Principles of General Physiology

Experimental evidence, in any case, shows that all the ions actually present are osmotically active. Vapour pressure measurements made by the method of Barger, described above (page 155), gave the same values as direct measurements with a parchment paper osmometer (Bayliss, 1911, p. 233). Now this vapour pressure method gives the total molar concentration of the solution, including that of the sodium ions, and therefore the parchment paper membrane does so also. A still simpler proof that the diffusible ions are really active, is to take the dye, " Chicago blue," in which the anion, like that of Congo-red, is a complex sulphonic acid, but in this case there are four sulphonic acid groups in the molecule, so that it combines with four sodium ions. If the latter were inactive, the osmot itpressure with a parchment paper membrane would be the same as that of an equally concentrated solution of Congo-red, since the concentration of the non-diffusible anion is identical ; in point of fact it is found to be double, hence the sodium ions play their part.

The matter is, however, not quite so simple. Although all the ions that are present must be osmotically active, the numerical values of the osmotic pressure, whether measured directly or by vapour pressure, are much less than would be the case if the dissociation were of the usual simple kind of such an inorganic salt as sodium chloride. The reason for this has not yet been satisfactorily made out, but there seems to be no doubt that it depends on the formation of complex, aggregated ions. The remarkable fact is that electrical conductivity measurements give no evidence of a less total number of charges than if no aggregation existed. The complex ions appear to possess the same number of charges as if their constituents were free.

The way in which the electrostatic forces at the membrane influence the distribution of diffusible saltc between the two sides of the membrane has been referred to above (page 120). It is found that, suppose the dye is a sodium salt, and the diffusible salt is sodium chloride, the distribution is such that there is always less of the sodium chloride within than without. The explanation is, no doubt, that in equilibrium, there must always be equal concentration of non-dissociated sodium chloride on both sides, since it is freely diffusible and there are no electrostatic forces to prevent its equal distribution. To ensure this, the total amount of sodium chloride present must be less inside, on account of the fact that its dissociation is lowered by the presence of an ion (Na'), which is common to the two salts within the membrane.

At first sight it seems strange that salts which have no ion in common with the dye are also affected in the same way. The reason is that, when equilibrium is established, there are present, inside and out, both kinds of the diffusible cation in the same ratio. The layer of Na' ions, arising from the dissociation of the dye salt, and situated on the outside of the double layer at the membrane, must not be thought to be composed of the same individual ions — there is perpetual interchange with those in the body of the solution. Suppose now we place a solution of potassium chloride outside ; the sodium ions, since they are kept in place merely by virtue of their positive charges, will naturally interchange with potassium ions of the outer solution, so that, to begin with, the outer layer at the membrane will consist of both K- and Na' ions ; these, in their turn, interchange with the Na' ions in the solution within the membrane, so that finally there will be the same relative distribution of total diffusible salt as if sodium chloride had been taken. Naturally there will also be present a certain proportion of the potassium salt of the dye in place of a part of the sodium salt originally present. An important point to be noticed is that the ratio of sodium to potassium will be the same inside and outside, as indeed I have found experimentally to be the case. It follows, as already pointed out, that a membrane impermeable merely to colloids will not account for the unequal ratio of sodium and potassium inside and outside the red blood corpuscles. The membrane must be impermeable to these also.

This formation of a double layer at the membrane, as pointed out by Laqueur and Sackur (1903, p. 203), should give rise to a considerable difference of potential between the two sides of the membrane. Theoretical considerations show that it will be expressed* by the same formula as that deduced by Nernst for the potential of metallic electrodes, viz. : — where R and T have their usual significance, q is the charge on one gram-equivalent of the diffusible ion concerned, n is the number of these gram-equiyalents, c.2 is the concentration of this ion inside the membrane, and Cj its concentration in the outside solution. Direct measurements made by myself (1911, pp. 243-248) confirm the correctness of the formula as applied to the case in question. The reader will recognise this formula as being the same as that for the isothermal compression of a gas or the concentration of a solution, the only difference being that, as we are dealing with electric charges, we have to introduce q, in order to give the correct numerical values to our result. In other words, the number in gram-equivalents of the ordinary formula has to be changed into the number of charges on these gram-equivalents. R, the gas constant, must also be expressed in electrical units. The way in which the formula is obtained is described below (Chapter XXII. ).

It is not to be forgotten that the results given in the present section apply not only to dyes, but to all salts of which one ion is held back by a membrane, permeable to the opposite ion. They apply to salts of proteins and also to noncolloidal electrolytes, if the membrane is impermeable to one only of their ions. This latter case has been discussed by Ostwald (1890). The considerations with regard to interchange of ions form also the explanation of the experiments of W. A. Osborne (1906) on the interchange of ions between colloids and salts.

Since dyes of the molecular weight of Congo-red give considerable osmotic pressures, owing to the fact that their molecules are only just sufficiently large to be unable to pass through parchment paper, they form very useful substances for the investigation of many problems relating to osmotic pressure. The difficulty of preparing reliable copper ferrocyanide membranes is avoided. A O'Ol molar solution of Congo-red has an osmotic pressure of 170 mm. of mercury, and that of Chicago blue is nearly double. There is one practical point to be taken care about, if permanent readings are to be expected, when dyes with an indiffusible anion are made use of. The free acid is insoluble, and although it forms a colloidal solution when free from electrolytes, it is precipitated by traces of them. If the outer water is exposed to the air, it will absorb carbon dioxide ; this, being diffusible, obtains access to the interior of the osmometer, and, although a weaker acid than that of the dye, it will slowly decompose the salt, by mass action, owing to the precipitation of the free acid out of solution, while the sodium carbonate diffuses away to the outer water. The fact

itself was noticed by Graham (1861, p. 217) in connection with the sodium salt of " albumen," where it was found that all the sodium diffused away in process of time and was found in the outer water in combination with carbon dioxide derived from the air. The same thing happens with the salts of caseinogen. It is necessary to give this warning, since various incorrect statements have been made on the basis of experiments in which this factor was ignored. It is, for example, no proof of hydrolytic dissociation when sodium is found to have diffused out.

The osmometer of Moore and Roaf (1907), with the additions described by myself (1909, i. p. 271), will be found suitable for the investigation of colloidal solutions. The platinum lining is rarely necessary ; it will be found sufficient to have the inside electrogilt. The membrane ma}' be of parchment paper, or of this impregnated with gelatine, collodion, etc. Many proteins, as we have seen (page 104 above), take up water by imbibition. In theory it would seem, therefore, when a certain molar solution is made, that the solution is really more concentrated than was intended, owing to the taking up of water by the colloid, which water is then no longer free as solvent. The osmotic pressure would, for this reason, be higher than the theoretical one. It is difficult to state how far this is actually the case, since we are so much in the dark as to the true molecular weight of proteins. The case of haemoglobin, which has an osmotic pressure in agreement with its molecular weight, suggests that the effect of imbibition is negligible. Some measurements of the osmotic pressure of the sodium salt of caseinogen made by myself (1911, i. p. 234) agree with the molecular weight assigned by Laqueur and Sackur (1903, p. 199). It may be that, although each molecule of the protein takes up a considerable number of water molecules, the total number of protein molecules present is too small to affect appreciably the molar fraction of the water, which is always present in excess. Pauli (1910, p. 485)j however, is of the opinion that the process of imbibition plays an important part in the apparent osmotic pressure of proteins.

Since the manifestation of osmotic pressure is an aspect of the kinetic energy of particles in motion, which also shows itself in the power of diffusion through a liquid, it is interesting to note that ovedberg (referred to by Arrhenius, 1912, p. 27) found that a certain gold hydrosol had a diffusion constant of 0'27 per day ; in the same units, chlorine, bromine, and iodine have respectively values of 1'22, 0'8, and 0'5. There is, then, more difference between the rates of chlorine and iodine than between those of iodine and of gold particles.

Living cells, as we saw in the previous chapter, are surrounded by a semipermeable membrane, so that it is obvious that the osmotic pressure of the solution outside, compared with their own osmotic pressure, is of great importance in many ways. The osmotic pressure in the interior of such an organism as an Amoeba must be higher than that of the fresh water in which it lives. Hence, if the cell is covered by a semi-permeable membrane, water is continually being taken up into its substance. According to Stempell (Zool. Jahrb. Abt. Zool., xxxiv. (1914) pp. 437-478), it is the function of the contractile vacuoles of these organisms to get rid of, periodically, the water which has entered in this way.

There is, we may note in the next place, an important difference between vegetable and animal cells. The former, surrounded by a tough cellulose envelope, are usually surrounded by water or by a considerably hypotonic solution ; in this way their internal osmotic pressure is uncompensated and maintains a state of tension or " lurgor " in the cell, serving to keep up the more or less rigid condition of living plant structures necessary for their satisfactory exposure to air and light.

Animal cells, on the contrary, are, as a rule, free to change their dimensions by taking or giving up water. In order that they may remain in a normal state, therefore, they must be surrounded by an isotonic solution. Now, any substance in appropriate concentration will make an isotonic solution, provided that the cell membrane is impermeable to it. On the other hand, there are very few substances which have no action on the cell beyond that due to their osmotic pressure. Perhaps cane-sugar has the least action, but, as we saw above (page 125), it is not a completely indifferent substance. The effects of solutions which are merely due to their osmotic pressure are accordingly rather difficult to investigate. In certain cases, however, the state of affairs is quite clear.

We may take first an interesting fact discovered by Dale (1913). The uterus of the guinea-pig is a very useful preparation for researches on the action of drugs on smooth muscle tissue. When suspended in isotonic saline solution (Ringer's fluid), it responds by contraction to the addition of various drugs, e.g., /2-iminazolylethylamine. Suppose that the concentration of the solution in sodium chloride is raised from the normal 0-9 per cent, to 1-1 per cent., the response is greatly decreased and is practically abolished at T3 per cent. If the osmotic pressure is raised by isotonic quantities of sodium sulphate or canesugar, the effect is identical, so that it appears to be one of tonicity alone. The reverse action may be produced by dilution, even from O9 per cent, to 0*85 per cent., so that the response to stimulant drugs is markedly increased. Dilution with isotonic cane-sugar has no effect, but urea solution acts as pure water, since the cells are permeable to it. When the action of a drug is to produce relaxation of a tonic state, as in the case of adrenaline on the virgin uterus of the cat, the effect of increase of osmotic pressure is to increase the inhibitory action and of decrease of osmotic pressure to diminish it. The tonus itself is also inhibited by rise of osmotic pressure and increased by addition of water.

We have already discussed briefly the two typical cases of the cells of the kidney and of striated muscle, as investigated respectively by Siebeck (1912) and by Beutner (1913, 1 and 3). The volume of the cells was found to be in exact relationship to the osmotic pressure of the solution outside them. Diminution in the volume of cells by loss of water must have the effect of increasing the internal concentration of substances to which the membrane is impermeable. By mass action, reactions of which these substances are components will be accelerated, and increase in volume by absorption of water will retard such reactions.

An interesting case is that of yeast. The cells of this organism, owing to the store of glycogen which they contain, undergo a process of auto-fermentation, the enzymes present acting on the glycogen, first to form sugar and then to convert it to alcohol and carbon dioxide. It was found by Harden and Paine (1911) that, if the cells are placed in solutions which cause plasmolysis, the rate of auto-fermentation is greatly increased, no doubt by increase of concentration both of enzymes and of substrate. Solutions of various substances, if their osmotic pressure was the same, caused equal increase. If no plasmolysis resulted, either because the solution was isotonic with the cell contents, or because the cell membrane was permeable to the solute, as urea, no effect was obtained.

Perhaps one of the most obvious phenomena in which osmotic pressure plays a part is that of secretion. Let us imagine a vertical tube, closed at the lower end by a semi-permeable membrane and open at the upper end. Let it be filled with a solution of cane-sugar and placed with its lower end in water. Water will enter the tube by osmosis and cause a continuous flow of liquid over the top as long as any osmotically-active substance is present inside it. It will clearly be without effect on the result if the top of the tube is closed

a permeable membrane, or even by a membrane through which cane-sugar can pass, however slowly, so long as it passes more quickly than through the semi - permeable membrane at the other end. If such a tube, with a permeable membrane at one end and a semi-permeable membrane at the other end, be totally immersed in water, or a solution of less osmotic pressure than that contained inside it, a current will flow through it, carrying out the solute, until the osmotic pressure is equal inside and outside.

A mechanism of this kind exists in certain organs of plants, in which drops of watery secretion are formed at the apex of a column of cells. These organs are known as " hydathodes " and the cells have been shown by plasmolytic methods to decrease in osmotic pressure as the apex is approached (Lepeschkin, 1906). The aerial hyphse of the fungus Pilobolus, which secrete drops at their tips, have been also investigated by Lepeschkin (1906) and a similar mechanism found.

The phenomenon of bleeding at cut ends of stems, or root pressure, receives its explanation in a similar manner. The liquids in the root have a higher osmotic pressure than the very dilute solution in the soil, and, since the cells are provided with semi-permeable membranes, a flow of liquid takes place as in our glass tube model. An important series of papers has been published by Demoor and his coadjutors (1907) on the relation of secretory organs, such as the liver, kidney, and subrnaxillary gland, to the osmotic pressure of the liquid perfusing their blood vessels. The discussion of some of these facts will be found in Chapter XI. ; in this place one or two suggestive points only will be referred to.

The cells lining blood vessels, like other living cells, are no doubt subject to changes of volume in response to changes in the osmotic pressure of the blood. According to Demoor, the effect of this change in volume will be to alter the lumen of the vessel, so that, other things being equal, a fall in the osmotic pressure of the blood causes a swelling of the lining cells of the blood vessels and a consequent narrowing of the lumen. This fact has special application to the function of the kidney. In the case of the liver (1907, p. 32) it was found that the rate at which 1*5 per cent, sodium chloride passed through was greater than that of 0'6 per cent., while 0*9 per cent, was intermediate. A solution of a concentration of 0'6 per cent, became more concentrated and one of 1'5 per cent, became diluted in its course. So that it is clear that the cells take up water from a hypotonic solution and that the swelling so caused obstructs the circulation, and vice versa. How far the effect is due to the liver cells themselves and how far to the lining cells of the blood vessels is not quite clear, but it seems probable that the former is the chief factor. We call to mind that the liver capillaries send branches into the substance of the liver cells (Schiifer, 1902), so that the capillaries are devoid of walls in certain places. The reactions described disappear when the semi-permeability of the cells is destroyed by sodium fluoride.

Similar phenomena were found in the pulmonary circulation (page 50), and it seems probable here that changes in the volume of the lining cells of the blood vessels might play the chief part. In the kidney, we meet with the same facts as regards the rate of flow of blood. The rate of secretion falls also with hypotonic solutions and rises with hypertonic, as had been observed by Starling (1899). But investigations on the changes of volume of the kidney show that the organ, as a whole, ,y//W/,s- when a hypertonic solution is perfused, and vice versa (page 69). Owing to the complexity of the factors involved here, discussion of the question will best be postponed to Chapter XI.

We have seen why it is necessary for animal cells to be in contact with a liquid of the same osmotic pressure as themselves. There is evidence, moreo\rr, that when exposed to the action of a liquid of a different osmotic pressure, they are able to accommodate themselves to a certain extent by change in their own osmotic concentration. For example, it appears that the cambium cells of trees, as the external pressure upon them increases, produce osmotically- / active substances in order to raise their own osmotic pressure.

The body fluids, including the blood, of marine invertebrates, have the same I osmotic pressure as the sea water in which they live. If certain of these organisms, ' Maia verrucosa, a crustacean, for example, is placed in concentrated or diluted sea water, it is found that the body fluid takes the same osmotic pressure as the solution; the following data from Fredericq's paper (1885) will show this: — The regulation is apparently effected through the cells of the gills. Since the changes in question do not permanently affect the animal, it is plain that

the cells must have altered their own osmotic pressure to compensate for the change in that of the body fluid. The same behaviour is shown by the lower fish, the Selachians. But, as we ascend the scale of evolution, we find that the blood is maintained at a nearly constant osmotic pressure by regulative mechanisms. The following values from Bottazzi's article (1908) apply to marine organisms: — In the Teleostean fish we find the regulative mechanism in process of development. Dakin (1908) found that the depression of the freezing point of the sea water at Kiel was 1°'09, while at Heligoland it had risen to 1°'9 ; correspondingly, that of the blood of the ray (a Selachian) rose in agreement. That of the plaice (a Teleostean), on the contrary, had risen from 00-66 to 0°'8 only, i.e., by 20 per cent., while that of the water had risen by 74 per cent. The cod is still more independent of the medium ; when the A of the sea water rose from l°-2 to 1°'9, that of the cod only rose from 0°'73 to 00>757, by 3'9 per cent. only.

We notice that, as the power of osmotic regulation becomes more manifest in the animal scale, the A of the blood tends to be fixed at about 00<6, which is the value of that of the higher land vertebrates. The advantage of a fixed osmotic pressure will be clear if we remember that it is due, almost entirely, to salts. Colloidal systems, such as protoplasm is, are especially sensitive to electrolytes, as we saw in Chapter IV., and fine adjustments of such processes are the more perfect, the greater the constancy of the electrolyte concentration of the medium in which they take place.

The regulation of the osmotic pressure of the blood to a constant value is shown in an interesting way by some observations of Cohnheim (1912, 1). Sweat contains a considerable amount of salts, having a A of about 0°'5, according to Tarugi and Tomasinelli (1908). Now Cohnheim found that he lost a certain considerable weight in this manner by performing a mountain ascent in hot weather. This weight could only permanently be replaced if he drank water containing sufficient salts to replace those lost. Distilled water was rapidly lost again through skin and kidneys.

The chemical changes associated with those cell activities which result in the setting free of energy usually consist in the splitting up of large complex molecules into a greater number of smaller ones, such, for example, as the oxidation of one molecule of glucose into six molecules of carbon dioxide and six molecules of water. The osmotic pressure of a solution being in proportion to its molar concentration, it is clear that, neglecting the water, the osmotic pressure of a glucose solution would be raised to six times its value. The bearing of this fact on the formation of lymph in active organs will be seen presently.

The mere addition of carbon dioxide to blood raises the osmotic pressure of the latter considerably more than the molar concentration of the added substance would account for. Kovacs (1902) states that addition of carbon dioxide to rabbit's blood raises the A in ten minutes from 0°'6to 0°'72. The effect is due to a complex reaction with the salts of blood, which will be discussed in the next chapter. It is rarely that the blood vessels lie in immediate contact with the tissue cells, whose food requirements the blood supplies and the products of whose metabolic changes it carries away. There intervenes a space, of varying dimensions, containing a fluid, the lymph, whose composition is very similar to that of the blood, minus its red corpuscles, although usually containing less protein. This lymph is being produced continually at a more or less rapid rate by transudation from the blood vessels, and carried back to the blood by means of the lymphatic vessels. Filtration is one of the factors concerned in its production, since the intra vascular pressure is greater than that in the tissue spaces ; but Starling

(1896 and 1912) has insisted on the importance of osmotic processes in addition. It is, in fact, clear that a rise in the osmotic pressure of the lymph, however this rise is produced, will result in the passage of water from the blood to the lymph, and an increase in the volume present. We see then why the amount of lymph flow from an organ is increased by activity of that organ. The energy required for activity is afforded by chemical processes which result in the production of a larger number of small molecules from larger ones, with a consequent increase in the molar concentration and osmotic pressure of the contents of the cells. These metabolic products diffuse from the cells into the lymph surrounding them, thus raising its osmotic pressure above that of the blood, with the result that water passes from the latter to the lymph and causes an increase in its volume.