Protoplasmic Action and Nervous Action

When any irritable organism or cell responds to stimulation, the energy for the response is derived from the chemical energy of the protoplasmic constituents, usually from the oxidation of carbohydrates. It is clear therefore that one of the essential effects of the stimulus is to alter the rate or character of cell-metab- oHsm. In many cases this effect may be indirect; e.g., in the voluntary muscle cell a large part of the heatproduction following a single stimulus succeeds the contraction^ (Hill); similarly in the turgor-motors of

plants and possibly in other motile organs. But in all cases the work performed in the response represents energy derived from metabolic breakdown, although this energy may act in the intervals between stimulation by developing a tension or turgor which is released only at the moment of stimulation. A fundamental problem thus arises with regard to the general nature of the conditions in living matter which render its rate of chemical reaction so readily alterable by physical changes in the system.

The most significant general fact is that it is only while the cell is living that its rate of metabolism is readily and quickly changed by a stimulating condition. In general, also, it is only during life that the energyyielding forms of metabolism have a high rate or intensity; typically COa-production, heat-production, and consumption of oxygen decrease greatly at death, although they may not cease entirely. One of the most remarkable peculiarities of living protoplasm, considered as a chemical reaction-system, is that its chief energyyielding reactions, e.g., oxidation of sugar, proceed rapidly at low temperatures, and in a medium which is approximately neutral. To produce a corresponding speed of reaction in vitro, high temperatures or strong reagents are required. It is probable that the conditions which determine the susceptibility to stimulation are the same as those which are responsible for the high velocity of the energy-yielding reactions. The nature of these conditions is imperfectly understood at present; but apparently they are especially favorable to certain types of oxidation; e.g., of carbohydrates.

The indications are that structural rather than purely chemical factors are of chief importance, since the oxidation-furthering enzymes (oxidases) extractable from the cell have a relatively slight influence on the physiologically important oxidations; e.g., of sugar. We may thus regard the possession of a certain type of structure, characteristic of the living state, as chiefly responsible for the facility with which chemical reactions proceed in living protoplasm, as well as for their modifiability under stimulating conditions. The synthetic reactions appear to be largely dependent upon the oxidations; this is indicated by the importance of oxygen for growth processes, as well as by various other facts, although the precise nature of this interdependence is not understood at present. The whole problem of the relations between the structure of protoplasm and its chemical activity is one of fundamental interest, and some of the more general facts and considerations bearing on this problem will now be briefly reviewed.

All Hving matter is characterized by the possession of a certain structural organization or permanent arrangement of components which is essential to its normal activity. ' If we destroy protoplasmic structure by heat, mechanical injury, or chemical treatment, the specific metabolic activity of the system and its responsiveness to stimulation are lost. In general, the chemical reactions of living matter may be grouped under two classes according to their relation to protoplasmic structure: (A) those reactions which continue in an essentially unaltered manner after the ^4ife" of the cell has been destroyed; e.g., in cell-

extracts or in the residue remaining after complete mechanical or other disintegration of the protoplasm; and (B) those which continue only while the protoplasm remains structurally intact and 'living." The former group (A) includes a large number of hydrolyses and some oxidations; e.g., those due to oxidases; but, as already indicated, the physiologically significant oxidations, especially of sugar and other energy-yielding compounds, cannot be accomplished, at least with anything like the normal velocity and completeness, under the influence of enzymes or cell-extracts. Yeast cells which have been mechanically destroyed, or even simple watery extracts of yeast, rapidly hydrolyze cane sugar, just as does the living cell, and autolyzing yeast cells split proteins rapidly into amino-acids. It has been found, however, that the alcoholic fermentation of sugar proceeds much more slowly in the press-juice of yeast than it does under the influence of the living protoplasm.^ Many other cases are known where biochemical reactions, although proceeding in dead cells or under the influence of cell-extracts, do so at a slower rate than in living protoplasm.

The latter group of reactions (B) include the specific syntheses, i.e., of protein, together with those syntheses which require the expenditure of considerable energy, like the building up of fats from carbohydrate, or of amino-acids and other compounds of high chemical potential from compounds of lower potential. The energy required for these syntheses is apparently de- ^ Cf . Harden, "Alcoholic Fermentation," in Monographs on Biochemistry, edited by Plimmer and Hopkins.

rived from the oxidation of other compounds, especially carbohydrates.^ It is especially signiiicant that in all cases the synthesis of specific proteins, the reactions essential to growth and maintenance, requires the intact protoplasmic structure. These syntheses constitute the chemical reactions most highly characteristic of the living state. At one time it was believed that all of the metabolic syntheses were the result of the special activity of living protoplasm, and that the chemical reactions of dead protoplasm or cell-extracts were always of a catabolic (splitting) kind; it is now known, however, that various syntheses involving little change of energy, e.g., the synthesis of esters and disaccharides, readily occur under the influence of enzymes alone. Yet the fundamental fact remains that the more important or specific part of the synthetic activity of protoplasm is exhibited only during life. A relation of the normal protoplasmic structure to certain types of chemical action, especially synthetic action, is thus indicated. With the alteration of structure occurring at death, as indicated by loss of semi-permeability, coagulation of cell-proteins, and other phenomena of disintegration, is associated a loss of synthetic power. Only the living yeast cell can build up from a solution of sugar, tartrates,

^ Hence the importance of carbohydrates for growth and assimilation; e.g., in plants, carbohydrate is indispensable for the assimilation of amino-acids by yeast and molds (cf. the series of papers by F. Ehrlich, Biochem. Zeitschrifi, I, VIII, XVIII, XXXVI (1906-11); similarly in higher plants the synthesis of proteins from amides in germination requires the presence of carbohydrates (cf. Jost's Physiology of Plants, p. 175, for a summary of the chief facts). The sequence of metabolic derangements associated with diabetes shows the fundamental importance of carbohydrate metabolism in higher animals.

and inorganic salts the various special compounds, definite and constant in number, proportions, and distribution, which compose the yeast protoplasm. Some of the changes in the chemical reactivity of protoplasm resulting from mechanical or other destruction of the living cells or tissue are well illustrated by Fletcher's and Hopkins' work on the formation and disappearance of lactic acid in muscle;^ also by the work of Harden and Maclean on oxidation by isolated animal tissues f and more recently by Warburg's determinations of the oxygen consumption in living cells (sea-urchin eggs, blood corpuscles, bacteria, etc.) as compared with that of the same cells after death or fine mechanical subdivision.^ In all of these cases chemical activity is greatly decreased when the protoplasmic structure is artificially destroyed. Warburg has also shown that when certain cells, the blood corpuscles of birds, are mechanically broken down by freezing and thawing, the oxygen consumption exhibited by the residue is associated with the more solid part of the complex — that which can be separated by centrifuging ; similarly, in liver cells the separable granules have a relatively high oxygen consumption."* A relation of oxidative activity to the solid part of the protoplasmic structure is thus indicated. In some cases it can be shown microchemically that certain oxidations (the indophenol reaction) occur most actively at the surfaces of solid

4 Warburg, cf. Biochem. Zeitschrift, CXIX (1921), 134, and references to earlier papers there given. structures, such as the nuclear and plasma membranes.^ It is interesting to note that a visible alteration or breakdown of protoplasmic structure seems always to be associated with the death process, however induced; even after natural death, coagulative or other alterations occur in most forms of protoplasm; death rigor, increased permeability, and loss of tensile strength in muscle cells, are examples of such effects. The death change involves a structural disintegration, with which is associated a loss of normal chemical activity.

A second class of cases, in which certain chemical reactions may be promoted instead of hindered by the breakdown of normal cell structure, also throws light upon the relation of structure to the chemical activity of protoplasm; an example is the autolytic breakdown of proteins or of glycogen in dead liver cells or other autolyzing cells. The rate of such breakdown is increased when the structure is altered by death, and still more so (according to Chiari's observations) in the presence of lipoid-solvent compounds like chloroform.^ Such facts illustrate another form of chemical control exercised by protoplasmic structure. Apparently they indicate that a partitioned or alveolar structure exists during life; enz}Tiie and substrate, for example, may thus be kept apart while this structure is intact, but on death the interalveolar partitions are broken down and interaction results. It has been suggested by Hofmeister^ that this chambered type of architecture is what renders it possible for a variety of chemical reactions to occur

within the limits of a single cell without mutual interference; different metabolic processes are thus localized* a necessary condition for a definite "chemical organization" of the cell. It is well known that when living protoplasm is acted upon by cytolytic agents or heat (40°) or is altered mechanically or osmotically (i.e., by hypertonic or hypotonic media) beyond a certain degree, chemical changes are induced in it which are absent or inappreciable under normal conditions. These changes are associated with profound structural alteration, as shown by coagulation of the cell-proteins, changes of permeability and water content, and loss of the normal tensile and other mechanical properties of the protoplasm. Thus in muscle, and probably in most other cells, lactic acid is formed in large quantity; in many cells autolytic changes are initiated; in oxidase-containing fruits and tubers (apple, potato) the browning reaction occurs; and in many cases (muscle) there is a marked temporary increase in the output of CO2. Of special interest is the fact that these changes are associated with a loss of the normal semi-permeability of the plasma membranes, coincidently with a loss of the characteristic water-immiscibility of the protoplasm as a whole; hence disintegration by diffusion processes follows rapidly. These effects are such as might be expected to result from a breakdown of the normal partitioned structure of the system. Materials which during life are kept apart by the interposition of films are thus enabled to interact; hence (as already cited) autolysis is accelerated by cytolytic compounds like chloroform. For a similar reason the minuter structural elements — which normally

are prevented by the pervading film-stnicture from fusing or otherwise losing their identity — undergo alteration or breakdown; a general coarsening or increase of opacity, indicating coagulative changes in the cell proteins, is characteristic of dying protoplasm, and is associated with the changes in mechanical properties described above/ The basis of these changes is insufficiently understood at present, but their ready production by lipoid-solvent compounds seems to indicate that the lipoid constituents of the protoplasm are specially involved. Apparently the lipoids have a relation to the protein constituents resembling that which a "protective colloid" (gelatine) added to a suspensoid hydrosol (gold) has to the colloidal particles of the suspensoid. In the presence of the protective substance the particles remain separate under conditions, such as the presence of salts or increase of H-ion concentration, which otherwise lead to fusion or precipitation;^ this stabilizing influence is apparently dependent on the formation of thin adsorption films about the particles. In the case of living protoplasm, the evidence from cytolysis and similar phenomena indicates that the normal fine subdivision of the structural proteins — shown by the characteristic translucency during life — is dependent on the presence of thin lipoid films (possibly soap) at the surface of the protein particles, fibrils, or other structural elements. When these films are broken down or destroyed, a coalescence of particles and a coarsening of structure result; these effects involve a loss of semi-permeability, together with the changes

^ The progress of structural changes of this kind can be followed by the microscope under dark ground illumination; cf. Aggazzotti, Z. allg. Physiol., XI (igio), 249. in mechanical and chemical properties already described. On such a view the cytolytic action of lipoid-alterant compounds may be explained. Such compounds act by destroying the film-structure; hence, in addition to destruction of semi-permeability, they break down the intracellular, partitions and induce chemical reactions of the above-described kind and cause coagulation of the cell-proteins. In irritable cells such compounds have also a strongly stimulating action of an irreversible kind, as shown in the contraction produced in muscle cells, and similar effects.

The above-described loss of translucency accompanying cytolytic or mortiferous processes is a phenomenon of much interest, which has an intimate bearing on the general problem of protoplasmic structure. This change is shown with great clearness in all of the more transparent forms of protoplasm; e.g., the eggs of marine animals (starfish, etc.), protozoa, and muscle cells. Some years ago while studying the conditions of activity in the ctenophore swimming plate — a beautiful example of a clear translucent protoplasm, consisting of parallel contractile fibrils (fused ciha) — I was struck with the constancy and definiteness of the relations existing between changes of translucency and changes of contractile activity. In dying animals the plates become partially clouded and adopt a rapid unintermittent movement, differing from the normal movement in being of quicker rhythm and in no longer showing the mechanical inhibition described above; this movement continues until finally the plate becomes white and opaque and all activity ceases.^

A similar cycle of alteration is passed through, only more rapidly, when the normal plates are transferred from sea water to various unbalanced solutions, such as pure isotonic NaCl; the plates then exhibit for a brief period (one or two minutes) an extremely active vibratory movement, which is associated with a progressive whitening or coagulation. In general the rate of coagulation is more rapid the more energetic the contractile activity; and it is especially noteworthy that the coagulative process does not begin until the plate starts vibrating; the vibration then continues until the whole structure is opaque. This change of structure is irreversible, and at the end the plate is so altered in consistency that it readily falls to pieces when shaken. Evidently the contractile activity is associated with the removal of some substance or condition which prevents the coalescence of the protein particles forming the fibrils. A film-structure of the kind suggested above seems indicated, which is broken down by the action of the solution with the production of both chemical and mechanical effects. The general relations between such effects and stimulation processes will be considered in more detail below. Apparently in the swimming plate the essential effect produced by the unbalanced solution is an acceleration or intensification of the normal processes of stimulation and contraction; a dependence of these processes on the alteration or removal of film material is thus indicated. The indications are that during the normal rhythm of contraction in sea water the film-structure is alternately broken down and reformed in each contractile cycle. Presumably under the abnormal conditions resulting from the action of the pure NaCl solution the rate of

breakdown is increased, and the restoration of filmstructure between successive contractions becomes imperfect, with the result that eventually the whole structure disintegrates. These effects may be compared with those of excessive fatigue, which also leads to irreparable structural breakdown.^ It will be evident from the preceding discussion that structure is only one factor in the chemical activity of protoplasm; undoubtedly many other factors — those entering in all chemical reactions, such as concentration, temperature, special affinities, catalysis — enter in determining the rate and character of the metabolic reactions. But the controlling factor — that which is subject to rapid and reversible alteration under the influence of stimulating agencies — appears to be the peculiar structure of the living substance. By the conception of ''structure" as applied to protoplasm is meant, generally speaking, the distribution of the physically stabler components, usually the solid components, of the system. Evidently, as already pointed out, this structure is itself a product of metabolism; but having once been formed, it influences the further course of metabolism — in the general manner of which Child's comparison of the living organism to the flowing river^^ gives a good illustration by analogy. In the living organism there is always structure of a definite kind; even the simplest ''undiffer-

^ Cf. the instances of structural alterations in the central nervous system described in Crile's recent book, A Physical Interpretation of Shock, Exhaustion, and Restoration, London (1921). ^ Cf. Child, The Regulatory Process in Organisms, Journal of Morphology, XXII (1911), 171; also Senescence and Rejuvenescence, chap. i. entiated" protoplasm is not homogeneous, and it is necessary to reach a clear conception of the essential nature of this structure in the most generalized forms of living substance if we are to be in a position to understand the fundamental conditions of physiological activity.

That metabolism is controlled by structure is seen in many well-known physiological facts already referred to in part; e.g., the course of development, with the associated constructive metabolism, may in many eggs or embryos be profoundly modified by artificially altering the structure of the system. Developmental processes are frequently initiated by mechanical means; cases of regeneration illustrate this, or cases where injury of the egg-surface (pricking in the case of the frog's egg,^ or any kind of cytolytic action in echinoderm eggs)"^ initiates cleavage and development. Mechanical treatment causes stimulation in innumerable instances; in others it causes inhibition. In all of these cases the energy for the developmental or other response comes directly or indirectly from metabolic processes. This sensitivity to the action of mechanical agents, which by their impact, pressure, or other effects locally modify cell structure, is perhaps the clearest proof of the intimate relations existing between structure and function in living protoplasm.

In physical chemistry the importance of structural conditions as modifying factors in chemical reactions is illustrated in the so-called heterogeneous catalyses. In these phenomena the acceleration of reaction is ^ Loeb, Artificial Parthenogenesis and Fertilization, University of Chicago Press (1913). dependent chiefly on surface effects, of which two classes appear to be especially important from the biological point of view: (i) adsorption effects, leading to increased concentration at surfaces and hence increased reactionvelocity; and (2) electrolytic effects, due to the existence of local potential differences between different regions of the surface separating the two phases; when both phases conduct electricity, local circuits may thus arise, furnishing the conditions for electrolysis. This latter effect may also be regarded as a form of catalysis, and is illustrated in the spreading of rust spots on iron surfaces, or the periodic catalysis of H2O2 by mercury. Both kinds of effects are of fundamental importance in protoplasmic processes, as will be shown in more detail later. Other conditions characteristic of surfaces may also enter (see pp. 217 ff.).

As already pointed out, all forms of protoplasm exhibit the power of specific synthesis characteristic of life. The constructive metabolism by which the specific structural elements are built up and maintained must, like other forms of metabolism, be under the control of structure. This synthetic activity, being a universal property of living matter, is undoubtedly to be correlated with the most general or fundamental type of structure exhibited by protoplasm. In correspondence with its uniformity of essential chemical composition and chemical behavior, protoplasm must also possess a uniformity in its essential type of physical structure; underlying the variety of structural detail must be some characteristic type of structural composition common to all forms of protoplasm, and determining the special features of its chemical activity. The traditional problem of the

structure of protoplasm is thus intimately bound up with the basic problem of general physiology. The problem relates to the nature of the structure in livmg protoplasm. All observers agree that cell structure is profoundly altered by death; disintegration then follows, accompanied by diffusion of the cell constituents into the surrounding medium. As already described, the death of the cell is associated with loss of its normal osmotic properties or semi-permeability; the normal electrical polarization also disappears at the same time; both phenomena are characteristic, and indicate interruption in the continuity of the protoplasmic boundary layer. The most obvious general structural changes occurring in the cell interior at death are of a coagulative kind ; the protoplasm loses its normal translucency and becomes more opaque (death rigor or death coagulation). This effect is seen in the greatest variety of cells and organisms, especially those with translucent protoplasm, as cited above. The protoplasm of muscle cells becomes more opaque and loses its coherency or tensile strength; dying swimming plates whiten and fall to pieces on shaking, and other phenomena of a similar kind are well known to all biologists. Many observations on the postmortem alterations of structure have been made since the introduction of the methods of microdissection. Kite and Chambers describe dying cells as losing their viscidity and as being easily torn to pieces. Chambers describes the isolated nerve-ganglion cells of the lobster as undergoing irreversible structural changes when mechanically injured; the protoplasm then ''sets into a coagulated non-viscous mass which may be broken into non-glutinous pieces." Taylor describes a similar break-

down of protoplasmic structures in Protozoa after injury with the microdissection needle.^ Facts of this kind show again that the maintenance of a certain characteristic type of structure is an essential part of normal protoplasmic activity. The structure of living protoplasm is not to be conceived as resulting from a combination of static parts like the structure of a machine; it is the product or expression of continual synthetic activity and persists only while metabolism persists; it expresses the constructive activity of metabolism, very much in the same manner as the structure of a flame or of a fountain expresses the dynamic activity of such a system. If the activity disappears, so also does the characteristic structure or configuration which is maintained by that activity. In this sense, structure in living protoplasm is to be conceived as continually in process of formation; i.e., as an index of the underlying synthetic reactions which, as already seen, are inseparable from the chemical activity of the system during life. The apparently static condition represents in reality a state of balance between construction and disintegration.

Yet a certain permanent or stable structural constitution (at least relatively permanent) has to be assumed, just as in the case of the fountain or candle flame. This is necessary if the dependent processes of chemical transformation are to exhibit constant characters. The physical nature of this permanent or persistent structural substratum of living protoplasm has first to be considered. It is not possible here to review in detail the numerous and frequently conflicting conceptions of protoplasmic structure. The details made visible by microscopical technique are of so varied a kind that none of the many attempts at unification have met with universal agreement. A chief difficulty has been that most histological investigators seem to have conceived of protoplasmic structure as existing independently of the chemical and physiological activities of the living system, and not as both dependent upon and determining these activities. Some conception of structure is required which will be general enough to apply to all of the forms of living matter, and which will at the same time enable us to understand the dependence of the fundamental vital properties of specific synthesis and irritability upon structure. It may be doubted whether we are yet in a position to form a clear and permanently valid conception of protoplasmic structure, but with the progress in our knowledge of the properties of colloidal systems has come what appears to be an increased insight into the possibilities. The problem may be defined in its essential terms, as follows: Can a system, with components of the kind which we find present in all living matter, be imagined which will exhibit, as a correlative of its structural composition, the above-described properties of specific growth, sensitivity to electrical conditions, catalytic

activity, and automatic regulation of composition and properties ? The experimental studies and observations of the last twenty years have led more and more to the conclusion that the general or fundamental structure of protoplasm corresponds more closely to that of an emulsion than to that of any other simple non-living physical system. The most general facts of its chemical composition are in agreement with this conclusion. Water-insoluble constituents (lipoids) occur in association with colloidal constituents which have water-combining powers (proteins). The whole resulting complex is during life immiscible with water, and typically is bounded from the external watery medium forming its immediate environment by a layer or surface-film having semipermeable properties. The semi-permeability and the water-immiscibility of the surface layer appear to be interdependent properties; they suggest the existence of a continuous external layer of water-insoluble material of fatty or similar nature.^ The unit of organic structure, the cell, would thus appear to be a system with an aqueous internal phase limited externally by a thin water-insoluble phase or boundary layer. The aqueous internal phase forms one component of a system, the cell protoplasm, which is structurally and chemically highly complex, and emulsion-like in its general physical constitution.

It is evident that the general properties of emulsions do not in themselves explain the properties of living matter. What seems highly probable, however, is that the original structural foundation upon which the properties of living matter have arisen — or which has made it possible for systems with vital properties to evolve — is that of an emulsion; i.e., a polyphasic system with thin interfacial films separating two or more component phases which have fluid or solvent properties. From general considerations it seems clear that some kind of pol}^hasic structure must be assumed in order to account for such a universal property as that of growth; the unit of living matter, even while it continues to increase in size, retains a complex and specific composition different from that of the surroundings; and this peculiarity is in itself incompatible with structural homogeneity, since the elementary need of providing against free diffusive interchange with the surroundings requires a surface layer with properties different from those of the internal protoplasm. This must be true even of the simplest forms of living matter. We cannot compare the protoplasm of ultra-microscopic organisms with self-propagating enzyme-like material (supposing such material possible), as has been done, since the physical conditions necessary for metabolism and growth must exist in even the simplest living systems; and this requires at the very least a differentiation between the more permanent or solid components of the system and the liquid components which contain in solution simpler materials (nutrients and oxygen) which are continually being renewed.

It has long been recognized that colloids form the basis of protoplasmic structure. Hardy's investigations showed that many of the characteristic structural appearances presented by fixed and stained protoplasm in microscopic preparations were incidental consequences of the colloidal composition of the system and not expressions of any distinctively vital condition or structure. He and others have demonstrated that similar appearances can be produced by fixation in apparently homogeneous colloidal solutions or gels (egg-white, gelatine).^ Hardy also pointed out various parallels between the processes of gelation in artificial colloidal systems and the changes of physical state in living protoplasm.^ From these and related facts it became clear that if we are to draw conclusions regarding protoplasmic structure from the appearances seen in microscopic preparations, the general nature of the changes produced in colloidal systems by physical and chemical agents must first be determined. Great impetus was thus given to the study of the physics and chemistry of colloids, a subject then in its early stages, and also to the study of the structure and physical properties of protoplasm in the living condition.

Various resemblances between living protoplasm and emulsions were long ago described by Biitschli.^ These resemblances relate both to structure and to certain peculiarities of behavior; e.g., amoeboid movement and modifications of activity by changes in the surroundings. Biitschli reached the conception that a ''foam structure," corresponding essentially to a filmpervaded or chambered structure, is the t>"pe most generally exhibited by living protoplasm."^

The subject of the physical chemistry of emulsions forms a part of the now extensively developed field of colloid chemistry, and cannot be considered here in any detail. Some of the more general facts relating to the structure and properties of emulsions must, however, be discussed briefly, since a clear conception of the physical conditions existing in these systems is necessary before proceeding to the consideration of the more complex types of structure and behavior which have evolved in living matter, apparently with emulsionsystems of a relatively simple kind as a basis.