Principles of General Physiology

Tyrode (1910) adds a small amount of a salt of magnesium and bicarbonate, obtaining the following solution : — This solution is a good one for the intestine of the rabbit, but the addition of magnesium does not appear to be of any advantage for the heart, as pointed out by Locke (1900). The last solution may be called Ringer-Tyrode's solution. The addition of bicarbonate is of value in assuring that the solution shall be only just on the alkaline side of neutrality, while possessing the capacity of neutralising acid products formed by the metabolism of the organ perfused. The description of the bicarbonate and phosphate systems, given above, has shown us how this may be done.

The work of Clark (1913, 2) on the heart of the frog has shown that continuous perfusion with renewed supplies of a pure saline solution removes some important constituent from the cells ; thus, a small volume of Ringer's solution, which has been repeatedly circulated through a heart, is capable of reviving another heart, whose beats are reduced owing to continuous perfusion (Fig. 64). The substance in question seems to be of a lipoid nature, since it has considerable power of lowering the surface tension of water, and a substance having a similar action can be extracted by ether from the residue left on evaporation of blood serum. The residue left on evaporation of the ether has a great effect in causing recovery of a heart which has become "hypodynamic" from prolonged perfusion with Ringer's solution (Fig. 65). This power is also present in lecithin. The interaction of calcium is necessary for the effect, which seems to be due to a change in the colloidal constitution of the cell membrane, which, as we have seen, undoubtedly contains a large proportion of lipoids.

A, Frog heart after perfusion for four hours with Ringer's solution. At the gap, where 10' is marked, 2 e.c. of Ringer's solution, which had circulated for twelve hours backwards and forwards through another heart, were added to the 2 c.c. already circulating. The result shows that some important substance is removed from the muscle by continuous perfusion with saline solutions. Antagonism of Salts. — In these physiological saline solutions we see how one salt alone is unable to preserve the excitability of living tissues, and we are reminded of the similar phenomena in the effect of salts on the permeability of the cell membrane. In this latter case we found that, as a rule, the action of a single

Fin. 65. EFFECT OF LIPOID CONSTITUENTS OF SERUM ON HYPODYN\MI< MKART. 1. Beats of heart rendered hypo<h nainic by prolonged ]>erfiisioi] with Ringer's solution. At A the constituents of serum which are insoluble in alcohol were added ; there is no effect. Tinjmlse volume (P.v.) increases merely from 0-026 to 0"033 c.c. •I. At B marked effect of the alcohol-soluble constituents of serum. I'ulxe volume increased in :5. Effect of ether extract of dry residue of alcoholic extract of serum, introduced at A. 4. Similar effect of sodium soap of the above ether extract.

salt is to cause a loss of the semi-permeable properties of the membrane, and it may well be that we have to do with related phenomena in the behaviour of cells to perfusion fluids. The way in which one salt is able to neutralise the toxic properties of another is, as yet, by no means clear. Loeb (1903) showed that a marine Gammarus dies in half an hour if transferred to cane sugar or pure sodium chloride isotonic with sea water, in fact, just as quickly as if placed in distilled water. The addition of either potassium chloride or of calcium chloride alone to distilled water or to cane-sugar does not improve it. In solutions containing sodium chloride plus either potassium or calcium chloride, the animals die also as soon as in pure water. Only in a solution containing Na', K' and Ca ' * ions in the proportion and concentration in which they exist in sea water are the animals able to remain alive. It seems clear that the normal semi-permeability of the colloidal constituents of the cell membrane is only kept intact when these three salts are present. Perhaps investigations on the physical properties of proteins and lipoids, as well as of other colloidal systems under the influence of these salts, separately and together, would throw light on the question.

A similar set of phenomena have been investigated by Loeb and Wasteneys (1911) in the case of the fish, Fundulus, which is not affected by the osmotic pressure of the solutions used in the experiments. In sodium chloride or potassium chloride solutions, of the concentration in which these salts exist in sea water, the fish only lived a few days ; whereas in calcium or magnesium chlorides they lived indefinitely. But, in contrast to what we have seen to be the case in the heart, it was found that salts of sodium and potassium, present together in certain proportion, mutually deprived one another of toxic action. In the heart, as will be remembered, the presence of calcium is necessary in addition. A further demand is made by the sea water plant, Ruppia maritima, which requires, according to the investigations of Osterhout (1906), no less than four salts, viz., all the cations present in any quantity in sea water. The following table shows this fact : —

Returning to the experiments of Loeb and Wasteneys, direct evidence is given that the effect is of one cation on another, and not of an opposite ion. The fact that above a certain concentration of potassium chloride, no neutralisation of its toxic effect by a sodium salt is possible, suggests a partition of some kind at the cell membrane and most probably an electrical adsorption. We have seen above (page 104) that there is no evidence for the formation of chemical compounds of protein with neutral salts, whether they be called " ion-proteids " or by other names. We may also call to mind that one substance may displace another from its state of adsorption, provided that in the process there is a further diminution of free energy of any kind.

If the proportion of sodium chloride to potassium chloride is much less than that in sea water, for example only eight molecules to one, the toxic action of the potassium is increased. It is also interesting to note that calcium chloride itself is not toxic for Fundulus, so that, when it neutralises the toxic action of sodium and potassium chlorides on this fish, it is a case of a non-toxic ion neutralising a toxic one. Calcium has a much more powerful action in neutralising potassium than sodium has, about 500 times as great. Like that of sodium, however, the antagonism is limited and the interesting point about the fact is that the limit is the same, viz., no stronger solution than 6'6 c.c. of 0'5 molar KC1 to 100 c.c. of water can be

neutralised by any amount of either calcium or sodium salt nor by both together. The fact that calcium has a much more powerful action than sodium has is not unexpected if we look upon the effect as exerted on the cell membrane. Ca* ', as a bivalent ion, has much greater action on colloidal aggregation than sodium has. Strontium chloride has an effect about equal to that of calcium chloride ; barium chloride has also a high value, but is very toxic. Magnesium chloride has relatively little action, so that the valency of the ion is not the only factor concerned.

Sodium chloride, in the concentration in which it exists in sea water, cannot be neutralised by potassium chloride alone, calcium must also be present. A further interesting fact is that the toxic action of acids is also abolished by sodium ions and still better by calcium ions. If electrical phenomena play a part in the action of ions in general, it is possible that the affinity of an ion for its charge may have to be taken into account, as insisted upon by A. P. Matthews (1904). The most active ions would be expected to be those which part with their charges most easily. Although we must admit, with this author, that physiological action has frequently no connection with chemical structure, for example, beryllium sulphate, lead acetate, sugar, phloroglucinol and saccharin all taste sweet, it is undoubtedly going too far to say that all actions of enzymes or toxins have nothing to do with chemical structure, or that the action of a lead or other salt on the living organism is determined by tincharacter and number of its electrical charges and by the ease with which it parts with these charges, and by nothing else.

In order to realise the many and various ways in which electrolytes intervene in physiological processes, it will be instructive to refer briefly to a few typical cases ; some of these will require more detailed treatment in future chapters, so that they may be merely mentioned here. The illustration by Hoeber (1911, p. 444) of our methods of regarding the combined effect of the various ways in which such actions may be exercised, is an apt one. He likens our conceptions to a mirror, which, in its present condition, does not give a sharp image. If the image appears to be a confused one, we must not jump to the conclusion that the mirror itself is an inappropriate one and distorts the object to be reflected, but that it is not sufficiently polished to show fine details as well as it does the coarser outlines. The physical chemistry of colloids, to mention one fact only, is still too full of gaps to answer all that it may be capable of.

It is perhaps well to name again the possible ways in which a salt or other electrolyte may act ; the electrical charge, as such, has its effect ; there is also the effect on the solvent, shown by lyotropic actions, and frequently expressed in the " Hofmeister series " ; finally, we may have effects, not included in any of these and more nearly related to purely chemical action, so that they are often exerted by the salts of one element alone, or by those of closely related ones.

The Sitjn of the Electrical Charge on Cell Membranes, as worked out by Mines (1912), is the first of these general effects to which we may call attention. On Adsorption by Surfaces. — When a substance with an electrical charge is adsorbed by the surface of a colloid, the amount adsorbed depends greatly on the sign of the charge of the surface, whether similar or opposite to that of the substance adsorbed. By electrolytes, the charge of the surface can be annulled or reversed.

Hamoglobin. — An important action of electrolytes on the dissociation of oxyhaemoglobin, described by Barcroft and Camis (1909), probably depends on the colloidal nature of this substance. At a given pressure of oxygen, less of this gas is taken up by haemoglobin in presence of salts than in pure water. For example, at 30 mm. of mercury of oxygen pressure, the percentage saturation in water is 85, and in Ringer's solution only 60. The effect is still more marked with acids, and is a delicate indication of the hydrogen ion concentration in blood. The importance of these facts will be seen later in connection with the supply of oxygen to the tissues.

Enzyme Action. — Many enzymes are inactive in the absence of electrolytes. In some cases, this appears to be due to the facilitation, by salts, of adsorption between enzymes and their respective substrates. Hwmolysin. — It was shown by Gengou (1908) that the hsemolysin of the serum of the eel is inactive without electrolytes. Secretion. — It will be seen later that the excitatory action of extracts of the duodenal mucous membrane in causing the pancreas to secrete is not shown in the absence of electrolytes.

Electrical Excitation. — Since salts are always present in living tissues, it is clear that the result of applying an electrical current must be the separation to a greater or less extent of the ions of opposite sign at the two electrodes. The exciting effect of the cathode and the inhibitory effect of the anode is, no doubt, connected with this fact. The opposite action of H* and OH' ions is a familiar fact and has been already referred to. Smooth Muscle. — Hooker (1911) shows in experiments on perfusion of the blood vessels of the frog with saline solutions, that calcium produces contraction of the muscular coat, while potassium and sodium cause relaxation. Gaskell (1880-82, pp. 55 and 56) had already shown that acids cause relaxation, and alkalies cause contraction.

Pigment Cells. — The fish, Fundulus, contains in its skin yellow and black pigment cells. It has been shown by Spaeth (1913) that potassium salts expand the former, contract the latter. Sodium salts have an opposite effect on both. By photographing the same cell, it is seen that the expansion and contraction does not concern the cell as a whole, since the processes remain permanently of the same form. The pigment granules migrate inside the processes to and from the centre of the cell.

Calcium. — Although, in certain processes, calcium can be replaced by other alkaline earths, there are others in which this is not so. Barium, for example, is especially toxic to the animal organism. The property of calcium to favour consolidation or stability in colloidal systems, in opposition to that of the alkali metals, which tend towards liquefaction in some cases, is, no doubt, rightly indicated by Hoeber (1911, p. 446) as being of great importance in the explanation of the physiological action of calcium. Moreover, the same author points out that the action of a bivalent ion is much less violent than that of a multivalent ion and is much more easily reversible.

We have already seen the necessity of calcium for the heart beat of the vertebrate, and Lovatt Evans (1912, 2, p. 410) has shown that the same statement applies to that of the snail. The latter, however, is able to stand a much higher concentration than that of the frog, beating quite normally in 2 per cent, calcium chloride. Barium is quite as toxic as to the vertebrate heart, one part in 20,000 causing a marked systolic condition. Locke showed (1894) that calcium is also necessary for the transference of the excitatory process from nerve to muscle and Overton (1904) showed that it is equally necessary for the transmission of the excitatory state through the synapse of a nerve fibre with a nerve cell. According to Busquet and Pachon (1908) when the action of the vagus nerve on the frog's heart has been stopped by perfusion with pure sodium chloride solution, as shown by Howell (1906), addition of calcium chloride in extremely small amount is sufficient to restore the inhibitory action to the vagus nerve.

Clark (1912, p. 12) has shown that digitoxin (the active substance of the foxglove) is inactive without calcium. In Fig. 66 the heart of the frog is seen to be at first beating normally in Ringer's solution. At A, calcium-free Ringer's solution is perfused to wash away calcium, and at A', repeated circulation of the same 3 c.c. of the same solution is established. The feeble beat seen in the tracing continues for hours under these conditions. At B, a trace of calcium chloride is added ; the beat returns to normal. At B', O'Ol mg. of digitoxin is added and at c, perfusion with calcium-free Ringer's solution is recommenced. It will be seen tJ although the beat is somewhat stronger and slower, the typical systolic tone, which the drug normally produces, is absent. At D, the normal amount of calcium chloride (0'02 per cent.) is

added ; the systolic arrest appears immediately. It is to be noted that the amount of calcium added is only just about the amount required to neutralise the potassium present in the solution, so that the effect is not due to the calcium but to the digitoxin, which had been in abeyance until the calcium was added. Hamburger (1910) finds that calcium increases the amoeboid movement of phagocytic leucocytes, while barium, strontium and magnesium have no such effect.

Chiari and Januschke (1910) describe a remarkable action of calcium. If sodium iodide is injected intravenously into dogs, fluid is exuded into the pleura and pericardium, and redenia of the lungs is produced. But, if calcium chloride is injected simultaneously, these cavities remain quite dry. Exudation produced in other ways is also subject to the same effect of calcium. Inflammation and swelling of the eyelids, caused by oil of mustard, is prevented by previous subcutaneous injection of calcium chloride. The action appears to be on the

FIG. 66. INACTIVITY OF DIUITOXIN ON THE FROG'S HEART IN- ABSENCE OK CAM 11 \i permeability of the walls of the blood vessels, increasing the " consistency " of the colloidal systems of the cell membranes. An interesting fact noted by Mines is that strontium can be a>itayoni#e,d by calcium in respect to its action on the heart muscle. The characteristic tonic effect of calcium is possessed to a greater degree by strontium ; but, if to a Ringer's solution, containing sufficient strontium to give a marked prolongation of the beat, there be added the normal amount of calcium salt, the beat returns to its correct form. It is difficult to say in what way this effect is produced ; possibly calcium lowers some form of surface energy to a greater extent than strontium does, and therefore displaces it from the cell membrane, or calcium may give up its electrical charge with greater ease than strontium does, and thus maintain the proper colloidal consistency of the membrane.

In the absence of calcium, the heart of the frog is unable to give contractions. The continuance of the electrical change shows that the excitatory process goes on (see Mines, 1913, 3, p. 231), and Locke and Rosenheim (1907) found that glucose is still consumed. It appears that there is some break in the mechanism of conversion of chemical energy to contractile stress. The state of the active surfaces (see below, page 448), in the absence of calcium, is such that the

increase of surface energy, normally produced by the liberation of lactic acid, cannot take place. If the blood vessels of the frog are perfused with Ringer's solution and a trace of adrenaline added, a marked constriction is shown by a slowing of the rate of flow. According to R. G. Pearce (with Asher, 1913, p. 274), if pure isotonic sodium chloride is used, adrenaline causes dilatation of the vessels. It appears that calcium is necessary for the normal effect of adrenaline on the sympathetic nerve-endings. In experiments of this kind caution is necessary on account of the spontaneous rhythmical changes which are apt to occur in the frog's blood vessels under saline perfusion, as I have described (1901, 1). In fact, I have been unable to confirm Pearce's results. This apparent reversal of an excitation to an inhibition will come up for discussion again in a later page.

There are two particular phenomena of physiological interest which appear to be colloidal aggregations. These are the coagulation of the blood and that of milk by rennet. For both, the presence of calcium is required. In the former case the fact was first definitely proved by Arthus et Pages (1890), although the favouring action of calcium salts had been noticed previously and it had been shown by Ringer and Sainsbury (1890) that barium and strontium had the same effect, but in less degree. Arthus et Pages, also, showed that strontium could replace calcium.

The clotting of milk by rennet is due to the peculiar properties of the calcium salt of caseinogen. Whether the salts with the other alkaline earths behave in the same way does not appear to have been investigated. Magnesium. — Meltzer and Auer (1905) describe how the subcutaneous injection into a rabbit of 1'7 g. of magnesium sulphate per kilogram produces in thirty to forty minutes deep anaesthesia and paralysis and (1908) how this effect is removed in a few seconds by the intravenous injection of about 8 c.c. of 3 per cent, calcium chloride. The same experimenters (1909) show that the application of a molar solution of magnesium sulphate to the surface of the medulla oblongata causes, within fifteen minutes, abolition of the functions of all the medullary centre?. Meltzer (1913) points out the value of a preliminary dose of magnesium sulphate in ether narcosis. If 0'6 g. of crystallised magnesium sulphate per kilogram of animal is given intramuscularly to rabbits (or 0'8 g. to dogs) a very small effect is produced ; but if ether be given, profound anaesthesia results from one-tenth of the dose usually required for mild anaesthesia.

Sodium. — It was shown by Overtoil (1904) that frog's muscle immersed in isotonic cane-sugar (7 per cent.) loses its excitability, and that restoration can be brought about by a sodium salt or, in a less degree, by a lithium salt, but not by salts of potassium or ammonium. Potassium. — The action of potassium is, in the main, but not always, a paralysing one, as seen in the case of the heart. At the same time, its presence is necessary to control the opposite action of calcium.

It is probable that the powerful physiological action of potassium may be connected with the rapid rate of migration of its ions. If the table on page 177 be consulted, it will be seen that these ions have a higher transport number than any other cation, with the exception of hydrogen. This fact will enable them to play a prominent part in the phenomena connected with the electric charge on surfaces. In the formation of a Helmholtz double layer, potassium ions will outdistance other cations and, therefore, tend to be in excess in the positively charged side of the layer.

Howell (1906) showed that, in the absence of potassium salts, the vagus nerve loses its power of inhibiting the beats of the heart, and the similarity between the action of potassium and that of the vagus nerve suggested to him (1906, p. 291) the hypothesis that the action of this nerve might depend on the setting free, in some way, of potassium. Howell and Duke (1908) found that an increase of potassium could be detected in a small amount of Ringer-Locke's solution which had passed repeatedly through a mammalian heart under vagus inhibition.