# Popular Science Monthly/Volume 84/June 1914/The General Physico-Chemical Conditions of Stimulation in Living Organisms

 THE GENERAL PHYSICO-CHEMICAL CONDITIONS OF STIMULATION IN LIVING ORGANISMS
By Professor RALPH S. LILLIE

CLARK UNIVERSITY

IT is customary to say that irritability, or the capability of responding to stimuli, is an essential characteristic of living beings. Whether this is true or not of the lowest organisms—certain bacteria or the filterable viruses—there is no doubt that it is preeminently so of the higher, and especially of those leading free and active lives, like most animals. If this were not the case it is difficult to see how such organisms could maintain themselves in their surroundings and continue to behave as living beings—i. e., show their characteristic activities, grow, and eventually reproduce themselves. At least it is clear that in order to do this they must react to the changes continually taking place in their particular environment in such a way as to favor their continued existence in that environment; if, for instance, any animal failed to respond to the presence of food—material that can serve it as source of energy—by capturing and incorporating enough to replace its own normal loss of substance, quite obviously its life would soon come to an end. And if it reacted in the same way to the poisonous or otherwise injurious substances in its surroundings as to food, and incorporated both classes of material indifferently, the same result would follow. Evidently there is needed some power of active and selective response to the changing conditions of the environment if the living organism is to continue to live; it must preserve a certain equilibrium with its surroundings; the materials and energy which it appropriates from those surroundings must in the long run at least equal those which it inevitably loses to them in the normal course of its vital processes. This is the physiological interpretation of Spencer's dictum that all life involves a continued adjustment between internal and external relations. The organism must continually alter its activities in correspondence with altered conditions in its surroundings, and in such a way as to preserve this adjustment—avoiding conditions likely to disturb or destroy the vital equilibrium and tending to place itself in those favorable to its continuance. Accordingly, we may say that living organisms in general, and especially animals, exhibit two broad classes of reactions, first those of a defensive or protective kind, including avoiding reactions and inhibitions of various kinds, and second, the more active group of what we may call self-seeking or acquisitive reactions; of these the chief are the reactions of food-seeking, by which the necessary supply of transformable energy and of building material is secured. Both kinds of reactions are equally responses to stimulation; and both are alike physiologically indispensable. In fact, the characteristic self-conserving or regulatory power of organisms, without which they could not continue to exist, depends essentially upon their ability to respond in this way, i. e., upon their irritability.

It is thus apparently not difficult to understand the general biological significance of irritability. What is still largely obscure, however, is the physico-chemical nature of the mechanism which renders possible the response of an organism to stimulation. Physiological experimentation has enabled us to simplify the problem to some degree. We find that not only the intact living organism, but many of its isolated tissues and even cells, react in characteristic ways to stimulation. This is especially true of the tissues that subserve the motor activities of the animal, the muscles, nerves and sense organs. Thus the problem of the nature of the stimulation-process becomes one of the general problems of cell-physiology, and may be stated as follows: What are the essential physicochemical peculiarities that render the irritable elements of these living tissues so sensitive to stimulation? and what is the physico-chemical nature of the process of stimulation itself? These are the questions which I shall attempt briefly to discuss in this paper. Any answers which can be given at present are incomplete and in part provisional. But recently some definite progress—as it seems to me—has been made toward their solution, and I shall try in what follows to give some account of this recent work and of the more important general conceptions to which it has led.

We have first to define more clearly what we mean by "stimulus" and what by "response." We find on reflection that it is a difficult matter to formulate definitions that are at once exact enough and comprehensive enough to characterize adequately all of the highly varied phenomena included under these terms. We may perhaps best define a stimulus as some change of condition that arouses a previously quiescent tissue or organism to activity, or appreciably modifies the activity of one already active; and the response as the resulting activity or change of activity. In many cases the response may be negative in kind; i. e.; the previous activity may be decreased or completely arrested; inhibition is in fact a very frequent mode of response and one perhaps fully equal in importance to the more positive or active modes as a means of biological adjustment. But what is perhaps the most characteristic peculiarity of the relation between stimulus and response is the fact that there is, broadly speaking, no definable relation of an energetic kind between the two. One of the most striking and distinctive features of stimulation is that an external event or change of condition which causes directly a very slight alteration in the irritable system or organism may yet arouse in the latter a process or series of processes in which the transformation of energy may be almost indefinitely large—out of all proportion to the exciting stimulus. We have therefore first to inquire into the general nature of the conditions that render possible such disparity between the stimulus—considered by itself as a particular chemical or physical process acting upon the irritable tissue—and the resulting special activity or response on the part of the tissue itself.

As all know, an irritable tissue like a nerve or muscle may be aroused to activity under the most various conditions; the effective stimulus may be an electric shock, a chemical substance, the action of light, change of temperature, loss of water, mechanical impact; and the tissue gives the same response to all of these. Now it is clear that such a stimulus can only act as some kind of a releasing agency—what Ostwald calls Anlass—which sets going some process all of the necessary conditions of which are already present, but which is held in check by some restraining condition which the releasing agency removes—as when a gun is fired, or an alarm-clock set off, or a mine exploded by the pressure of a button—which closes an electrical circuit, thus enabling a spark to pass, which raises the temperature of the explosive to the critical point. The connection between Anlass and resulting event may be highly indirect, and there need be no resemblance or other relation than that of interconnection between the two. In all cases the system is, as it were, "wound up"; the potential energy is there, ready to become kinetic; once the process is started or activated by the releasing event, it proceeds of its own accord to its conclusion, i. e., till a second state of equilibrium is reached. In the case of a living irritable tissue or organism we are evidently dealing with a physico-chemical system belonging—as regards the relations between the initiating conditions and the resulting process itself—to this general class. If we press the end of a nerve connected with a muscle, or pass through it an electrical current of sufficient intensity for a sufficient length of time, or dip it into a solution of some appropriate chemical substance, there is initiated at the site of stimulus a "physiological" process which is propagated with unaltered intensity along the nerve to the muscle and there calls forth a complex variety of interdependent physical and chemical changes, of which the contraction is the most conspicuous and physiologically important. Thus a process specific to the tissue, unique and obviously highly complex, is initiated by the relatively insignificant change which the stimulus causes directly. We ought particularly to note that in any special tissue the physiological process remains the same in kind, whatever the nature of the stimulus. The latter merely causes some critical or releasing change which initiates the physiological sequence of events; the latter then proceeds automatically in its characteristic way to its conclusion.

Let us now consider more particularly the physiological part of the whole series of processes—i. e., the response of the living system to stimulation. First we must note that since in any special case the response as a whole is constant, all of its single component stages or separate processes must also be constant, both in their character and their interconnections. There must therefore be some one constant initial process which is directly caused by the external event or stimulus, and upon which the others automatically and inevitably follow. This initial process thus constitutes the critical or activating event in the physiological sequence. It alone is directly dependent on the stimulus; the others are dependent upon it. What is remarkable is that it should be produced by such a variety of different agents. The problem first to be considered may therefore be put somewhat as follows: What is the nature of the initial change produced in the irritable living tissue by the action of the external agent, and how does it happen that it can be caused by such diverse agencies? This problem has evident relations to a wide group of physiological and psychological problems; thus the question of the basis of the "specific energies" of the special sensory apparatus belongs here. In this case also the response—the conscious affective state or sensation—is distinctive and its quality independent (within certain limits) of the character of the stimulus. This is in fact characteristic of all cases of stimulation. How this can be possible I shall now attempt to indicate.

Let us take the case of the simplest of the irritable tissues of higher animals, one in which the excitation-process occurs in a highly characteristic form, but unaccompanied by highly specialized physiological effects like contraction or secretion. Such a tissue is nerve. What are the essential features in the response of this tissue to stimulation? It is first to be noted that the process set up by the external stimulus is self-propagating. The disturbance, whatever its nature, which originates at the point of stimulus is of such a kind that it imparts a stimulus to the adjoining regions of the nerve beyond the original point of stimulus; these on becoming active stimulate the next stretch of nerve, and in this way the state of excitation passes along the entire nerve to its termination. Evidently there is an active change of some kind, forming an essential component of the local nerve process, that acts as stimulus to adjoining regions. Now there is no mechanical change in a nerve as the impulse passes, little or no production of heat,[1] apparently a slight physical or chemical change involving a loss of carbon dioxide; but none of these is in itself sufficient to act as stimulus. There is, however, another definite physical change which has this power: namely, the electrical variation—the bioelectric process or action-current—which always accompanies the activity of a nerve or indeed of any other irritable tissue. The stimulated region undergoes a rapid change of electrical potential, becoming externally negative relatively to its resting condition; the neighboring still inactive regions being thus positive relatively to the active region, the conditions for the flow of an electrical current between stimulated and unstimulated regions arise. This current is undoubtedly of sufficient strength to stimulate the tissue for some distance beyond the immediate site of stimulation. The voltage of the action-current of frog's nerve is at least thirty millivolts; and a current between platinum electrodes two or three centimeters apart differing in potential by this degree is amply sufficient to stimulate an irritable nerve. The conditions when the two regions of different potential are not externally applied electrodes but portions of the nerve itself are not essentially different; in either case a current flows along the nerve; and if this current is intense enough and arises suddenly enough it must stimulate the latter. There is thus reason to believe that the electrical variation accompanying stimulation is the main condition of propagation of the excited state. This conclusion is supported by various experimental facts; for instance, it is found that the rate of development of the electric variation and the rate of passage of the impulse are influenced to the same degree by changes of temperature, and by certain chemical substances such as the anesthetics. There are various other facts pointing in the same direction, and there are also certain difficulties in the way of this conception; but into these we cannot enter here. The fact remains that the electrical variation is the only known peculiarity of the local process that can account for its self-propagating character; and recent determinations.of the minimal current needed for excitation indicate that the bioelectric currents are of sufficient intensity to serve as the basis for this propagation.

It is clear that propagation of the state of excitation from the immediate site of stimulus over the entire cell or nerve fibre is indispensable to stimulation of any irritable element as a whole by any local stimulus; so that if the above view is correct we must regard the electrical variation as perhaps the most essential feature of the stimulation process. If so, we can understand why the electrical current has such universal stimulating action. In passing a current through a tissue we are artifically setting up differences of electrical potential between different portions of the irritable elements, and according to the above conception this should always cause excitation if the current is strong enough and rises to its maximum with sufficient rapidity. That this is in fact the case needs no emphasis. The electrical current is recognized as the most universal form of stimulus; and all irritable cells and elements, virtually without exception, respond to its action.

We conclude then that the critical or initiatory event in stimulation is an electrical change, consisting essentially in a sudden decrease in the electrical potential of the external surface of the irritable element at the site of stimulation; a difference of electrical potential is thus set up between one portion of the irritable element and another. The problem thus becomes clearer: how is it possible that, e. g., a slight mechanical pressure, or the action of a chemical substance or ray of light, may have this effect on an irritable tissue—i. e., may cause a negative electrical variation and so stimulate? and why do electrical changes, of all the processes in nature, bear this distinctive relation to stimulation?

The answer to these questions is far from complete at present, and their consideration brings us at once to some of the most fundamental questions of general physiology. All of the evidence indicates that the bioelectric processes are of critical importance in the life of the cell; they are associated with the most various physiological activities, and accompany the process of stimulation in all irritable tissues; and it is clear that we must understand their controlling conditions before we are in a position to answer the above questions. Now there is one very general peculiarity of living cells which is intimately connected with their power of responding to stimuli—namely, the possession by the surface-layer of protoplasm of peculiar properties in relation to the diffusion of dissolved substances. Living protoplasm is an aqueous solution, chemically complex and containing a high proportion (10-20 per cent.) of colloidal substances, chiefly proteins and lipoids. Experiment has shown that not all soluble substances readily enter the protoplasm of living cells; thus neutral salts like ${\displaystyle {\ce {NaCl}}}$, sugars and amino-acids (the chief elementary constituents of proteins) diffuse into unaltered cells with difficulty if at all; the surface-film of the protoplasm typically acts toward such substances as a semi-permeable membrane. It is for this reason that the cells of plants remain during life turgid or distended with water, often under high pressure. Osmotic effects, dependent on the semi-permeability of the protoplasmic membranes, are the direct cause of this turgor. The living cell, in other words, is typically enclosed by a modified protoplasmic surface-film or membrane, the plasma membrane, which allows water to pass readily but not dissolved substances of the above kinds. The presence of this membrane makes it possible for the dissolved substances within and without the cell to be very different in character and concentration, and upon this condition the integrity of the living cell undoubtedly very largely depends. We find in fact that when the cell dies many substances, confined during life within its interior, diffuse out into the surroundings; the plasma membrane loses its osmotip properties; the plant loses turgor, wilts and withers; analogous changes occur in animal cells, the colloids coagulate and the cell disintegrates. Conversely if we alter the plasma membranes by chemical substances (poisons), so as irreversibly to destroy their semipermeability, death inevitably follows. Semipermeability is thus for many if not for all cells an essential condition of continued life.

Semipermeability also forms the condition of another fundamentally important property of the living cell, namely, its possession of highly characteristic electrical properties. Physical chemists find in general that if a solid film or other partition consisting of any sufficiently impermeable material—e. g., glass, an organic membrane or a precipitation membrane of copper ferrocyanide or similar material—is interposed between two different solutions containing electrolytes which are thus prevented from mixing, a permanent difference of electrical potential arises between the two solutions. The same appears also to be true of the protoplasmic surface-films or membranes. Apparently, so long as the plasma membrane preserves its normal semi-permeability there exists a considerable difference of electrical potential between its external and internal surfaces—i. e., between the exterior and the interior of the cell—dependent on the difference in composition between the protoplasm and its surroundings. Thus the exterior of a resting muscle cell or nerve fibre is always found positive relatively to its interior. But with the loss of semipermeability at death this potential difference—or demarcation-current potential—also disappears. It thus evidently depends upon the semipermeability of the plasma membrane; and since this electrically polarized condition of the membrane is undoubtedly a factor of prime importance in many cell activities, including stimulation, we see again how physiologically essential a property this semipermeability of the plasma membrane may be.[2]

Further, there is little doubt that this property is one of the essential conditions on which the possibility of stimulation depends. Nernst has shown that an electric current stimulates by changing the concentration of ions at the semipermeable membranes of the irritable tissue; this is equivalent to producing a potential-difference or electrical polarization between the outer and inner surfaces of the membrane. The normal preexisting or physiological potential difference is thus altered—in stimulation is typically diminished—and when this change is sufficiently extensive and rapid the tissue gives its characteristic response. Now these polarization-effects depend on semipermeability, since if the membrane allowed all ions to pass freely the differences of concentration in which the polarization depends evidently could not arise. We find in fact that the cell whose membranes have lost their semipermeability does not respond to stimulation. Such a cell is "dead"; this however need not mean that all vital manifestations have ceased; many metabolic processes may in fact continue in dead cells and lead to far-reaching chemical transformation of the cell-constituents; such changes are called "autolytic." What is lost is the power of responding to stimulation; hence the automatic regulation of the vital processes ceases, and presently these come to an end. We have seen that such a cell has also lost the characteristic vital potential difference between exterior and interior. Response to stimulation thus depends on semipermeability, which implies polarizability of the membranes bounding the irritable cells or elements. This conclusion is one of far-reaching importance, because it localizes the primary change in electrical and hence in other forms of stimulation at the plasma membranes. Some membrane-process forms the first stage of the response to stimulation. The membrane is thus not to be regarded as a mere passive diffusion-preventing barrier between living substance and surroundings, but as the essentially sensitive and controlling portion of the cell.

Although there is much evidence that the initial event in stimulation is a surface-process and involves a change in the chemical and physical properties of the plasma membrane, the precise nature of this change is imperfectly understood at present. It seems, however, clear that it involves a temporary loss or lowering of semipermeability: i. e., the osmotic properties of the membrane are altered, and along with these its state of electrical polarization. This change forms the condition of the other and more complex changes in the interior of the stimulated cell. Evidence of a temporary loss of semipermeability comes from a number of sides, and is seen in the irritable tissues of both animals and plants. Many motor mechanisms in plants depend on this change; e. g., the movements of the sensitive plant, of the Venus' fly-trap, the tentacles of the sundew, etc. Turgid cells arranged in special ways lose their turgor on stimulation and collapse; the resulting movements may be so rapid—e. g., in the Venus' fly-trap—as to simulate muscular contraction. Yet the effect is undoubtedly due to a loss of water caused by a change in the osmotic properties of the plasma membranes.[3] Phenomena of just this kind are not seen in animal cells, where osmotic distension or turgor plays a less important part than in plants; but in gland-cells, many of which are under nervous control, closely similar changes follow upon stimulation. Water and dissolved substances are rapidly lost from the cells, which in many cases shrink at the same time. Electrical variations accompany these processes in both the plant and the animal, and are probably directly due to the change in the membranes. An especially clear parallelism between increase of membrane-permeability and stimulation as seen in the larva? of the marine annelid Arenicola; these larvæ are minute worm-like organisms a third of a millimeter long, actively muscular, and swimming freely by their cilia. When brought into pure sodium chloride or other appropriate salt solution the muscles instantly contract strongly and the contraction is invariably accompanied by a rapid loss of a yellow coloring matter from certain cells forming part of the body. Apparently any change of condition, chemical or other, that increases the permeability of these cells sufficiently to cause a rapid loss of pigment causes also strong stimulation of the irritable elements. It is possible to prevent the stimulating effect of the salt solution by anesthetics or by certain other salts, e. g., calcium or magnesium chloride; and at the same time the change in the pigment-containing cells is also prevented. Rapid increase of permeability and strong stimulation thus show a definite parallelism. Other widespread phenomena, such as the refractory or inexcitable period shown by all irritable tissues immediately after stimulation, point in the same direction. There is indeed an unusually broad basis of biological fact for the inference that in irritable tissues the plasma membranes undergo a sudden and well-marked increase of permeability during stimulation—i. e., lose their semipermeability for a brief time, the exact duration of which varies characteristically for different tissues.

Stimulation appears always to be accompanied by a change in the electrical properties of the irritable elements; and there is every indication that the characteristic negative variation or action current is an expression or consequence of the above change in the membranes. As already pointed out, any semipermeable partition or membrane separating two electrolyte solutions becomes the seat of an electrical polarization, whose degree depends on the nature and concentration of the dissolved substances and on the nature of the partition. Under these conditions any sudden increase of permeability—sufficient to abolish semipermeability—must have the same effect as if the partition were suddenly to disappear; the potential difference between the two solutions then falls to what it would be if no partition separated them. The variation in the electrical potential of the cell-surface during stimulation has in fact the characteristics that we should expect to find if just this change occurs. The electrical variation is always in the direction of an increased negativity of the stimulated region; similarly the dead or injured region where the membranes have lost their normal properties always becomes negative, only permanently instead of transitorily so. In stimulation the membrane-change is reversible, in death irreversible. But the direction of the transitory electrical variation of stimulation indicates a temporary-change in the osmotic properties of the membranes of the same general nature as that associated with death or permanent injury.

We conclude therefore that during stimulation there occurs a temporary and well-marked increase in the permeability of the limiting membranes or protoplasmic surface-films; with this change is associated an electrical depolarization. Experiments with the class of substances known as anesthetics confirms this point of view. When present in the proper proportions these substances render an irritable tissue irresponsive to stimulation. I have recently found that they also change the properties of the plasma membranes in Arenicola larvæ and sea-urchin eggs in such a way as to make them more resistant to increase of permeability under the influence of salt-solutions. A definite parallelism appears to exist; if we render the membranes more resistant to alteration than formerly, we render the tissue less irritable. This influence of anesthetics on plasma-membranes is a very general if not universal characteristic of living organisms. Thus the permeability of plant-cells to salts is decreased by these substances, as Lepeschkin and Osterhout have found, and Loewe has recently shown that artificial lipoid-impregnated membranes are similarly affected. These facts explain why anesthetics counteract the effects of stimulating agencies—which cause temporary increase of permeability; and since most anesthetics are lipoid-solvents, we are led to the conclusion that they cause their effects by changing the state of the lipoid components of the membrane; thus the properties of this structure are altered—particularly the readiness with which its permeability is changed by external conditions acting upon it.

Irritability would thus appear to depend on a peculiar state of the plasma membranes—one in which under slight variations of external conditions these structures undergo automatically a rapid and pronounced increase of permeability. A certain state of physico-chemical instability or lability of the protoplasmic surface-film seems to be the essential condition on which a highly developed irritability depends. Such a membrane appears to retain its properties unaltered only if the external and internal conditions remain approximately constant, especially the state of electrical polarization. If this latter is suddenly changed, as by an external even slight electrical disturbance, some hindrance to interaction seems to be removed, and a chemical process is initiated which instantly alters the character of the membrane and stimulation follows. This, or something closely similar, appears to be the condition in tissues whose irritability is sensitive and rate of response rapid. Apparently all variations occur in the rate at which this change takes place. The electrical variation, whose rate of appearance and subsidence is an index of the rate of the surface-change, is highly rapid in some tissues and slow in others. Thus it lasts for about a thousandth of a second in a frog's motor nerve, and for several seconds in a slowly responding tissue like smooth muscle; and all intermediate conditions are known to exist. These variations in the speed and sensitivity of the response depend primarily on the specific peculiarities of the plasma membranes of the different tissues. What determines the differences between different irritable tissues and organisms in these respects is a subject for future investigation.

These peculiarities of the plasma membranes enable us to understand why the same tissue may respond in the same way to so many different stimulating agencies. Any agency that alters the surface-film to a degree and at a rate sufficient to cause a critical change in its electrical polarization will stimulate. The membrane may be directly altered by mechanical agencies, or by heat or the direct action of chemical substances; or it may contain photosensitive substances and hence be sensitive to light, or special chemical substances and show a specific chemical sensitivity. Whatever alters it in such a way as to change, even momentarily and locally, its permeability and electrical polarization to the critical degree may thus stimulate, i. e., may originate a depolarization which spreads and affects the entire cell. The response which then follows is independent of the nature of the stimulating agency and is determined by special peculiarities of the irritable tissue itself.

The processes which take place in the interior of the stimulated cell are too various and complicated to be considered here. Their nature depends entirely on the specific peculiarities of the cell, and any general characterization is impossible. Usually there is an increase of oxidations and hence of heat-production—in addition to the special physiological manifestation which is evoked—but this is not always the case; thus in nerve, although there is increased loss of carbon dioxide, the heat produced during activity is almost inappreciable; and in other cases there may be a decrease or even complete cessation of all outward activities, e. g., in structures that give an inhibitory response to stimulation; such an instance is seen in the swimming plates of ctenophores which stop movement instantly on slight mechanical stimulation. Facts like these again illustrate the extreme diversity which the entire sequence of events forming the response may show in different irritable tissues, in spite of the essential uniformity of the first stage of the process. This uniformity is the most remarkable feature of physiological stimulation. Nature has apparently found in the variations of permeability and of electrical polarization which external changes may cause in the protoplasmic surface-films the most effective and reliable means of which the internal processes of the protoplasmic system can be made to vary in response to variations in the environment; and in the course of evolution this mechanism has acquired a degree of perfection that still largely baffles physiological analysis.

1. Such, e. g., as causes the transmission of the chemical change along a train of gunpowder.
2. Cf. my article on the rôle of membranes in cell processes in The Popular Science Monthly for February, 1913.
3. The term plasma membrane is applied by some botanists to the entire layer of protoplasm between cell-surface and vacuole-surface. The most external surface-layer, to which ordinarily the term is applied, can not in fact be sharply separated from the inner protoplasm.