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ary state which results when the opposing light and "dark" reactions become balanced, and no fresh decomposition products can be formed by the light. Dark adaptation very obviously is a clear function of the unopposed "dark" reaction.

More than this, however. Certain predictions may be made on the basis of this reversible reaction. Several of these have been investigated with complete success. To mention just a simple example: the "dark" reaction, PAS, is an ordinary chemical reaction; its temperature coefficient should therefore lie between 2 and 3. This is equivalent to saying that the temperature coefficient of dark adaptation should lie between 2 and 3 for 10° C. This is precisely what has been found to be true. The temperature coefficient of dark adaptation for Mya is 2.4. This concept of a reversible photochemical reaction has therefore been fruitful in accounting for the known properties of photosensory stimulation, and has served to suggest the investigation of other properties. The results of these have in turn corroborated the original explanation.

VIII

So far we have considered the events which take place during the sensitization period only. The photosensory responses of these animals, however, involve the very definite existence of a latent period. In fact, in the case of Mya, most of the reaction time is merely latent period and nothing more. Fortunately this part of the reaction time has also yielded to quantitative methods of analysis, and as a result we can now offer an explanation of photoreception which covers not only the sensitization period, but the latent period as well.

At the beginning of this paper, in defining the different parts of the reaction time, I pointed out a significant fact. It is that if the exposure of an animal to light is made shorter than the sensitization period at that intensity, the reaction time and consequently the latent period-is prolonged. This indicates that there is some interrelation between the two portions of the reaction time. Ex

periments were therefore made in which animals were exposed for varying periods of time, all less than the sensitization period. It was found that the duration of the latent period varies inversely with the length of the exposure to light.

The latent period, being the interval during which the animal may remain in the dark following the exposure, is certainly not a time during which nothing happens. We may be sure that a process takes place during the latent period which is in some way a vital link in the chain of events between the incidence of the light and the appearance of the response. Whatever this process may be, we can consider its velocity as proportional to the reciprocal of the duration of the latent period. When this is done, we find that the velocity of the latent period process is a linear function of the duration of the initial exposure to light.

During the exposure we know that the photosensitive substance S is decomposed. We may assume that for, these extremely small exposures, the photochemical effect is directly proportional to the time of action of the light. It therefore follows that the velocity of the latent period process is a linear function of the photochemical effect during the exposure. In other words, the velocity of the latent period reaction is directly proportional to the concentration of freshly formed precursor substances P and A.

Such a relationship may be explained in several ways. The one finally chosen assumes that during the latent period an inert substance, L, is changed into a chemically active material, T, which then acts upon the nerve to produce the outgoing sensory impulse. This reaction, LT, is catalyzed by the presence of the freshly formed photochemical decomposition products, P and A, formed during the exposure to the light. The linear relation between velocity of reaction and concentration of catalyst is a very common one in catalyzed reactions.

In terms of this conception the latent period assumes a position of prime importance in the photosensory mechanism. The latent

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period reaction is all set and ready to go, and requires only that the light change S into P and A so that the latter can catalyze the transformation of L into T, which is the endproduct of the sensory process. The whole photosensory mechanism may then be summed up in the two reactions

S-PA; LIP+AT,

in which the symbol || PA || means catalysis by one or both of the precursor substances. The first of the two reactions occurs during the sensitization period; the second during the latent period.

IX

This hypothesis of photoreception is rather concrete. The concreteness of the conceptions has however proved a useful tool in the acquisition of knowledge in this field. Time does not permit the description of experiments designed to test the hypothesis in numerous ways. I can, however, mention just a few to indicate its fruitfulness.

The latent period is assumed to be a simple, chemical reaction, perhaps as hydrolysis or an oxidation. Its behavior with the temperature should therefore follow quantitatively the rule deduced by Arrhenius for the relation between the velocity constant of a reaction and the absolute temperature. This means more than a mere determination of the temperature coefficient for 10 degrees; it means a continuous relationship between temperature and velocity, following certain theoretical considerations. Experiments showed that the reaction L→T follows this prediction accurately, and that the value of the constant, μ= 19,680, for our reaction is in accord with those usually found for hydrolyses, saponifications, etc., in pure chemistry.

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Another test concerns the interrelations between the exposure and the latent period. I have mentioned that the velocity of the latent period reaction is directly proportional to the exposure (t), provided the intensity (I) is kept constant. This may be written

V = k1t.

If now we keep the time of exposure constant and vary the intensity we find that V = k2 log I

or that the velocity is proportional to the logarithm of the intensity. Ordinary mathematical reasoning indicates that if we combine these two equations-which means experimentally that we vary simultaneously both the time and the intensity-it should be true that

V =

kt log 1. Experiments prove that this expected relationship indeed holds good.

Still another and perhaps more significant application of the proposed hypothesis has been made. This concerns the dark adaptation of the human eye. A careful analysis of the data of dark adaptation in terms of the principles discovered in these investigations has shown that dark adaptation and protoreception in the human retina are fundamentally similar in principle to the process in Mya and Ciona. As a result there has been opened up a new field of investigation in retinal photochemistry which may some day enable us to possess a reasonable theory of vision. SELIG HECHT

PHYSIOLOGICAL LABORATORY,
CREIGHTON MEDICAL COLLEGE,
OMAHA, NEB.

THE MECHANISM OF INJURY AND RECOVERY OF THE CELL1

SOME of the fundamental ideas of biology are extraordinarily difficult to analyze or define in any precise fashion. This is true of such conceptions as life, vitality, injury, recovery and death. To place these conceptions upon a more definite basis it is necessary to investigate them by quantitative methods. To illustrate this we may consider some experiments which have been made upon Laminaria, one of the common kelps of the Atlantic coast.

1 Address for the Symposium on General Physiol. ogy at the meeting of the American Society of Naturalists, December, 1920.

It has been found that the electrical resistance of this plant is an excellent index of what may be called its normal condition of vitality. Agents which are known to be injurious to the plant change its electrical resistance at once. If, for example, it is taken from the sea water and placed in a solution of pure sodium chloride it is quickly injured, and if the exposure be sufficiently prolonged it is killed. During the whole time of exposure to the solution of sodium chloride the electrical resistance falls steadily until the death point is reached; after which there is no further change. If we study the time curve of this process, we find that it corresponds to a monomolecular reaction (slightly inhibited at the start).

This and other facts lead to the assumption that the resistance is proportional to a substance, M, formed and decomposed by a series of consecutive reactions. On the basis of this assumption we can write an equation which allows us to predict the curve of the death process under various conditions. This involves the ability to state when the process will reach a definite stage, i.e., when it will be one fourth or one half completed. This can be determined experimentally with considerable accuracy.

This curve is of practical, as well as of theoretical importance, since it allows us to compare the degree of toxicity of injurious substances with a precision not otherwise attainable. The best way of doing this is to proceed as a chemist might in such cases and express the degree of toxicity by the velocity constants of the reaction (i.e., of the death process) under various conditions.

From this point of view we must regard the death process as one which is always going on, even in an actively growing normal cell. In other words the death process is a normal part of the life process. It is only when it is unduly accelerated by a toxic substance (or other injurious agent) that the normal balance is disturbed and injury or death ensues.

If we wish to put this into chemical terms we may say that the normal life process con

sists of a series of reactions in which a substance O is broken down into S, this in turn breaks down into A, M, B and so on. Under nomal conditions M is formed as rapidly as it is decomposed and this results in a constant condition of the electrical resistance and other properties of the cell. When, however, conditions are changed so that M is decomposed more rapidly than it is formed the electrical resistance decreases and we find that other important properties of the cell are simultaneously altered.

Hence it is evident that injury and death may result from a disturbance in the relative rates of the reactions which continually go on in the living cells.

It is evident that we can follow the process of death in the organism in the same manner that we follow the progress of a chemical reaction in vitro. In both cases we obtain curves which may be subjected to mathematical analysis, from which we may draw conclusions as to the nature of the process. This method has been fruitful in chemistry and it seems possible that it may be equally useful in biology.

If we suppose that resistance depends on a substance, M, it may be desirable to discuss briefly certain assumptions which have been made in regard to it. The protoplasts of Laminaria are imbedded in a gelatinous matrix (cell wall) which offers about the same electrical resistance as sea water or dead tissue. Since the electrical resistance of the living tissue is about ten times as great as when it is killed it is evident that the living protoplasm must be responsible for the increased resistance. The living cells consist for the most part of a large central vacuole surrounded by a delicate layer of protoplasm: the sap which fills the vacuole seems to have about the same resistance as the sea water. The high resistance of the living tissue must therefore be due to the layer of protoplasm surrounding the vacuole, a layer so extremely thin as to be comparable to what is commonly called the plasma membrane." Since the current is due to the passage of ions through

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this extremely thin layer of protoplasm2 it would seem that the electrical resistance may be regarded as a measure of the permeability of the protoplasm to ions. It is of interest in this connection to find that the measurements of the permeability of the protoplasm by a variety of other methods (plasmolysis, exosmosis, diffusion of salts through the tissue, entrance of dyes, etc.) confirm the results obtained by electrical measurement.

In view of these facts the simplest assumption which we can make concerning M is that it is a substance at the surface of the protoplasm which determines the resistance: as M increases in amount and forms a thicker layer the resistance increases, and vice versa.

Tissue which has developed under normal circumstances is found to be rather constant in its electrical resistance. This is of considerable practical importance as it enables us to test material as it comes into the laboratory and to reject any which has been injured or is in any way abnormal.

We may therefore speak of a normal degree of resistance as indicating a normal state of the tissue. If injury occurs and the resistance falls we may consider that the loss of resistance gives a measure of the amount of injury. Thus if the tissue loses ten per cent. of its normal resistance we may say that the injury amounts to ten per cent. This enables us to place the study of injury upon a quantitative basis.

In the case of Laminaria we find that if the injury in a solution of sodium chloride amounts to five per cent. the tissue recovers its normal resistance when replaced in sea water. If however the injury amounts to twenty-five per cent. the recovery is incomplete: instead of rising to the normal it recovers to only ninety per cent. of the normal. The greater the injury the less complete the recovery. When injury amounts to ninety per cent. there is no recovery at all.

2 Some of the current passes between the masses of protoplasm (i.e., in the cell wall) but allowance can be made for this since the relative proportion of cell wall and protoplasm remains unaltered throughout the experiment.

This is of practical interest in view of the fact that in physiological literature it seems to be generally assumed that when recovery occurs it is always complete, or practically so, as if it obeyed an "all or none" law. It is evident that partial recovery may be easily overlooked unless accurate measurements are possible. This may serve as another illustration of the fact that quantitative methods are indispensable in the study of fundamental processes.

It is evident that injury presents two aspects. One is the temporary loss of resistance which disappears, wholly or in part, when the tissue is placed under normal conditions: this may be called temporary injury. The other is the permanent loss of a part of the resistance which is observed after more prolonged exposure: this may be called permanent injury. By exposing tissue for various lengths of time to a toxic solution and observing the amount of recovery each time we may construct a time curve of permanent injury. This curve may be subjected to the same kind of mathematical treatment as the time curve of temporary injury, already discussed. The mathematical analysis leads to the conclusion that if we adopt the scheme OS→ A→M-B we must regard temporary injury as due to the loss of M while permanent injury is due to the loss of O. Recovery occurs when the loss of M is replaced by a fresh supply of M derived from O, but if O is itself depleted recovery will be incomplete.

It may be added that an equation has been found which enables us to predict the recovery curves under a great variety of conditions with considerable accuracy.

If we accept the conclusions stated above we are obliged to look upon recovery in a somewhat different fashion from that which is customary. Recovery is usually regarded as due to the reversal of the reaction which produces injury. The conception of the writer is fundamentally different; it assumes that the reactions involved are irreversible (or practically so) and that injury and recovery differ only in the relative speed at

which certain reactions take place. Thus in the series of reactions

O→SAM → B,

if the rate of OS becomes slower than the normal, injury will occur, while a return to the normal rate will result in recovery. Injury could also be produced by increasing the rate of M→ B, or decreasing the rate of SA or A→ M.

If life is dependent upon a series of reactions which normally proceed at rates bearing a definite relation to each other, it is clear that a disturbance of these rate-relations may have profound effects upon the organism, and may produce such diverse phenomena as stimulation, development, injury and death. It is evident that such a disturbance might be produced by changes of temperature (in case the temperature coefficients of the reactions differ) or by chemical agents. The same result might be brought about by physical means, especially where structural changes occur which alter the permeability of the plasma membrane or of internal structures (such as the nucleus and plastids) in such a way as to bring together substances which do not normally interact.

In the case of Laminaria death may occur in two ways. In the first there is a loss of resistance which continues until the death point is reached, as, for example, in sodium chloride. In the second, as in calcium chloride, there is an increase of resistance followed by a decrease. Both of these methods may be predicted by means of the scheme already outlined.

If we mix sodium chloride with calcium chloride we do not get a result which is merely intermediate for we find that long after death has occurred in pure sodium chloride or pure calcium chloride the tissue still survives in a mixture of these salts (made in certain definite proportions). The facts lead us to assume that both sodium and calcium combine with a constituent, X, of the protoplasm, forming a compound Na,XCa. According to the laws of mass action we may

calculate the amount of this compound which will be formed in each mixture of sodium and calcium chlorides. These calculations indicate that the speed of all the reactions is regulated by the amount of Na,XCa (it is also found that certain reactions are accelerated by calcium chloride).

This enables us, by means of the equations already mentioned, to predict the time curves of injury and death in mixtures (in addition to those in pure salts) as well as the recovery curves when tissue is transferred from such mixtures to sea water.

It is evident therefore that the theory not only explains why pure sodium chloride and calcium chloride are toxic but also why they antagonize each other in mixtures. Moreover the explanation which it furnishes is a quantitative one, i.e., it shows just what degree of antagonism is to be expected in each mixture.

Extremely interesting results are obtained when the tissue is first exposed to sodium chloride, then to calcium chloride, then to sodium chloride or to sea water and so on. By varying the conditions of the experiment a very complicated set of curves may be obtained. It is rather remarkable to find that all of these may be predicted with considerable accuracy by means of the equations already referred to. A detailed statement of the results will be found in recent papers in the Journal of General Physiology.

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Throughout these investigations the aim has been to apply to the study of living matter the methods which have proved useful in physics and chemistry. The attempt presented no serious difficulties after accurate methods of measurement had been devised: nor does there seem to be any real obstacle a general use of methods which lead biology in the direction of the exact sciences. It is evident that if the facts have been correctly stated such fundamental conceptions as vitality, injury, recovery and death may be investigated by quantitative methods. This leads us to a quantitative theory of these phenomena and a set of equations by which they can be predicted. It may be added that the

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