PROTOPLASMIC POTENTIALS IN HALICYSTIS : III. THE EFFECTS OF AMMONIA

The nature and origin of the large "protoplasmic" potential in Halicystis must be studied by altering conditions, not only in external solutions, but in the sap and the protoplasm itself. Such interior alteration caused by the penetration of ammonia is described. Concentrations of NH₄Cl in the sea water were varied from 0.00001 M to above 0.01 M. At pH 8.1 there is little effect below 0.0005 M NH₄Cl. At about 0.001 M a sudden reversal of the potential difference across the protoplasm occurs, from about 68 mv. outside positive to 30 to 40 mv. outside negative. At this threshold value the time curve is characteristically S-shaped, with a slow beginning, a rapid reversal, and then an irregularly wavering negative value. There are characteristic cusps at the first application of the NH₄Cl, also immediately after the reversal. The application of higher NH₄Cl concentrations causes a more rapid reversal, and also a somewhat higher negative value. Conversely the reduction of NH₄Cl concentrations causes recovery of the normal positive potential, but the threshold for recovery is at a lower concentration than for the original reversal. A temporary overshooting or increase of the positive potential usually occurs on recovery. The reversals may be repeated many times on the same cell without injury. The plot of P.D. against the log of ammonium ion concentration is not the straight line characteristic of ionic concentration effects, but has a break of 100 mv. or more at the threshold value. Further evidence that the potential is not greatly influenced by ammonium ions is obtained by altering the pH of the sea water. At pH 5, no reversal occurs with 0.1 M NH₄Cl, while at pH 10.3, the NH₄Cl threshold is 0.0001 M or less. This indicates that the reversal is due to undissociated ammonia. The penetration of NH₃ into the cells increases both the internal ammonia and the pH. The actual concentration of ammonium salt in the sap is again shown to have little effect on the _P.D. The pH is therefore the governing factor. But assuming that NH₃ enters the cells until it is in equilibrium between sap and sea water, no sudden break of pH should occur, pH being instead directly proportional to log NH₃ for any constant (NH₄) concentration. Experimentally, a linear relation is found between the pH of the sap and the log NH₃ in sea water. The sudden change of P.D. must therefore be ascribed to some system in the cell upon which the pH change operates. The pH value of the sap at the NH₃ threshold is between 6.0 and 6.5 which corresponds well with the pH value found to cause reversal of P.D. by direct perfusion of solutions in the vacuole.     

A MICRO INJECTION STUDY ON THE PERMEABILITY OF THE STARFISH EGG

The experiments with the NH₄Cl are similar to, and corroborate micro injection experiments performed in connection with some work on mustard gas in which the writer collaborated. Eggs immersed in sea water containing decomposed mustard gas, at a certain low concentration are not affected. If, however, the solution be injected, the egg quickly cytolyzes owing to the free HCl present. A similar impermeability of the protoplasmic surface film to certain substances was also encountered in injection work on Amœba. Amœbœ immersed in an aqueous solution of eosin will not take the stain till after death. On the other hand, the eosin, when injected into the Amœba, quickly permeates the protoplasm, to be arrested only at the surface. The semipermeability of a living cell appears primarily to be a function of its surface film. It is immaterial whether this film be that of the original cortex of the cell, a film newly formed over a cut surface, or a film that surrounds an artificially induced vacuole within the cell. As long as such a surface film exists neither the acid group of the NH₄Cl nor the alkaline group of the NaHCO₃ can, within certain concentration limits, penetrate the protoplasm. These solutions, if injected beneath the surface film, however, will produce their characteristic effects upon the protoplasm.     

BIOELECTRIC POTENTIALS IN VALONIA : II. EFFECTS OF ARTIFICIAL SEA WATERS CONTAINING LiCl,CsCl, RbCl,OR NH4Cl

In their influence on the P.D. across the protoplasm of Valonia macrophysa, Kütz., Li⁺ and Cs⁺ resemble Na⁺, while Rb⁺ and NH₄ ⁺ resemble K⁺. The apparent mobilities of the ions in the external surface layer of Valonia protoplasm increase in the order: Cs⁺, Na⁺, Li⁺ < Cl^- < Rb⁺ < K⁺ < NH₄ ⁺.     

NATURE OF THE ACTION CURRENT IN NITELLA : IV. PRODUCTION OF QUICK ACTION CURRENTS BY EXPOSURE TO NACl

Treatment of Nitella with NaCl greatly reduces the time required for the action current and produces an action curve with one peak instead of the customary two. The time may be reduced to 0.6 second in place of the usual 15 to 30 seconds. This might be expected if the treatment increased the conductivity of the aqueous part of the protoplasm. The experiments favor this idea although they do not prove its correctness. This effect is prevented by calcium, possibly because calcium inhibits penetration of salts. That penetration is an important factor is indicated by the fact that salts which might be expected to penetrate rapidly have the most effect. Thus NaSCN is more effective than NaCl but Na₂SO₄ has little or no effect. The action of NH₄Cl and LiCl is similar to that of NaCl.     

BIOELECTRIC POTENTIALS IN VALONIA : THE EFFECT OF SUBSTITUTING KCl FOR NaCl IN ARTIFICIAL SEA WATER

The P.D. across the protoplasm of Valonia macrophysa has been studied while the cells were exposed to artificial solutions resembling sea water in which the concentration of KCl was varied from 0 to 0.500 mol per liter. The P.D. across the protoplasm is decreased by lowering and increased by raising the concentration of KCl in the external solution. Changes in P.D. with time when the cell is treated with KCl-rich sea water resemble those observed with cells exposed to Valonia sap. Varying the reaction of natural sea water from pH 5 to pH 10 has no appreciable effect on the P.D. across Valonia protoplasm. Similarly, varying the pH of KCl-rich sea water within these limits does not alter the height of the first maximum in the P.D.-time curve. The subsequent behavior of the P.D., however, is considerably affected by the pH of the KCl-rich sea water. These changes in the shape of the P.D.-time curve have been interpreted as indicating that potassium enters Valonia protoplasm more rapidly from alkaline than from acidified KCl-rich sea water. This conclusion is discussed in relation to certain theories which have been proposed to explain the accumulation of KCl in Valonia sap. The initial rise in P.D. when a Valonia cell is transferred from natural sea water to KCl-rich sea water has been correlated with the concentrations of KCl in the sea waters. It is assumed that the observed P.D. change represents a diffusion potential in the external surface layer of the protoplasm, where the relative mobilities of ions may be supposed to differ greatly from their values in water. Starting with either Planck''s or Henderson''s formula, an equation has been derived which expresses satisfactorily the observed relationship between P.D. change and concentration of KCl. The constants of this equation are interpreted as the relative mobilities of K⁺, Na⁺, and Cl^- in the outer surface layer of the protoplasm. The apparent relative mobility of K⁺ has been calculated by inserting in this equation the values for the relative mobilities of Na⁺ (0.20) and Cl^- (1.00) determined from earlier measurements of concentration effect with natural sea water. The average value for the relative mobility of K⁺ is found to be about 20. The relative mobility may vary considerably among different individual cells, and sometimes also in the same individual under different conditions. Calculation of the observed P.D. changes as phase-boundary potentials proved unsatisfactory.     

CHANGES OF APPARENT IONIC MOBILITIES IN PROTOPLASM : I. EFFECTS OF GUAIACOL ON VALONIA

In normal cells of Valonia the order of the apparent mobilities of the ions in the non-aqueous protoplasmic surface is K > Cl > Na. After treatment with 0.01 M guaiacol (which does not injure the cell) the order becomes Na > Cl > K. As it does not seem probable that such a reversal could occur with simple ions we may assume provisionally that in the protoplasmic surface we have to do with charged complexes of the type (KX _I)⁺, (KX _II)⁺, where X _I and X _II are elements or radicals, or with chemical compounds formed in the protoplasm. When 0.01 M guaiacol is added to sea water or to 0.6 M NaCl (both at pH 6.4, where the concentration of the guaiacol ion is negligible) the P.D. of the cell changes (after a short latent period) from about 10 mv. negative to about 28 mv. positive and then slowly returns approximately to its original value (Fig. 1, p. 14). This appears to depend chiefly on changes in the apparent mobilities of organic ions in the protoplasm. The protoplasmic surface is capable of so much change that it does not seem probable that it is a monomolecular layer. It does not behave like a collodion nor a protein film since the apparent mobility of Na⁺ can increase while that of K⁺ is decreasing under the influence of guaiacol.     

THE PENETRATION OF BASIC DYE INTO NITELLA AND VALONIA IN THE PRESENCE OF CERTAIN ACIDS,BUFFER MIXTURES,AND SALTS

When living cells of Nitella are exposed to an acetate buffer solution until the pH value of the sap is decreased and subsequently placed in a solution of brilliant cresyl blue, the rate of penetration of dye into the vacuole is found to decrease in the majority of cases, and increase in other cases, as compared with the control cells which are transferred to the dye solution directly from tap water. This decrease in the rate is not due to the lowering of the pH value of the solution just outside the cell wall, as a result of diffusion of acetic acid from the cell when cells are removed from the buffer solution and placed in the dye solution, because the relative amount of decrease (as compared with the control) is the same whether the external solution is stirred or not. Such a decrease in the rate may be brought about without a change in the pH value of the sap if the cells are placed in the dye solution after exposure to a phosphate buffer solution in which the pH value of the sap remains normal. The rate of penetration of dye is then found to decrease. The extent of this decrease is the greater the lower the pH value of the solution. It is found that hydrochloric acid and boric acid have no effect while phosphoric acid has an inhibiting effect at pH 4.8 on stirring. Experiments with neutral salt solutions indicate that a direct effect on the cell (decreasing penetration) is due to monovalent base cations, while there is no such effect directly on the dye. It is assumed that the effect of the phosphate and acetate buffer solutions on the cell, decreasing the rate of penetration, is due (1) to the penetration of these acids into the protoplasm as undissociated molecules, which dissociate upon entrance and lower the pH value of the protoplasm or to their action on the surface of the protoplasm, (2) to the effect of base cations on the protoplasm (either at the surface or in the interior), and (3) possibly to the effect of certain anions. In this case the action of the buffer solution is not due to its hydrogen ions. In the case of living cells of Valonia under the same experimental conditions as Nitella it is found that the rate of penetration of dye decreases when the pH value of the sap increases in presence of NH₃, and also when the pH value of the sap is decreased in the presence of acetic acid. Such a decrease may be brought about even when the cells are previously exposed to sea water containing HCl, in which the pH value of the sap remains normal.     

CALCULATIONS OF BIOELECTRIC POTENTIALS : V. POTENTIALS IN HALICYSTIS

Interest in the study of Halicystis and of Valonia has been stimulated by discoveries of marked contrasts and striking similarities existing side by side. This is illustrated by new experiments with the alkali metals and alkaline earths. In Halicystis the apparent mobilities of K⁺, Rb⁺, Cs⁺, and Li⁺ (calculated by means of Henderson''s equation from changes in P.D. produced by replacing sea water by a mixture of equal parts of sea water and 0.6 M of various chlorides) are as follows, u _K, = 16, u _Rb = 16, u _Cs = 4.4, and u _Li = 0.2; u _Na is taken as 0.2. These values resemble those in Valonia except that in the latter u _Cs is about 0.2. No calculation is made for u _NHNH4, because in these experiments even at low pH so much NH₃ is present that the sign of the P.D. may reverse. This does not happen with Valonia. According to Blinks, NH₄ ⁺ at pH 5 in low concentrations acts like K⁺. The calculation gives u _Mg = 1.9 which is similar to the value found for Valonia. No calculation can be made for CaCl₂ since it produces protoplasmic alterations and in consequence Henderson''s equation does not apply. This differs from Valonia. Evidently these plants agree closely in some aspects of electrical behavior but differ widely in others.     

DISSIMILARITY OF INNER AND OUTER PROTOPLASMIC SURFACES IN VALONIA. II

In measurements of P.D. across the protoplasm in single cells, the presence of parallel circuits along the cell wall may cause serious difficulty. This is particularly the case with marine algae, such as Valonia, where the cell wall is imbibed with a highly conducting solution (sea water), and hence has low electrical resistance. In potential measurements on such material, it is undesirable to use methods in which the surface of the cell is brought in contact with more than one solution at a time. The effect of a second solution wetting a part of the cell surface is discussed, and demonstrated by experiment. From further measurements with improved technique, we find that the value previously reported for the P.D. of the chain Valonia sap | Valonia protoplasm | Valonia sap is too low, and also that the P.D. undergoes characteristic changes during experiments lasting several hours. The maximum P.D. observed is usually between 25 and 35 mv., but occasionally higher values (up to 82 mv.) are found. The appearance of the cells several days after the experiment, and the P.D.''s which they give with sea water, indicate that no permanent injury has been received as a result of exposure to artificial sap. If such cells are used in a second measurement with artificial sap, however, the form of the P.D.-time curve indicates that the cells have undergone an alteration which persists for a long time. On the basis of the theory of protoplasmic layers, an attempt has been made to explain the observed changes in P.D. with time, assuming that these changes are due to penetration of KCl into the main body of the protoplasm.     

THE ACCUMULATION OF ELECTROLYTES : III. BEHAVIOR OF SODIUM,POTASSIUM, AND AMMONIUM IN VALONIA

When 0.001 M NH₄Cl is added to sea water containing Valonia macrophysa there seems to be a rapid penetration of undissociated NH₃ (or NH₄OH) which raises the pH value of the sap so that the thermodynamic potential of KOH becomes greater inside than outside and in consequence K leaves the cell: NaOH continues to go in because its thermodynamic potential is greater outside than inside. NH₄Cl accumulates, reaching a much higher concentration inside than outside. This might be explained on the ground that NH₃, after entering, combines with a weak organic acid produced in the cell whose anion is exchanged for the Cl^- of the sea water, or (more probably) the organic acid is exchanged for HCl.     

THE ACCUMULATION OF ELECTROLYTES : I. THE ENTRANCE OF AMMONIA INTO VALONIA MACROPHYSA

When 0.005 M NH₄Cl is added to sea water containing cells of Valonia macrophysa ammonia soon appears in the sap and may reach a concentration inside over 40 times as great as outside. It appears to enter as undissociated NH₃ (or NH₄OH) and tends to reach a pseudoequilibrium in which the activity of undissociated NH₃ (or NH₄OH) is the same inside and outside. When ammonia first enters, the pH value of the sap rapidly rises but it soon reaches a maximum and subsequently falls off. At the same time there is an increase of halide in the sap which, however, does not run a parallel course to the ammonia accumulation, but it comes to a new equilibrium value and remains constant. The increase in NH₃ in the sap is accompanied by a decrease in the concentration of K. As NH₃ enters the specific gravity of the sap decreases and the cells rise to the surface and continue to grow as floating organisms. The growth of the cells is increased.     

SOME ASPECTS OF SELECTIVE ABSORPTION

1. A mechanism exists in Valonia which prevents certain substances (Na, Mg, Ca, SO₄) from reaching as high a concentration inside the cell as in the sea water which surrounds it. 2. A trapping mechanism also exists which causes K to accumulate in the cell in a concentration far in excess of that found in sea water. Practically all the K in the cell exists in the form of KCl. 3. The concentration of Cl does not differ greatly within and without. 4. These facts are not in harmony with present theories regarding the accumulation of K in living cells.     

PROTOPLASMIC ASYMMETRY IN NITELLA AS SHOWN BY BIOELECTRIC MEASUREMENTS

Using multinucleate cells of Nitella 2 or 3 inches in length it is possible to kill one end with chloroform without producing at the other any immediate alteration which can be detected by our present methods. When a spot in external contact with sap is killed its potential difference falls approximately to zero and it is therefore possible to measure the potential difference across the protoplasm at any desired point merely by leading off from that point to the one where the protoplasm has been killed. The results indicate that the inner and outer protoplasmic surfaces differ, for when both surfaces are in contact with the same solution (cell sap) there is an electromotive force of about 15.9 millivolts, the inner surface being positive to the outer (i.e. the positive current tends to flow from the inner surface through the electrometer to the outer surface). The situation resembles that in Valonia where the corresponding value (with Valonia sap applied to the outside) has been reported as about 14.5 millivolt (the inner surface being positive to the outer). It would seem appropriate to designate this as radial polarity.     

THE KINETICS OF PENETRATION : XVI. THE ACCUMULATION OF AMMONIA IN LIGHT AND DARKNESS

The accumulation of ammonia takes place more rapidly in light than in darkness. The accumulation appears to go on until a steady state is attained. The steady state concentration of ammonia in the sap is about twice as great in light as in darkness. Both effects are possibly due to the fact that the external pH (and hence the concentration of undissociated ammonia) outside is raised by photosynthesis. Certain "permeability constants" have been calculated. These indicate that the rate is proportional to the concentration gradient across the protoplasm of NH₄ X which is formed by the interaction of NH₃ or NH₄OH and HX, an acid elaborated in the protoplasm. The results are interpreted to mean that HX is produced only at the sap-protoplasm interface and that on the average its concentration there is about 7 times as great as at the sea water-protoplasm interface. This ratio of HX at the two surfaces also explains why the concentration of undissociated ammonia in the steady state is about 7 times as great in the sea water as in the sap. The permeability constant P'''''' appears to be greater in the dark. This is possibly associated with an increase in the concentration of HX at both interfaces, the ratio at the two surfaces, however, remaining about the same. The pH of sap has been determined by a new method which avoids the loss of gas (CO₂), an important source of error. The results indicate that the pH rises during accumulation but the extent of this rise is smaller than has hitherto been supposed. As in previous experiments, the entering ammonia displaced a practically equivalent amount of potassium from the sap and the sodium concentration remained fairly constant. It seems probable that the pH increase is due to the entrance of small amounts of NH₃ or NH₄OH in excess of the potassium lost as a base.     

The penetration of radioactive sodium intoValonia andHalicystis

Conclusions In comparing the uptake of Na^* byValonia and that byHalicystis, it was found that the greater proportion was taken up by the protoplasm in the former case, and by the sap in the latter case.In former experiments only the sap was tested for penetration and found to contain negligible concentrations of Na inValonia and comparatively larger concentrations inHalicystis. It is therefore of interest to show that Na^* does penetrateValonia but is taken up by the protoplasm considerable concentrations; but that it does not pass into the sap under normal conditions, thereby showing that the semi-permeable membrane between the sap and the protoplasm is the region of non-penetration. InHalicystis this is not the case since Na^* was found in both the sap and the protoplasm of this cell.Further work on the difference between these two membranes would be of interest in elucidating the movement of K and Na ions through membranes.This paper was assembled by Matilda M. Brooks.     

Amine Transport in Riccia fluitans: Cytoplasmic and Vacuolar pH Recorded by a pH-Sensitive Microelectrode

The cytoplasmic and vacuolar pH and changes thereof in the presence of ammonia (NH₄Cl) and methylamine (CH₃NH₃Cl) have been measured in rhizoid cells of Riccia fluitans by means of a pH-sensitive microelectrode.

On addition of 1 micromolar NH₄Cl, the cytoplasmic pH of 7.2 to 7.4 drops by 0.1 to 0.2 pH units, but shifts to pH 7.8 in the presence of 50 micromolar NH₄Cl or 500 micromolar CH₃NH₃Cl. The pH of the vacuole increases drastically from 4.5 to 5.7 with these latter concentrations. Since a NH₄⁺/CH₃NH₃⁺ uniporter has been demonstrated in the plasmalemma of R. fluitans previously (Felle 1983 Biochim Biophys Acta 602:181-195), the concentration-dependent shifts of cytoplasmic pH are interpreted as results of two processes: first, acidification through deprotonation of the actively transported NH₄⁺; and second, alkalinization through protonation of NH₃ which is taken up to a significant extent from high external concentrations. Furthermore, it is concluded that the determination of intracellular pH by means of methylamine distribution is not a reliable method for eucaryotic systems.

     

ON THE NATURE OF THE DYE PENETRATING THE VACUOLE OF VALONIA FROM SOLUTIONS OF METHYLENE BLUE

When uninjured cells of Valonia are placed in methylene blue dissolved in sea water it is found, after 1 to 3 hours, that at pH 5.5 practically no dye penetrates, while at pH 9.5 more enters the vacuole. As the cells become injured more dye enters at pH 5.5, as well as at pH 9.5. No dye in reduced form is found in the sap of uninjured cells exposed from 1 to 3 hours to methylene blue in sea water at both pH values. When uninjured cells are placed in azure B solution, the rate of penetration of dye into the vacuole is found to increase with the rise in the pH value of the external dye solution. The partition coefficient of the dye between chloroform and sea water is higher at pH 9.5 than at pH 5.5 with both methylene blue and azure B. The color of the dye in chloroform absorbed from methylene blue or from azure B in sea water at pH 5.5 is blue, while it is reddish purple when absorbed from methylene blue and azure B at pH 9.5. Dry salt of methylene blue and azure B dissolved in chloroform appears blue. It is shown that chiefly azure B in form of free base is absorbed by chloroform from methylene blue or azure B dissolved in sea water at pH 9.5, but possibly a mixture of methylene blue and azure B in form of salt is absorbed from methylene blue at pH 5.5, and azure B in form of salt is absorbed from azure B in sea water at pH 5.5. Spectrophotometric analysis of the dye shows the following facts. 1. The dye which is absorbed by the cell wall from methylene blue solution is found to be chiefly methylene blue. 2. The dye which has penetrated from methylene blue solution into the vacuole of uninjured cells is found to be azure B or trimethyl thionine, a small amount of which may be present in a solution of methylene blue especially at a high pH value. 3. The dye which has penetrated from methylene blue solution into the vacuole of injured cells is either methylene blue or a mixture of methylene blue and azure B. 4. The dye which is absorbed by chloroform from methylene blue dissolved in sea water is also found to be azure B, when the pH value of the sea water is at 9.5, but it consists of azure B and to a less extent of methylene blue when the pH value is at 5.5. 5. Methylene blue employed for these experiments, when dissolved in sea water, in sap of Valonia, or in artificial sap, gives absorption maxima characteristic of methylene blue. Azure B found in the sap collected from the vacuole cannot be due to the transformation of methylene blue into this dye after methylene blue has penetrated into the vacuole from the external solution because no such transformation detectable by this method is found to take place within 3 hours after dissolving methylene blue in the sap of Valonia. These experiments indicate that the penetration of dye into the vacuole from methylene blue solution represents a diffusion of azure B in the form of free base. This result agrees with the theory that a basic dye penetrates the vacuole of living cells chiefly in the form of free base and only very slightly in the form of salt. But as soon as the cells are injured the methylene blue (in form of salt) enters the vacuole. It is suggested that these experiments do not show that methylene blue does not enter the protoplasm, but they point out the danger of basing any theoretical conclusion as to permeability on oxidation-reduction potential of living cells from experiments made or the penetration of dye from methylene blue solution into the vacuole, without determining the nature of the dye inside and outside the cell.     

PROTOPLASMIC POTENTIALS IN HALICYSTIS

The cells of Halicystis impaled on capillaries reach a steady P.D. of 60 to 80 millivolts across the protoplasm from sap to sea water. The outer surface of the protoplasm is positive in the electrometer to the inner surface. The P.D. is reduced by contact with sap and balanced NaCl-CaCl₂ mixtures; it is abolished completely in solutions of NaCl, CaCl₂, KCl, MgSO₄, and MgCl₂. There is prompt recovery of P.D. in sea water after these exposures.     

THE COMPOSITION OF THE CELL SAP OF THE PLANT IN RELATION TO THE ABSORPTION OF IONS

1. Chemical examination of the cell sap of Nitella showed that the concentrations of all the principal inorganic elements, K, SO₄, Ca, Mg, PO₄, Cl, and Na, were very much higher than in the water in which the plants were growing. 2. Conductivity measurements and other considerations lead to the conclusion that all or nearly all of the inorganic elements present in the cell sap exist in ionic state. 3. The insoluble or combined elements found in the cell wall or protoplasm included Ca, Mg, S, Si, Fe, and Al. No potassium was present in insoluble form. Calcium was predominant. 4. The hydrogen ion concentration of healthy cells was found to be approximately constant, at pH 5.2. This value was not changed even when the outside solution varied from pH 5.0 to 9.0. 5. The penetration of NO₃ ion into the cell sap from dilute solutions was definitely influenced by the hydrogen ion concentration of the solution. Penetration was much more rapid from a slightly acid solution than from an alkaline one. It is possible that the NO₃ forms a combination with some constituent of the cell wall or of the protoplasm. 6. The exosmosis of chlorine from Nitella cells was found to be a delicate test for injury or altered permeability. 7. Dilute solutions of ammonium salts caused the reaction of the cell sap to increase its pH value. This change was accompanied by injury and exosmosis of chlorine. 8. Apparently the penetration of ions into the cell may take place from a solution of low concentration into a solution of higher concentration. 9. Various comparisons with higher plants are drawn, with reference to buffer systems, solubility of potassium, removal of nitrate from solution, etc.