Injury, Recovery and Death in Relation to Conductivity and Permeability

The temperature was controlled in short experiments so that the fluctuations did not amount to more than ±2°C. In longer experiments (lasting several days) greater fluctuations were unavoidable, but the effect of these was minimized by starting all the experiments of a series at the same time so that the fluctuations affected 30 Water distilled from a copper still should never be used. all of them equally. This answers very well as long as we are comparing experiments which last about the same length of time, but it may happen that one of the series lasts but a short time and after its completion the others proceed at a different temperature. In this case the whole series should be rejected unless the difference in temperature is small.

The temperature coefficient of the electrical conduc- tivity of living Laminaria22 is about 1.331 ; this is higher than that of dead tissue (1.26) which is very close to that of sea water. This coefficient may be employed to correct readings which are not made at the standard temperature, provided the deviation in temperature does not exceed two or three degrees. IN studying Laminaria it is found that toxic sub- stances may be divided into two classes according to their effects upon the conductivity of the tissue. The first class includes those which cause a progressive loss of resistance, ending in death ;* the second class produces a rise in resistance,2 followed by a fall which continues until the death point is reached.

The first group includes salts of monovalent metals.3 The investigations of Eaber (1920) have shown that the higher the valency of the anion (which is combined with the monovalent kation) the more rapid is the fall in resistance.4 In subsequent studies5 it was found that the trivalent arsenate anion is more efficient than the bivalent molyb- date and sulphate and these in turn are more efficient than the univalent formate and chlorate.16 Further 1 Effects of this sort are also produced by hypo- and hypertonic solu- tions, by drying, by moderate heat (e.g. 35° C), by lack of oxygen, or by exposure to ordinary laboratory conditions.

* In some cases a temporary rise is observed, due to the fact that ions diffuse out of the tissue faster than they diffuse in. This is easily recog- nized because it is as pronounced with dead tissue as it is with living. See Osterhout (1918, D}. 6 In these studies the solutions were not of the same concentration, but all had the conductivity of sea water, except the molybdate, which had the conductivity of 75% sea water plus 25% distilled water. Hence the conclusions stated above should be taken in a qualitative rather than a quantitative sense.

studies by Raber7 indicate that the rise in resistance which is produced by bivalent and trivalent kations is Fia. 13. — Curves of net electrical resistance of Laminaria agardhii in 1793 c.c. NaCl 0.52 M+207 c.c. CaCh 0.279 M with the addition of 0.01, 0.02, and 0.03 M NaOH. 'The per- centages were calculated on the basis of the net resistance of the control. All readings were taken at 18° C. or corrected to this temperature. Each curve represents a single experiment

less when they are combined with trivalent anions than when combined with univalent. The writer8 has found that OH is more effective than any other anion in producing a fall in resistance. Since the addition of alkali to sea water produces a precipitate of Mg(OH)2 the experiments were made by adding vari- FIG. 14. — Curves of net electrical resistance of Laminaria agardhii in 1793 c.c. NaCl 0.52 M +207 c.c. 0.279 M containing various amounts of NaOH. The abscissae represent the concentration of NaOH in the solution: the ordinates represent the percentage of electrical net resistance calculated on the basis of the net resistance of the control. All readings were taken at 18° C. or corrected to this temperature. Each curve represents a single

sea water until a slight precipitate of Mg(OH)2 was formed. When tissue was placed in this its resistance FIG. 15. — Curve of net electrical resistance of Laminaria agardhii in 1975 c.c. sea water plus 48 c.c. NaOH 0.22 M (pH about 10), unbroken line, and of a control in sea water, broken line. The percentages were calculated on the basis of the net resistance in sea water at the beginning of the experiment. The readings were taken at 18° C. or corrected to this figure.

steadily decreased, falling to 68% in about six hours. (Fig. 15). Haas9 has shown that in this case the pH • Haas, A. R. C. (1916, A ) . From the table given by Haas it is evident that when the concentration of added NaOH is about 0.005 M (the writer used 0.0052 M) the burette reading has risen from 7.28 to about 7.33, giving a pH of about 10. value is about 10. This is evident from Fig. 16, which shows the increase of pH value as alkali is added to sea water.10 It is therefore evident that small amounts of alkali affect the resistance.

FIG. 16. — Curve showing changes in the hydrogen ion concentration of sea water upon the addition of alkali at 21° C. Ordinates show the hydrogen ion concentration. In passing from 1X1Q-8 to 1X10-9 the successive divisions are read as follows: 9,8, 7, 6, 5, 4, 3, 2, 1, 5, 1, each multiplied by 10-9. Abscissa? show burette readings beginning at 7.28 cc. and ending at 9 c.c. The curve shows that on adding alkali to sea water the hydrogen ion concentration at first falls rapidly and then very slowly until the magnesium hydrate has all been precipi- tated. After this further additions of alkali cause a more rapid fall in the concentration of the hydrogen ion, but this is soon checked by the precipitation of the calcium hydroxide. After the calcium hydroxide is all precipitated further additions of alkali will cause a corresponding decrease in the concentration of the hydrogen ion.

The efficiency of OH in decreasing resistance is strik- ingly shown in Fig. 17, which illustrates the rapid fall of resistance in NaCl + Ca(OH)2 as compared with Since the alkaline solution contains fewer calcium ions (though the concentration of calcium molecules is the 10 The sea water was obtained from Woods Hole and was the same as that used in the writer's experiments. same) an experiment was made in 10O which the concentration of cal- cium ions was kept undiminished. For this purpose there was added to a s a t u r a t e d solution of Ca(OH)2 (in distilled water) suffi- cient CaCl2 1.42 M to make the conductivity equal to that of sea water. Tissue was placed in this and also in CaCl2 0.278 M. In spite of the fact that the concen- tration of calcium ions was practi- cally the same in the two solutions the behavior of the tissue was markedly different. In pure CaCl2 the net resistance rose to 171% of the original net resis- tance while in CaCl2 + Ca(OH)o it rose to only 113%. At the end of forty-five minutes the resis- tance in CaCl2 was 146%, while in CaCl2+Ca(OH)2itwasonly23%.

These experiments make it evident that small amounts of NaOH are able to produce a con- siderable increase in permea- bility.11 Let us now consider those sub- stances which increase the resis- of the tissue. In general we Fiq. 17. — Curves of net electrical resistance of Laminaria agardhii in a solution containing NaCl 97.2 mols of NaCl to 2.8 mols of CaCh (uppermost curve) ; a solution con- taining 97.2 mols NaCl to 2.8 mols Ca(OH)2 (middle curve) and in NaCl 0.52 M (lowest curve). All the solutions had the conductivity of sea water. All readings were taken at 18° C. or corrected to this temperature. Each curve repre- sents a single typical experiment.

find thatbivalentkations are very effective in this respect12 and trivalent still more so.13 In most cases the effect is so striking that the addition of the solid salt to the sea water, although decreasing the resistance of the solution, nevertheless increases the resistance of the living tissue so greatly that the net result is an increase in the resist- ance of the tissue plus solution. As this does not happen FIG. 18. — Curve showing the net electrical resistance of Laminariaagardhii in sea water to which was added sufficient cobalt chloride (in the form of dry salt) to make the concentration 0.005 M . Solution neutral to litmus. All readings were taken at 18° C. or corrected to this temperature. The curve represents a single experiment. Dead tissue showed no rise.

with dead tissue the increase must be due to an alteration in the living protoplasm.14 It is therefore evident that the current must flow in part through the living proto- plasm, as well as through the cell walls. "It might be suggested that the increase in resistance is due to a de- crease of the spaces between the protoplasmic masses brought about by an expansion of the protoplasm or by a shrinkage of the cell wall. Micro- scopic and macroscopic examination shows that this does not occur. The tendency is, on the other hand, to increase the spaces between the cells, as the result of incipient plasmolysis.

When dead Laminaria is transferred from sea water to CaCla 0.278 M there is a very slight shrinkage which, however, is entirely inadequate to cause a noticeable rise in resistance if it occurs in the cell walls of living tissue. Zn) the addition to sea water of sufficient dry salt to make the concentration 0.005 M kept the resistance above that of the control for several hours (Fig. 18). In other cases (FeS04 and SnCl2) the resistance rose, but soon fell below that of the control.15

Fia. 19. — Curve showing the net electrical resistance of Laminaria agardhii in 1000 c.c. sea water plus 10 c.c. CaCh 5.0 M. Upper curve, living tissue; lower curve, dead tissue. All readings were taken at 18° C. or corrected to this temperature. Each curve represents a kations it was found that while the resistance remained above that of the control for ten hours or more in the case of La (N03)3 and Y (N03)3 this was not the case16 with Fe2(S04)3 and Th (NO3)4.

These experiments were varied by adding strong solutions to the sea water in place of the dry salt. The "In the case of SnCl2 this may be due to the acidity of the solution. The concentration is .005 if in the case of each of these salts. result of such an experiment with CaCL is shown in Fig. 19. It will be observed that while the resistance of FIG. 20. — Curves showing the net electrical resistance of Laminaria agardhii in solutions of the same conductivity as sea water, i.e., in La(NOs)3 about 0.126 M , CaCta 0.278 M., MnClt about 0.317 M, MgCh about 0.28 M and in NaCl 0.52 M. The curves for La(NOa)3, MnCh and MgCh represent single typical experiments (all readings were taken at 18° C. or cor- rected to this temperature) ; the curves for CaCh and NaCl represent the averages of six or more experiments; probable error of the mean less than 10% of the mean (all readings were taken at 18° C. or corrected to this temperature).

17 The temporary fall at the start is due to the increased conductivity of the solution contained in the apparatus and in the cell walls. tissue falls from 15.2% to 13.53%, thereby losing 11% of the resistance it had in sea water. This corresponds quite closely to the loss of resistance of the sea water itself upon the addition of this amount of CaCl2. Experiments were also performed by placing tissue in solutions (having the same conductivity as sea water) which contained only one salt. It was found (Fig. 20) that the bivalent Ca, Ba, Sr, and Mn raised the resistance (increasing it to 160% or more) while the trivalent La and Ce gave a much greater rise (increasing it to 220% or more). On the other hand, Mg gave very little rise (increasing the resistance as a rule to not more than 115%). In this respect its behavior is not unexpected, since it is usually less effective than other bivalent kations (as for example in antagonizing the effects of Na).18

It was found that Ulva (sea lettuce) and Zoster a (eel grass) resemble Laminaria in showing a rise with MgCL and a much greater rise with CaCl2.19 Rhodymenia palmata (dulse) agrees with Laminaria in showing a rise in resistance in 20CaCl2, BaCl2, SrCl2, MnCL, and NiCl2, and a greater rise in alum, Ce(N03)3, and La(N03)3. The rise with MgCl2 is less than with CaCL, but the latter is less than that found with Lami- naria (Fig. 21). Experiments on frog skin21 showed a rapid rise in CaCl2 (resistance increased to 140%, or less) followed by a fall. In La(N03)3 the rise is greater (resistance increased to 190% or less) and in MgCl2 it is less (resistance increased to 110%, or less). It is evident

that the behavior of frog skin in these solutions resem- bles that of Laminaria. On the other hand, such substances as NaCl and KC1 produce in frog skin and in Rhodymenia no rise, but only a fall of resistance, just as in Laminaria. FIQ. 21. — Curves showing the effect of CaCb 0.278 M on the net electrical resistance of Laminaria agardhii (upper curve) and of Rhodymenia palmata (lower curve) . The ordinates denote net electrical resistance. Temperature 17 =*= 2° C. Average of six experiment!. Probable error of the mean less than 3% of the mean.

duced in echinoderm eggs by La and Ce. Shearer (1919, B) was unable to find such an effect in bacteria. It was of especial interest to investigate the effect of the hydrogen ion, which in many respects behaves unlike other monovalent kations. The first experiments were made by adding to sea water a solution of HC1 of the same conductivity as the sea water (about 0.119 M). The results are shown in Fig. 22. concentrations the maximum was reached earlier, while in the lower concentrations it occurred later. It is evident that as the concentration increases, the rise in resistance is more rapid and the maximum point is passed more quickly. If the concentration be sufficiently increased,

Fio. 22. — CurvoB showing net electrical resistance of Laminaria agardhii in sea water con- taining rarious amounts of HC1, as shown by the figures attached to the curves. Each curve represent* a single experiment. All readings were taken at 18° C. or corrected to this temperature. Each curve represents a single typical experiment. the period of increased resistance becomes less and less, until it becomes difficult to detect it. The relation between concentration and changes in resistance is better shown in Fig. 23.

Experiments with acetic acid also showed a rise in resistance. FIG. 23. — Curves of net, electrical resistance of Laminaria agardhii in sea water containing various amounts of HC1. The ordinates represent net resistance. The abscissse represent concentrations of HC1. All readings are taken at 18° C. or corrected to this temperature. necessary to employ a solution containing NaCl+CaCl2, since the addition of alkali to sea water causes a precipi- tate of Mg(OH)2. In order to compare the effect of acid with that of alkali a solution of HC1 having the same

FIG. 24. — Curves showing net electrical resistance of Laminaria agardhii in 1793 o.c. NaCl figures attached to the curves. Each curve represents a single experiment. All readings conductivity as the sea water was added to a solution containing 1793 cc. NaCl 0.52 M + 207 cc. CaCl2 0.278 M (this solution had the same conductivity as sea water). The results are shown in Fig. 24; it will be noted that they are in good agreement with those obtained by adding

Fio. 25. — Curves of net electrical resistance of Laminaria agardhii in (1793 o.c. NaCl 0.52M-T-207 c.c. CaCli 0.278 Af) containing various amounts of HC1. The ordinatea repre- sent net resistance ; the absciss® represent concentrations of HC1. All readings were taken at 18° C. or corrected to this temperature. Each curve represents a single typical experiment. acid to sea water. These relations are still more clearly shown when the results are plotted as shown in Fig. 25.

These results present a marked contrast to those obtained by the use of alkali ; with the latter there is no rise in resistance, but, on the contrary, a fairly rapid fall which continues until the death point is reached. In view of the great importance of acid and alkali in life processes these results deserve especial consideration since it would seem that slight changes in the reaction of the medium affect conductivity and permeability. It may be added that experiments with frog skin23 showed that in this case also HC1 produces a rapid rise in resistance followed by a fall. Shearer (1919, A) found a rise in the case of bacteria.

The writer has also found24 that high concentrations of KCN (0.01 to 0.381 M) produce a slight temporary rise in the resistance of Laminaria*5 It is of considerable interest to find that certain organic substances are able to increase resistance. As an example of this we may consider experiments with bile salts.26 In these investigations Na-taurocholate was added to the sea water, which was then restored to its normal conductivity and made approximately neutral to litmus.27 All concentrations employed produced an immediate increase in resistance followed by a fall, as illustrated in Fig. 26. Under the conditions .of the experiment, the rise lasted about an hour. An increase in resistance was also observed with TJlva rigida (sea

" Possibly this might have been greater had not the solution been alkaline. "The amounts varied from 0.8 to 1.5 gm. added to 1000 c.c. of sea water. If the Na-taurocholate were pure 1 gm. in 1000 c.c. would make the concentration about 0.002 M, but its purity is doubtful. lettuce) and Rliodymenia palmata (dulse) ; in the latter case it was much less than in Laminaria. An increase has also been observed in experiments with ether, chloroform, chloral hydrate, and alcohol.28

jj'- ^' — Curve showing rise in net electrical resistance of Laminaria anardhii produced by adding 1 gm. of Na-taurocholate to 1000 c.c. of sea water (solid line). Control in sea water, dotted line. Average of two experiments; probable error of the mean less than 2.3% of the The question arises whether the rise due to these organic substances differs from that produced by inor- ganic salts and by acids. The writer is inclined to believe that this may be the case, but prefers to await the results of additional experimentation before reaching a definite decision.

The fact that in all these cases there is a rise in resistance followed by a fall suggests that there are two processes at work, one producing an increase and the other a decrease. In order to picture a mechanism which would account for this the writer has assumed that at the surface of the cell there is a substance, M, forming a continuous layer29 whose thickness determines the amount of resistance. It is assumed that the thickness of this layer is increased by the breaking down of a substance, A, to form M, according to the monomolecular reaction A — > M. At the same time M breaks down to form a substance, B, according to the monomolecular reaction, M — >B. The two reactions go on simultan- eously according to the scheme30 A-r-*-M — >B.

It is obvious that if the rates of the reactions are such that M is formed as rapidly as it is decomposed, it will remain constant in amount; and that an increase in the velocity of the first reaction will cause M to increase, while an increase in the velocity of the second reaction will cause M to decrease. The nature of this process is evident from a considera- tion of Fig. 27. If the reservoir A be filled with water while M and B are empty, and if water be allowed to flow from A into M, the amount of water in M (for convenience this amount is called y) will first increase and then decrease. The rate of increase and decrease and the maximum attained will depend on the relation between the two outlets Kl and K*. We may suppose that if K! is equal in diameter to K2, we get the upper