate or aluminium-chloride solution does not as a rule cause such a reaction. This hypersensitiveness of the skin can be done away with by putting the feet subsequently into a normal or 2 n solution of cane sugar. Urea acts similarly, but not so well. I consider it possible that a number of cases of abnormal sensibility, such as accompany certain neuroses, may have their basis in a change in the proportion of metal proteids or soaplike compounds in a tissue. As far as the action of salts on motor nerves is concerned, I have made only the observation mentioned above, that those sodium salts whose ions are liable to form insoluble Ca compounds are liable to cause an increased irritability in a motor nerve. Mathews f has made a long series of investigations on the concentration at which the various salts produce rhythmical contractions when applied to a motor nerve.

His results practically coincide with the statements made in regard to the muscles ; his interpretation, however, is different. From the fact that lithium or potassium citrate causes the muscle to contract in a rather low concentration when applied to the motor nerve, he concludes that it is the anion which stimulates. I consider it more probable that the citrate in this case acts on the Ca in the nerve in the way mentioned above, and that this causes the stimulation. The formation of the Ca- citrate is the essential feature, and this will occur no matter whether the citrate is introduced into the muscle in the form of the sodium or lithium salt. The nerve, however, differs from the muscle in that the former can be stimulated through the loss of water, which is not possible in the case of the muscle. It makes no difference whether the nerve loses water through evaporation, or whether the water is withdrawn from the muscle by a hypertonic solution. According to Mathews, cane sugar and

urea will bring about this effect in a — solution, while for KC1 this con- centration lies at - - or — . Considering the dissociation of the latter I had noticed in my experiments that those salts which produce the abnormal or increased irritability in muscles or nerves are identical with those which are commonly used as purgatives. The action of the purgative salts has been explained by Schmiedeberg and Cushny in this way, that these salts inhibit the absorption of liquid from the intestine, and the excess of fluid in the intestine causes the purgative effect. It seemed to me that the increase in irritability caused by these salts in the muscles and nerves of the intestine must suffice to favor an increase in the peristaltic motions of the intestine, and that this must cause the purgative effect. If this were correct, cathartics should be just as effective if given subcutaneously or intravenously as if given per os ; and moreover, it should be possible to stop these effects by giving cal- cium salts. MacCallum* investigated this point and found indeed that barium salts or citrates, sulphates, fluorides, etc., have cathartic effects if injected under the skin or in the blood vessels. The quickest peristaltic

effects could be produced by applying — solutions of these salts to the peritoneal surface of the intestine. Application of a solution of CaCl2 or MgCl2 inhibited these effects. MacCallum found, in addition, that the watery character of the stools in this case is due to an active secretion of fluid into the intestine (and not as had been assumed to a retention of fluid in the intestine). When he isolated an empty loop of the small intestine in a rabbit, it was filled in a short time with a clear liquid, after a series of drops of a sodium-citrate or barium-chloride solution had been applied to the peritoneal surface of the intestine. It was thus possible to obtain in a short time 20 c.c. or more of a perfectly clear fluid from the small intestine of a small rabbit. This secretion of liquid into the intestine could also be inhibited by CaCl2 or MgCl2.

MacCallum t showed also that the secretion of other glands can be accelerated by the addition of the above-mentioned salts, and can be transitorily retarded by the addition of Ca-salts. The same salts, e.g. BaCl2 or Na-citrate, which accelerate the secretion of fluid into the intestine also accelerate the secretion through the kidneys, and this acceleration can be counteracted by solutions of CaCl2. We have t MacCallum, University of California Publications, Physiology, Vol. I, p. 8l, 1903.

already pointed out that these facts may aid us in obtaining a theory of secretion, i.e. an understanding of the additional forces besides osmotic pressure which must be at work in the process of secretion. It is obvious from all these observations that the salts, especially the Na-, K-, and Ca-salts, play a dominating role in the regulation of those life phenomena which fall generally under the head of irritability or stimulation and inhibition. To give a further idea of how far-reaching the influence of salts in this direction is, I may mention the following fact : Bock and Hoffmann * found, and other authors confirmed the fact, that solutions of sodium salts, e.g. NaCl, NaBr, etc., when injected into the blood, cause glycosuria in a rabbit. It seemed to me that in this case, too, the accelerating effect of citrates and the inhibiting effect of Ca might be found. I asked Dr. M. H. Fischer, who was then a mem- ber of the laboratory, to investigate this question. He found that it is necessary in this case to infuse a NaCl solution of a higher concen-

tration than — . The higher the concentration the quicker, according o to Fischer, the glycosuria ensues.f Fischer found, also, that the citrates, etc., act more powerfully than NaCl, and that CaCl2 was, to a certain extent, able to counteract this effect of NaCl. These observations support the idea of PflugerJ that this is a case of nervous glycosuria produced through the influence of the salts upon certain nervous elements in the medulla oblongata. It is possible that through this influence the concentration of sugar in the blood is raised transitorily, while the CaCl2 has the opposite effect. MacCallum § has made the interesting observation that in this case sugar is secreted not only through the kidney but also into the intestine.

If we finally summarize the results of these observations, we come to the conclusion that all those phenomena which depend on the action of muscles, nerves, or glands seem to be influenced to a large extent by the salts, and that especially changes in the proportion of Na or K to the Ca-ions in the tissues seem to affect their properties and their actions. The idea to which we have given preference, namely, that the substitution of Na or K for Ca, or vice versa, in certain organic compounds gives rise to a contraction, may possibly have to be modified in detail, and undoubtedly many new facts will be required and found before we are ready for a final theory ; but I am inclined to believe that the main structure will remain such as intimated in my papers in 1899

and 1900; namely, that the normal qualities, especially the normal irritability, of animal tissues depend upon the presence in these tissues of Na-, K-, Ca-, and Mg-ions in the right proportion ; that these ions are at least partly in combination with colloids (proteids or higher fatty acids or possibly carbohydrates), and that any sudden change in the relative proportions of these ion lipoids or ion proteids or ion carbo- hydrates alters the properties of the tissues and gives rise to an activity or an inhibition of the activity, according to the sense in which the change takes place. Finally, I believe that the natural rhythmical processes such as heartbeat, respiration, etc., are due to a substitution of certain metal ions for others, these substitutions being caused by the enzymatic processes going on continually, and by which, among others, metal ions are freed from certain combinations, and rendered available for others, as seems to be the case in the action of rennet in the coagulation of milk. We certainly understand by this hypothesis why the combina- tion of the Na-, K-, Ca-, and possibly Mg-salts is so important for life phenomena, especially those of animals.

3. THE REACTION OF LIVING * MATTER AND THE ROLE OF BICAR- BONATES FOR THE PRESERVATION OF LlFE Not only the life of the aquatic animals but the life of every cell is passed in a solution of electrolytes. It had generally been assumed that the liquids in the animal tissues, as well as the sea water, had an alkaline reaction, while the liquids of the tissues of plants had an acid reaction. This assumption was founded upon the titration method. Physical chemistry altered the conception of alkalinity, and measured it by the concentration of the free hydroxyl-ions. Hober* was the first to make use of the methods required to determine the concentration of the hydroxyl-ions in the blood, and found with the aid of gas batteries that the blood was slightly alkaline. His method was defective in a detail, and later Friedenthal,f Franckel,J Farkas, and Hober himself showed that the concentration of the hydroxyl-ions in the blood is not higher than in distilled water. Cottrell and I found the same for sea water. § Friedenthal showed also that the liquids of the tissues of ani- mals and plants are practically neutral. We therefore may draw the conclusion that life phenomena occur in a neutral liquid. The forma- tion of CO2 is one of the most general processes in living tissues. Be- sides, other acids (e.g. lactic acid in the muscle) are formed in metabo-

lism. Respiration eliminates the CO2 in part, but there would be danger that every organism would finally perish through its own produc- tion of acid were these acids not constantly neutralized. This is partly done by the carbonates of the blood. The proteids of the blood are also capable of neutralizing a considerable amount of acid by com- bining with it,* and I believe this role of the proteids in the blood should not be overlooked. The fact can be demonstrated in a striking manner

by putting a frog's muscle into 100 c.c. of ox blood, to which 10 c.c — HC1 or 10 c.c. — NaHO have been added. The muscle does not absorb water in such a solution, and remains alive for several days. If the muscle be put into an isotonic solution of icoNaCl, 2 CaCl2, 2 KCL, to which the same amounts of acid or alkali are added, the muscle absorbs considerable quantities of water and dies rapidly. A third means of keeping the reaction of the liquids of the tissues neutral is probably the compensatory production of bases in the body, possibly induced by the acids.

It is easy to show that marine animals are able to develop and grow only in such solutions as are capable of neutralizing the acids which might be formed. If the polyp be cut off from stems of Tubularia crocea, new polyps are formed in about two days if the temperature is about 20° C. As soon as the polyp is formed, growth begins. If a solution be prepared of 100 molecules NaCl, 2 molecules KC1, 2 molecules CaCl2, 7.8 molecules MgCl2, 3.8 molecules MgSO4, isotonic with sea water, the formation of polyps occurs more slowly than in sea water, and growth is slight. If, however, to such a solution be added from 0.5

tion of NaHO, regeneration and growth occur with normal velocity. f These three substances have the property in common that they are able to neutralize acids, and I am inclined to ascribe it to this peculiarity that they are capable of accelerating growth in Tubularians. It har- monizes with this view that NaHCO3 acts better than NaHO. If, however, a trace of acid instead of alkali is added to the original solution, growth is still more retarded. The addition of o.i to o.i c; c.c. — HC1

solution to 100 c.c. of the above-mentioned solution suffices to suppress growth entirely; this corresponds to a concentration of HC1 of — — - to Similar conditions exist for the development of the eggs of a sea urchin, Arbacia. I found that in neutral solutions of NaCl, KC1, CaCl2, MgCl2, MgSO4, the eggs of Arbacia can reach the pluteus stage, but that no normal skeletons are formed. If, however, NaHCO3 is added, normal skeletons are formed. Similar results were previously obtained by Herbst, and in this case also the NaHCO3 serves for the neutralization of an acid. Experiments in Strongylocentrotus purpura- tus were still more surprising in this respect.* When these eggs were put immediately after fertilization into a van't Hoff solution (100 NaCl,

2 KC1, 2 CaCl2, 7.8 MgCl2, 3.8 MgSO4), only few eggs went beyond the two-cell stage, and only very few reached the pluteus stage. If, however, to 100 c.c. of such a solution 0.5 to i.o c.c. f m NaHCO3 were added, almost all the eggs went into the pluteus stage. In such a solution the development also occurred just as fast as in normal sea water. The addition of o.i c.c. f m Na2CO3 solution caused also some eggs to reach the pluteus stage. These plutei, however, did not live as long

as when the bicarbonate was added. When 0.2 to 0.4 — NaHO was added to 100 c.c. of the van't Hoff solution, the majority developed, but no skeletons were formed. We must, however, take into consideration the fact that in such solutions, as I found, NaHO is neutralized in a few hours or in less than a day by the CO2 of the air and the CO2 formed by the eggs, while the formation of a skeleton occurs only after from /yvL forty-eight to seventy-two hours. The addition of 0.8 c.c. of a —

I think these examples may suffice to show the importance of a regulator which is capable of keeping the solution in which marine animals live neutral. The same is true for the liquids in which tissues live; this point was investigated by Rogers in experiments on the heart of the crab. Here, also, the addition of bicarbonate made a great differ- ence. This is also, as I believe, the explanation of the fact that Ringer's solution is improved through the addition of a trace of NaHCO3. Gaule f was the first to point out the necessity of neutralizing the acid formed in the heart if we wish to make it beat in an artificial solution. In fresh- water animals, Wolfgang Ostwald made the interesting observation that, if Gammarus be put into a salt solution of a comparatively high osmotic pressure, life could be prolonged considerably by adding NaHCO3 to the solution. He was able to show that without the addition of the

bicarbonate, the hypertonic solution was rendered acid by the animals.* During some of the above-mentioned experiments Osterhout and I found that marine algae under the influence of light make the sea water slightly alkaline, while in the dark they do not act in this way. They produce also this alkalinity under the influence of light in a solution of NaCl, KC1, CaCl2, which shows that the act of assimilation is accom- panied by the excretion of a base. It is possible that this is one of the means by which the reaction of the ocean is kept neutral in spite of the animal life. It is also possible that this is one of the reasons why ani- mals keep better in a well-lighted aquarium if green plants are added.