THE DEATH WAVE IN NITELLA : II. APPLICATIONS OF UNLIKE SOLUTIONS.

The hypothesis of protoplasmic layers enables us to predict the bioelectrical behavior of the cell under a great variety of conditions. It is shown in the present paper that this is clearly the case when a death wave passes through different points in contact with unlike solutions.     

THE DEATH WAVE IN NITELLA : III. TRANSMISSION

Cutting a cell of Nitella sets up a series of rapid electrical responses, transmitted at a rate too rapid to be measured by means of our records. These are followed by slower responses whose speed falls off as the distance from the cut increases, as though they were caused by a mechanical disturbance whose intensity falls off as it travels. The faster responses seem to be due to the motion of sap past protoplasmic surfaces which have suffered little or no alteration (they seem to be similar to the electrical changes following a blow on the end of a soft rubber tube containing Ag-AgCl electrodes). The slower responses appear to be due to alterations in the protoplasm and are usually irreversible.     

NATURE OF THE ACTION CURRENT IN NITELLA : I. GENERAL CONSIDERATIONS

The outstanding features of the action curve in Nitella are explained as due to the movement of potassium ions accompanied by increase of permeability. This may be useful as a working hypothesis since it accounts not only for the normal behavior but also for many striking deviations which will be treated in subsequent papers. The views here set forth are in harmony with the local circuit theory of stimulation.     

WATER RELATIONS IN THE CELL : I. THE CHLOROPLASTS OF NITELLA AND OF SPIROGYRA

Chloroplasts may contract under natural conditions and give up water to the rest of the cell, thus indicating changes in metabolism or constitution. Such contractions may be produced experimentally. In Nitella the chloroplasts are ellipsoid bodies which, under natural conditions, may contract to spheres with a loss of volume. This may be brought about by lead acetate, ferric chloride, and digitonin: the contraction may occur while the cell is alive. The contraction in lead acetate is reversible (in lead nitrate little or no contraction occurs). In Spirogyra the chloroplast is a long, spirally coiled ribbon which may contract under natural conditions to a short nearly straight rod with a loss of volume. This can be brought about by inorganic salts and in other ways while the cell is still alive.     

DIFFERING RATES OF DEATH AT INNER AND OUTER SURFACES OF THE PROTOPLASM : I. EFFECTS OF FORMALDEHYDE ON NITELLA

When protoplasm dies it becomes completely and irreversibly permeable and this may be used as a criterion of death. On this basis we may say that when 0.2 M formaldehyde plus 0.001 M NaCl is applied to Nitella death arrives sooner at the inner protoplasmic surface than at the outer. If, however, we apply 0.17 M formaldehyde plus 0.01 M KCl death arrives sooner at the outer protoplasmic surface. The difference appears to be due largely to the conditions at the two surfaces. With 0.2 M formaldehyde plus 0.001 M NaCl the inner surface is subject to a greater electrical pressure than the outer and is in contact with a higher concentration of KCl. In the other case these conditions are more nearly equal so that the layer first reached by the reagent is the first to become permeable. The outer protoplasmic surface has the ability to distinguish electrically between K⁺ and Na⁺ (potassium effect). Under the influence of formaldehyde this ability is lost. This is chiefly due to a falling off in the partition coefficient of KCl in the outer protoplasmic surface. At about the same time the inner protoplasmic surface becomes completely permeable. But the outer protoplasmic surface retains its ability to distinguish electrically between different concentrations of the same salt, showing that it has not become completely permeable. After the potential has disappeared the turgidity (hydrostatic pressure inside the cell) persists for some time, probably because the outer protoplasmic surface has not become completely permeable.     

STUDIES ON NITELLA HAVING ARTIFICIAL CELL SAP I. REPLACEMENT OF THE CELL SAP WITH ARTIFICIAL SOLUTIONS

A method for replacing the cell sap of Nitella with an artificialsolution was introduced. The technique, which is a modificationof KAMIYA and KURODA'S (1, 2), is applicable not only for isotonicbut also for hypertonic or hypotonic solutions. Photometricdeterminations of K⁺, Na⁺, Ca⁺⁺ and Cl^– proved that thereplacement of the cell sap with the present method is satisfactory.The internodal cell of Nitella, whose cell sap was replacedwith an isotonic solution with a simple composition such asa mixture of KCl, NaCl and CaCl₂, can be kept living at leastfor several days, sometimes even for more than one month. (Received September 6, 1963; )     

DIFFERING RATES OF DEATH AT INNER AND OUTER SURFACES OF THE PROTOPLASM : II. NEGATIVE POTENTIAL IN NITELLA CAUSED BY FORMALDEHYDE

A previous paper showed that when the inner protoplasmic surface has lost its potential under the influence of formaldehyde the outer surface can still respond to changes in the concentration of electrolytes. The present paper indicates that after the inner surface has lost its potential there may be a sudden development of negative potential at the outer surface due to substances coming out of the sap and combining with formaldehyde.     

PACEMAKERS IN NITELLA : I. TEMPORARY LOCAL DIFFERENCES IN RHYTHM

A series of negative variations passing along the cell may reach a region where only every other variation registers. This condition may be temporary. It would seem to depend on a local change in the refractory period.     

ON THE ACCUMULATION OF DYE IN NITELLA

Living cells of Nitella were placed in different concentrations of brilliant cresyl blue solutions at pH 6.9. It was found that the greater the concentration of the external dye solution, the greater was the speed of accumulation of the dye in the cell sap and higher was the concentration of dye found in the sap at equilibrium. Analysis of the time curves showed that the process may be regarded as a reversible pseudounimolecular reaction. When the concentration in the sap is plotted as ordinates and the concentration in the outside solution as abscissae the curve is convex toward the abscissae. There is reason to believe that secondary changes involving injury take place as the dye accumulates and that if these changes did not occur the curve would be concave toward the abscissae. The process may be explained as a chemical combination of the dye with a constituent of the cell. This harmonizes with the fact that the temperature coefficient is high (about 4.9). Various other possible explanations are discussed.     

THE BEHAVIOR OF CHLORIDES IN THE CELL SAP OF NITELLA

1. A method is given for determining the chloride content in a drop (less than 0.03 cc.) of the cell sap of Nitella. 2. Chlorides accumulate in the sap to the extent of 0.128 M; this accumulation can be followed during the growth of the cell. The chloride content does not increase when the cell is placed for 2 days in solutions (at pH 6.2) containing chlorides up to 0.128 M. 3. The exosmosis of chlorides from injured cells can be followed quantitatively. When one end of the cell is cut off a wave of injury progresses toward the other end; this is accompanied by a progressive exosmosis of chlorides.     

CALCULATIONS OF BIOELECTRIC POTENTIALS : II. THE CONCENTRATION POTENTIAL OF KCl IN NITELLA

Cells of Nitella have been studied which behave differently from those described in earlier papers. They show unexpectedly large changes in P.D. with certain concentrations of KCl. This is due to the production of action currents (these are recorded at the spot where KCl is applied). A method is given for the separate evaluation of changes of P.D. due to partition coefficients and those due to mobilities. A new amplifier and an improved flowing contact are described.     

NATURE OF THE ACTION CURRENT IN NITELLA : II. SPECIAL CASES

The action curve involves four movements each of which shows considerable variation. These variations can be accounted for on the assumption that the action curve is due to the movement of potassium ions accompanied by an increase in permeability.     

NATURE OF THE ACTION CURRENT IN NITELLA : III. SOME ADDITIONAL FEATURES

Several forms of the action curve are described which might be accounted for on the ground that the outer protoplasmic surface shows no rapid electrical change. This may be due to the fact that the longitudinal flow of the outgoing current of action is in the protoplasm instead of in the cellulose wall. Hence the action curve has a short period with a single peak which does not reach zero. On this basis we can estimate the P.D. across the inner and outer protoplasmic surfaces separately. These P.D.''s can vary independently. In many cases there are successive action currents with incomplete recovery (with an increase or decrease or no change of magnitude). Some of the records resemble those obtained with nerve (including bursts of action currents and after-positivity).     

CYTOTAXONOMY OF THE CHARACEAE: KARYOTYPE ANALYSIS OF NITELLA OPACA AND NITELLA FLEXILIS

The haploid chromosome complement of Nitella flexilis (n = 12) is composed of two quite different basic karyotypes. One of these is symmetrical and appears to be identical to the karyotype of N. opaca (n = 6), and the other is quite asymmetrical and seems to be identical to that of an apparently undescribed dioecious Nitella from Kansas (n = 6). This may indicate that the monoecious N. flexilis has arisen through hybridization between two dioecious species. Although heteromorphic sex chromosomes were not observed in either species, it appears that female and male potentialities are confined to separate basic chromosome complements and mechanisms determining the monoecious and dioecious states are in close relation to ploidy.     

WATER PERMEABILITY OF THE CELL WALL IN NITELLA

1. A method has been developed to measure the hydraulic conductivityof the wall of the internodal cell of Nitella flexilis.
2. Therate of water penetration through the cell wall varies linearlywith the hydrostatic pressure difference between the two sidesof the wall, showing that water permeability of the cell wallremains independent of the pressure difference applied.
3. Waterpermeability of the cell wall is inversely proportionalto itsthickness It is 30µµmin^–3{dot}atm^–3when the thickness of the wall is 10 µ.
4. Water permeabilityof the cell wall is the same for inward andoutward water flow.The polar water permeability of the entiremembrane system (walland protoplasmic part) of the living celldemonstrated by KAMIYAand TAZAWA (1) is, therefore, due tothe living protoplasmicpart.
5. The ratio of the inward to outward permeability constantsofthe protoplasmic layer alone is higher than that of the entiremembrane system composed of protoplasmic layer and cell wall.

¹ Dedicated to Prof. H. TAMIYA on the occasion of his 60th birthday.The present work was supported in part by a Grant-in-Aid forFundamental Scientific Research from the Ministry of Education. ² Present address: Sh?in Women's College, Kobe. (Received July 21, 1962; )     

THE EFFECTS OF COLCHICINE ON SPERMATOGENESIS IN NITELLA

Treatment of Nitella antheridia with colchicine results in various sperm abnormalities, depending upon duration of exposure and subsequent recovery. Early effects of treatment include disappearance of spindle fibers and a cessation of ordered cell wall formation in dividing cells. Sperm released from antheridia treated for 24 hr and allowed to recover for 4–5 days possess branched flagella. After a recovery period of 6–10 days the sperm appear normal; however, following longer recovery periods, the sperm exhibit variations in size and number of flagella. Branched flagella contain a variety of microtubule patterns ranging from branches containing a single microtubule to flagella with an excess of microtubules. Spermatids which differentiate in the presence of colchicine lack flagella and a microtubular sheath. Nuclear contents undergo condensation stages; however, the nucleus as a whole does not undergo the orderly elongation and coiling characteristic of untreated Nitella spermatids. Long-term colchicine treatment followed by a recovery period produces atypical microtubules and microtubular aggregations in the spermatid. The results indicate that colchicine affects not only polymerization of microtubule subunits but also factors responsible for their ordered spatial relationships in the cell. The presence of microtubules is a prerequisite for normal morphological changes during spermiogenesis.     

THE CONCENTRATION EFFECT IN NITELLA

A method distinguishing between the concentration effect due to the cell wall and that due to the protoplasm is described: the importance of this lies in the fact that if the protoplasm shows a concentration effect one or both ions of the salt must tend to enter its outer surface. Studies on the concentration effect of KCl with living protoplasm of Nitella show that when P.D. is plotted as ordinates and the logarithm of concentration as abscissæ the graph is not the straight line demanded in the ideal case by theory but has less slope and is somewhat concave to the axis of the abscissæ. With a variety of salts the dilute solution is positive, which indicates that the cation has a greater mobility in the protoplasm than the anion or that the partition coefficient of the cation (A_c) increases faster than that of the anion (A_a) as the concentration increases. If the result depended on the partition coefficients we should say that when A_c ÷ A_a increases with concentration the dilute solution is positive. When A_c ÷ A_a decreases as the concentration increases the dilute solution is negative. In either case the increase in concentration may be accompanied by an increase or by a decrease in the relative amount of salt taken up. Theoretically therefore there need be no relation between the sign of the dilute solution and the relative amount of salt taken up with increasing concentration. Hypothetical diagrams of the electrical conditions in the cell are given. If we define the chemical effect as the P.D. observed in leading off at two points with equivalent concentrations of different salts we may say that the chemical effect of the protoplasm is very much greater than that of the cell wall.     

NATURE OF THE ACTION CURRENT IN NITELLA : V. PARTIAL RESPONSE AND THE ALL-OR-NONE LAW

When a stimulus arrives before recovery is complete there may be no response or only a partial response. A typical response appears to involve an immediate loss of potential at the inner protoplasmic surface but not at the outer surface. As long as recovery is incomplete only a part of the total potential is located at the inner protoplasmic surface and the loss of this part of the total potential can cause only a partial response; i.e., one of smaller magnitude than the normal. Even after the action curve has returned to the base line recovery may be incomplete and the response only a partial one. The return of the action curve to the base line means a recovery of total potential but if part of this is located at the outer protoplasmic surface and if this part is not lost when stimulation occurs the response can be only a partial one. During recovery there is a shift of potential from the outer to the inner protoplasmic surface. Not until this shift is completed can recovery be called complete. The response to stimulation then becomes normal because the loss of potential reaches the normal amount. In many cases the partial responses appear to conform to the all-or-none law. In other cases this is doubtful.     

ON GUAIACOL SOLUTIONS : I. THE ELECTRICAL CONDUCTIVITY OF SODIUM AND POTASSIUM GUAIACOLATES IN GUAIACOL

1. Measurements on the densities, viscosities, dielectric constants, and specific conductances of pure anhydrous and water-saturated guaiacol at 25°C. are reported. 2. The solubility of water in guaiacol at 25°C., and its effect on the electrical conductivity of a sodium guaiacolate solution is given. 3. Electrical conductivity measurements are reported on solutions of sodium and potassium guaiacolates in water-saturated guaiacol at 25°C. 4. The decrease of electrical conductivity with increasing concentration for these salts is explained on the basis of an ionic equilibrium combined with the interionic attraction theory of Debye and Hückel. 5. The limiting equivalent conductances of sodium and potassium guaiacolates in water-saturated guaiacol at 25°C., the corresponding limiting ionic mobilities, and the dissociation constants are computed from the conductivity measurements. The salts are found to be weak electrolytes with dissociation constants of the order of 5 x 10^–6.