MICRURGICAL STUDIES IN CELL PHYSIOLOGY : V. THE ANTAGONISM OF CATIONS IN THEIR ACTIONS ON THE PROTOPLASM OF AM?BA DUBIA.

I. The Plasmalemma. 1. On the plasmalemma of amebæ CaCl₂ antagonizes the toxic action of LiCl better than it does NaCl, and still better than it does KCl. MgCl₂ antagonizes the toxic action of NaCl better than it does LiCl and still better than it does KCl. 2. CaCl₂ antagonizes the toxic action of LiCl and of KCl better than does MgCl₂: MgCl₂ antagonizes NaCl better than does CaCl₂. II. The Internal Protoplasm. 3. The antagonizing efficiency of CaCl₂ and of MgCl₂ are highest against the toxic action of KCl on the internal protoplasm, less against that of NaCl, and least against that of LiCl. 4. CaCl₂ antagonizes the toxic action of LiCl better than does MgCl₂: MgCl₂ antagonizes the toxic action of NaCl and of KCl better than does CaCl₂. 5. LiCl antagonizes the toxic action of MgCl₂ on the internal protoplasm more effectively than do NaCl or KCl, which have an equal antagonizing effect on the MgCl₂ action. III. The Nature of Antagonism. 6. When the concentration of an antagonizing salt is increased to a toxic value, it acts synergistically with a toxic salt. 7. No case was found in which a potentially antagonistic salt abolishes the toxic action of a salt unless it is present at the site (surface or interior) of toxic action. 8. Antagonistic actions of the salts used in these experiments are of differing effectiveness on the internal protoplasm and on the surface membrane.     

A THEORY OF INJURY AND RECOVERY : II. EXPERIMENTS WITH MIXTURES.

1. The equations which serve to predict the injury of tissue in 0.52 M NaCl and in 0.278 M CaCl₂ and its subsequent recovery (when it is replaced in sea water) also enable us to predict the behavior of tissue in mixtures of these solutions, as well as its recovery in sea water after exposure to mixtures. 2. The reactions which are assumed in order to account for the behavior of the tissue proceed as if they were inhibited by a salt compound formed by the union of NaCl and CaCl₂ with some constituent of the protoplasm (certain of these reactions are accelerated by CaCl₂). 3. In this and preceding papers a quantitative theory is developed in order to explain: (a) the toxicity of NaCl and CaCl₂; (b) the antagonism between these substances; (c) the fact that recovery (in sea water) may be partial or complete, depending on the length of exposure to the toxic solution.     

THE EFFECT OF CERTAIN ELECTROLYTES AND NON-ELECTROLYTES ON PERMEABILITY OF LIVING CELLS TO WATER

1. Permeability to water in unfertilized eggs of the sea urchin, Arbacia punctulata, is found to be greater in hypotonic solutions of dextrose, saccharose and glycocoll than in sea water of the same osmotic pressure. 2. The addition to dextrose solution of small amounts of CaCl₂ or MgCl₂ restores the permeability approximately to the value obtained in sea water. 3. This effect of CaCl₂ and MgCl₂ is antagonized by the further addition of NaCl or KCl. 4. It is concluded that the NaCl and KCl tend to increase the permeability of the cell to water, CaCl₂ and MgCl₂ to decrease it. 5. The method here employed can be used for quantitative study of salt antagonism.     

CALCULATIONS OF BIOELECTRIC POTENTIALS : IV. SOME EFFECTS OF CALCIUM ON POTENTIALS IN NITELLA

In Nitella the substitution of KCl for NaCl changes the P.D. in a negative direction. In some cases this change is lessened by adding solid CaCl₂ to the solution of KCl. This may be due to lessening the partition coefficient of KCl or to decreasing the solubility of an organic substance which sensitizes the cell to the action of KCl. Little or no correlation exists between this effect of calcium and its ordinary antagonistic action in producing a balanced solution which preserves the life of the cell indefinitely. CaCl₂ is negative to NaCl but positive to KCl. The effects of mixtures of KCl, NaCl, and CaCl₂ are discussed. The concentration effect of a mixture of KCl + CaCl₂ shows certain peculiarities due to action currents: these resemble those found with pure KCl. These studies and others on Nitella, Valonia, and Halicystis indicate that mobilities and partition coefficients are variable and can be brought under experimental control.     

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.     

MICRURGICAL STUDIES IN CELL PHYSIOLOGY : I. THE ACTION OF THE CHLORIDES OF NA,K, CA,AND MG ON THE PROTOPLASM OF AM?BA PROTEUS.

By means of micro-dissection and injection Amœba proteus was treated with the chlorides of Na, K, Ca, and Mg alone, in combination, and with variations of pH. I. The Plasmalemma. 1. NaCl weakens and disrupts the surface membrane of the ameba. Tearing the membrane accelerates the disruption which spreads rapidly from the site of the tear. KCl has no disruptive effect on the membrane but renders it adhesive. 2. MgCl₂ and CaCl₂ have no appreciable effect on the integrity of the surface membrane of the ameba when applied on the outside. No spread of disruption occurs when the membrane is torn in these salts. When these salts are introduced into the ameba they render the pellicle of the involved region rigid. II. The Internal Protoplasm. 3. Injected water either diffuses through the protoplasm or becomes localized in a hyaline blister. Large amounts when rapidly injected produce a "rushing effect". 4. HCl at pH 1.8 solidifies the internal protoplasm and at pH 2.2 causes solidification only after several successive injections. The effect of the subsequent injections may be due to the neutralization of the cell-buffers by the first injection. 5. NaCl and KCl increase the fluidity of the internal protoplasm and induce quiescence. 6. CaCl₂ and MgCl₂ to a lesser extent solidify the internal protoplasm. With CaCl₂ the solidification tends to be localized. With MgCl₂ it tends to spread. The injection of CaCl₂ accelerates movement in the regions not solidified whereas the injection of MgCl₂ induces quiescence. III. Pinching-Off Reaction. 7. A hyaline blister produced by the injection of water may be pinched off. The pinched-off blister is a liquid sphere surrounded by a pellicle. 8. Pinching off always takes place with injections of HCl when the injected region is solidified. 9. The injection of CaCl₂ usually results in the pinching off of the portion solidified. The rate of pinching off varies with the concentration of the salt. The injection of MgCl₂ does not cause pinching off. IV. Reparability of Torn Surfaces. 10. The repair of a torn surface takes place readily in distilled water. In the different salt solutions, reparability varies specifically with each salt, with the concentration of the salt, and with the extent of the tear. In NaCl and in KCl repair occurs less readily than in water. In MgCl₂ repair takes place with great difficulty. In CaCl₂ a proper estimate of the process of repair is complicated by the pinching-off phenomenon. However, CaCl₂ is the only salt found to increase the mobility of the plasmalemma, and this presumably enhances its reparability. 11. The repair of the surface is probably a function of the internal protoplasm and depends upon an interaction of the protoplasm with the surrounding medium. V. Permeability. 12. NaCl and KCl readily penetrate the ameba from the exterior. CaCl₂ and MgCl₂ do not. 13. All four salts when injected into an ameba readily diffuse through the internal protoplasm. In the case of CaCl₂ the diffusion may be arrested by the pinching-off process. VI. Toxicity. 14. NaCl and KCl are more toxic to the exterior of the cell than to the interior, and the reverse is true for CaCl₂ and MgCl₂. 15. The relative non-toxicity of injected NaCl to the interior of the ameba is not necessarily due to its diffusion outward from the cell. 16. HCl is much more toxic to the exterior of a cell than to the interior; at pH 5.5 it is toxic to the surface whereas at pH 2.5 it is not toxic to the interior. NaOH to pH 9.8 is not toxic either to the surface or to the interior. VII. Antagonism. 17. The toxic effects of NaCl and of KCl on the exterior of the cell can be antagonized by CaCl₂ and this antagonism occurs at the surface. Although the lethal effect of NaCl is thus antagonized, NaCl still penetrates but at a slower rate than if the ameba were immersed in a solution of this salt alone. 18. NaCl and HCl are mutually antagonistic in the interior of the ameba. No antagonism between the salts and HCl was found on the exterior of the ameba. No antagonism between the salts and NaOH was found on the interior or exterior of the ameba. 19. The pinching-off phenomenon can be antagonized by NaCl or by KCl, and the rate of the retardation of the pinching-off process varies with the concentration of the antagonizing salt. 20. The prevention of repair of a torn membrane by toxic solutions of NaCl or KCl can be antagonized by CaCl₂. These experiments show directly the marked difference between the interior and the exterior of the cell in their behavior toward the chlorides of Na, K, Ca, and Mg.     

SODIUM CHLORIDE AND SELECTIVE DIFFUSION IN LIVING ORGANISMS

1. It is shown that NaCl acts like CaCl₂ or LaCl₃ in preventing the diffusion of strong acids through the membrane of the egg of Fundulus with this difference only that a M/8 solution of NaCl acts like a M/1,000 solution of CaCl₂ and like a M/30,000 solution of LaCl₃. 2. It is shown that these salts inhibit the diffusion of non-dissociated weak acid through the membrane of the Fundulus egg but slightly if at all. 3. Both NaCl and CaCl₂ accelerate the diffusion of dissociated strong alkali through the egg membrane of Fundulus and CaCl₂ is more efficient in this respect than NaCl. 4. It is shown that in moderate concentrations NaCl accelerates the rate of diffusion of KCl through the membrane of the egg of Fundulus while CaCl₂ does not.     

Involvement of an acrosin-like enzyme in the acrosome reaction of sea urchin sperm

Extracts of the jelly coat of eggs of several marine invertebrates are known to induce in homologous sperm morphological changes known as the acrosome reaction. When sperm of the sea urchin Strongylocentrotus purpuratus are treated with low concentrations (0.2 μg fucose/ml) of egg jelly coat or 30 mM CaCl₂ in artificial seawater the acrosome reaction does not occur. However, either of these treatments causes the exposure of an acrosin-like enzyme to exogenous substrate and inhibitors. Subsequent addition of jelly coat to 3.7 μg fucose/ml to sperm in this “initial stage” induces the acrosome reaction (as judged by the appearance of an acrosomal filament). This concentration is also effective for untreated sperm. If inhibitors of the enzyme (diisopropylphosphofluoridate or phenylmethanesulfonyl fluoride) are added to sperm in the initial stage, no acrosomal filaments are observed when the high concentration of jelly coat is added. Whether other morphological changes occur in these sperm has not been examined. If phenylmethanesulfonyl fluoride is added 4 sec after the jelly coat, the acrosomal filaments are observed, but the sperm still fail to fertilize eggs. These results suggest a dual role for the acrosin-like enzyme(s), first in the mechanism of the acrosomal filament formation and then in a subsequent event in the fertilization process.     

THE ELECTRICAL CONDUCTIVITY OF PURE PROTOPLASM

The electrical conductivity of the plasmodium of the slime-mold Brefeldia maxima (Fr.) Rost., which constitutes practically pure protoplasm, was found to be approximately equivalent under normal conditions to that of a 0.00145 N NaCl solution, and about 2.8 times that of the liquid in contact with which it developed. When bathed in 1 per cent sea water, the conductivity was much increased, becoming greater than that of the surrounding fluid. These preliminary tests are in agreement with the supposition that the protoplasm is permeable to and in equilibrium with its environment in so far as electrolytes are concerned.     

Alleviation of the adverse effects of NaCl on germination of maize grains by calcium

The lengths of roots and shoots, fresh and dry matter yield, and the contents of insoluble saccharides and free amino acids were reduced with the rise in NaCl concentration. However, under combination of NaCl with Ca²⁺ ions, these parameters generally raised. Contents of soluble saccharides, proline and quaternary ammonium compounds increased with increasing NaCl concentration, but under addition of CaCl₂ or CaSO₄, contents of these compounds were decreased. Low concentrations of NaCl stimulated soluble proteins, production, but higher concentrations decreased the content of soluble proteins. Addition of Ca²⁺ in the media did not improve the soluble protein production. Insoluble proteins content was increased with the rise of salinity level, but these effects were more pronounced with NaCl and CaCl₂ or CaSO₄ than with NaCl only.     

Studies on the permeability of living cells

Summary The penetration of the dye, dahlia, into the sap ofNitella has been determined in the presence of NaCl, KCl, MgCl₂ and CaCl₂ at various concentrations.NaCl is the least effective and MgCl₂ was the most effective in preventing penetration of the dye.It was found that NaCl antagonizes the action of CaCl₂ in certain proportions to a small degree but not sufficiently to permit the dye to penetrate at a normal rate.Published by permission of the Surgeon General.     

COMPARATIVE STUDIES ON RESPIRATION : XVI. EFFECTS OF HYPOTONIC AND HYPERTONIC SOLUTIONS UPON RESPIRATION.

1. In highly hypertonic solutions of sea water the rate of respiration of Laminaria agardhii is rapidly reduced. 2. In highly hypotonic solutions the rate of respiration of Laminaria agardhii is reduced somewhat less rapidly than in the case of hypertonic solutions. 3. Hypertonic solutions of NaCl, CaCl₂, and of mixtures of NaCl, and CaCl₂ in the proportion of 50:1, all caused a decrease in the rate of respiration of wheat seedlings.     

Artificial fertilization of gametes from the South African clawed frog,Xenopus laevis

A reproducible and effective method for fertilization eggs of Xenopus laevis was developed based of systematic manipulation of environmental factors. The effects of varying concentrations of individual components of a fertilization medium were tested by measuring jelly swelling, sperm motility, and sperm longevity. Results were used to develop an improved medium for fertilization, consisting of 41.25 mM NaCl, 1.25 mM KCl, 0.25 mM CaCl₂, 0.0625 mM MgCl₂, 0.5 mM Na₂HPO₄, 2.5 mM HEPES, 1.9 mM NaOH, final pH(2°) 7.8.     

CHEMICAL ANTAGONISM OF IONS : IV. EFFECT OF SALT MIXTURES ON GLYCINE ACTIVITY.

The pH of a 0.01 molar solution of glycine, half neutralized with NaOH, is 9.685. Addition of only one of the salts NaCl, KCl, MgCl₂, or CaCl₂ will lower the pH of the solution (at least up to 1 µ). If a given amount of KCl is added to a glycine solution, the subsequent addition of increasing amounts of NaCl will first raise the pH (up to 0.007 M NaCl). Further addition of NaCl (up to 0.035 M NaCl) will lower the pH, and further additions slightly raise the pH. The same type of curve is obtained by adding NaCl to glycine solution containing MgCl₂ or CaCl₂ except that the first and second breaks occur at 0.015 M and 0.085 M NaCl, respectively. Addition of CaCl₂ to a glycine solution containing MgCl₂ gives the same phenomena with breaks at 0.005 M and 0.025 M CaCl; or at ionic strengths of 0.015 µCaCl₂ and 0.075 µCaCl₂. This indicates that the effect is a function of the ionic strength of the added salt. These effects are sharp and unmistakable. They are almost identical with the effects produced by the same salt mixtures on the pH of gelatin solutions. They are very suggestive of physiological antagonisms, and at the same time cannot be attributed to colloidal phenomena.     

NOTE ON THE EXCITATION AND INHIBITION OF LUMINESCENCE IN BER?

1. The ions of Ca and K condition general luminescence, and are therefore necessary to the conduction of the impulse. 2. In van''t Hoff''s solution from which Mg is omitted, Berœ shows hyperirritability with respect to luminescence. This is the result of the action of Ca and K ions unantagonized by Mg. 3. The luminescent material spread on filter paper does not show luminescence in sea water, NaCl, MgCl₂, or saccharose solutions isotonic with sea water. In solutions of CaCl₂, SrCl₂, BaCl₂, KCl, and K₂SO₄ the indicator paper glows with a bright luminescence. 4. In dark adapted Berœ, luminescence is inhibited by a certain quantity of light. This quantity has an average value of 57,285 meter-candle-minutes, which is twelve times the value given by Mnemiopsis.     

Influence of Electrolytes on the Thicknesses of the Phospholipid Bilayers of Lamellar Lecithin Mesophases

Over a wide range of water contents, aqueous lecithin-water mixtures are mesophases in which lecithin bilayers alternate with water layers. This paper reports on low-angle X-ray diffraction measurements of the effects of electrolytes, at 1.0 N concentration, on the thicknesses of the bilayers in mesophases formed by the synthetic lecithin: 1-octadec-9-enyl-2-hexadecylglycerophosphocholine. With solutions of LiCl, NaCl, Na₂SO₄, KCl, and CsCl, the bilayer thicknesses are less than with pure water. The maximum reduction in bilayer thickness with these electrolytes is about 10% and occurs with mesophases of high content of KCl and CsCl solutions. With HCl solutions the bilayer thicknesses are about 5% greater than with pure water, and with CaCl₂ solutions the bilayer thicknesses are about the same as with pure water. The maximum amount of solution which can be mixed with lecithin before a second, purely aqueous phase is formed is also affected by electrolytes, the order for the various 1.0 N solutions being CsCl = KCl > NaCl > Na₂SO₄ > (pure water) = LiCl > CaCl₂.     

Polar lobe formation and cytokinesis in fertilized eggs of Ilyanassa obsoleta. II. Large bleb formation caused by high concentrations of exogenous calcium ions

Fertilized eggs of the mollusk Ilyanassa obsoleta (Nassarius obsoletus) form large blebs resembling polar lobes within 12 min of exposure to solutions of isotonic CaCl₂, whereas control eggs in sea water remain spherical. Under identical conditions, fertilized eggs of the sea urchin, Strongylocentrotus purpuratus, an organism which normally does not form polar lobes, do not form blebs upon exposure to solutions of isotonic CaCl₂. The calcium-induced blebbing in Ilyanassa still occurs if other cations such as Na⁺, Mg²⁺, or Mn²⁺ are present in addition to Ca²⁺, but not if comparable concentrations of K⁺ are present. Cytochalasin B prevents the calcium-induced blebbing, whereas colchicine does not. Cytokinesis in both Ilyanassa and Strongylocentrotus and normal polar lobe formation in Ilyanassa appear to require exogenous K⁺ but not exogenous Ca²⁺. Preliminary electron microscopy of Ilyanassa eggs exposed to isotonic solutions of CaCl₂ has shown microfilaments in the cortical cytoplasm in the region of the bleb constriction but no microfilaments in spherical control eggs in sea water. These data suggest that high concentrations of exogenous Ca²⁺ trigger the polymerization and contraction of a ring of microfilaments in the cortical cytoplasm of the Ilyanassa egg which results in the formation of a lobelike bleb of cytoplasm. The observation that K⁺ antagonizes this Ca²⁺-induced blebbing has led to the formulation of a theory which postulates that the ratio of intracellular Ca²⁺ to intracellular K⁺ is critical in the control of polar lobe formation and cytokinesis.     

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.     

The role of water in protoplasmic permeability and in antagonism

The behavior of the cell depends to a large extent on the permeability of the outer non-aqueous surface layer of the protoplasm. This layer is immiscible with water but may be quite permeable to it. It seems possible that a reversible increase or decrease in permeability may be due to a corresponding increase or decrease in the water content of the non-aqueous surface layer. Irreversible increase in permeability need not be due primarily to increase in the water content of the surface layer but may be caused chiefly by changes in the protoplasm on which the surface layer rests. It may include desiccation, precipitation, and other alterations. An artificial cell is described in which the outer protoplasmic surface layer is represented by a layer of guaiacol on one side of which is a solution of KOH + KCl representing the external medium and on the other side is a solution of CO₂ representing the protoplasm. The K⁺ unites with guaiacol and diffuses across to the artificial protoplasm where its concentration becomes higher than in the external solution. The guaiacol molecule thus acts as a carrier molecule which transports K⁺ from the external medium across the protoplasmic surface. The outer part of the protoplasm may contain relatively few potassium ions so that the outwardly directed potential at the outer protoplasmic surface may be small but the inner part of the protoplasm may contain more potassium ions. This may happen when potassium enters in combination with carrier molecules which do not completely dissociate until they reach the vacuole. Injury and recovery from injury may be studied by measuring the movements of water into and out of the cell. Metabolism by producing CO₂ and other acids may lower the pH and cause local shrinkage of the protoplasm which may lead to protoplasmic motion. Antagonism between Na⁺ and Ca⁺⁺ appears to be due to the fact that in solutions of NaCl the surface layer takes up an excessive amount of water and this may be prevented by the addition of suitable amounts of CaCl₂. In Nitella the outer non-aqueous surface layer may be rendered irreversibly permeable by sharply bending the cell without permanent damage to the inner non-aqueous surface layer surrounding the vacuole. The formation of contractile vacuoles may be imitated in non-living systems. An extract of the sperm of the marine worm Nereis which contains a highly surface-active substance can cause the egg to divide. It seems possible that this substance may affect the surface layer of the egg and cause it to take up water. A surface-active substance has been found in all the seminal fluids examined including those of trout, rooster, bull, and man. Duponol which is highly surface-active causes the protoplasm of Spirogyra to take up water and finally dissolve but it can be restored to the gel state by treatment with Lugol solution (KI + I). The transition from gel to sol and back again can be repeated many times in succession. The behavior of water in the surface layer of the protoplasm presents important problems which deserve careful examination.