Injury, Recovery and Death in Relation to Conductivity and Permeability

A reading taken at the end of 18 hours showed that the tissue was dead in all the solutions. The plants can be kept alive much longer than this in mixtures of NaCl + CaCl2 ; it is also noteworthy that the degree of antagon- ism, as shown by the electrical resistance, is greater in NaCl + CaCL than in NaCl + HC1.34 The hypothesis was further tested by investigations on other salts, the most interesting of which are those which (in contrast to those just mentioned) are more effective than CaCL in decreasing permeability, such as La (N03)3, Ce (N03)3, etc. Here also it was found that the degree of antagonistic action could be foretold by observing the amount of decrease of permeability pro- duced by the pure salts. The results of these investiga- tions afford strong support to the hypothesis.

The soundness of this point of view is indicated not only by the fact that we are able to predict both qualita- tively (and to a considerable extent quantitatively) the effect of combinations of salts35 but also by the very 88 It should be noted that mixing solutions of two salts which belong to different classes does not produce an effect which is merely intermediate between the two. For example, tissue may be killed by an exposure of 24 hours to NaCl or to CaCL, but not in a mixture of these in the proper proportions.

The writer has found cases in which two substances which can decrease permeability are able to antagonize each other. So far as the writer's experiments with Laminaria have gone there is no great amount of antagonism in such cases and what there is may perhaps be correlated with the fact that all substances which decrease permeability do not act alike, some producing a much greater decrease than others. Moreover these substances will, if the exposure be sufficiently prolonged, alter their action and increase permeability. The rapidity of this change varies with different substances, and this may be related to the fact that some of these substances antagonize each other to some degree.

Experiments on some plants (in which the criterion of antagonism is not electrical resistance, but growth) show a fairly strong antagonism between magnesium and calcium. It is possible that for these plants mag- nesium belongs in the first class. It will be noted that the hypothesis, as here set forth, says nothing about the mutual relations of substances belonging to the same class, but merely states that substances of one class will antagonize those of the other. In this form the hypothesis is completely justified by all the experiments, including those on organic substances.

significant fact that we are able to extend this conception to organic compounds and to show that non-electrolytes which decrease permeability can also antagonize such substances as NaCl. These facts indicate that the hypoth- esis may perhaps be applied in a general manner so as to include both electrolytes and non-electrolytes. As an example of antagonism between salts and organic substances we may cite some experiments with bile salts. The writer found, very early in the course of his experiments, that Na-taurocholate increases the elec- trical resistance of Laminaria. This was somewhat striking in view of the fact that agents which increase permeability have long been known, but the discovery of substances which have the opposite effect, is compara- tively recent. The number of such substances known at present (especially organic substances) is very small and it is therefore of interest to find that bile salts possess this property.

The experiments were made by determining the elec- trical conductivity of Laminaria in solutions to which Na-taurocholate was added.36 In the first experiments the bile salt was dissolved in sea water. The amounts added to 1,000 cc. of sea water va- ried from 0.8 to 1.5 gm. If the Na-taurocholate were pure, 1 gm. in 1,000 cc., would make the concentration about 0.002 M, but as its purity is doubtful the concentration can- not be accurately determined.

After dissolving the Na-taurocholate the sea water was restored to the normal conductivity and made approximately neutral to litmus. At all the concentrations employed there was an immediate increase in resistance37 followed by a fall. Under the conditions of the experiment (temperature 19°±2°C.) the rise lasted about an hour. The effect is comparable with that of anesthetics38 (ether, chloroform, and alcohol) as described by the writer. An increase in resistance was also observed with Ulva rigida and with Rhodymenia palmata.

In the experiments on antagonism the tissue was placed in a solution of NaCl 0.52 M to which various amounts of Na-taurocholate were added (all the solu- tions having the same conductivity as the sea water and being approximately neutral to litmus). The tem- perature was 18.5±2.5°C. The results are shown in Fig. 72. There is a gradual fall of resistance in all the solutions which continues until the death point (10%) is reached. In the solution containing 1,000 c.c. of NaCl 0.52 M + 0.52 gm. of Na- taurocholate the fall of resistance is much slower, indi- cating that this is the most favorable mixture.

It should be emphasized that the effect is not an intermediate but an antagonistic one. By this is meant that the resistance is not merely the algebraic mean between a rise in resistance produced by the bile salt and a fall produced by NaCl. A consideration of the lowest curve shows that at 180 minutes the tissue is dead in NaCl 0.52 M as well as in 1,000 c.c. of NaCl 0.52 M + 10 gm. of Na-taurocholate, but in the mixture containing only 0.5 gm. of taurocholate it is not yet half dead, its resistance being much higher than in the other mixtures.89

The result serves as a striking confirmation of the idea that antagonistic relations can be predicted, to a considerable extent at least, by ascertaining the effect 88 At the end of 180 minutes the resistance of the control in sea water upon permeability of each substance taken by itself, inasmuch as substances which decrease permeability antagonize those which increase it. Similar investigations were made upon alkaloids. Antagonism between salts and alkaloids has been reported

FIG. 72. — Curves showing antagonism between NaCl and Na-taurocholate. The ordinates represent the net electrical resistance of Laminaria agardhii (expressed as per cent, of the control in sea water which is taken as 100%). The abscissae represent the amount of Na-taurocholate added to 1000 c.c. of NaCl 0.52 M. Average of two experiments; probable error of the mean less than 5% of the mean. by several authors, the most extensive investigation being that of Eobertson.40

The alkaloids studied were nicotine, caffeine and cevadine. They were added in varying amounts to NaCl 0.52 7lf41, and their effect upon the electrical conductivity of Laminaria was determined. 41 All the solutions had the same conductivity as sea water. 73. The lower curve shows that after 18% hours the resistance of the tissue has dropped to 10% (the death FIG. 73. — Curves showing antagonism between NaCl and nicotine. Ordinates represent net electrical resistance of Laminaria agardhii (expressed as per cent, of the normal) ; abscissae represent concentrations of nicotine added to 0.52 M NaCl. The resistance of the control at 18X hours was 94%. Average of two experiments; probable error of the mean less than

point) in 0.52 M NaCl, as well as in 0.52 M NaCl to which sufficient nicotine has been added to make the concentra- tion 0.01 M. In NaCl 0.52 M plus nicotine 0.002 M the resistance has dropped to 49.5% (i.e., the tissue is about half dead). It is evident that nicotine antagonizes the action of NaCl by inhibiting the fall of resistance which occurs in pure NaCl. The upper curve (2 hours) shows even more pronounced antagonism. The results with caffeine (Fig. 74) are similar except that the curve does not fall as rapidly with increasing

FIG. 74. — Curves showing antagonism between NaCl and caffeine. Ordinates represent net electrical resistance of JLaminaria agardhii (expressed as per cent, of the normal); abscissae represent concentrations of caffeine added to 0.52 M NaCl. The resistance of the control at 18^2 hours was 96%. Average of two experiments; probable error of the mean less than concentrations of alkaloid. With cevadine (Fig. 75) the curve falls much more rapidly, the maximum being in the neighborhood of 0.005 M cevadine sulfate. Here death

is more rapid, the tissue being killed in 18 hours or less, even in the most favorable solution. FIG. 75. — Curves showing antagonism between NaCl and cevadine sulfate. Ordinates represent net electrical resistance of Laminaria af/ardhii (expressed as per cent, of the normal) ; abscissae represent concentrations of cevadine sulfate added to 0.52 M NaCl. The resistance of the control at 150 minutes was 100%. Average of two experiments; probable error of the

ing the day at 15 ±2°, and the time curves in the various solutions follow more or less closely a monomolecular course. In the case of nicotine and caffeine (where the experiment ran during the day and the following night) this is not the case, except in the earlier part of the reac- tion. This is perhaps explained by the fall of tempera- ture which occurred during the night and retarded the speed of the process. It should bd noted that all the experi- ments in any set were begun at the same time, so that all shared equally in the variations of temperature; in consequence the form of the antagonism curve is not greatly affected by such variations.

In order to determine whether these alkaloids pro- duce a decrease in permeability they were added to sea water. The experiment was not successful in the case of nicotine, owing to the formation of a visible precipitate, which was apparently due to the presence of calcium and magnesium in the sea water. In the case of caffeine (0.01 to 0.04 M) and of cevadine sulfate42 (0.0006 to 0.0025 M) a distinct decrease in permeability was found (as shown by the rise in resistance) ; this was followed by an increase. In this respect they resemble CaCl2 which also produces a decrease in permeability when added to sea water.

The idea that substances which have opposite effects on permeability can antagonize each other seems to apply to alkaloids as well as to salts. The question arises whether the decrease of conduc- tivity and the antagonistic action produced by organic substances are of the same nature as those produced by salts. Are they, in terms of the theory outlined above, due to an increase in the thickness of the layer of M at the surface of the cell? The writer is not prepared to answer this question at present, but there is no reason to suppose that their effects may not differ from those produced by

43 This is regarded as two molecules of cevadine united to one molecule of H2S04. It was purchased from Merck under the name of veratrine sulfate ( C32H40N09 ) 2.H2S04. Cf. Osterhout ( 1919.Z) ) . Fio. 76. — Curves showing antagonism (after an exposure of 24 hours) between NaCl 0.52 Af and CaCh 0.278 M in Laminaria agardhii (upper curve) and Rhodymenia palmata (lower curve). The ordinates denote net electrical resistance. The abscissae denote molecular proportions of the solutions (all the solutions having the conductivity of sea water). Thus NaCl 85, CaCh 15 signifies a mixture of 75 c.c. NaCl 0.52 M + 25 c.c. CaCk 0.278 M in which the molecular proportions of Na to Ca are as 85 to 15. Temperature 17.5° ± 5°C. During the 24 hours the resistance of Laminaria in sea water remained practically unaltered while that of Rhodymenia fell to 84.5%. Average of six experiments. Probable error of the

era! validity, experiments were made upon other plants and upon animals. In general the outcome (as far as the experiments have gone) is similar to what has been described for Laminaria. Thus antagonism between NaCl and CaCl2 was observed in the cases of TJlva (sea lettuce), Rhodymenia (dulse) and Zoster a (eel grass).43 As was to be expected, the most favorable proportions were not always exactly the same for the different plants. Thus it was found that in the case of Rhodymenia it required more Ca to antagonize Na than it did in the case of Laminaria. It was also observed that in the case of Rhodymenia (Fig. 76) the antagonism was not so great as in Laminaria and this appears to be correlated with the fact that less decrease of permeability is produced by Ca in Rhodymenia. In other words, the effect of such a substance as Ca upon permeability not only indicates what substances it will antagonize but also, to some degree at least, the amount of antagonism.

It may be added that Rhodymenia affords an interest- ing confirmation of the value of the electrical method in measuring antagonism, since the plants begin to change color soon after injury occurs. It was found that the relative rates of death as indicated by color changes in NaCl, CaClo, and the various mixtures, correspond with the results obtained by determining conductivity. Antagonism between NaCl and CaCl2 was also observed in the case of frog skin.44

Shearer (1919) in an experiment on bacteria finds that NaCl and KC1 decrease, and that CaCL increases the resistance, but that a mixture of these (Ringer's solution) in the proper proportions preserves the normal resistance. Thus far, we have confined ourselves to the consid- eration of antagonism among kations. Numerous cases of this are known, but the search for similar relations among anions has achieved little result. Some cases have been described by Loeb45 and Miss Moore (1901, 1902). Lipman and his associates46 have reported antagonistic action of anions as the result of studies on bacteria in

which salts were added to the soil, but it is very difficult to separate the effects of the added salts from those FIQ. 77. — Curves showing the resistance of Laminaria agardhii in 1.1 M sodium acetate, in 0.36 M sodium sulfate, and in mixtures of both: A in equal parts (by volume) of acetate and sulfate; B in acetate 75, sulfate 25; C in acetate; D in acetate 25, sulfate 75; E in sulfate. Ordinates represent net electrical resistance (expressed as per cent, of the original resistance in aea water which is taken as 100%). Each point represents the average of ten experiments: probable error of the mean less than 5% of the mean.

of salts already present hi the soil. Miyake (1913) found some antagonism among anions in studying the growth of rice. Fenn (1918) has called attention to the fact that this kind of antagonism is commonly met with in experiments on gelatin. Using electrical conductivity as a criterion, Raber (1920) has found well marked antagonism between FIG. 78. — Antagonism curves showing the net electrical resistance of Laminaria agardhii in 1.1 M sodium acetate, in 0.36 M sodium sulfate, and in mixtures of both. Ordinates repre- sent resistance (expressed as per cent, of the original resistance in sea water which is taken as 100%); abscissae represent volumetric proportions of the two salts. The dotted line connecting the ends of each curve shows the approximate additive effect; the vertical dis- tance of the curve above this dotted line may be regarded as a measure of antagonism.

Na-acetate and Na2S04 in experiments on Laminaria, as shown in Fig. 77. On placing tissue in the pure acetate we observe that at the end of 1% hours, the resistance has fallen to about 40% of the original and in the pure sulfate it has fallen to about 25% of the original, while in the mixture com- posed of equal volumes of the solution of each salt, the resistance has fallen only to about 60%. If no antagon- FIG. 79. — Increased toxicity shown by curves of the electrical resistance of Laminaria ayardhii in NaCl 0.52 M, Na-citrate 0.58 M (approximate) and in mixtures of these (the proportions, representing c.c. of the component solutions, are indicated on the abscissae. Curve A, observed values, after an exposure of 15 minutes to the solution, Curve B values, expected on the supposition that neither salt affects the action of the other (additive effect). The increase of toxicity is measured by the vertical distance between the curves. All readings were made at 23° C. or corrected to this temperature. Each observed point pre- sents the average of 10 experiments: probable error of the mean less than 10% of the mean.

ism were present, the resistance in the mixture should drop to about 35% (additive effect). Fig. 78 shows the antagonism curves after various intervals, using resistance for ordinates and salt propor- tions as abscissae. Here the antagonism is clearly evident. Eaber (1917) gave quite the opposite result. In this case a distinct increase of toxicity occurs on mixing the com- ponents. This is evident from Fig. 79. Similar results were obtained when Na-citrate was combined with Nal, NaSCN, NaN03 or Na2S04.

Cases which show increased toxicity (as judged by other criteria) have been reported by Lipman47 and by Loeb.48 It may be of interest to call attention to certain phenomena in non-living matter which bear at least a superficial resemblance to some of the facts dis- cussed above. In the course of experiments on Laminaria, the writer frequently observed that fronds kept in NaCl become softer,49 but that in CaCl2, and in LaCl:{, they become harder. The changes in viscosity are so great as to suggest that they are fully capable of explaining the fall of electrical resistance which occurs when tissue is placed in NaCl and the rise of resistance which occurs in CaCl2 and LaCl3 (which is always followed by a fall of resistance).

In the hope of throwing some light upon this process, sections of tissue were observed in CaCL under the microscope. It was then seen that after a time the proto- plasm assumed a coagulated appearance: it seemed obvious that the process which increased the viscosity might produce a coagulation of the protoplasm or some other change in its structure whereby it became more permeable. This conception led the writer to expect decreased resistance in tissues placed in NaCl (because of decreased

viscosity) while in CaCl2 we should expect to find increased resistance (due to increased viscosity) followed by a fall of resistance (due to coagulation or other struc- tural change in the protoplasm). It soon became apparent that there were several serious objections to this conception. The most impor- tant of these may be briefly stated as follows :50 1. If to a solution of NaCl we add CaCl2 until the increase of viscosity produced by one salt is just balanced by the decrease produced by the other, the resistance should remain stationary. This is not the case: there is always a fall, or a rise followed by a fall, of resistance.

2. If more CaCl2 be added there should be a rise of resistance : this should after a while become stationary, provided there is not enough CaCl2 to produce the coagu- lation or other structural change which decreases the resistance. This does not occur: the tissue never maintains its increased resistance, but shows a fall of resistance which begins soon after the maximum is reached. 3. If still more CaCl2 be added, so as to produce the coagulation or other structural change which decreases resistance, we should expect to find in all cases the same viscosity (and consequently the same maximum of resist- ance) just before the fall begins. Still further increase of CaCl2 would only hasten this process without changing the maximum. This does not correspond with the facts. The maximum steadily rises as the proportion of CaCL increases, so that the greatest maximum is found in pure CaCL.