Principles of General Physiology

To begin with, we must remember that the film covering the outer surface of protoplasm, or, in fact, any surface where it is in contact with another phase, is not of such a nature that it can be separated off, even optically, from the rest of the cell. After death, under the action of toxic substances, it seems that a distinct membrane may be visible. There are, of course, membranes covering whole organs, which can be separated from the cells beneath them, such as the interesting one on the barley corn, whose properties have been investigated by Adrian Brown (1909). Such membranes play an important part in the physiology of organisms, but are to be distinguished from those with which we are immediately concerned.

Suppose that a mass of protoplasm, such as an Amotba, is immersed in water. By the principle of Willard Gibbs, any constituent of the protoplasm which lowers surface energy will be concentrated at the interface between the two phases, forming already a kind of membrane. Further, as shown by Kamsdm (1904) and described on page 55 above, many of the substances present in cells, especially the proteins, suffer a kind of coagulation when subjected to such concentration. Now, substances of a fatty nature, the so-called lipoids, such as lecithin, and the fats themselves, are normal constituents of cells and, as we saw in Chapter III., have a particularly powerful action in decreasing surface energy and will naturally take a large share in the formation of a membrane of the kind in question.

An interesting experiment by Nageli (1855, i. pp. 9 and 10), discussed also by Pfeffer (1897, i. p. 92, and 1877, p. 127, etc.), shows that such membranes are formed on any free protoplasmic surface. A root hair of Jlydrochari* (a water plant with relatively long root hairs, which are processes of the root cells themselves) is placed under a cover-glass in a solution of a dye, such as aniline-blue, to which the normal cells are impermeable. The root hair is then crushed by pressure and, from the places where the cell wall is torn, masses of protoplasm exude and form into little balls. These balls show similar osmotic phenomena to those of the entire cell. The protoplasm remains unstained by the dye. Kiihne, also (1864, p. 39), describes the formation of a similar membrane on protoplasm pressed out from Stentor, a ciliate protozoon. Further observations will be found in Pfeffer's paper (1890, p. 193, etc.).

It seems probable that the observations of Kite (1913), in which solutions injected into the substance of certain cells, so as to form vacuoles, which behaved as if surrounded by a similar membrane to that on the outside of the cell, are to be explained by this formation of a surface condensation at the interface between the solution in the vacuole and the surrounding protoplasm. It may be noted here that the clear surface layer of protoplasm, noticed in Amvzba, leucocytes, Mycetozoa and similar organisms, and known as " hyaloplasm," also owes its origin to surface forces. When the cell changes in dimensions, as by taking up or losing water, it is found that the thickness of the layer of hyaloplasm does not change, so that its total volume must have altered. It is constantly maintained so as to extend to a particular depth below the surface. It is not to be thought that this clear layer is the cell membrane itself, to which the semipermeability is due. This is to be seen from the fact that a dye, which is unable to enter a cell, is stopped before it reaches the hyaloplasm, which remains unstained, like the rest of the cell (Hober, 1911, p. 59).

The new formation of a cell membrane on fresh surfaces of protoplasm, referred to in the preceding paragraphs, occurs only in the " living " state, although, under certain conditions, it remains intact after the death of the cell, as shown by the following experiment of Pfeffer (1877, p. 136). A root hair of Hydrocharis is mounted in an isotonic solution of cane-sugar, placed under the microscope, and a trace of hydrochloric acid added. The protoplasm becomes granular and opaque, and its movement ceases, that is, the cell is killed. But if cherry juice or other dye, to which the normal cell is impermeable, be added, it will be seen that, although the cell is dead, the membrane remains impermeable, since the dye does not enter. But suppose that we now replace the coloured isotonic solution by a hypotonic one. The cell expands by taking up water, but, contrary to what happens in the living cell, the membrane does not expand also, so that it gives way at one place or another ; the defect is not made good, the dye enters and slowly stains the whole of the protoplasm. One must not, however, hastily draw the conclusion that this semi-permeable membrane, after the action of hydrochloric acid, is the ' same thing as the natural one. The experiment -merely shows the possibility of producing a membrane similar to the natural one in its properties and situation.

Under certain circumstances the existence of an actual membrane can be made visible, although there is no proof that the membrane was in existence in the living cell in the same state as that seen. As already said, a membrane similar to that on the outer surface of the cell protoplasm exists also on the surfaces of the vacuoles enclosed within it. De Vries (1885) takes Spiroyyra and plasmolyses by immersion in 10 per cent, potassium nitrate coloured with eosin. After about an hour, the cells die, become stained and the red shrunken protoplast lies in a rose-coloured liquid situated between it and the cell wall. The vacuoles alone remain unstained and sometimes shell out of the cell as colourless balls, which slowly take up the dye. In this process it is seen that the surface layer becomes very deeply stained before the dye penetrates to the interior liquid.

At the beginning of the present section, it was pointed out that contents of the protoplasm, capable of lowering surface energy, are concentrated on the surface and are, in all probability, the origin of the cell membrane. The experiment just described suggests a further important point. The interface between two phases may be regarded as belonging to both phases, so that constituents of both phases will be concentrated there if they lower surface energy. This circumstance does not much concern the protoplasm of organisms like Amoeba or the cells of plants, for the most part, where the external phase is nearly pure water, but is of considerable importance in the higher animals, where the fluids in contact with the cells are of a highly complex composition. The difference seen in the experiment of De Vries, quoted above, between the outer cell membrane, which has been killed, and allows potassium nitrate and dye to pass freely, and the membrane of the vacuole, which is not for some time made permeable to them, suggests that the composition or structure of the membrane in contact with the contents of the vacuole is not the same as that of the outer cell membrane. This difference is probably due to the presence of substances in the vacuole, which contribute to the formation of the membrane.

It will be noticed that the view here taken as to the nature of the cell membrane implies that it is a variable thing as regards its composition, since this depends on the substances present in the protoplasm of the cell, and in the surrounding medium, at any given time. In a certain sense, it is, indeed, a part of the protoplasm, so that it is not to be wondered at that its permeability is capable of change with varying functional states of the cell. The fact that it is readily formed is shown by the experiment of Nageli, described above, where a new surface of protoplasm becomes rapidly covered with a membrane, having apparently the same properties as concerns permeability as the original one. That it can be reabsorbed is shown by the facts that pseudopodia of protozoa will fuse together, and that a number of amoeboid organisms, as in Mycetozoa, will unite to form a plasmodium. In the above sense, we may accept the view taken by Hbber (1911, p. 264), that the cell membrane is a living structure. In the way in which I regard it, it may be said to be a local concentration of integral parts of the cell protoplasm.

There is a certain amount of optical evidence of the existence of something on the surface of protoplasm distinct from the inner mass. Gaidukov (1910, p. 51, and Fig. 3s on plate v.) describes, in a germinating spore of a mycetozoon, the appearance under dark ground illumination of a reticulated appearance on the surface of the protoplasm ; this network had a violet colour, while particles in the endoplasm had a yellow colour. Osterhout (1912, ii. p. 114), also, saw an obvious change on the surface of protoplasm under the action of calcium. It is well to be cautious in the interpretation of these phenomena, owing to the possibility of diffraction effects.

The question of the chemical composition of the cell membrane has excited much discussion. Since lipoid substances, with cholesterol, are universal constituents of protoplasm, while they possess in a marked degree the power of lowering surface tension, it is practically certain that they must form an important part of the membrane. Now, Overton (1899) has advocated the view that the limiting membrane of the cell is essentially of a lipoid nature, and has supported this hypothesis by a large amount of powerful evidence, which it is important, as well as instructive, to examine somewhat closely. It is, in the first place, a very remarkable fact that, in the case of cells of the most various kinds, in the state in which they are usually investigated, the substances which easily obtain entrance into the cell are just those which are soluble in lipoids. In view of certain facts, to be spoken of later, it is, perhaps, more correct to say, that those substances in which lipoids are soluble, such as alcohol, chloroform, benzene, etc., and those which are themselves soluble

in liquids which dissolve lipoids, such as urea, fatty acids, some ammonium salts, etc., are found to penetrate the cell membrane. Those to which the membrane is impermeable are not dissolved by lipoid solvents; such are sugar, amiim acids, inorganic salts, mineral acids, etc. We note, for example, that sodium hydroxide, insoluble in benzene, does not penetrate, while ammonium hydroxide, which is soluble in benzene, readily does so. But it will doubtless occur to the reader that these two bases differ in many other ways besides that of solubility in benzene.

Again, Loeb (1909) showed that the lower fatty acids are more effective in modifying certain cell processes, such asthose involved in the fertilisation of ova, than the mineral acids are. The fact can be explained on the ground of the " lipoid solubility " of the former. The aniline dyes were made extensive use of by Overtoil (1899, ii.) to test the hypothesis, and it was found that those soluble in lipoids, that is, the salts of colour bases, passed into the cell, while those not soluble, salts of colour acids, did not. The meaning to be attached to the phrase " lipoid -solubility " in this connection will appear hereafter. We may note also that the "basic" dyes which enter, are uniformly electropositive as regards the coloured substance to which we direct our attention, while the "acid" dyes are electro-negative, so that lipoid solubility cannot be adduced as the only difference between the two classes. Further, just as remarked above with reference to the hydroxides of sodium and ammonium, it cannot be held that " lipoid-solubility " is the only difference between acetic and hydrochloric acids.

In fact, a layer of benzene shows the same selective permeability in respect of organic or weak bases and acids, on the one hand, and strong inorganic bases and acids on the other hand, as the cell membrane does, but no one* supposes that this membrane is composed of benzene. Benzene, however, does not dissolve even the "basic" dyes, although solutions of certain lipoids in chloroform, etc., appear to do so. It will be seen presently, however, that there is strong evidence that this js really an adsorption on the surface of the lipoid, which is only in colloidal solution.

Notwithstanding what has just been said, it seems from the work of Overton that we must admit that " lipoids " play an important part in the properties of the cell membrane, although we shall see later that it is impossible to assign the total composition of the cell membrane to them. Moreover, we shall find that there are difficulties in looking upon them as solvents in the ordinary sense. At this point, then, we may profitably consider some of the chemical and physical properties of the cell constituents to which the name " lipoids " has been somewhat loosely applied.

We find sometimes that all those substances extracted by alcohol are called lipoids. This is clearly calculated to cause confusion. Glucose, urea, free bases, such as choline, may be mentioned as being soluble in alcohol, but not of a lipoid nature. Overton himself includes cholesterol, although, strictly speaking, the name should be restricted to substances chemically related to the fats proper. For the present purpose, perhaps, it may be allowed to remain in the class of lipoids, owing to the similarity of its physical properties.

The simple ordinary fats, glycerol esters of both saturated and unsaturated higher fatty acids, are common constituents of the cell, but the most interesting are those complex fats, to which the name " lipines," with its derivatives, has been given by Leathes (1910). Lipines themselves are compounds of fatty acids with a nitrogen-containing group, but contain no phosphorus nor carbohydrate. Phospholipines contain phosphorus in addition, and are sometimes called " phosphatides," while " galactolipines " contain no phosphorus, but a carbohydrate group, galacto.se, and correspond to the cerebrins or cerebrosides of some authors. The most familiar of these lipoids is the phospholipine, lecithin, of which the formula is usually given thus : —

It may be looked upon as glycero-phosphoric acid combined up with one molecule each of oleic and palmitic acids, on the one hand, and with choline, a base, with the constitution of a tertiary amine, on the other hand. It is to be remembered, however, that other fatty acid radicals may take the place of oleyl or palmityl, and other bases the place of choline. The physical properties of this substance are the most important in the present connection, and they are somewhat remarkable. It is a soft, waxy, substance, soluble (probably in colloidal form) in chloroform, benzene, oil, and alcohol, rather less so in ether ; insoluble in cold acetone or ethyl acetate. Placed in contact with water, it tends to disperse, assuming the so-called "myelin" forms, like the pseudopodia of amoeboid organisms. If shaken up with water, it forms a colloidal solution of the emulsoid type, in which the internal phase consists of lecithin containing " imbibed " water.

Although the physical properties are the most striking, the chemical composition suggests important functions of a chemical nature, but what these are is at present very uncertain. When alcoholic solutions containing lecithin and glucose or certain proteins are evaporated to dryness, it is found that ether takes up from the residue adsorption compounds of lecithin with glucose or protein, substances normally insoluble in ether. It was at one time supposed that these were definite chemical compounds, but it has been shown that the proportion of the constituents varies with that in the original mixture and is never definite. The cases of "jecorin," which contains glucose, and of " vitellin," have been already discussed (page 66 above).

The relationship of lecithin to water is of much importance as regards the cell membrane. This membrane, with very rare exceptions, is freely permeable to water. Now, the true fats are not so, while lecithin, as stated above, easily swells up in water, and is therefore permeable to it. But, as Nathansohn (1904, p. 640) points out, in this state it has lost its power of being a solvent for lipoidsoluble substances only ; dry lecithin in solution in benzene dissolves the " basic " dyes only, but moist lecithin in benzene is also a "solvent" for the sulphonic acid dyes, to which the cell membrane is normally impermeable. Buhland (1909, p. 34) prepared membranes of lecithin and cholesterol in the manner of Pascucci (1905), and found that no dye, "basic" or "acid," diffused through cholesterol at all. Neither did this happen through lecithin membranes, which were completely impermeable until saturated with water : when this occurred, as could be seen from the fall of the water column in the cell, both kinds of dyes began to come through.

Loewe (1912) has recently published important work on the physical chemistry of these "lipoids." Kephalin is a substance closely related in its composition to lecithin and present in considerable amount in brain. As already mentioned, lecithin forms an obviously colloidal solution in water, and Loewe shows that kephalin, even in chloroform or benzene, is also in the colloidal state. The solutions show the Faraday-Tyndall phenomenon and an illuminated cone under the ultra-microscope. In solution in chloroform, the raising of boiling point is • too small to be detected, showing that the solute is in large aggregates, a fact also evident from vapour pressure measurements. Further, when swollen by the action of water, it becomes insoluble in ether. The reader will probably remember that, in the old Hoppe-Seyler method of extracting the lipoids from brain, it was necessary to dehydrate first with alcohol in order to make them soluble in ether. The meaning of this insolubility in ether will be apparent when it is remembered that presence of water does not affect true solubility in ether, such as that of picric acid, which is extracted by ether from its solution in water. Kephalin, then, is not in true solution in these various so-called "solvents" for lipoids.

This fact raises considerable difficulty in the interpretation of Overton's experiments with " lipoid-soluble " dyes and other substances. According to his view, a substance obtains admission to the cell because it is soluble in lecithin and similar substances. Take the case of methylene blue. This is insoluble, except to a minute degree, in chloroform, but, if kephalin be present in the chloroform, the chloroform-lipoid phase becomes deeply coloured when brought into contact with a watery solution of methylene blue. The explanation given by the adherents of the lipoid-membrane theory is that methylene blue is more soluble in kephalin than in water and that the staining of the lipoid is due to a true solution of the dye in it. Now Loewe brings strong evidence against this interpretation of the fact. Suppose that the dye is dissolved in true solution ; there is a certain ratio between its concentration in the water phase and that in the chloroform-lipoid phase, known as the " partition coefficient," and, if the molecular weight is the same in both solvents, this ratio will not vary with the concentration. Loewe finds, on the contrary, that the ratio varies very considerably with the concentration, but that it follows the parabolic law of adsorption, viz., the ratio varies as some power of the concentration. The exponent 1/n has values between O35 and 0'16, according to the particular lipoid used, kephalin, cholesterol, residual brain lipoids, etc. On the other hand, for each individual lipoid, the value is fairly constant. The conclusion drawn is that we are dealing with a case of adsorption, but it must not be forgotten, as Loewe appears to have done, that the partition between solvents also has an exponential ratio if the molecular weight of the solute is not the same in the two. Take, for example, acetic acid dissolved in benzene and in water ; in the former the molecular weight is double that in the latter, owing to the association of two molecules together. In such cases, the exponent expresses the ratio of the molecular weight in the two solvents, so that it must be a whole number.

Now, in the case of methylene blue in lipoid and water, a ratio of whole numbers can only be obtained by assuming a very large association in both solvents ; a quite impossible degree in fact as regards water, where, judging by its electrical conductivity, there is no association. It appears, then, that Loewe's interpretation is correct. Moreover, as this investigator poinN out, if the phenomenon is a partition owing to different solubility, the dye would readily be removed when the lipoid phase is put into contact with pure water. But this is not so, and the case is precisely similar to that of paper stained with the dye. It will be remembered that paper, owing to its negative charge, has a strong adsorptive power for electro-positive dyes and is in equilibrium only when a very deeply-stained paper is in contact with a very dilute solution of dye. So that, as Freundlich points out, very little dye is removed by pure water. Another fact observed by Loewe, which shows the staining of lipoid by methylene blue to be a surface condensation only, is that, if a mass of kephalin be placed in contact with a watery solution of methylene blue, the dye does not diffuse into the lipoid. Further, if a solution of dye, to which gelatine has been added in order to prevent mixing of the various layers, be covered with a layer of lipoid and over this water be placed, no dye passes into the water. Similar facts were noticed with regard to other substances supposed to be soluble in lipoids, such as narcotics, nicotine and tetanus toxin. As concerns other lipoids, cerebroside (a galactolipine) and the lipoid residue from brain after removal of kephalin and cerebroside, all behaved like kephalin. Cholesterol was found to obey the partition law, but dissolved very little dye.

Thymol in chloroform was found to be partly in a colloidal form, partly in true solution, but obeyed the adsorption law and not the partition law. In this last case, apparently, only the colloidal particles took up the dye. There is, finally, another difficulty involved in the acceptance of the solubility partition theory. If we take a particular case of Loewe's, say the first on Table II. (p. 161 of his paper), we see that the final concentration of the dye is greater in the lipoidchloroform phase than in the water phase. Remembering that the dye is practically insoluble, j^ chloroform itself, the result means that the solvent power of chloroform for £he dye has been raised by the addition of 0'5 per cent, of kephalin to at least that of water. If we compare this effect with the increase of the solvent power of alcohol for cane-sugar, produced by the addition of as much as 3'28 per cent, of water, which was found by Scheibler (1872) to be raised only to 0'36 per cent., we are compelled to admit the inherent improbability of explanation on these lines.

So far as Loewe's experiments go, it appears that a lipoid membrane, so far from being an assistance to the passage of " lipoid - soluble " substances into the cell, is rather of the nature of a hindrance, since it holds fast the substances instead of passing them on. At the same time, the fact has to be explained why it is just these particular things that enter the cell so easily, although some property other than partition according to solubility will have to be brought into the account.

Before proceeding further, a few words may be said as to cholesterol. The ubiquitous presence of this chemically-inert substance is a remarkable fact and suggests that it must have some important part to play in the regulation of the mechanisms of the cell. In strictness, it is not a lipoid, although for convenience usually reckoned with them. In chemical constitution, it is the monatomic alcohol of a substance related to the terpenes ; according to Windaus and Stein (1904), the complex terpene in question is methyl-isopropyl-phenanthrene. The most familiar terpenes are the essential oils of plants, cymene, for example, from oil of caraway seed and from oil of eucalyptus is methyl-isopropyl benzene. It is of some interest to find in the animal a representative of this class of substances so widely spread in the vegetable kingdom. Cholesterol is soluble in ether, benzene, chloroform and fats ; insoluble in water and in cold alcohol. In the work of Loewe, above mentioned, it was found that it could take up lipoid-soluble dyes to a very small degree only and apparently in accordance with the partition law and not with that of adsorption.

Although it is necessary to hold that lipoids form a part of the constituents of the cell membrane, there is reason to doubt that they are the sole substances taking part in it. For one thing, it is very difficult to understand how the permeability is capable of regulation by processes occurring inside and outside of the cell unless the membrane has a very complex composition. There is, also, more direct evidence that a more complex structure than a mere lipoid one is concerned, as we shall see presently. But, whatever explanation may be given of the fact, it seems certain that cells are always permeable to substances soluble in lipoid solvents, while being only at times permeable to those not so soluble, such as sugars, amino-acids and most salts. There are, as it were, two kinds of permeability, of which the latter one alone is subject to functional change.

The presence of more than one constituent in the case of the membrane of the red blood corpuscles is shown by the experiments of Ryvosh (1907) on haemolysis by saponin. This glucoside causes the corpuscles to break up by a kind of solvent or dispersive action on the cell membrane. It has remarkable powers of being adsorbed at interfaces between phases, driving out most other substances from this situation. At the same time, it is difficult to demonstrate that it lowers surface tension to any considerable degree. It is probable that this difficulty arises from the fact, discovered by Ramsden (1904), of the formation of a rigid filfti at the surface where its solution is in contact with another phase. We have seen that haemolysis is also brought about by mixing the blood corpuscles with a hypotonic solution. The phenomenon in this case is due to swelling by osmosis. Now the corpuscles of different animals have a different relative power of resistance towards these two methods of haemolysis, and in such a way that the corpuscles of some animals require the difference between their own osmotic pressure and that of the hypotonic solution, in order that haemolysis may occur, to be greater than those of other animals. Also those of certain species require a higher concentration of saponin than in the case of other species. The important point is that the more resistant a particular kind of corpuscle is towards saponin, the more sensitive it is to a hypotonic solution and vice versa. The two series below illustrate this fact, the most resistant species in both columns being at the top : —

The rabbit alone of all the animals tested fails to come into the corresponding place in the two series. It is evident that the constituent acted on by saponin is of a kind different from that which gives way when distended by osmosis. Again, while most lipoid solvents do, as a matter of fact, cause haemolysis, the absence of an effect on the part of pure olein, while a mere trace of an oleatc is sufficient, shows that the solvent action exerted on the lipoids of the cell membrane is not the chief factor. A surface tension and adsorption effect is rather suggested, leading to modifications in the colloidal state of the membrane.

Mines (1912, p. 226) has shown that red blood corpuscles behave to the agglutinating action of trivalent ions as if coated with an emulsoid colloid. Now a suspension of lecithin in water behaves rather as a suspensoid towards electrolytes (Forges and Neubauer, 1907), being precipitated by bivalent ions in low concentrations. These facts suggest that the composition of the cell membrane is rather that of protein than of lecithin. According to Pascucci (1905, p. 551), the stroma, or colourless portion, of the red blood corpuscles consists of protein, lecithin, cholesterol and a cerebrosidc. The greater part of this stroma forms the outer membrane. Various reasons are given for the belief that there is very little, if any, protoplasmic skeleton within the corpuscle, the chief reason being the separation, in certain conditions, of the whole of the haemoglobin in large crystals within the corpuscle, while nothing is to be seen of any protoplasmic substance between the crystals.

The same investigator made artificial membranes of lecithin and cholesterol (p. 555) by impregnation of a fine silk tissue, tied over the end of a glass tube, with the fnsed lipoid or mixture of the two. Such membranes were found to be attacked by haemolytic agents, saponin, cobra venom and tetanus toxin, in a similar way to blood corpuscles, becoming permeable to haemoglobin. Lecithin was much more readily attacked than was cholesterol. Lipoid solvents attacked both, as would be expected, but dilute sulphuric acid had no action. On the other hand alkalies, both ammonium and sodium hydroxides, rendered them permeable. In this latter respect they differed from the normal cell membrane, which, as we have seen, is permeable to the former, impermeable to the latter.