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.     

PROTOPLASMIC POTENTIALS IN HALICYSTIS : IV. VACUOLAR PERFUSION WITH ARTIFICIAL SAP AND SEA WATER

Perfusion of the vacuole of living cells of Halicystis is described, the method employing two longitudinally fused capillaries as entrance and exit tubes. Natural sap, artificial sap, and sea water have been successfully perfused, with various additions and deficiencies, within the limits of physiological balance. In H. ovalis the P.D. remains positive and scarcely reduced in value when normal sea water, at pH 8.1, is perfused in the vacuole. In H. Osterhoutii the P.D. reverses in sign when the perfused solution has a higher pH than 6.5. In both cases a large P.D. persists when the solutions are the same on both sides of the protoplasm. In the absence of external gradients, there must be some internal gradient or asymmetry of the protoplasm itself to account for the P.D. Since appreciable currents are produced, there must be some metabolic activity as a source of energy. The higher normal P.D. in H. ovalis is not due to the higher KCl content of its sap (as earlier suggested by the author) since it persists nearly unchanged when sea water is substituted for sap.     

PROTOPLASMIC POTENTIALS IN HALICYSTIS : II. THE EFFECTS OF POTASSIUM ON TWO SPECIES WITH DIFFERENT SAPS

The potential difference across the protoplasm of impaled cells of two American species of Halicystis is compared. The mean value for H. Osterhoutii is 68.4 mv.; that for H. ovalis is 79.7 mv., the sea water being positive to the sap in both. The higher potential of H. ovalis is apparently due to the higher concentration of KCl (0.3 M) in its vacuolar sap. When the KCl content of H. Osterhoutii sap (normally 0.01 M or less) is experimentally raised to 0.3 M, the potential rises to values about equal to those in H. ovalis. The external application of solutions high in potassium temporarily lowers the potential of both, probably by the high mobility of K⁺ ions. But a large potential is soon regained, representing the characteristic potential of the protoplasm. This is about 20 mv. lower than in sea water. The accumulation of KCl in the sap of H. ovalis is apparently not due to the higher mobility of K⁺ ion in its protoplasm, since the electrical effects of potassium are practically identical in H. Osterhoutii, where KCl is not accumulated.     

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.     

BIOELECTRIC POTENTIALS IN HALICYSTIS : VIII. THE EFFECTS OF LIGHT

The effects of light upon the potential difference across the protoplasm of impaled Halicystis cells are described. These effects are very slight upon the normal P.D., increasing it 3 or 4 per cent, or at most 10 per cent, with a characteristic cusped time course, and a corresponding decrease on darkening. Light effects become much greater when the P.D. has been decreased by low O₂ content of the sea water; light restores the P.D. in much the same time course as aeration, and doubtless acts by the photosynthetic production of O₂. There are in both cases anomalous cusps which decrease the P.D. before it rises. Short light exposures may give only this anomaly. Over part of the potential range the light effects are dependent upon intensity. Increased CO₂ content of the sea water likewise depresses the P.D. in the dark, and light overcomes this depression if it is not carried too far. Recovery is probably due to photosynthetic consumption of CO₂, unless there is too much present. Again there are anomalous cusps during the first moments of illumination, and these may be the only effect if the P.D. is too low. The presence of ammonium salts in the sea water markedly sensitizes the cells to light. Subthreshold NH₄ concentrations in the dark become effective in the light, and the P.D. reverses to a negative sign on illumination, recovering again in the dark. This is due to increase of pH outside the cell as CO₂ is photosynthetically reduced, with increase of undissociated NH₃ which penetrates the cell. Anomalous cusps which first carry the P.D. in the opposite direction to the later drift are very marked in the presence of ammonia, and may represent an increased acidity which precedes the alkaline drift of photosynthesis. This acid gush seems to be primarily within the protoplasm, persisting when the sea water is buffered. Glass electrode measurements also indicate anomalies in the pH drift. There are contrary cusps on darkening which suggest temporarily increased alkalinity. Even more complex time courses are given by combining low O₂ and NH₄ exposures with light; these may have three or more cusps, with reversal, recovery, and new reversal. The ultimate cause of the light effects is to be found in an alteration of the surface properties by the treatments, which is overcome (low O₂, high CO₂), or aided (NH₄) by light. This alteration causes the surface to lose much of its ionic discrimination, and increases its electrical resistance. Tests with various anion substitutions indicate this, with recovery of normal response in the light. A theory of the P.D. in Halicystis is proposed, based on low mobility of the organic anions of the protoplasm, with differences in the two surfaces with respect to these, and the more mobile Na and K. ions.     

BIOELECTRIC POTENTIALS IN HALICYSTIS : VII. THE EFFECTS OF LOW OXYGEN TENSION

The potential difference across the protoplasm of impaled cells of Halicystis is not affected by increase of oxygen tension in equilibrium with the sea water, nor with decrease down to about 1/10 its tension in the air (2 per cent O₂ in N₂). When bubbling of 2 per cent O₂ is stopped, the P.D. drifts downward, to be restored on stirring the sea water, or rebubbling the gas. Bubbling 0.2 per cent O₂ causes the P.D. to drop to 20 mv. or less; 1.1 per cent O₂ to about 50 mv. Restoration of 2 per cent or higher O₂ causes recovery to 70 or 80 mv. often with a preliminary cusp which decreases the P.D. before it rises. Perfusion of aerated sea water through the vacuole is just as effective in restoring the P.D. as external aeration, indicating that the direction of the oxygen gradient is not significant. Low O₂ tension also inhibits the reversed, negative P.D. produced by adding NH₄Cl to sea water, 0.2 per cent O₂ bringing this P.D. back to the same low positive values found without ammonia. Restoration of 2 per cent O₂ or air, restores this latent negativity. At slightly below the threshold for ammonia reversal, low O₂ may induce a temporary negativity when first bubbled, and a negative cusp may occur on aeration before positive P.D. is regained. This may be due to a decreased consumption of ammonia, or to intermediate pH changes. The locus of the P.D. alteration was tested by applying increased KCl concentrations to the cell exterior; the large cusps produced in aerated solutions become greatly decreased when the P.D. has fallen in 0.2 per cent O₂. This indicates that the originally high relative mobility or concentration of K⁺ ion has approached that of Na⁺ in the external protoplasmic surface under reduced O₂ tension. Results obtained with sulfate sea water indicate that Na⁺ mobility approaches that of SO₄ ^— in 0.2 per cent O₂. P.D. measurements alone cannot tell whether this is due to an increase of the slower ion or a decrease of the faster ion. A decrease of all ionic permeability is indicated, however, by a greatly increased effective resistance to direct current during low O₂. Low resistance is regained on aeration. The resistance increase resembles that produced by weak acids, cresol, etc. Acids or other substances produced in anaerobiosis may be responsible for the alteration. Or a deficiency of some surface constituent may develop. In addition to the surface changes there may be alterations in gradients of inorganic or organic ions within the protoplasm, but there is at present no evidence on this point. The surface changes are probably sufficient to account for the phenomena.     

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.     

DISSIMILARITY OF INNER AND OUTER PROTOPLASMIC SURFACES IN VALONIA

The protoplasm of Valonia macrophysa forms a delicate layer, only a few microns in thickness, which contains numerous chloroplasts and nuclei. The outer surface is in contact with the cell wall, the inner with the vacuolar sap. As far as microscopic observation goes, these two surfaces seem alike; but measurements of potential difference indicate that they are decidedly different. We find that the chain sap | protoplasm | sap gives about 14.5 millivolts, the inner surface being positive to the outer. In order to explain this we may assume that the protoplasm consists of layers, the outer surface, X, differing from the inner surface, Y, and from the body of the protoplasm, W. We should then have the unsymmetrical chain sap | X | W | Y | sap which could produce an electromotive force. If the two surfaces of such a very thin layer of protoplasm can be different, it is of fundamental significance for the theory of the nature of living matter.     

CHANGES IN PROTOPLASMIC CONSISTENCY AND THEIR RELATION TO CELL DIVISION

1. The development of the amphiaster is associated with the formation of two semisolid masses within the more fluid egg substance. 2. The elongation of the egg during cleavage is possibly produced as a consequence of the mutual pressure of these two growing semisolid masses. 3. The division of the egg into two blastomeres consists essentially in a growth, within the egg, of two masses of material at the expense of the surrounding cytoplasm. When all the cytoplasm of the egg is incorporated in these two masses cleavage occurs. 4. After a certain period of time the semisolid masses revert to a more fluid state. In the eggs studied this normally occurs after the cleavage furrow has completed the separation of the two blastomeres. The formation of the furrow, however, may be prevented in various ways, upon which the egg reverts to a single spherical semifluid mass containing two nuclei. 5. An egg mutilated during its semisolid state (amphiaster stage) may or may not revert to a more fluid state. If the more solid state is maintained, the cleavage furrow persists and proceeds till cleavage is completed. If the mutilation causes the egg to revert to the more fluid state the furrow becomes obliterated and a new cleavage plane is subsequently adopted. 6. The nuclei of eggs in the semifluid state are able to alter their positions. In semifluid mutilated eggs the nuclei tend to move to positions which may assure symmetry in aster formation and cleavage.     

DIFFUSION POTENTIALS IN MODELS AND IN LIVING CELLS

The behavior of guaiacol resembles that of certain protoplasmic surfaces to such an extent that it can be advantageously used in models designed to imitate certain aspects of protoplasmic behavior. In these models the electrical potentials appear to consist of diffusion potentials and this may be true of certain living cells. In dealing with models we determine ionic mobilities and use these to predict potentials. In studying living cells we measure potentials and from these calculate ionic mobilities. The question arises, how far is this method justified. To test this we have treated guaiacol like a living cell, measuring potentials and from these estimating ionic mobilities. The results Justify the use of this method. This is of interest because the method is most useful in studying protoplasmic activity. In its extended form it enables us to follow changes in mobilities and in partition coefficients due to applied reagents and to metabolism.     

REPETITIVE ACTION POTENTIALS IN NITELLA INTERNODES

Typical spontaneous action potentials can be elicited in 10–100mM NaCl or LiCl solution. The period of repetition is 0.5–2seconds and the action potential generally consists of a rapidspike alone. Similar spontaneous action potentials are alsodemonstrated by adding either 1 mM EDTA (pH 6.6) or 2 mM ATP(pH 6.6) to the artificial pond water. In these cases, however,the period of repetition is much longer and the action potentialis of a normal shape, a rapid spike being followed by a slowtransient depolarization. The period of repetition and the sizeof the action potential decrease with the elevation of the vacuolarpotential level. The cause of the spontaneous firing is supposedto be the removal of Ca⁺⁺ from the outer surface of the Nitellamembrane. (Received May 18, 1966; )     

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 DISCOVERY OF HALICYSTIS OVALIS (LYNGBYE ARESCHOUG IN NEW ENGLAND1,2

Halicystis ovalis is recorded for the first time on the northeast coast of North America, from 12–24 m on the exposed open coast of New Hampshire.