Principles of General Physiology

These enzymes are all in the colloidal state, that is, they form a separate phase of the heterogeneous system. Their action is exerted on their surface, and is controlled by the amount of reagents adsorbed. The substances acted on by enzymes are usually called " substrates." The heterogeneous nature of the systems in which enzymic reactions occur is, in all probability, the reason why the equilibrium position is not quite the same under the action of enzyme and under that of acid. But, owing to the almost complete thermo-neutrality of the hydrolytic reactions in question, an extremely small amount of energy is all that is necessary to change the equilibrium to the extent found.

Whenever the equilibrium position under the action of an enzyme, or other catalyst, is anywhere except at complete change, either in the direction of hydrolysis or synthesis, the enzyme must accelerate both reactions, although not necessarily to an equal extent. This relative degree of acceleration depends on the respective chemical difficulty of the two reactions of hydrolysis and synthesis. Enzymes, therefore, bring about synthesis as well as hydrolysis.

There is no sufficient evidence that the enzyme forms a constituent of the final chemical equilibrium. Since the essential property of an enzyme or catalyst is to change the rate at which a reaction proceeds, a discussion of the formulae of velocity of reactions, deduced from the law of mass action, so far as applicable to the case, is introduced into the text. When the velocity constant of an enzymic reaction is calculated by the appropriate formula, it is found that it suffers, as a rule, considerable diminution as the reaction progresses. This means that the activity of the enzyme is decreasing. In some cases there is a spontaneous destruction of the enzyme, in others it is merely temporarily paralysed by some products of the reaction. In the latter case, the most frequent cause is change of the hydrogen ion concentration to a point above or below the optimal one. Enzymes, in fact, are very sensitive to such changes.

Some evidence has been brought forward to show that there is a special chemical affinity on the part of the enzyme for the substrate or for some constituent of it ; but, at present, the evidence is not very convincing. The relation between the concentration of an enzyme and its degree of activity is an exponential one, as would be expected from the fact of its acting by its surface. Small concentrations are, relatively, more active than larger ones. This is to be accounted for by the fact that the rate of the reaction is determined by the amount adsorbed. It is impossible to assign definite numerical values to the exponents of different reactions, since they vary with the relative concentration of enzyme and substrate. In the middle of the reaction, with the usual amount of enzyme, it is generally just below - 2, or thereabouts. This is the " square-root law," which is a rough approximation for a particular stage of the reaction.

There are certain agents which affect the rate of enzymic reactions by a special action on the catalyst. Such are electrolytes, co-enzymes, and "anti -enzymes." Electrolytes, especially hydrogen and hydroxyl ions, have a powerful effect. Neutral salts also have an influence, as a rule, of a favourable kind. Some enzymes require the presence of another substance in order to exert their activity. This other substance, known as " co-enzyme," acts in a different manner in different cases. In some it increases the active surface of the enzyme

by greater dispersion, in others its mode of action is more specific and as yet obscure. There is considerable doubt whether true anti-enzymes, whose nature is explained in the text, have any existence. Some of the effects described as being due to them are to be accounted for by changes of hydrogen ion concentration, others to adsorption of the enzyme by a colloid. That of intestinal worms is a peculiar substance, having none of the properties of an antibody in the sense of the theory of immunity.

Contrary to what mass action would predict, it is only in moderate concentrations of substrate that the rate of reaction is proportional to this concentration. Above a certain value, differing according to the case, the velocity of the reaction either remains constant or may even decrease. The cause appears to be of various nature, viscosity, adsorption-saturation of the enzyme, or removal of water. But, where the question has been investigated, the composition of the system in equilibrium is as the law of mass action requires, so that the anomalous effect of increase of concentration only relates to the rate of change.

The rate of enzymic reactions is greatly accelerated by rise of temperature. The optimum temperature is merely that at which the increased rate due to the rise is in greatest preponderance over the simultaneous increased rate of destruction of the enzyme. The importance of regarding reversible or balanced reactions from the dynamic point of view is insisted upon. The law of mass action shows that, in order to obtain much synthesis, concentration of water must be decreased as far as possible. In the living cell there are probably effective mechanisms for doing this. At the same time, if the synthetic products are continually removed in any way, a small degree of synthesis may result in a considerable amount of products, since the reaction is always going on towards its equilibrium.

There is no evidence for the existence of enzymes which either hydrolyse only or synthesise only. In fact, if enzymes are catalysts, the one agent must do both. It is possible that an intermediate chemical compound may be formed between the surface of the enzyme and the substrate preliminary to decomposition, but there is no actual evidence that such is the case. The reacting substances, such as water and substrate in a hydrolytic reaction, are certainly brought into very intimate contact by adsorption on the surface of the enzyme, and the question is still an open one as to whether this fact, combined with the special nature of the surface itself, is not a sufficient explanation of the increased rate of reaction. The special nature of the surface referred to may be merely physical, but the action of particular enzymes on particular substrates has to be accounted for.

The interaction of electrical forces in the action of neutral salts on adsorption is to be taken into account. The chemical nature of enzymes is probably of very different kinds. There is direct evidence that some are not proteins, and it is doubtful whether any are. Some appear to be complex systems of colloids with inorganic components, or other simple compounds. There are three stages in heterogeneous reactions — diffusion, adsorption, and chemical reaction. The actual rate of the reaction depends on the slowest member of the series. In colloidal solutions, diffusion and adsorption are rapid, so that the chemical reaction proper is the determining one, a fact which accounts for the high temperature coefficient. But the rate of the chemical change itself is determined by the amount of substrate adsorbed at a given moment, according to the law of mass action.

Some enzymes can be obtained in a stage of formation in which they are inactive, and are then known as " xyniogens.'' These are converted by certain agents into the active enzymes, a change which does not appear to be reversible. While many enzymes seem to be very "specific," or selective, in that their effect on one particular substrate is very much greater than on any other, it is necessary to be cautious in assuming this as being unconditionally true. Further investigations are needed of the changes in the action of en/.yme> produced by different conditions. There is also, in many cases, evidence that the same enzyme may act on different substrates at such different rates that it appeals to act only on one, unless prolonged observations are made, but the reason why the rate is faster in the one case requires elucidation. Optical isomerism certainly plays a part.

WE have seen in the preceding chapters how important is the function of enzymes in the regulation of the chemical changes of living organisms. Now there is a liquid, one amongst others of the same class, and known as the pancreatic juice, which is formed by the cells of a certain organ and poured into the cavity of the intestine. Its chief properties are due to the variety of enzymes which it contains, although there are other substances present. It may be considered as a typical case of secretion. The cells of the pancreas produce substances which are not present in the blood bathing them and, at the same time, they separate water from the blood in order to carry off these substances in solution. Along with the water we find, as a rule, some other constituents of the blood transferred to the secretion, especially diffusible salts, such as sodium chloride.

There are, however, included under the general name of secretion, the activities of such an organ as the kidney, whose chief function is to separate from the blood certain products of metabolism, such as urea, which would be injurious to the organism unless removed. There are, moreover, the so-called "internal secretions," where substances having special actions on other parts of the organism are formed, but, instead of leaving the cells in which they are produced by a surface in connection with a special channel, the duct of the gland, they are sent in the other direction into the blood current. In such cases, materials supplied by the blood are converted by the organ in question into " chemical messengers " or " hormones," and returned to the blood in this altered form.

It will thus be seen that, xinder certain aspects, the process of secretion is a part of the general cell metabolism, especially in the case of the internal secretions The manner in which the passage of water is effected in the case of the typical external secretions is a question of much interest, together with the way in which it is regulated. The influences at work causing the production of the specific contents of the secretion will also require our consideration.

In the present state of knowledge, it is impossible to treat the subject from a really general point of view. Perhaps we may look upon the transfer of water from the blood to the secretion as a property common to the majority of cases, so that this phenomenon will be discussed in the first place. It will afterwards be necessary to take special instances, and, as far as possible, our chief attention will be given to those points of most general application.

The most obvious hypothesis to make is that the layer of cells forming the membrane intervening between the blood vessels and the lumen of the duct has the properties of a semi-permeable membrane, so that, supposing the pressure in the blood vessels to be higher than the osmotic pressure of the blood, pure water will be forced through. But the osmotic pressure of the blood, as we have seen (page 165), is as high as 6'5 atmospheres, or 5,000 mm. of mercury, whereas 200 mm. of mercury is a high value for the blood pressure. Such a hypothesis is clearly an impossible one. But it is very rarely, if ever, that a secretion consists of pure water, so that the difference of osmotic pressures is not so great as that given. If the membrane, or one of the membranes, intervening is permeable only to the colloids of the blood, such as a gelatine membrane, a very much lower arterial pressure will suffice to filter off a solution containing all the crystalloid components of the blood, in the same concentration as in it. We shall presently see reason to believe that this is the case with the "glomerulus" of the kidney,

FNJ. 88. ALVEOLI OF SEROUS GLAND OF RABBIT. — Fresh, without any addition. All figures from the same gland. The boundaries between the cells are made too obvious in all. B, 1-4S hours later, after 3'65 c.c. of saliva had been secreted under the influence of C, Five hours later than A, after stimulation of the sympathetic nerve for alxnit two hours, with intervals of rest. It! saliva secreted. The nuclei should not be shown so clearly, although they are unobscured by granules.

where the liquid secreted is blood plasma minus its colloids, with perhaps certain crystalloids adsorbed thereon. Although the secretion of water in general is not a simple process, there are grounds for holding that osmotic phenomena play an important part in it. We have seen (page 163) how a tube containing a solution of some substance, cl< ><••<! at one end by a membrane impel - meable to the solute, and at the other end by a membrane permeable to it, and i mmersed in water, gives a continuous current of water, or rather

solution, issuing from the permeable end, as long as any osmotically active substance is left in the tube. Such a mechanism has been described by Lepeschkin (1906) in the fungus Pilobolus, and in the hydathodes of higher plants. If, therefore, we are justified in assuming that the secreting cells of such organs as the salivary glands or the pancreas are possessed of a membrane on the ends next the blood vessels of such a kind as to be impermeable to some substances produced in the cells, while on the ends next the duct the membrane is permeable to these substances, we can account for a flow of water as long as these osmotically active substances are being formed. They are, of course, carried out with the secretion through the membrane permeable to them.

FIG. 89. SEROUS GLAND OF THE HUMAN TONOFE. — Fixed preparation. Diagrams of the series of functional states (a to (j) from the charged resting state through activity to the state of rest again (h). According to the work of Zimmermann. When the secretion has an osmotic pressure mechanism as simple as higher than that of the blood, it is clear that a that described is insufficient, and additional complications, so-called " protoplasmic activities," must intervene, in order to afford the energy necessary to raise the osmotic pressure. Moreover, in any case, except simple filtration, the mechanism in question requires the continuous production in the cells of osmotically active substances. The osmotic pressure of milk, bile,

saliva, and sweat is, in fact, lower than that of the blood, but urine, as it leaves the kidney, has an osmotic pressure considerably higher than that of the blood, owing The reader will probably note that a mechanism the reverse of that sketched above, namely, cells with their permeable and semipermeable membranes interchanged in position, would account for absorption of water from such cavities as that of the intestine. &, Outer transparent zone, with faint striation in B. e, Lumen of alveolus, obvious in B, indistinct in A. d, Indentation at junction of two cells, resulting from decrease in volume.

A fact which points to the intervention of osmotic processes in the secretion of water is the discovery of Ludwig (1851) that the pressure in the duct under which saliva still continues to be secreted is considerably higher than that in the arteries. Hill and Flack (1912) found, with an arterial pressure of 130 mm. of mercury, a pressure of saliva as high as 240 mm. of mercury. This pressure might be brought about either by the produc tion within the cells of osmqtically active substances, in conjunction with a membrane impermeable to them, and situated next the blood vessels, or by some process of imbibition, by which swelling of some constituents of the cells takes place. It is more difficult, on the latter view, to see how a continuous secretion could be provided for.

Changes in the Microscopic Appearance of gland cells might perhaps be expected to throw some light on the question before us, but the interpretation of those phenomena which have been observed is not an easy matter. For details, the reader is referred to the article by Metzner (1907, 1). It must suffice to mention here that it appears to be almost universal that granules (" zymogen " granules) make their appearance in the more or less homogeneous protoplasm of the resting cell, and slowly increase in size. When the gland is excited to secretion, these granules usually undergo a process of solution, and are regarded as the source of the special constituents of the secreted fluid. Sometimes the granules pass directly into the secretion without preliminary solution. It is conceivable that, in the breaking up of these granules, substances of a much smaller molecular weight and higher osmotic pressure than themselves might be formed. During the process of continuous secretion, it is clear that the granules must be renewed, although it is not difficult to obtain, by artificial stimulation of the gland, a nearly complete disappearance of the granules, those that are left being situated next the lumen of the duct. Figs. 88 to 90 show this result in different cases. See also Fig. 92 on page 347 below.

CUTED).— Fixed and stained by Flemming's method. Obj. 2 mm., Oc. 8. After activity the cells are seen to be diminished in volume. Bunch (1900) showed that, when the submaxillary gland is caused to secrete, there is a rapid decrease in volume of the whole gland, although the simultaneous vascular dilatation in itself produces an increased volume. In cells which have been fixed, the granules referred to stain with the so-called "acid," that is, electro-negative dyes, such as eosin and acid fuchsin. They behave, therefore, like electro-positive colloids, and in an opposite way to the general mass of the cell protoplasm ami the nucleus. There is also frequently to be seen what appears to be a specially differentiated part of the cytoplasm, known as " kinoplasm " or "ergastoplasm," staining deeply with "basic" dyes. This is shown in Fig. 91. According to Laguesse and Delieyre (1912), the dye known as Janus-green brings out the filaments of ergastoplasm in the fresh cell, so that they seem to be present in the living cell, and not to be produced by the fixation pn This dye also stains a little cap of matter on each zymogen granule, which is itself unstained. Some further particulars with regard to morphological changes in gland cells will l)e found below, in the discussion of the pancreatic secretion.

That there is a change of permeability in the secreting cell is indicated by the experiments of Garmus (1912) referred to above (page 140). Under atropine, which paralyses the secretory process, the gland cells are less permeable to dyes than when secreting under the influence of pilocarpine. According to Gildemeister (1913), the cell membrane of the sweat glands becomes more permeable when activity is brought about by stimulation of the nerves to the glands. Thi:. is indicated by the diminution of galvanic polarisation, presumably due to increase of permeability of the membrane to ions, in a way similar to that described above in the case of Congo-red and parchment paper (page 161).

According to Macallum (1911, p. 644), differences of adsorption, due to surface tension, play a part in secretory processes. Taking the distribution of potassium as an index to that of the other cell constituents which lower surface tension, he finds, in secretory cells, that there is considerable accumulation of this substance at the cell surface next the lumen. It seems possible that this fact may play a part in the transfer of substances from the body of the cell to the lumen of the duct, although it is difficult to understand how adsorbed substances can play a part in the processes of osmosis or diffusion, since they are held by sin-face force* In connection with the remarks made above (page 335) on the possible relation between secretion, and absorption, it is interesting to note that, in intestinal cells engaged in absorption, the greater accumulation of potassium is at the end ojip<>xit'~> to the lumen of the intestine. During absorption of fat, also, it has been noticed that the cells of the intestinal villi show a greater accumulation of fat droplets at their attached ends.

It has long been known that when the arterial blood pressure falls below some 30 to 40 mm. of mercury the secretion of urine ceases. It occurred to Starling (1899) that, if the liquid leaving the glomerular capsule is a filtrate from the blood plasma, containing all the constituents of the plasma with the exception of the colloids, then the blood pressure would be insufficient to effect the filtration unless it were higher than the osmotic pressure of the colloids. This process would then be similar to the filtration of a colloidal solution through Martin's gelatine filter. Accordingly Starling prepared the filtrate of serum through such a filter, and compared the osmotic pressure of the original serum against it, thus obtaining the osmotic pressure of the colloids, which amounted to about 30 mm. of mercury. As pointed out above, this was the first definite proof that colloids could hav»> a measurable osmotic pressure. Further support was given to Starling's view by measurements of the difference between the pressure in the ureter and the arterial pressure, when the former was gradually raised until secretion ceased. At this point the ureter pressure was 92 mm. of mercury when the arterial pressure was 133 mm. of mercury, a difference of 41 mm. of mercury. It will readily be seen that the rate of filtration under a given blood pressure will be increased by reducing the osmotic pressure of the colloids by dilution, for example. Such a dilution can be produced by the injection of hypertonic solutions, say of glucose, into a vein ; the effect is the withdrawal of water from the tissues into the blood,

a state known as " hydrcemia " or "hydraemic plethora." The same result can be brought about more simply by the injection of a quantity of isotonic saline. In both cases a large rise in the rate of urinary secretion results. It was further shown by Knowlton (1911) that if a colloid, such as gelatine or gum acacia, was added to the saline, so that the osmotic pressure of the colloid added was equal to that of the serum colloids, injection of such solutions caused scarcely any increase of flow. Again, Barer oft and IStraub (1910), by the ingenious device of replacing a great part of the blood plasma by Ringer's solution, retaining the blood corpuscles to supply oxygen, were able to obtain a greatly increased rate of secretion without rise of blood pressure.

The experiment is of sufficient interest to be described in more detail. A rabbit with a blood pressure of 95 mm. of mercury was secreting 0'05 c.c. of urine per minute; 22 c.c. of blood were removed, and replaced by 25 c.c. of Ringer's solution. The secretion rose to 0'4 c.c. per minute, with a blood pressure of 52 mm. of mercury. The blood which had been removed was centrifuged, the corpuscles washed, and made up with Ringer's solution to the volume of the blood withdrawn. This was then injected ; the blood pressure rose to 84 mm. of mercury, nearly as high as it was originally, while the flow of urine rose to 2 '35 c.c. per minute, or nearly fifty times as rapid as the original one, and six times as rapid as that after simple saline injection to replace the volume of the blood taken out. The authors point out the possibility of great variations in the necessary filtration pressure by comparatively small changes in the osmotic pressure of the colloids. Thus, suppose the pressure in the glomerular blood vessels to be 27 mm., and the osmotic pressure of the colloids to be 25 mm., the pressure available for nitration would be 2 mm. Suppose the osmotic piessure of the colloids to be reduced by one-fifth, so that it becomes 20 mm., then the filtration pressure becomes 7 mm., or 3 "5 times as great as before.

From these various experiments it is clear that the filtration hypothesis is capable of accounting for the production of a urine which is equivalent to the, blood plasma minus its colloids. If the glomerular process is merely a filtration, it is clear that what work is required for it is afforded by the blood pressure, in other words by the heart, so that the cells of the glomeruli take no part in the performance of work. Barcroft and Straub (1910) have made observations on the oxygen consumption of the kidney which confirm this point of view. No increase in the oxygen consumed occurred when the increase of secretion was brought about merely by dilution of the blood. We shall see presently that where work has to be done, increased oxygen is consumed in order to give energy by oxidation.