Principles of General Physiology

It is scarcely necessary to remark that, as yet, it is not possible to explain satisfactorily why changes of permeability should give rise to the various phenomena connected with the state of excitation or of narcosis ; further investigation is required and it seems probable that a more intimate knowledge of the electrical conditions of the surface of the cell will give valuable information. We have seen (page 120) how the impermeability of the membrane to one only of the ions of a salt prevents the escape of the other, diffusible, ion, giving rise to a difference of potential between the two sides of the membrane, and how this can be changed by the presence of salts of which both ions are diffusible. But whether such changes in the polarisation of the membrane are sufficient to account for the change in permeability, as Lillie appears to hold, or whether the change in permeability is itself the primary factor, will come up for discussion later in Chapter XIII.

The effect of the substances to which Armstrong (1910) has applied the name " hormones " is clearly allied to the increase of permeability produced by fatal quantities of anaesthetics. These " hormones " are lipoid-soluble and coincide very closely with those substances which are known to abolish the semi-permeability of the membrane, such as ether, alcohol, toluene, etc. Their main obvious action is to set up an enzymic process which was previously in abeyance, such, for example, as the action of emulsin in the leaf of the cherry-laurel on a cyanogenetic glucoside also present. This is regarded by Armstrong as being due to an exciting action on the part of the " hormone" after entering the cell ; but it seems to me that it falls T>ett«r into line with other similar processes if it be looked upon as due essentially to the removal of some such obstacle as that of a membrane, which prevented the access of the enzyme to the glucoside.

ffcemolysis is of two kinds. One in which the surface membrane of the corpuscles is acted on by various haemolytic agents, such as saponin, the other in which the corpuscle is broken up by osmotic swelling, as in the action of water. Substances acting on lipoids produce the first effect ; hypotonic solutions, the second. Ryvosh (1913) holds, with Hamburger, that, in haemolysis by hypotonic solutions, the membrane is not destroyed, but merely stretched to such a degree that the pigment can escape. The ground for this view is, that, after treatment with water, or with 0'3 per cent, sodium chloride, although the relative volume of the deposit, after centrifuging, is 0"2 in the first case as against 0'8 in the second, yet, on placing in 2 per cent, sodium chloride and again centrifuging, the volumes became practically equal, 0"2 to 0'25. That is, although the corpuscles were greatly swollen in 0'3 per cent, sodium chloride, they could still contract under the influence of a hypertonic solution, showing that they retained their semipermeability as regards sodium chloride.

Further details are beyond the space at our disposal and may be obtained from the general summary by Stewart (1909). Secretion. — It is clear that constituents formed in gland cells must leave these cells by the side turned towards the lumen of the alveolus in connection with the duct. Apart from the actual chemical processes in connection with secretion, to be described in a subsequent chapter, changes in the permeability of the cell membranes must be taken into account. Various researches by Asher and his co-workers have brought out a number of facts, interesting in this connection. It is well known that atropine has the property of stopping the activity of secreting cells in general, and Garmus (1912) shows that, under the action of this alkaloid, the cells of the glands of the frog's skin take up less dye than normally. There is no reason to suppose that the actual stainable material is diminished, so that the result must be ascribed to a diminution "of permeability, especially as pilocarpine, which excites the cells, has the opposite effect on staining. We saw previously that, in the excited muscle cell there is also an increase of permeability. It has been shown by Straub (1912, p. 22) that the action of atropine in antagonising that of muscarine on the heart is

due to the diminution of permeability towards muscariue, brought about by atropine. The Nerve Synapse. — According to the view advocated by Sherrington (1906, p. 16) the communication of a nerve impulse to the cell body of another neurone takes place across a membrane, the " synaptic membrane." It is, therefore, owing to changes in the permeability of this membrane that impulses are allowed to pass or not. Whatever may be the actual chemical substance that diffuses through, or whether only a physical process is involved, there is every probability that the ions of dissociated salts play a large part in the transmission of nerve processes, so that it is a matter of importance to see what kind of action may be looked for.

We have as yet very little direct evidence on the question, but there are some observations which are of interest. Locke (1894) found that immersion of the sartorius muscle of the frog in 0'7 per cent, sodium chloride had the effect of preventing the muscle from contracting when the nerve to it was excited, although its direct excitability was not abolished and the effect was not produced if the nerve alone were immersed. Addition of traces of calcium salt to the solution restored the normal state. According to Overton (1904, p. 280), reflex excitability is lost in the absence of calcium from the central nervous system and one is obviously reminded of the action of calcium salts on colloids. Whether the synaptic membrane requires to be more or less semi-permeable, in the osmotic sense, in order to permit the excitatory process to pass, cannot be answered until we know more as to the nature of this process.

Certain facts to be described below with respect to reciprocal innervation in reflex action are made more explicable if we could imagine a membrane permeable to certain ions in one direction only. There is some evidence that the skin of the frog is permeable to sodium ions from without in, but not from within out. The most satisfactory evidence seems to be that the skin acts as a rectifier for alternating currents, that is, it allows the one part of the period, in which the current is flowing in one direction, to pass through more easily than that in which the current flows in the opposite direction (Bayliss, 1908, p. 235).

This result would also be obtained, as Hober justly points out (1911, p. 493), if the cell membrane on the inner side of the skin were permeable to one only of the ions of the salt. I found, in fact, that similar phenomena are shown by a system consisting of a solution of Congored inside a parchment paper membrane, which is permeable to the sodium ion of the salt only. Since a current can only pass continuously when a quantity of positive ions can pass to the negative pole equal to the negative ions passing to the positive pole, it follows that, if the positive pole is outside the membrane, which is impermeable to the negative ions, these can never get to the positive electrode outside at all ; while, if this electrode is on the same side of the membrane as the anions, so that they can reach it without hindrance, the current will pass readily, because the cations can pass through the membrane.

It does not seem necessary, therefore, to assume an irreciprocal permeability, which is ditncult to conceive. In any case, it would only be possible in the case of a living membrane, to which energy was being supplied by cell activity. Otherwise, there would be a spontaneous difference of potential kept up between the two sides of the membrane and the possibility of a perpetual motion machine. Fertilisation of the Egg Cell. — In this process, it has been shown by M'Clendon (1910, p. 256) that the membrane becomes considerably more permeable to electrolytes, evidenced by the increase of electrical conductivity of a mass of eggs of the sea urchin on fertilisation. There is other evidence of increased permeability in the escape of pigment observed by Lillie, who regards the essential element in the artificial segmentation under the influence of certain salts as an increase in the permeability of the cell membrane.

Gray (1913), also, found diminution of electrical resistance in Echinus eggs in the process of fertilisation, followed by return to or towards the normal. The Permeability of the Blood Vessels.— It is plain that all substances necessary for the nutrition of cells and all those produced by the cells, so far as they pass into the blood stream, have to pass through the wall of the capillaries (except, perhaps, in the case of the liver— Schafer, 1902). Some of these substances are in the colloidal state, and therefore, unless the cells are permeable to colloids, which does not seem probable, these colloids must escape between the cells, by a process like that of filtration. This question will come up for discussion later, but it may be remarked here that, as far as the blood proteins are concerned,

evidence already referred to (page 107 above) indicates that they do not serve for the nutrition of cells. It is certain, on the other hand, that the permeability of the capillary wall may let through proteins, especially in pathological conditions. In dropsy the continuous flow of lymph, which is obtained from a canula in the subcutaneous tissue, contains protein and must have been filtered from the blood capillaries. Direct evidence on the question at issue is, naturally, difficult to obtain.

There are many substances which exercise a powerful action on cell pro< • but which can be proved in certain cases not to enter the cell at all, and in other cases, although they do enter the cell, they exercise no action after having obtained entrance. One of these cases has been referred to in another connection, viz., the experiments of O. Warburg (1910, p. 313) on the action of alkalies on the oxidation processes in the developing egg of the sea urchin, in which it was found that the consumption of oxygen could be doubled by the addition of very small amounts of sodium hydroxide to the sea water in which the cells were immersed. Ammonium hydroxide, on the other hand, produced scarcely any effect. By previously stain ing the cells with neutral red, it could be shown that no sodium hydroxide entered the cell ; whereas, if ammonium hydroxide was used, a rapid change of the dye to yellow showed that the alkali had entered the cell. The action of alkali on oxidation must, therefore, be exerted on the cell membrane itself.

The following observations of the same experimenter (1911, p. 425) are of interest in several ways. The young red blood corpuscles of the goose are distinguished by considerable consumption of oxygen. This process, unlike that of the sea urchin eggs, is not affected by salts. If, however, the cell membrane is destroyed by careful freezing and thawing, which does not affect the total consumption of oxygen, then the process becomes sensitive to salts, especially to barium chloride. The unavoidable conclusion is, that, as long as the membrane is intact, barium chloride cannot enter. In those cases in which it produces its effect on the intact cell, it must do so by intermediation of the membrane, since it cannot pass any further.

Newton Harvey (1911, p. 546), working on Paramtecium, found that the action of sodium hydroxide on the changes in behaviour, the formation of vesicles, cessation of movement, and final death were all produced without the entrance of the alkali into the cell substance. The same investigator later (1913), in a special series of experiments, showed that the method used was free from objection. The experiments of Bethe (1909) on Medusce showed that acids had an accelerating action on their movements, although no change of dye indicator within the cells occurred.

An experiment of Overton's (1904, p. 202) shows'that the action of potassium on muscle is also on the surface only. A sartorius muscle is transferred from Ringer's solution, through 6 per cent, cane-sugar, to 2 per cent, potassium tartrate, in which no change of weight takes place, showing that the cells are completely impermeable to the salt, since the solution is isotonic with the cane-sugar. Nevertheless, the muscle is totally paralysed. On placing in Ringer's solution again, the excitability is quickly regained. This latter fact confirms the view taken of the action of tlie potassium salt as being on the cell membrane, since, if it had penetrated into the interior, it is difficult to understand how it could pass out again with such rapidity.

Overton also showed (1902, 2), as will be remembered, that, if all the sodium chloride be washed out of a muscle, it becomes inexcitable until more sodium chloride is supplied. Now Fahr (1909) states that the only satisfactory explanation of the results of his experiments is that the muscle cells themselves normally contain no sodium at all. But since sodium is necessary for their activity, it follows that it must act on the membrane, as this is the only part of the cell with which it comes into relation.

Conclusions of the same kind are drawn by Straub (1912, p. 14) with regard to the action of calcium on the heart muscle, which is impermeable to this substance big. 46 illustrates this point. In the tracing, the first three beats are under the action of Ringer's solution containing sodium, potassium, and calcium. At the signal, the solution is suddenly changed for a similar one without calcium. It is seen how the want of calcium is shown in the first beat succeeding the change. At the end of the signal, the normal Ringer's solution is replaced, with its action on the very next beat. Straub reckons that the effect must be manifested in less than 0-1 second. It is to be remembered that the interior of the cells contain calcium, which could not diffuse out by the time at which the action of a calciumfree solution is manifest. The calcium could merely be removed from the outer surface of the cells.

The experiments of the same investigator on the effect of muscarine on the heart of Aplysia (1907) have already been described. When this organ is allowed to lie in a small quantity of a solution of the drug, it is noticed that the effects TION OF CALCIUM. — The first three beats are normal in Ringer's solution (which contains calcium). At the beginning of the white space, this solution is suddenly changed for one otherwise similar, but from which the calcium has been omitted. The effect is shown at once on the first beat after the change. At the end of the white space, the normal solution is replaced, with an immediate effect. Time in seconds. Note that less than O'l second is required to affect the muscle cells, so that the action must be exerted on the colloidal system of the cell membrane. This is the only part of the cell which could be deprived of calcium with such rapidity.

of the poison are only seen while there is a particular concentration of it left in the solution ; the heart then recovers, although it is found that muscarine has been stored inside its cells and remains there. It is plain that the effect was only to be seen while the poison was in the act of passing through the membrane, so that it must be supposed that it leaves the membrane, and passes into the cell substance as soon as there is less than a certain minimal concentration in the outer liquid.

Somewhat similar results were obtained by Neukirch (1912) with the action of pilocarpine on the excised small intestine of the rabbit, immersed in warm oxygenated saline solution. The addition of pilocarpine to this solution causes a great increase of tonus, which slowly decreases but does not disappear, even in several hours. But, if the pilocarpine solution be changed for fresh saline, a second increase of tonus occurs as the alkaloid diffuses out from the cells into the saline. A remarkable fact is that if, as soon as this tonus has developed, the pure saline be exchanged for the original pilocarpine saline, so that diffusion ceases on account of equality of concentration inside and outside of the cells, the tonus also disappears. It seems, then, that in this case, the actual presence of

equally concentrated solutions of the alkaloid on both sides of the membrane is of no effect or a minimal one. The characteristic effect is manifested only while the drug is in the act of passing through the membrane. As long as we remember that the cell membrane is a modifiable part of the cell system, the various facts described above need not cause surprise. Since protoplasm has the properties of a liquid and can also be shown to contain free, uncombined salts, there must be some means by which free diffusion between the contents of a cell or organism and the surrounding medium is controlled.

There is every reason to suppose that the regulation of the passage of substances between the inside and outside of a cell is effected by means of a film or membrane. The membrane of the cell must allow water to pass freely, but hold back dissolved substances. Such a membrane is known as a semi-permeable one. Artificial membranes can be made of various degrees of permeability ; thus, some will only hold back colloids, others will allow certain crystalloids to pass, but not sugar, and so on.

Different views are held as to that property of a membrane which makes it permeable to some solutes and impermeable to others. Reasons are given in the text for accepting, with some modifications, the original sieve theory of Traube, according to which the passage of a solute through a particular membrane, depends on the size of the pores in the membrane in relation to the molecular, or particulate, dimensions of the solute. The hydration of solutes must be taken into account. In a few cases, the question of solubility in the substance of the membrane appears to play a part.

The protoplasmic substance of the cell is capable of forming a new membrane on a fresh surface. The substances present in the protoplasm which lower surface energy, and there are a large number of them, will be concentrated at the interface between protoplasm and external phase, and some of them may be coagulated. In this way a membrane is formed. It is to be noted that the cell membrane is, accordingly, an integral part of the cell system, and capable of modification with changes in the composition of the cell contents.

In this way a difficulty is overcome. If the cells are always impermeable to such solutes as sugar, amino-acids, and salts, how is growth to take place or the functions of the cell to be performed ? We must conclude that the permeability of the membrane is not always the same ; a fact which is also demonstrated by experiment. The difficulty alluded to has caused certain investigators to deny altogether the existence of a semi-permeable l^^fcrane covering the cell protoplasm. Evidence of various kinds is given in the text, which shows that, in the condition in which cells are usually met with, they are actually impermeable to crystalloids.

This evidence consists in the permanent change of volume which cells undergo under the action of various dissolved crystalloids, in the difference between the concentration and nature of crystalloids in the interior of the cell and in the outer medium, and, lastly, in the resistance opposed by living cells to the passage of electrical currents, notwithstanding the fact that they contain free electrolytes. Although this may be regarded as the usual state of cells at rest, their permeability may be altered, without killing them, and therefore reversibly, by the action of various substances on the membrane. Of these we may mention electrolytes in particular. Narcotics and light are also found to have an influence.

The chemical nature of the membrane depends on the constituents of the protoplasm which lower surface energy. As fatty or lipoid substances possess this power in a marked degree, it is to be expected that the membrane will manifest many of the properties of lipoids. At the same time, reasons are given for not accepting the view that the cell membrane consists of lipoids alone, and still less that it consists of protein alone. The various substances of which it is composed exist in a complex colloidal intermixture in a more intimate connection than a mere mosaic of lipoid and protein.

It appears that, as a general rule, it may be stated that the cell membrane is always permeable to substances soluble in lipoids, but whether this fact is essentially due to the solubility itself, or to some other property, such as surface tension or molecular dimensions, is uncertain. The apparent solubility of many dyes and other substances in solutions of lipoids is not a true solution, but a surface adsorption on the colloidal particles of the lipoid. These dyes are insoluble in the lipoid itself. As regards substances insoluble in lipoids, the permeability of the membrane is capable of variation, so that, while being usually impermeable to salts, sugar, etc., it may sometimes become permeable to them. This latter fact necessitates a complex structure.

Various instances are given in the text which show that changes of permeability do actually take place in functional processes. Tlie state of excitation of muscle, narcosis, secretion, the passage of the nerve impulse from one neurone to another or to a muscle cell, the fertilisation of the ovum and changes in the walls of the blood vessels are referred to. Certain cases are known where substances produce profound changes in cell processes without passing beyond the membrane. The action of alkali on the oxidation process of sea urchin eggs and on the movements of medusae, and that of potassium, sodium, and calcium ions on muscle are of such a kind. In other cases, the substance, muscarine or pilocarpine, only produces its effect during its passage through the membrane. \,

In brief, the cell membrane is a local concentration of constituents of the cell protoplasm due to their property of lowering surface energy of some kind. Substances present in the external medium, if possessing the same property, may also take part. The properties of the membrane are, therefore, not fixed, but capable of modification according to the chemical processes taking place in the cell, or they may be changed by influences on the outside. It is to be regarded as a part of what we may, for the present, call the "living system" of the cell. In its resting state, as usually investigated, it is impermeable both to colloids and to the majority of crystalloids, but may become, temporarily, permeable to all crystalloids and perhaps to some colloids.