Principles of General Physiology

The experiments of Garmus (1912) on the living skin glands of the frog lead him to the conclusion that the penetration of dyes into the cells of these glands has no relation to their solubility in lipoids, since some of those that obtain entrance are insoluble in lipoids. Moreover, poisons like saponin, sodium fluoride and ether, which attack lipoids, do not affect the vital staining of the gland cells. It is possible, however, that secreting cells behave in a different way from the majority of other kinds of cells.

Peskind (1903, p. 420) comes to the conclusion, from experimental results which are not very convincing, that a "nucleo-protein" forms a constituent of the cell membrane, in conjunction with lipoids. In respect of the question as to the penetration of substances into cells on account of their solubility in lipoids, a certain confusion is apt to be made in tininterpretation of the action of such lipoid-soluble substances. It appears to be assumed sometimes that, if a particular substance, say chloroform, is more soluble in the lipoid membrane than it is in a watery liquid, the result will be that there is a greater concentration of the chloroform in the interior of the cell than in the surrounding liquid. On the contrary, if the solution inside the cell is the same as that outside, the concentration will be identical ; the fact of greater solubility in the lipoid only means that the concentration in the cell membrane itself is higher. The meaning of the " partition coefficient " is that there is a particular ratio between the j concentration of a substance in two phases, according to its relative solubility in them, so that, unless the interior of the cell has the same sol vent power as the lipoid itself, the "partition coefficient" applies to the membrane only and not to the cell as a whole. Whether the concentration is higher in the cell protoplasm depends on the amount of lipoids which this contains. When it is found, for example, that narcotics as a class

are taken up by the nervous system in greater proportion than they are by other tissues, this must be understood to mean that lipoid constituents are in greater proportion in this tissue. It does not necessarily mean that the protoplasmic substance of the nerve cell itself is exposed to a greater concentration of narcotic than that of other cells is. Experiments by Osterhout (1911) on Spirogyra show that the cells of this alga are permeable to both sodium chloride and to calcium chloride, as well as to many other salts, when present alone • the conclusion is drawn that the membrane is not lipoid, since these salts are insoluble in such substances. The remarkable fact was observed that a mixture of chlorides of sodium and calcium renders the membrane impermeable to both. These results are regarded as indicating a protein constitution. It appears to me, however, that caution must be exercised in interpreting them and that they indicate rather that a pure salt affects the membrane in such a way as to produce an abnormal permeability, which is of a temporary nature, since, in the experiments referred to, the cells were not permanently injured. From the fact that the natural cells were found to be isotonic with O375 molar sodium chloride, it must be concluded that they contain a considerable amount of osmotically-active crystalloids, which would diffuse out into the nearly pure water in which the cells normally live, unless the membrane were impermeable to salts.

That a simple protein membrane is insufficient to account for such impermeability is shown by the behaviour of some interesting protein membranes prepared by Newton Harvey (1912). When chloroform is shaken with solutions of eggalbumen, a membrane is formed on the surface of the drops by condensation of the protein in the manner described by Ramsden (1904). If these globules are allowed to stand in water, the chloroform diffuses out faster than water enters, so that they shrink ; if lecithin be dissolved in the chloroform previously to the shaking with the egg-white, water is taken up sufficiently rapidly to prevent shrinking and, if left in an open vessel, the chloroform disappears entirely in the course of an hour or two and there remains, inside the delicate protein membranes, a colloidal solution of lecithin, partly in the form of granules which are visible under the microscope. When a dilute solution of neutral red is added to a suspension of these artificial cells in water, the lecithin granules take up the dye by adsorption and become red, so that an opportunity is given to test the permeability of the protein membrane as regards alkalies. It is found, contrary to the behaviour of the living cell, which is impermeable to sodium hydroxide but permeable to ammonium hydroxide, that the two alkalies pass through the protein membrane at an equal rate. The membrane of the living cell is therefore of quite a different composition from that which condenses, on chloroform drops in a solution of egg-white.

Another instructive experiment made by the same observer is to take a solution of lecithin in benzene, instead of in chloroform, and to repeat the above procedures. The benzene of the droplets cannot, of course, diffuse away into water, but, if they be stained with neutral red and placed in ammonium hydroxide of O'OOOl molar concentration, the change to yellow is almost instantaneous, while even in O'l molar sodium hydroxide, it takes twenty minutes to produce the change. Ammonium hydroxide, in fact, is readily soluble in benzene-lecithin solution, while sodium hydroxide is not. But it is easy to show that wet benzene itself behaves in the same way.

Gelatine, stained with neutral red, is allowed to set in the bottom of an Erlenmeyer flask, which is then filled with water and inverted in a vessel of water. By means of a bent tube, benzene is passed up into the flask, where it forms a layer between the water and the gelatine. Various alkalies and acids can be added to the water in the flask by the same tube, and it will be found that benzene is permeable to ammonium hydroxide and to acetic acid, impermeable to sodium hydroxide and to hydrochloric acid, in fact it behaves like the cell membrane. According to these experiments, the cell membrane should be composed of benzene, which is absurd. Newton Harvey's experiment, in fact, tells us nothing as to the properties of lecithin when saturated with water ; according to Pascucci, as we saw above, a lecithin membrane is attacked both by ammonium and sodium hydroxides. None of these experiments, indeed, affords proof that the cell membrane is composed only of lipoid material.

When cells are killed by various means, their semi-permeability is, as a rule, converted into complete permeability. But there are some agents which, when dilute, have not this effect, although they kill the cell. Formaldehyde in 4 per cent, solution destroys the semi-permeability, but in 0*2 per cent, solution, this property is preserved in an apparently normal state for a considerable time ; so that, for example, Stewart (1901) was able to show that blood corpuscles, treated with dilute formaldehyde, retain their normal permeability for ammonium chloride and their normal impermeability for sodium chloride. Moreover, saponin and water cause the same change in permeability to ions that they do in living blood, although no laking takes place. It follows from these facts that the action of saponin or of water does not depend on liberation of haemoglobin, but must be exerted on the cell membrane. When the corpuscles, after fixation by formaldehyde, are extracted with ether, which presumably removes the lipoids, the conductivity of the corpuscles is increased and saponin has no further effect in this direction. The inference seems to be that lipoid substances are an integral part of the membrane and that the action of saponin is on these substances, although the possibility is not to be forgotten that ether may produce other alterations in the nature of the membrane, apart from abstraction of lipoids.

A remarkable effect on blood corpuscles produced by cobra venom has been described by Noguchi (1905). Like snake poisons in general, this is haemolytic in low concentrations, but different species of animals vary much in their sensitiveness to this effect. In great e\ cobra venom is not hsemolytic ; on the contrary, it prevents the hsemolytic action of saponin. Even water, several times renewed, has no action on corpuscles subjected to the action of large quantities of cobra venom. It seems impossible to explain this fact except on the hypothesis that the membrane has become actually impermeable to water, as if converted into wax or india-rubber. When washed with sodium chloride, their normal behaviour to water is restored. It seems that some constituent of the membrane enters into combination with the poison, forming a substance which is insoluble in water, but decomposed by sodium chloride.

Ether and chloroform, like formaldehyde, have a different action in dilute and in concentrated solutions. In the latter, they increase permeability, in the former, they decrease it. Osterhout (1913) has shown this in the case of Laminaria by conductivity measurements. It is to be noted, however, that the decreased permeability is the reversible one and not associated with permanent injury to the cells, so that it seems to be the normal narcotic effect. Further facts will be found under the head of narcosis below.

A point to be remembered is that it is not to be assumed that, when a cell is killed, the semi-permeability of its membrane is necessaiily lost. It may be fixed in some way. A cell, dying naturally, may become surrounded by a tough impenetrable membrane. Penard (1890) made the following interesting observation. An Amoeba, while living, had taken in the egg of a small worm. After the death of the Amoeba, the egg hatched, but the worm was unable to escape through the surrounding membrane.

Heat, applied gradually, destroys the semi-permeability of the membrane. EVen at 40° the pigment escapes from the red beet. A sudden rise of temperature to 100° appears to be a useful fixing method for certain histological purposes, but what its effect on the membrane may be, I am unable to state. What conclusions may we, justifiably, draw from the various experimental data of the preceding pages? In the first place, it seems certain that the membrane consists of substances in the colloidal state. The marked effect of electrolytes shows this, especially the fact that valency plays an important part.

The following observations of Szucs (1910) on the diminution of the permeability of Spirogyra to methyl violet are of interest. In order to produce a particular depth of staining in eight minutes, the concentrations required were of potassium nitrate, 0'08 molar ; of calcium nitrate, 0-04 molar ; of aluminium nitrate, O'OOOo molar. It will bo seen that the effect is in relation to the valency of the cation, which probably acts in a coagulating manner

on the colloids of the cell membrane. Another important fact in this connection is that blood corpuscles are much more sensitive to saponin when suspended in isotonic sodium chloride than in isotonic cane-sugar, as found by Handovsky (1912, p. 413). For example, 0'002 per cent, saponin produced 98 per cent, haemolysis in the former cafe, but only 20 per cent., under similar conditions, in the latter. The way in which this effect is produced is not quite clear. Although saponin may not be in colloidal solution in water, the experiments of Dumanski on molybdenum oxide, referred to on page 95 above, suggest that the presence of electrolytes may cause it to assume the necessary aggregated condition, and thus the electrolyte, also changing the sign of the charge on the corpuscles, may facilitate adsorption by electrical means.

r In the second place, there are reasons, as we have seen, for rejecting the "•Hypothesis of a membrane consisting of a simple kind of substances, lipoid or protein, alone, and for regarding it as a .complex colloidal system of all cell constituents, together with those of the outer liquid, which diminish the surface Lepeschkin (1911), in fact, comes to the conclusion, as the result of an elaborate series of experiments, that a simple mosaic structure of lipoid and protein is not a satisfactory hypothesis, but that a colloidal complex is necessary. The effect of the addition of varying proportions of glycerol or castor oil to the collodion of which an artificial membrane is made will occur to the reader (page 95 above). The function of lipoids is suggested by Lillie (1912, 2, p. 17) to be that of increasing the stability of the other colloids, in fact as a protection from excessive aggregation, as described in the preceding Chapter (page 97).

Although lipoids must enter into the composition of the membrane, it seems evident that their relationship to substances which are supposed to be "lipoidsoluble is not that of solvents, in which case the laws of partition would be obeyed, but rather that of surface adsorption, owing to their state of colloidal dispersion. A colloidal solution of lecithin in benzene behaves quite differently from one of benzene in lecithin, that is, according to which is the external or continuous phase and which the internal or dispersed phase. .

Ruhland (1913) gives strong evidence that, at all events as regards dyes and enzymes, permeability is not a question of solubility in the membrane, but of the dimensions of particles or molecules; that is, the membrane may be looked upon as a sieve. In this paper a full account of the literature on the subject is given. An important point to remember is that the membrane must not be looked upon as an invariable permanent structure. Its permeability can be changed by reagents applied to the outside, as in the experiments of Osterhout, where sodium salts make it permeable to the Na ion, while the addition of calcium re-establishes the normal state of semi-permeability ; other cases have been given above. Functional changes of the cell itself are also associated with changes in permeability, as will be shown in the next section. If, however, we look upon the cell membrane as an integral part of the protoplasmic system, as locally concentrated constituents of the cell, this behaviour will not seem so difficult to understand.

Supposing that the cell membrane becomes impermeable to substances to which it was previously permeable, what effects may be expected to follow ? We know that, in a reversible reaction, the position of equilibrium depends on the relative concentration of the constituents of the system. Such a reaction will therefore continue to take place in one direction if the products are allowed to escape from the cell, but, if the membrane becomes impermeable to them, the reaction will come to an equilibrium and cease.

Take the case of starch or glycogen stored in a cell, which cell also contains an enzyme capable of causing their hydrolysis to sugar ; if the membrane is impermeable to this sugar, the reaction soon comes to an end, partly on account of the back reaction, -partly because the action of the enzyme is more or less paralysed by the accumulation of the products of i activity, as we shall see in Chapter X. But, as soon as the products are allowed to escape again, the reaction starts afresh. This consideration applies to any reversible reaction taking place in the cell.

From the powerful action of electrolytes on colloidal systems, such as that of protoplasm, it will readily be understood how important are changes in the permeability of the membrane to these substances. That such changes occur is indicated, amongst other facts, by the experiments of M'Clendon (1912, 2), who found in excited muscle an increase of electrical conductivity, an index of increased permeability to ions, such as we have seen to happen in Laminaria under the influence of substances which increase the permeability of the cells.

It might be thought, perhaps, that the separation of electrolytes from an adsorbed state, owing to diminution of the active surface of the colloids in the cell by aggregation, as suggested by Macdonald (1909, p. 44), would account for this. But we know that the cell membrane is, under normal conditions, impermeable to ions, and acts as a non-conductor, so that increased production of ions inside the cell, apart from increased permeability of the membrane, would have no effect on the electrical conductivity of the tissue.

Lillie (1911), also, has brought forward evidence to show that all agents which cause increased permeability of the cell membrane act in an exciting manner. This is very noticeable in the case of the larva of Arenicola, which contains a yellow pigment to which the membrane is normally impermeable. When placed in pure sodium chloride, isotonic with sea water, the cells become tonically contracted, while at the same time pigment leaves them. This action of sodium chloride is prevented by calcium or magnesium ions, just as the increased permeability of Laminaria produced by sodium ions is prevented by calcium. Further discussion of the mechanism of muscular contraction will be found in Chapter XIII. One interesting consequence may be noted here. If the state of capability of being exeited to contraction is connected with the semi-permeability of the membrane, it follows that when this state is changed into one of permeability the cell will be inexcitable as long as the state lasts; hence the "refractory period."

The observations of PfefFer (1873) on the movements of the sensitive plant showed their cause to be the sudden disappearance of turgor in the cells of the pulvinus, due to loss of semi-permeability and, therefore, of osmotic pressure, due to escape of osmotically active substances, so that water also is pressed out. Narcosis. — There is a group of substances which act on living cells in such a way as to abolish temporarily those activities which we regard as manifestations of life. These are called " narcotics " or " anaesthetics." The former name means " making numb " or paralysing, while the latter obviously refers to abolition of conscious sensation, so that, in general use, the former is used to apply to the abolition of all forms of protoplasmic activity, including those of the nervous system, while the latter, strictly speaking, should refer only to consciousness. But, in point of fact, the substances themselves form one and the same group and the names are frequently used interchangeably.

As first pointed out by Hans Meyer (1899) and by Overton (1901), independently, the intensity of the narcotic action of a substance stands in relation to its partition coefficient between fats or lipoids and watery liquids ; the more soluble it is in the former, the greater its effect. Now, although this fact shows how a narcotic obtains access to the interior of a cell, it does nothing more in explanation of its action than to suggest that it is in some way exerted on the boundary membrane. As pointed out above, the greater solubility in the membrane would only entail a greater degree of activity if this were due to some direct action on the membrane itself. The concentration in the water phase of the cell would not be increased by mere increase of solubility in the membrane alone.

What evidence, then, have we as to the action of narcotics on the permeability of this membrane ? As pointed out by Lillie (1912, 1), the property of rendering cells temporarily irresponsive to stimuli belongs to the most diverse classes of chemical compounds. The action of isotonic cane-sugar on muscle (see page 125 above) may be mentioned. At the same time, the particular group known as " anaesthetics," par excellence, such as ether, chloroform, alcohol, etc., are characterised by special activity of this kind, which seems undoubtedly to be connected with lipoid solubility. On the other hand, the mere fact of lipoid solubility does not make a substance an anaesthetic.

For example, capryl alcohol is a powerful anaesthetic, its " critical concentration " (Overton) being 0'0004 molar, compared with ethyl alcohol at O3 molar, that is, it is 750 times as powerful as ethyl alcohol. But benzene is a very poor anaesthetic, although its lipoid solubility is as great as that of capryl alcohol. The fact that benzene is only slightly soluble in water does not account for the fact, since, according to Rothmund (1907, p. 75), it is soluble in water to the extent of 1 '4 parts in 2,000, while capryl alcohol is only soluble to the extent of 1 part in 2,000.

If lipoid solubility were the only condition making a particular substance a narcotic, it would be expected that the greater the lipoid content of 'an organ, the less would be the concentration of a certain narcotic required to produce its effect. Although this applies when we compare the central nervous system with other tissues, it has been shown by Choquard (1913) that it does not hold in the case of the heart muscle and skeletal muscle. The former, according to Erlandsen (1907), is considerably richer in lipoids than the latter and should therefore be more sensitive to all lipoid-soluble narcotics. Choquard's experiments show numerous exceptions.

We turn now to experiments with regard to the effect of anaesthetics on permeability. According to Lillie (1911), there is a general parallelism between the effect of agents in producing a state of excitation and their power of increasing the permeability of the cell membrane. If this is so, we should expect the opposite effect on the membrane to be produced by narcotics, which abolish the excitability. Lillie himself (1912, 2) has shown that the action of sodium chloride in causing excitation in the Arenicola larva, along with escape of pigment, is prevented by ether, alcohol, chloroform, and chloretone. He draws the conclusion that the characteristic effect of these substances is produced by an action on the cell membrane, making it more resistant to the action of substances which tend to increase its permeability. Although lipoid-soluble anaesthetics enter the cell immediately, the fact that magnesium chloride is a powerful anaesthetic, although it enters the cell with extreme slowness, indicates that the effect is essentially on the boundary membrane itself. Further evidence of the same nature is given in the experiments of Osterhout (1913) on Laminaria. In ether of 1 per cent., the electrical resistance of the cells rises, showing a decrease of permeability to salts, in 3 per cent., after a preliminary rise, the resistance falls and the tissue is killed. After exposure to 1 per cent, ether, recovery is complete ; since recovery is a distinctive mark of anaesthetic action proper, it is reasonable to hold that it is the diminution of permeability which is associated with this effect.

As to the way in which the state of the lipoid constituents of the membrane is modified, we are as yet in the dark. A purely solvent action is precluded, since the lipoids would be washed away and the state be irreversible. This has been pointed out by Overton (1901, p. 51). There is no evidence that the state of colloidal dispersion of the lipoid is altered, that is as regards the number of particles. Lillie holds (1912, 1, p. 395) that the lipoid particles must increase in size by taking up the anaesthetic. In fact, Calugareanu (1910, p. 100) has seen this to occur in lecithin suspensions when ether or chloroform is added. It is evident that such a process would tend to decrease the interstices or pores of the membrane, if such a sieve-like structure be accepted, and that it would be reversible. According to Loewe (1913), narcotics change lipoids from lyophile into lyophobe colloids by surrounding them with an impermeable layer. Hence, such agents would diminish permeability so far as the water in the colloidal particles acted as a solvent, or carrier for solutes. No excitation would be possible in this state, because the membrane cannot be made permeable. It is found, in fact, experimentally, that narcotics lower permeability. Are the lipoid particles robbed of their water or not? The decrease of permeability indicates the latter, for otherwise they would shrink and allow more watery space for diffusion. On the other hand, the irreversible increase of permeability, leading to death, may well be due to actual dissolving away of the lipoids. The investigations of Czapek (1911) have shown that there is a close parallelism between the power of the various alcohols in killing cells and their power of lowering surface tension. As already mentioned (page 52), when the surface tension at the cell membrane is lowered to a certain degree, death results.

This phenomenon is undoubtedly connected with the various degrees of lipoid solubility shown by the series of alcohols. It is difficult to say whether the surface tension as such plays any important pa,rt. There is no doubt that adsorption of active substances, including narcotics, by the constituents of the cell membrane must play a considerable part, and indeed Straub (1912, p. 11) regards the adsorption theory as the most satisfactory one in respect to alkaloids.

An experiment by Calugareanu (1910, p. 101) shows that lecithin does not distribute it.-dt between water and chloroform according to the usual rules of relative solubility. If chloroform be shaken up with an equal volume of a 0 '5 per cent, watery lecithin "solution," instead of the lecithin oeing extracted by the chloroform, in which it is greatly more soluble than in water, what happens is that the chloroform layer only contains 8 per cent, of the total lipoid present, the rest is still present in the watery phase, but has taken up 50 per cent, of the chloroform. No doubt this behaviour is connected with the state of the lecithin as an emulsoid colloid, especially in presence of chloroform.