Principles of General Physiology

more than likely that chemical facts will sooner or later find their description in terms of molecular physics. The enormous molecules and aggregates of molecules which play so large a part in vital phenomena differ from simple small molecules in that they already begin to show the properties of matter in mass, especially those connected with the development of surface. This fact will l)e found to account for many otherwise puzzling phenomena, and cannot be ignored with impunity. Instances will be found in later pages of this book.

A few words are advisable with respect to the separation by chemical methods of various cell constituents. The view is held by Kanitz (1910, p. 234) that it is impossible to obtain any such substance in the form in which it existed in the living cell. He calls attention to the fact that, in the living cell, reactions must be supposed to be in continual progress, never actually arriving at equilibrium. A system in equilibrium is, in fact, dead, as will be seen better in the next chapter. But when a cell is acted upon by the reagents necessary to extract its constituents, the various reactions are supposed by Kanitz to be brought at once into equilibrium. The researches of Fletcher and Hopkins on lactic acid formation in muscle, already quoted, show that this is not necessarily the case. When muscle is heated to 40° C. so that it passes into heat rigor, the maximum amount of lactic acid to be obtained is formed (p. 266), about 0'52 per cent, as zinc lactate. When, on the contrary, the resting muscle is crushed under ice-cold alcohol, only 0'02 per cent, is obtained (p. 260). This is sufficient to show that the reaction producing lactic acid is stopped, practically at once, by destruction of the muscle structure at a low temperature. The manipulation requisite to extract it does not cause the reaction to proceed to completion. We may also call to mind that a reaction may be stopped at once by the addition of a chemical agent ; as, for example, the hydrolysis of cane-sugar by the enzyme invertase, when a mercury salt is added, by which the enzyme is destroyed (see Chapter X.).

The following considerations, due to Hopkins (1912, p. 218), will show that the amount of a particular substance extracted from a cell is no index to its importance in the series of reactions going on in the living cell. The metabolism of the cell undoubtedly takes place in such a series of reactions that the products of one form the starting point of the next following. The various component reactions of this chain will almost certainly not progress at the same rate. Suppose, then, that the first component is kept constant in concentration by continuous supply, as will usually be the case. Then the amount of the products of each reaction present at any given moment will be in inverse ratio to the rate at which they change into the next member of the chain. It is clear that, in such a state of " dynamic equilibrium" the actual amount of chemical change taking place in each reaction must be the same; so that, if the rate at which any particular step is decomposed into the succeeding one is less than that at which it is produced from the preceding one, there will be a heaping up until the larger quantity reacting will compensate for the lesser rate of change. In symbolic form : —

where Kj, K2, K«, K4, etc., are the respective velocity constants of the reactions, and [A], [B], [C], [D], etc., are the corresponding concentrations, iir accordance with the law of mass action. It is plain that, if Kj is small and K., large, [A] must be large and [B] small, and so on. One important result of this fact is that, when a cell is killed, the amount of any particular body present may be very small, although all the members of the chain of reactions may have passed through this stage.

The movements of naked protoplasm have already been referred to incidentally. For more detailed description, memoirs such as those of Jensen (1902) Kiihne (1884), or Ewart (1903), may be consulted. The observation of the phenomenon, as shown in the staminal hairs of Tradescantia , should be made by every student. The hairs have only to be mounted in water under a cover-glass. The ordinary species, T. virginica, is grown in most gardens. If the flowers of the greenhouse species, T. discolor, are available, it will be easier to see the protoplasmic filaments, since the cell-sap is colourless, instead of being of a purple colour as in T. virginica.

The immediate cause of these movements seems to be changes of surface tension, produced either by outside influences or in the A fact to be borne in mind, in discussing the behaviour of any organism to external stimuli, is that the response to similar stimuli is not always precisely the same. There is, so to speak, no fatal necessity about the reaction. This will be dealt with more fully in Chapter XVI., but the remark must be made here that we are not thereby compelled to assume the presence of a controlling "soul" or "Psyche." The state of the organism itself is by no means always identical. No stimulus, in other words, meets with a reacting system in precisely the same condition as a previous one did.

A sea anemone, which has been without food for some time, reacts rapidly to bits of crab meat, seizing them with its tentacles and pushing them into its gastric cavity. As repeated portions are presented, the reaction becomes gradually more inert, until finally no reaction is obtained at all. The presence of food in some way prevents the taking of more (Jennings, 1906, pp. 225-236). We are irresistibly reminded of a reversible, or balanced, chemical reaction becoming slower and slower as equilibrium is approached (see Chapters VIII. and X.). This is made the more striking by the fact that pieces of filter paper, which produce no chemical change in the organism, continue to be pushed into the gastric cavity as long as they are presented, although there is no room for them, and they are immediately disgorged.

Pages 111-127 of Jennings' Carnegie publication (1904), dealing with "Physiological States as Determining Factors in the Behaviour of Lower Organisms," should be read. We have seen how portions of a protoplasmic organism, such as Badhamia, when separated by passing through cotton wool, subsequently coalesce again. The same thing occurs when the separate amoebae, proceeding from germinating B, the same cell, after moderate, local, electrical stimulation. The

region of the excited protoplasm extends from a to b. c, protoplasm contracted to round lumps and balls, d, pale vesicles. Length of cell, 0'2 mm. spores, unite to form a plasmodium. On the other hand, it appears that the pseudopodia of an individual amoeba, or other rhizopod, never unite with pseudopodia of another individual (Jensen, 1895, and v. Uexkiill, 1909, pp. 16 and 38). The reason of this is not clearly understood. When the surface of such an organism comes into contact with food, it appears to soften and become sticky, so that the food substance adheres and is more readily taken in. The same thing seems to happen when a protoplasmic process comes into contact with another part of the same individual, but why it does not usually occur when portions of different individuals come into contact, is not easy to explain. Of course, no two individuals will be in precisely the same state, chemical and physical, at the same time, owing to different states of digestion of food and so on ; but the power of discrimination possessed by protoplasm must be very great to appreciate these differences. There are, indeed, many other reasons for believing that living cells are extremely sensitive to minute changes in their environment.

When structures consisting of naked protoplasm, such as leucocytes or the streaming substance of vegetable cells, are exposed to an electric shock from an induction coil, their movements cease, and they draw themselves together into spheres or series of spheres as shown in Fig. 19 (see Kiihne's description, 1864, p. 30). The way in which this effect is produced is not quite clear. Perhaps the colloids are temporarily sent into the "gel," or coagulated state, but it seems also necessary to assume that some kind of contraction of the surface layer occurs, in order to account for the spheroidal forms produced. If the " gel " state were brought about, it is probable that observations on the Brownian movement of particles in the protoplasm would throw light on the matter. In the "gel " state these movements cease, owing to the particles being held in the rigid framework of the separated solid phase. This fact, as we shall see later, has been used to facilitate the counting of particles in colloidal solutions. Some observations by Kiihne himself (1864, pp. 31, 75, 95) point to the stoppage of these movements on excitation, and I have myself recently seen in protoplasmic structures under dark ground illumination that Brownian movements cease under the action of induction shocks too weak to kill the organism.

The effects due to the anode and cathode of the constant current can, in the main, be explained by electrolytic changes. Details of these effects are beyond the scope of this book, since they do not appear to throw much light on the problems with which we are concerned. It has long been known that various organs of cold-blooded animals will continue their activities for a considerable time when separated from the rest of the body, but the corresponding fact in the case of warm-blooded animals has only been established by experiments of comparatively recent date. If artificial circulation of blood, sufficiently oxygenated and at the correct temperature, be maintained, it seems clear that the only experimental difficulty should be with regard to the lapse of time during which the organ is deprived of oxygen, during the necessary operative procedures.

An important step was taken when Locke (1901, p. 490) showed that the heart of the rabbit continued to beat for several hours if fed with a warm saline solution saturated with oxygen. The method has also been applied to the kidney and salivary glands, although it has, as yet, been found impossible to preserve all their activities. This will be discussed further when we are considering the mechanism of secretion. Other cases of isolated warm-blooded tissues, more especially smooth muscle, continuing their contractions immersed in similar solutions, will be found under the head of intestinal movements (Magnus). Blood vessels and the uterus can also be investigated by this method.

Further ^advance was made in 1907 by Ross Harrison (1907, p. 140, and 1910, p. 787), who found that cells separated from frog embryos and immoral in lymph continued to grow. Particularly valuable results were obtained as regards the growth of nerve fibres from cells. Burrows (1911, p. 63) extended the method to the chick embryo, and Carrel and Burrows (1910) to FIG. 20. GROWTH OF NERVE FIBRES IN CLOTTED LYMPH. — Medullary cord tissue of embryo Rana palnstris, 3 "3 mm. long. Lymph from Rana pipiens.

7. Apparently single fibre («/) growing from a pointed cell (c<j) which projects from a mass of cells (ma). One 9 Same group of fibres, 10.25 P.M. Four distant fibres (nf\ — 71/4) now visible. Fibrin filaments (thr) were also present in the earlier stages, but omitted in sketches. 11. Same group, 10.30 P.M. Continuation of Ji/j and upper branch of »>/a unfortunately left out of sketch. Note migration of cell ct.^. Identity of other isolated cells uncertain.

Total interval between first and last figures — thirty-four hours. the adult cat and dog, showing that fragments of various organs, such as kidney, spleen, bone marrow, thyroid, cartilage, etc, placed in coagulated blood plasma, form new cells of the appropriate kind. Rena tubules for example are to be seen increasing in length, and cartilage cells in number, while forming new cartilaginous substance, of a nerve fibre of the chick. For the details of the method the reader is referred to the articles by Carrel and Burrows (1912), and by Carrel (1912). Further, Carrel (1913) finds that the culture medium is greatly improved by the addition of tissue juice of an adult or embryo animal, the younger the better, but this favourable effect is only shown when the tissue comes from the same species. Pieces

of the heart from a chick embryo continued to beat for two to three days after each removal to fresh medium, until unfortunately lost on the 104th day. Embryonic connective tissue could be made into subcultures repeatedly, since it grew so fast. After 14 months, tests grew to 30-40 times their bulk in 5 or 6 days. After 15 months the tissue was still living, although it had been transplanted 172 times. It is important to remember that, when isolated tissues grow normally, we have satisfactory evidence of the preservation of their vital activity.

Some recent experiments by Champy (1913) have given interesting results. Confining our attention, to begin with, to the growth of kidney tissue, taken from an embryo rabbit at full term, we notice certain facts. In Fig. 21, fixed after nine hours of culture, we see a portion of new growth on the right and upper part of the figure, while the cells of the original portion are clearly degenerated ; this degeneration appears to be due to failure of sufficient supply of oxygen. In the new growth there are tubules in the part first formed, but whereas, even in the degenerated state, it is easy to distinguish different kinds of tubules in the original tissue, the new tubules are all alike and of a primitive epithelial type (shown also in Fig. 22). As growth proceeds, we notice that the production even of these primitive tubules ceases, and there is merely a mass of indifferent cells, like those of the embryo before

If adult tissue is taken, such as smooth muscle, which no longer undergoes cell division in the organism, it is found that mitotic figures are produced in vitro and embryonic cells split off. We see thus that differentiated cells, which undergo no further division as long as they are part of a complete organism, when cultivated in plasma outside of the organism, are set off on a course of multiplication, forming cells similar to those from which the differentiated cells were first formed. If it were possible to preserve these cells alive for a sufficient time, it would be extremely interesting to know whether they would ultimately be subject to differentiation into cells similar to those of the tissue from which they grew.

It seems that cells, when they have taken on special functions in the organism, are normally prevented, by some means, from continuing their primitive multiplication, and that, when this influence which restrains their growth is removed, they start afresh and produce simple embryonic tissue. There is significance in these facts in connection with the formation of malignant tumours. The same investigator finds later (1914) that if the fragment of tissue happens to be composed of both epithelium and connective tissue, the new cells growing from the epithelium remain like those from which they grow; whereas, if by chance some of the epithelium leaves the connective tissue and grows towards the outside of the plasma, its cells loose their typical aspect and arrangement,

FIGURE. — After seven hours. Fixed in Bouin's fluid, stained with iron haematoxylin. and cannot be identified as of epithelial nature. Similarly, in a fragment of retina, a typical proliferation of the connective tissue fibres does not occur as long as any nervous cells remain alive. This mutual effect is not universal, it does not occur in the case of muscle and connective tissue. The facts, as a whole, tend to confirm the point of view expressed above as to the effect of one part of the organism on the growth of other parts.

Some further details, especially as to the absence of specific influence of the plasma of the same species of animal, will be found on page 288, and in Chapter XXIV. The numerous and valuable results obtained by the investigation of chemical changes in •surviving tissues and organs, such as muscle and liver, will be dealt with in later pages, when the particular functions in question are under consideration. Protoplasm in the living state has the properties of a liquid system, containing, however, particles of solids and droplets of immiscible liquids in a freely movable state. The protoplasm itself is structureless to the highest powers of the microscope, with ordinary forms of illumination. To the ultra-microscope it presents the characteristics of a colloidal system.

It forms " organs " for particular purposes ; these organs appear and disappear, according to need. But there is no necessity, at present, for the assumption of unknowable " supermechanical " properties in living cells. Many of the properties referred to can lie explained by known laws, such as those of surface tension, while the time element itself is shown by inorganic colloids. By fixing reagents, structure of various kinds, networks, alveoli, and so forth, can be produced. But these structures have no resemblance to the living condition. Obviously, they must be produced from constituents already present, so that certain conclusions are admissible from the examination of fixed cells.

There is very little ground for the view that protoplasm consists of " biogens " or "giant molecules," in the chemical sense. It is rather a complex of substances of various chemical natures and in various states of aggregation, associated together by forces of surface tension, electrical charge, and so forth. The liquid state enables an elaborate play of forces to take place. Chemical reactions can evidently proceed simultaneously in different parts of a cell, so that there is some mechanism by which one part is isolated from another part, at all events temporarily. After death, this separation ceases to be effective. The activities of the cell are regulated by reversible changes in the distribution of the phases of the complex heterogeneous system of colloids, crystalloids, and solvents.

For further information on the subjects dealt with in the preceding chapter, the following works may be consulted : — The student is advised to read the preceding chapter a second time after having read the following eight or nine chapters. THE most striking characteristic of living organisms is the perpetual state of change which they show, as will have been clear from the previous chapter. It is a matter of general experience that, in order to effect changes, work must be done. This capacity of doing work is due to the possession of something which is called energy, and is frequently defined in these very words.

There are two great laws dealing with changes of energy, known as the first and second laws of Thermodynamics or Energetics. The reason of the name thermodynamics, used in this connection, is that the laws were first arrived at, in the main, from considerations of heat energy. The first law tells us that, while energy may be of many kinds, kinetic, thermal, chemical, electrical, and so on, which can be converted into one another, there is never any gain or loss. This fact, derived from universal experience, is known as the " conservation of energy."

It may be noted here that the observation that energy of motion can be transformed into heat suggested the thought that the latter is itself a form of movement, and ultimately that the other forms of energy which can be derived from heat are also kinetic in nature, not excepting chemical energy itself. The second law is somewhat more abstruse, and deals with the " quantitative relations which restrict the convertibility of energy,'1 as Nernst puts it (1911, p. 16). Thus, "while external work and the kinetic energy of moving bodies can be transformed into one another completely and in many ways, and can also be converted into heat, as by applying brakes to a railway train in motion, the reverse change of heat into work is only possible under certain conditions." This is the principle of Carnot and Clausius in one of its forms.

For example, in the case of a steam engine, the part of the energy given out by the fuel which is available for work is given by the ratio of the difference of temperature between the boiler and condenser to the absolute temperature of the latter ; this means, of course, that only a certain part of the heat energy given out by the burning coal can be utilised even in the most perfect steam engine. The fact just referred to led to the important distinction made by Helmholtz (1882, p. 33) between "free" and "bound" energy. It is plain that, of the energy contained in a system, only that part which can do work is of value.

As an illustration, imagine a system of two similar copper balls, isolated completely from the surroundings, one of which is initially at a higher temperature than the other. The system as a whole contains a definite quantity of heat energy, given by temperatures and thermal capacities of the constituents of the total mass. If left to itself, a part of this energy will pass from the warmer to the cooler body, until both are at the same temperature. During this process a certain fraction of the energy transferred may be used to perform work. When the two balls have arrived at the same temperature, although no loss of energy has occurred, no more work can be got out of the system in itself, but only when brought into relation with another system at a lower temperature. In this state, so far as the system itself is concerned, its energy content is not free, but bound and useless.

A further important fact, also arising from experience, is that free energy always decreases, if it possibly can, but never increases. In the above illustration, energy passes from the hot body to the cooler one, so that the difference of temperature diminishes, and with it the free energy ; the reverse passage from a cool to a hot body never occurs. This fact has various applications, as we shall find later. It follows from it, for instance, that if a process resulting in a diminution of free energy can take place, it will invariably do so. This principle was applied by Willard Gibbs (1878, pp. 216, etc.) to the investigation of the deposition of substances from solution on the surfaces of bodies immersed therein, and will be discussed in the next chapter.

Clausius, at the end of a fundamental paper (Pogg. Annalen, cxxv. p. 400, 1865), formulates the two laws of energetics as follows :— I. The energy content of the universe is a constant quantity. II. The entropy of the universe is always striving to a maximum. The word "entropy" is here used as having essentially the same meaning as the "bound" energy of Helmholtz. The law is therefore equivalent to the statement that " free " energy is always striving to a minimum.