Principles of General Physiology

An important fact emerges from the above experiments of Bethe and Warburg. That is, that acid and alkali can produce their characteristic effects without entrance into the substance of the cell. This question will be referred to again later. Jacques Loeb (1909), in investigating the effect of acids on the formation of the fertilisation membrane in the eggs of the sea urchin, found that this effect was not in proportion to the strength of the acids, but to their permeability or

lipoid solubility. In fact, the mineral acids were far less active than the fatty acids. • Hustin (1912, p. 334), in perfusion of the pancreas with saline solutions, found that, if these were hypotonic with respect to the normal blood, the concentration was increased by passing through the blood vessels of the gland. If hypertonic, the concentration was diminished. The explanation on the basis of semi-permeability of the gland cells is simple ; these cells would take up water from a hypotonic solution in order to equalise their osmotic pressure to it and give up water to a hypertonic solution. No satisfactory explanation is apparent on any other view. No change takes place in the composition of the perfused fluid if the cells have been killed by sodium fluoride, so that their semi-permeability is abolished.

The ratio of the sugar content of blood corpuscles to that of the plasma is very variable, although as a rule higher in the plasma than in the corpuscles. The addition of glucose to the blood sometimes raises the content of the corpuscles, sometimes not (Hober, 1912, 1). It is difficult to give an explanation of these facts. It seems that the conclusion must be drawn that the corpuscles are capable of being made permeable or impermeable to glucose, but that their usual condition is that of impermeability.

At this point it is well to call attention to the remarks justly made by Hober (1911, p. 244) to the effect that it is impossible to account for the constant difference in the ratio of potassium to sodium in the blood corpuscle and other cells compared with that in the plasma, which bathes them, except on the hypothesis of complete semi-permeability. If these salts were able to diffuse out, however slowly, equilibrium must result sooner or later, unless the extremely improbable assumption be made that the corpuscles and other cells obtain a continuous supply of salts from some source other than the blood and that the latter is able to get rid of them as fast as they pass in.

We now come to the third set of facts proving the semi-permeability of cells towards salts, namely, those connected with the electrical conductivity of cells. A few preliminary words of explanation are desirable. When an electrical current is passed through a solution of a salt by means of wires dipped into it, the transport of electricity from one wire to the other is effected by means of atoms or molecules, each carrying a definite amount. These along with their charges, which ditlcr according to the valence of the carrier, are called ions. The unit charge, carried by a univali-nt ion, is known as an electron. A bivalent ion carries two electrons and so on. Imagine a flock of sheep at one side of a field and that they start to run to the other side ; the amount of wool (= electricity) which arrives at the other side in unit of time depends on the number of sheep and on the freedom of the course. Suppose that there are a number of square pens in the middle of the field, each fenced round and separated from the neighbouring pen by a narrow interval, the number of sheep now getting across in unit time will be much less than before, because they have to wait for each other to get through the openings, or rather, they obstruct one another.in their efforts to get through. We may say that less wool passes across per unit time, or in electrical terms, the conductivity is less. Further, matters would not be improved if the closed pens were full of sheep, since these sheep would not be able to help in the transport. On the other hand, suppose the cross-fences were removed, the enclosed sheep could get out and cross the field, while the originally free sheep would have as clear a course as if no pens were there.

Living cells, as regards the transport of electricity, are like the enclosed pens with sheep in them and are in the same way obstructive to the passage of ions by filling up part of the channel. Whereas, if we make their membranes permeable to salts, the resistance is removed. This fact, in the case of the blood corpuscles, was described in detail by G. N. Stewart (1897) and made the basis of a method of determining the relative proportion of corpuscles and plasma in blood (1899).

Osterhout (1912) also finds that living cells of Laminaria are impermeable to the salts of sea water, as shown by their taking no part in the conduction of an electrical current. They are made conductors by any agent which kills the protoplasm, such as heat, chloroform, and so on. The'ir permeability also can be changed reversibly, as will be seen later. M'Clendon (1910, p. 255) finds that the eggs of sea urchins massed together have a conductivity greatly inferior to that

of sea water, and regards the fact as being due to impermeability of the cell membrane to ions. The fact that a membrane being impermeable to salts makes it a non-conductor is shown in an interesting way in the method used by Morse and Horn (1901) in preparing copper ferrocyanide cells. By passing an electrical current through the membrane from copper sulphate outside to potassium ferrocyanide inside, the imperfect places are filled up and the resistance of the membrane gradually rises ; for example, in one case reported by Berkeley and Hartley (1906, p. 487) the resistance of a membrane rose from 2,700 ohms to 300,000 ohms.

Although the resistance offered by living cells to the passage of a current of electricity is explained simply and satisfactorily by the existence of a membrane which is impermeable to salts, it must not be overlooked that other explanations have been advocated. It is very difficult or impossible to prove experimentally that cells are complete non-conductors, owing to the practical impossibility of removing all external electrolytes from the solution bathing them, except by means which affect the normal state of the membrane. We cannot, therefore, make the definite statement that cells are actual non-conductors, so that there is a possibility that their high resistance may be due to the presence of electrolytically dissociated colloids, enclosed in a membrane impermeable only to colloids. This circumstance would, as we shall see more in detail later, oppose the passage of a current in one direction entering the cell, and in the opposite direction on leaving it, since the one ion is imprisoned. It may be objected to this view that the presence of such colloids in the blood corpuscles has not been proved.

If the electrolytes within the cell were combined with the cell-proteins, in the form of non-dissociated salts, they would be non-conductors, since ions only can convey a current. But there is no experimental evidence to warrant an explanation of the facts of the case on such an assumption. Reasons have also been given previously to show that adsorption is insufficient as an explanation, since an adsorption compound exists only in presence of free electrolytes in the liquid phase with which it is in contact. Free electrolytes must, therefore, be present in the interior of living cells. Their existence in that situation has been, in fact, demonstrated experimentally by Hober in two ways.

The first of these (1910, 2) depends upon the fact that the capacity of a condenser is increased when a conducting stratum is introduced into the dielectric between the plates, and the amount of the increase is proportional to the conductivity of the stratum. It will be clear that there is no question of ions being able to leave the cells in such a case. By this method, the internal conductivity of blood corpuscles, after repeated washing with cane sugar solution, was found to be about the same as that of a decinormal potassium chloride solution. The second method (1912, 2) is founded on an experiment by J. J. Thomson (1895). A conducting body, placed in the axis of a coil of wire through which a rapidly-alternating current is passed, diminishes the strength of this current by damping the vibrations, and it does this in proportion to its own conductivity. By this more sensitive method, the content of blood corpuscles in free electrolytes showed itself to be equal to that of a O'l to 0'4 per cent, solution of potassium chloride. The method was afterwards improved (1913) so as to require less material, and at the same time to be increased in sensibility. Frog muscles were also investigated by its means, and found to have an internal conductivity equal to 0*1 to 0-2 per cent, sodium chloride.

Comparing this number with the analyses of Fahr (1909), we note that a part of the salts must be adsorbed on the colloid surfaces, or in chemical combination in some form other than a dissociated salt, so that this part does not contribute to the conductivity, which is less than what would be given by the total salts of Fahr's results. It is also of interest to note^that the above value of the internal conductivity of muscle cells was obtained after six hours' soaking in isotonic cane-sugar, so that the membrane had not allowed the electrolytes to escape from the cell.

It has been suggested by Roaf (1912, i. p. 146), as indicated above, that the properties of a colloidal salt, in allowing a current to pass through a membrane in one direction only, might account for the high resistance of cells, without the necessity of a membrane impermeable to crystalloids. I showed indeed (1911, u. p. 242) that if a salt, of which one ion only is in the colloidal state, be separated from water by means of a parchment paper membrane, and an electrical potential difference established by placing electrodes, one inside, the other outside the membrane, then it depends on the sign of the electrode compared with that of the colloidal ion whether a current passes or not. Suppose we have a sodium salt of a colloidal acid, such as caseinogen or Congo-red, and that the electrode in this solution is the positive one or anode. The current must pass through the membrane from inside to outside ; that is, positively charged ions must pass through to the negative electrode and negative ions from outside to inside and be discharged there ; unless this can happen, no current will pass. Now, sodium ions can freely pass through the membrane and the opposite negative ions are already inside, so that current will flow when the internal electrode is the anode. On the contrary, if the outer electrode is the anode, in order that a current shall pass, the negative ions must reach it. This cannot happen, since there is an impassable barrier between them and the electrode.

Such conditions would clearly account for the resistance of cells to the passage of currents. The boundary surface on the one side of the cell would oppose currents in one direction, and that on the other side, those in the opposite direction. They would appear to be non-conductors. But it is to be remembered that this state of affairs holds only as long as the colloidal ion is the only one available of the right sign. Jf any diffusible ion is present, the current will pass by means of it, and we know that there are in the cells inorganic ions of both signs. A high resistance might be accounted for by the existence of most of the inorganic constituents of the cell in the form of salts with colloids, while the noncolloidal salts of the cells and the plasma of the blood were freely diffusible. But, as we have shown (page 1 20), if this were the case, the ratio of the different cations, say of potassium and sodium, must be the same inside and outside the blood corpuscles, and this is not what is actually found.

It appears from the preceding section that we must regard the surface membrane of cellSj.at all eveiftsTin the condition in which they are usually investigated, as Detligmipermeable both to colloids and to the majority of crystalloids. There are, however, certain substances— ammonium salts, urea, glycerol, alcohol, etc. — to which the membrane is more or less permeable at all times. When placed in hypertonic solutions of these, there is a preliminary plasmolysis or shrinking of the cell, greater or less according to the diffusibility of the solute, but this disappears as the concentration becomes equal on the two sides of the membrane.

On the other hand, we know that it is necessary for cell processes that such things as glucose and amino-acids, which are usually unable to pass the membrane, should get into the cell. For this reason certain recent work, showing that it is possible to produce reversible changes of permeability without killing the cell, are of great importance. Osterhout (1912) showed, as already stated, that the cells of Laminaria are impermeable to the ions of sea water, when immersed therein. But, if immersed in pure sodium chloride of the same conductivity (and temperature) as sea water, their conductivity rapidly rises, until they oppose very little more resistance to the passage of the current than the salt solution itself does. If the exposure to the sodium chloride has not been too prolonged, the normal state of the cells is recovered on return to sea water.

It may be remarked, in passing, that this fact seems impossible to account for on the view of the membrane being only semi -permeable as regards colloids ; for it would be necessary t<> assume that it becomes permeable to colloids under the action of sodium chloride ; in \vhirh case the protoplasmic substance of the cells would diffuse away and no recovery be possible on replacing in pure sea water. Lillie (1909) found that the larva of Arenicola, if placed in pure sodium chloride, isotonic with sea water, constricts up and the pigment contained in its cells diffuses out freely. This pigment is soluble in water, and does not appear to be in colloidal solution. The addition of one volume of 0*5 molar calcium chloride

to 24 volumes of the 0*5 molar sodium chloride prevents the contraction, and also the loss of pigment. Fluri (1909), again, found that three days' immersion in O01 per cent, solution of aluminium sulphate makes Spirogyra permeable for most salts as well as glucose, and that the effect can be removed, so that the cells become normal again, by return to pure water. Newton Harvey (1911, p. 546) states that sodium salts makes the membrane of Spirogyra and of Elodea permeable to sodium hydroxide, to which, as we have seen in Warburg's experiments, it is normally impermeable.

Another fact which may be mentioned is that M'Clendon (1912, i. p. 296) found that the eggs of Fundulus lose magnesium in pure sodium chloride solutions. Siebeck (1913) showed that frog's muscle, if immersed in isotonic potassium chloride, swells, showing that the action of the potassium salt is to diminish or abolish the impermeability to potassium, which the muscle normally possesses in the presence of sodium and calcium. Wachter (1905) showed that the passage of sugars from the cells of the onion was inhibited by the presence of potassium nitrate.

Osterhout (1910) shows that the root hairs of Dianthus barbatus, grown in distilled water, contain no crystals of calcium oxalate. If the water be changed for a solution containing calcium salts, the crystals soon make their appearance. They may easily be detected by observation between crossed Nicols in the polarising microscope. Gerard (1912) found that, on feeding animals with excess of potassium salts, the blood maintains its constant composition, while the cells of the tissues lose sodium.

-These various facts are given in order that the reader may grasp the fact that the cell membrane is capable of changes in its permeability. Instructive experiments may easily be made with slices of the root of the red beet. It will be found that the pigment does not leave the cells when immersed in tap water. (It is well to rinse the slices previously for a minute or two in tap water in order to remove the contents of the cells which have been injured in the process of cutting the slices.) If, on the contrary, they be placed in pure sodium chloride of O31 molar ( = 1 '82 per cent. ) strength, which is about isotonic with the cell contents, the pigment will gradually come out. Addition of 0'17 per cent, of calcium chloride to the pure sodium salt prevents this effect. It is convenient to take 3 '64 per cent, solution of sodium chloride and to dilute it with an equal volume of water or of 0'34 per cent, calcium chloride as the case may be. Many other experiments on permeability may be made with the red beet ; chloroform, bile salts, soap, warming to 50°, all cause loss of pigment, but in most cases the cells are killed. If it be desired to make quantitative experiments, the cane-sugar, which escapes along with the pigment, may be estimated by an appropriate method. In this case, the slices to be compared must, of course, be of equal dimensions.

It seems evident from the various instances quoted that calcium must produce some change in the properties of the cell membrane and of such a kind as to make it less permeable, and that sodium has the opposite effect. Osterhout (1912, ii. p. 114), in fact, states that visible effects are to be detected under the action of calcium. This antagonistic nature of calcium and other ions is of much importance and will require treatment in Chapter VII. A matter of some practical importance is the action of cane-sugar on the cell membrane. For the investigation of the effect of various salts, it is necessary to have cells suspended in an isotonic solution of a non-electrolyte. Now, while cane-sugar appears to be the least injurious, and at the same time convenient, especially if not in contact with the cells for too long a time, there are several facts which show that it increases the permeability of the membrane if the contact is prolonged. Bethe (1908, p. 560) found that the contractions of meduste were slowed if one part of isotonic cane-sugar was added to nineteen of sea water. Magnus (1904, p. 131) found that the movements of the excised intestine in Ringer's solution were weakened by the addition of cane-sugar above 0'02 per cent. Kiister (1909) noticed that, on plasmolysis of the cells of the onion in hypertonic cane-sugar, the protoplasm broke up into separate clumps and that, on placing in water, these clumps did not fuse together again, while the surface membrane seemed to be fixed or coagulated. According to Bang (1909, p. 263) blood corpuscles give up salts to isotonic cane-sugar, after prolonged contact with it. Muscle, on the contrary, is relatively resistant to the action of cane-sugar, giving up in twenty-two hours to repeated changes scarcely more salts than those contained in the spaces between the cells (Fahr, 1909). Overton (1902, ii. p. 349) showed that a muscle, which had lost its excitability by lying in cane-sugar solution, owing to removal of sodium salts from between the cells, quickly regains its excitability when placed in sodium chloride, so that no permanent injury is inflicted.

A further practical point of some importance is that, when a substance is found to penetrate into a cell, the conclusion must not hastily be drawn that the cell is normally permeable to this substance. The experiments of Osterhout (1912), in which the cells of Laminaria were found freely permeable to sodium chloride when this salt was present alone, but impermeable to it when calcium was also present, are sufficient to prove the contrary. In fact, statements regarding permeability to any particular substance can only be held to be valid when the proof is given that the membrane is in its normal state, a proof that is not always given, and one which, as must be confessed, it is not always easy to give.

There are certain other substances, in addition to electrolytes, which produce changes in permeability. The most important of these are those known as anaesthetics or narcotics and will be discussed in a succeeding section of this chapter. Certain functional states of the cell are known to be accompanied by changes of permeability ; the state of excitation produced by stimuli in contractile tissues appears to be accompanied by increased permeability to electrolytes ; this will be discussed later.

Lepeschkin (1908) finds that the permeability of plant cells is increased by exposure to light. The question was worked out further by Trondle (1910), especially with respect to the relation between the amount of change and the intensity of the illumination. The bearing of this fact on the explanation of the movements which take place under the action of light is obvious. Diminution in permeability produces a fall in the concentration of osmotically-active substances in the cell, the osmotic pressure and turgor consequently fall in value, so that opposing forces are able to bend the side of a stem exposed to light. Hence the heliotropic curvature. V. H. Blackman (1914) also finds that light causes increase of permeability in the pulvinus of the sensitive plant, described on page 431 below.

Again, the great variation in the relative concentration of sugar in the blood corpuscles and the plasma, and the manner in which changes in the concentration in the plasma affect that in the corpuscles, serve to show that the permeability of blood corpuscles is not a fixed and unalterable thing. The following data from a paper by Sober (1912, i.) will illustrate the point: — An increase of glucose concentration in the blood was produced in various ways, adding glucose to shed blood, and determining the distribution between plasma and corpuscles after standing, giving adrenaline to the living animal, extirpation of the pancreas, or a large amount of glucose introduced into the stomach.

There is clearly no question of parallelism, as would be the case if the corpuscles were always permeable to glucose; neither does the content of the corpuscles remain constant, as would be the case if they were always impermeable. As a rule, rise in the content of the plasma is associated with a rise in that of the corpuscles, but not in invariable proportion. The facts suggest the possibility that the normal semi-permeability of the membrane to glucose is connected with a particular difference of concentration on the two sides, but that the actual value of this difference may be changed by other influences. The membranes may be, as it were, tuned to different concentrations of glucose by the action of other substances. Similar conditions may perhaps apply to cells in general, but the data as yet available are not sufficiently decisive.

The action of electrolytes on the permeability of the membrane suggests that electrical forces play a part in the phenomena. The relation to precipitation of colloids will occur to the reader. It seems also possible that the presence or absence of an electrical charge on the membrane itself may be of importance in determining the permeability to ions. Suppose that a membrane has a negative charge, it would, to a certain extent, oppose the passage of electro-negative ions. Certain experiments by Girard (1910, p. 479) seem to support this view. A membrane of gelatine allowed magnesium chloride to pass more freely when given a positive charge by the presence of a trace of acid. The change produced in the structure of the membrane, however, must be taken into consideration. In any case, it is difficult to see how the presence of an electrical charge could exercise a permanent influence on the distribution of an electrolyte between the two sides of a membrane, although the time taken to attain equilibrium might be affected, a factor of importance in rapid changes of state. The experiments of Mines (1912) on the production of potential difference will be referred to in Chapter XXII.

The work of Overton (1899, i. and ii.) has shown a striking correspondence between the nature of a dye, as the salt of a colour-acid or a colour-base, and its passage into cells. While the cell membrane is impermeable to the former, it is readily permeable to the latter. The fact is brought by this investigator into relation with lipoid solubility and the lipoid nature of the membrane, a question to be discussed presently. Here, we may direct attention to the fact that these two classes of dyes, or the coloured ions into which they dissociate, have opposite electrical charges. The so-called " acid " dyes, that is, those in which the coloured part of the salt is the acid radical, are electro-negative, while the " basic " dyes are electro-positive, a fact which would undoubtedly have much influence on their adsorption by constituents of the membrane and of the cell itself. In fact, Endler (1912) has shown that the rate at which the diffusible dyes enter the cell is greatly affected by the presence of various electrolytes and brings the fact into relation with changes in electric charge, although it does not seem quite clear whether the effects described by him are not rather, of a "lyotropic " origin.

Hardy and Harvey (1911, p. 220) find that unicellular plants and animals possess, as a rule, a surface charge, which varies with functional activity. This latter fact is shown by the circumstance that different individuals of the same species in a mixed culture were found to migrate in an electric field at different rates. Red blood corpuscles, on the other hand, have a markedly uniform rate of migration and may be regarded as having very slight chemical activity, although living. The activity they possess is also very, uniformly distributed between individuals.