Injury, Recovery and Death in Relation to Conductivity and Permeability

changing from net resistance to protoplasmic resistance we merely shift the value of the constants. The question arises whether this affects the general conclusions drawn from the study of net resistance. In order to decide this question the constants for CaCL and for various mixtures of NaCl and CaCL, were ascertained; these are given in Table XII.34 There are two points of principal importance in the consideration of these constants: (1) It has been shown35 that the value of KA-^-KM increases regularly as the per

cent, of CaCl2 in the surface of the cell increases. That this is also true in the case of protoplasmic resistance is evident from Fig. 94. (2) It was also pointed out that as the per cent, of CaCl2 in the solution decreases from 62 to 1.41% the value of KM first decreases (reaching a mini- mum at 4.76% ) and then increases. It was found that the 34 These are approximate values, obtained graphically. The con- stants of the curves of protoplasmic resistance are designated as &Ap (corresponding to KA ) and KMP (corresponding to RM)- The curves of protoplasmic resistance may show less inhibition at the start than those of net resistance.

amount of decrease corresponds to the amount of a hypo- thetical salt compound (Na4XCa). This is also true in the case of protoplasmic resistance, as shown in Fig. 95.36 It would therefore appear that we arrive at the same conclusions whether we study net resistance or protoplas- mic resistance. When the solution is changed the con- FIG. 94. — Ordinates represent the increase in value of KA -f- K^and K^p -=- K MP. In each case the value given represents the increase over the corresponding value in the solution con- taining 1.41% CaCh (the per cent, in the surface being 12.5). Abscissae represent per cent. of CaCh in the surface. In order to facilitate comparison the values of KAP -r- K^!P have

stants change in a corresponding manner in both cases, the only difference being in their absolute values, but it is evident that in this case differences in absolute values are of no importance. It should be emphasized that this general conclusion would remain valid in case it should be found that the values given here for Cp and Cw are incorrect. There seems to be no doubt that the value of Cw is constant under the conditions of these experiments and as long as

A rough calculation shows that this is also true of (corresponding to the KN and KO mentioned on page 98)- FIG. 95. — Ordinates represent the amount of Na^XCa and also the decrease in the value of K Af/\ and of ^J,//>Q as compared with the corresponding value in the solution containing 62%CaCl2. Absciss® represent per 'cent, of CaCh in the solution. In order to facilitate comparison the values of KM have been multiplied by 0.251 and those of K Mp by 0.321.

as follows : An electrical current passing through a liv- ing plant flows partly through the cell wall and partly through the protoplasm. The relative amounts of these two portions of the current can be calculated. The outcome of such calculations shows that the conclusions drawn from the study of the resistance of the tissue as a whole apply also to the resistance of the protoplasm, and consequently to the permeability of the protoplasm to ions.

If these conclusions are sound it is evident that per- meability may be measured with considerable accuracy. Measurements under a variety of conditions indicate that marked fluctuations of permeability are possible, and, when their duration is brief, no permanent injury results. It is obvious that the effect of such fluctuations on metabolism may be of great importance. Some writers37 seem to think that under normal con- ditions the cell is quite impermeable to salts. This is at va- riance with the results obtained from measurements of permeability by the method outlined above. If the net resistance of the tissue under normal circumstances is taken as 100, we find that in certain solutions (having the conductivity of sea water) it may rise to 300. The proto- plasmic resistance under normal conditions may be taken as 140. When the net resistance of the tissue rises to only 250, the protoplasmic resistance increases to 874.89, a gain of 524.92%.

It is therefore evident that the permeability of the cell is by no means at a minimum under normal conditions. This conclusion is borne out by the results of experiments on plasmolysis38 carried out by the writer as well as by the investigations of Brooks cited above. It would seem that it is well founded, since if the cell were impermeable, it could not obtain the salts necessary for its existence.31* It is, of course, true that the electrical resistance of the cell is much higher when alive than when dead, as is shown by the work of Roth (1897), Bugarsky and Tangl (1897), Stewart (1897), and Woelfel (1908), on blood corpuscles, that of McClendon (1910), and Gray (1913, 1916) on sea urchin eggs, that of Shearer (1919) on bacteria, as well as the results of the writer.

The seat of this higher resistance might be sought in the interior of the cell, or at the surface. Plant cells offer especially good material for this sort of investigation, since in most cases the protoplasm forms a thin layer surrounding a large central vacuole filled with cell sap. It has been shown above, (pp. 198 and 199) that, in the cases investigated, the sap has a conductivity which does not differ greatly from that of the external solution. It would therefore seem that the cause of the high resistance is to be sought in or near the surface. Hober (1914, pp. 383, 442) has reached this conclusion as the result of experi- ments on red blood corpuscles and muscles. He employed two methods for measuring the conductivity of the interio.r of the cell. The first depends on the fact that a conducting body increases the capacity of a condenser when inserted between the plates. The second is based upon the fact that a conductor placed in the centre of a coil of wire diminishes the strength of an alternating current in the coil. Using these methods, Hober finds that the conductivity is higher than when it is measured in the usual way (in which the current passes through the cell ) . He therefore concludes that the surface

has a higher resistance than the interior. It should be noted, however, that in these methods the experimental errors are so great that the results must be accepted with caution. The view that the surface layer of the protoplasm is less permeable than the interior has long been current. Such a layer need not be a visible membrane:40 on the contrary, it need only have the thickness of a single layer41 of molecules. This surface layer is commonly spoken of as the plasma membrane, but the writer prefers the term cell surface (since a morphologically distinct membrane is not necessary in order to ensure selective permeability). If it is not necessarily a visible structure, we may ask what evidence there is for its existence and whether it is any- thing more than a convenient fiction.

It is easy to understand how the idea of the plasma membrane was accepted by botanists. In many cases the interior of the plant cell is filled with cell sap around which the protoplasm forms a layer, so thin as to be almost invisible under the microscope, except under the most favorable conditions. In such a case the whole of the protoplasm might be looked upon as constituting the plasma membrane. When the layer of protoplasm is thicker, it may be shown that there are differences between the permeability of its inner and outer surfaces, de Yries found that

'A layer of liquid may serve as in the experiment of Nernst (1904) where a layer of water is interposed between pure ether and benzene dis- solved in ether; such a layer is permeable to ether, but not to benzene. In the same way a layer of air may be employed, e.g., the layer of air over an aqueous solution of cane sugar is permeable to water molecules, but not to sugar. If we place under a bell jar two beakers, one con- taining pure water and the other sugar solution, the water will pass over, in the form of vapor, into the sugar solution. 41 Cf. Langmuir, I. (1917).

certain dyes penetrated the42 outer surface much more readily than the inner.43 The experiments of the writer show a difference in the two surfaces and emphasize the conception that the permeability of the protoplasm is not alike in all its parts.44 The real question is whether a special layer exists at the outermost surface of the cell which admits some sub- stances, but not others. Furthermore, is it possible that substances which penetrate the outermost layer with difficulty can spread freely throughout the cell when they have passed the outer layer?

It is a well-known fact that substances, which, like protoplasm, contain a considerable amount of protein readily form films at their surfaces when brought into contact with liquid.45 By means of the ultra-microscope Gaidukov46 observed a differentiated film at the surface of the cell. Such films or membranes have been sho\vn to exist in some cells by micro-dissection and there are indi- cations that they also exist at the surfaces of vacuoles,47 and of nuclei.48 The surface of Amoeba and of some other protozoa is covered with a thin membrane capable of

*3 The objection might be made that the dye cannot penetrate the inner surface until the protoplasm has become saturated with it, and this might be confused with a difference in permeability. forming wrinkles. What part these films or membranes play in permeability is not known. When a cell is crushed, so that drops of protoplasm are extruded, it is often observed that each drop behaves as if surrounded with a plasma membrane, and a rupture is in most cases instantly repaired (as long as the cell remains in the normal condition). This might be ex- plained as due to the formation of films upon contact with liquid.

Kiister (1909; 1910 A, B.) found that when the proto- plasm of a cell was separated into several pieces by plasmolysis the parts would fuse if brought together at once, but if left for a time would no longer do so, indicat- ing that a change had taken place in the surface film. Kite (1913) states that a dye which could not pene- trate the cell was able to spread freely in its interior, when introduced by a Barber pipette.49 The nature of the cell surface has been the subject of much dispute. Overton based Ins view that it is lipoid in nature on the ground that lipoid-soluble substances readily penetrate, while those which are not soluble in lipoid do not enter the cell, and stated that this was particularly the case with inorganic salts. It was subse- quently found, however, that cells are permeable to salts,50 and to other substances insoluble in lipoid. He found51 an apparent confirmation of his theory in the behavior of dyes. It had been shown by Ehrlich52 that basic dyes are taken up by nerves and by lipoid substances. Overton extended this notion to living cells in general and assumed

that the penetration of dyes is dependent on their solu- bility in lipoid. Subsequent investigations have brought to light so many exceptions to this rule that it can no longer be regarded as conclusive evidence in favor of Overton 's views.53 Overton 's views gained wide support through their application to the explanation of narcosis. Overton54 and Meyer55 independently arrived at the conclusion that the more soluble a substance is in lipoid, the more effective it is as a narcotic. They explained this by saying that the more soluble the anesthetic is in lipoid, the more easily it penetrates the lipoid membrane.

Although this hypothesis has found wide acceptance, there are serious objections to it. If it be true that anesthetics are generally effective in proportion to their solubility in lipoid,56 it does not by any means follow that the plasma membrane is lipoid. As we have already seen,57 the effectiveness of a dye in color- ing the cell does not depend on its rate of penetration, but on its ability to accumulate within the cell by combining with substances in the protoplasm. If this is also the case with anesthetics, lipoids in the interior of the cell may be the determining factor, and there is no necessity for the assumption of a lipoid membrane.

M There are some substances which act as anesthetics ( e.g., magnesium salts) which are only slightly soluble in lipoid. discussion of the nature of the cell surface.58 Enough has been said to show that there is considerable evidence that there is a layer at the surface which is different from the underlying protoplasm and that some substances pene- trate it more rapidly than others. It is doubtful, whether there are many substances to which it can be regarded as wholly impermeable. It is, however, able to protect the metabolism of the cell from various kinds of interference from without, and to provide for the differentiation of multicellular organisms by making it possible to keep various processes separate. The principal advantage of cell division may consist in providing the semiper- meable membranes, which make differentiation possible.

A good illustration of this differentiation is seen in those cases where diverse chemical operations go on in adjoining cells without mutual interference. In many plants deeply colored cells are surrounded by colorless ones, and the soluble coloring matter does not show any tendency to diffuse into the surrounding cells. We may even observe that the color is confined to the vacuole of the cell, and does not diffuse into the surrounding proto- plasm. In the same way we observe in some plant cells colored plastids (chromatophores) containing soluble pigments which do not diffuse out into the cytoplasm. A cell of this sort is shown in Fig. 96.

In the case of Griffithsia, each of these plastids is surrounded by a semipermeable membrane which retains Czapek (1914) has suggested that the plasma membrane is com- posed of soaps. Nathanson (1914) regards it as mosaic of lipoid and non-lipoid particles. This would not provide an entrance for lipoid- soluble and lipoid-insoluble substances into the cell-sap unless each ele- ment of the mosaic extended continuously, without a break, from the outer surface to the vacuole. For a general summary see Bayliss (1915), Hober (1914), and McClendon (1917).

the pigment. This can easily be shown by killing the cell, whereupon the semipermeable membranes are destroyed and the pigment at once begins to diffuse out. In this case, we have to do with variety of semipermeable mem- branes, such as the plasma membrane, the surfaces of the plastids, the vacuolar surface/'9 and the nuclear surface. It is to be expected that these surfaces may differ some- what in permeability. Each of them is in contact with Fia. 96. — A cell of Griffithsia Bornetiana (in optical section), a, cell wall; b, protoplasm

c, chromatophore containing chlorophyll and a red pigment (phycoerythrin) which is soluble a somewhat different environment, and this, as we have already seen, might produce differences in permeability. That such differences really exist is indicated by treating the cells with NH4C1 (neutralized by adding NH4OH) which is not strong enough to plasmolyze. The vacuolar surface then contracts while the plasma membrane main- tains its original position. At the same time the surfaces of the plastid become permeable and the red pigment comes out: it cannot, however, pass through the plasma membrane or the vacuolar surface. We see that all three sorts of surfaces act differently, and to these we may add

69 de Vries (1885) states that certain dyes penetrate the outer sur- face more easily that the surface of the vacuole. It has been objected that the dye may combine with the protoplasm and hence cannot penetrate the vacuole until the protoplasm is saturated. This might cause an appearance of a difference in permeability. a fourth, the nuclear surface, which does not agree in behavior with any of the others.60 It is quite possible that there are other surfaces within the cell which likewise differ in their behavior.

If we suppose that these surfaces not only differ among themselves, but that their permeability fluctuates under normal circumstances, we shall probably get a fairly cor- rect picture of the complex relations which obtain in the cell. This conception is not as simple as that of the "reaction chamber " hypothesis of Hofmeister,61 but it agrees more nearly with our present knowledge.62 The conception that the cell contains a variety of mem- branes which are capable of alterations in permeability, is capable of explaining some important phenomena. Among these may be mentioned certain effects of injury. It is well known that mechanical injury is followed by increased respiration:63 this may be explained by the increased permeability of membranes which have pre- viously kept the oxidizable material from being attacked. Increased respiration due to chemical agents64 might be explained in the same way.

An illustration of a different reaction is the bitter almond, which evolves HCN upon injury. In this case a glucoside and an enzyme are brought together when 62 It may be added that while changes in the permeability of internal membranes may affect the electrical conductivity of cells which are filled with protoplasm (as in the case of most animal cells) they can hardly play an important role in cells like those of Laminaria (and most plant cells) in which the interior of the cell is occupied by a large central vacuole. In the latter, however, the permeability of the vacuole mem- brane must be of importance.

injury occurs, and the resulting reaction produces HCN. Such illustrations might be multiplied indefinitely. Another important question which may be considered in this connection is that of mechanical stimulation. The effects of certain kinds of stimuli can be referred directly to chemical changes which they produce in the proto- plasm, but there are other kinds which appear to operate by physical means only. In the latter category are such stimuli as contact, mechanical shock and gravitation. While their action appears at first sight to be purely mechanical, they are able to produce effects so much like those of chemical stimuli that it appears prob- able that in every case their action must involve chemical changes.

The chief difficulty which confronts a theory of mechanical stimulation appears to be this : How can purely physical alterations in the protoplasm give rise to chem- ical changes? It would seem that a satisfactory solution of this problem might serve to bring all kinds of stimu- lation under a common point of view, by showing that a stimulus acts in every case by the production of chemical reactions. The writer has observed when one of the larger cells of Griffithsia (Fig. 96) is placed under the microscope (with- out a cover glass) and touched near one end (with a needle or a glass rod or a splinter of wood) a change occurs in the chromatophores directly beneath the spot which is touched. The surfaces of the chromatophores in this region become permeable to the red pigment, which begins to diffuse out into the surrounding protoplasm. This change begins soon after the cell is touched. As the red pigment diffuses through the protoplasm it soon reaches neighboring chromatophores and it may then be seen that their surfaces also become permeable and their pigment

begins to diffuse out. In this way a wave — which may be compared to a wave of stimulation — progresses along the cell until the opposite end is reached. The rate of propagation of this wave corresponds to that of the diffusion of the pigment. It would seem that at the point where the cell is touched, pigment, and prob- ably other substances, are set free, diffuse out and set up secondary changes as they progress. These changes are doubtless chemical in nature.

The important question then arises: How does the contact initiate the outward diffusion of the pigment or other substances! It seems to the writer that this may be due to a mechanical rupture of the surface layer of the chromato- phore which is either not repaired at all or only very slowly. Many cases are now known in which the surface layers of protoplasmic structures behave in this way.65 If, therefore, such structures exist within the cell, it is evident that any deformation of the protoplasm which is sufficient to rupture their surface layers, will permit their contents to diffuse out into the surrounding protoplasm. A great variety of cellular structures (plastids, vacuoles, "microsomes," inclusions, etc.), possess surface layers of great delicacy, and it is easy to see how some of these might be ruptured by even the slightest mechani- cal disturbance.