THE KINETICS OF PENETRATION : XVII. THE EXIT OF AMMONIA IN LIGHT AND DARKNESS

The exit of accumulated ammonia from the sap of Valonia macrophysa, Kütz., into normal (nearly ammonia-free) sea water, has been studied in light (alternation of daylight and darkness) and in darkness. Exit is always preceded by an induction period lasting 1 or more days. This is longer in darkness. After exit starts the rate is greater in light than in darkness. The pH of the sap drops off soon after the cells are exposed to normal sea water even before any definite decrease in the ammonia concentration of the sap has occurred. This suggests that the decrease in the pH is due to the loss of a very small amount of NH₃ or NH4OH without a corresponding gain of sodium as a base. In most cases sodium replaced the ammonia lost during exit, but there is some evidence that potassium may also replace ammonia. To account for the induction period it is suggested that other species than NH₄ X are concerned in the transport of ammonia, for example urea or amino acids.     

THE KINETICS OF PENETRATION : VIII. TEMPORARY ACCUMULATION

A model is described which throws light on the mechanism of accumulation. In the model used an external aqueous phase A is separated by a non-aqueous phase B (representing the protoplasm) from the artificial sap in C. A contains KOH and C contains HCl: they tend to mix by passing through the non-aqueous layer but much more KOH moves so that most of the KCl is formed in C, where the concentration of potassium becomes much greater than in A. This accumulation is only temporary for as the system approaches equilibrium the composition of A approaches identity with that of C, since all the substances present can pass through the non-aqueous layer. Such an approach to equilibrium may be compared to the death of the cell as the result of which accumulation disappears. During the earlier stages of the experiment potassium tends to go in as KOH and at the same time to go out as KCl. These opposing tendencies do not balance until the concentration of potassium inside becomes much greater than outside (hence potassium accumulates). The reason is that KCl, although its driving force be great, moves very slowly in B because its partition coefficient is low and in consequence its concentration gradient in B is small. This illustrates the importance of partition coefficients for penetration in models and in living cells. It also indicates that accumulation depends on the fact that permeability is greater for the ingoing compound of the accumulating substance than for the outgoing compound. Other things being equal, accumulation is increased by maintaining a low pH in C. Hence we may infer that anything which checks the production of acid in the living cell may be expected to check accumulation and growth. This model recalls the situation in Valonia and in most living cells where potassium accumulates as KCl, perhaps because it enters as KOH and forms KA in the sap (where A is an organic anion). In some plants potassium accumulates as KA but when HCl exists in the external solution it will tend to enter and displace the weaker acid HA (if this be carbonic it can readily escape): hence potassium may accumulate to a greater or less extent as KCl. Injury of the cell may produce a twofold effect, (1) increase of permeability, (2) lessened accumulation. The total amount of electrolyte taken up in a given time will be influenced by these factors and may be greater than normal in the injured cell or less, depending somewhat on the length of the interval of time chosen.     

THE KINETICS OF PENETRATION : XX. EFFECT OF pH AND OF LIGHT ON ABSORPTION IN IMPALED HALICYSTIS

The rate of entrance of electrolyte and of water into impaled cells of Halicystis Osterhoutii is unaffected by raising the pH of the sea water to 9.2 or lowering it to 7.0. It is quite possible that sodium enters by combining with an organic acid HX produced by the protoplasm. If the pK'' of this acid is sufficiently low the change in external pH would not produce much effect on the rate of entrance of sodium. The rate of entrance of electrolytes is affected by light. In normal light (i.e. natural succession of daylight and darkness) the rate is about twice as great as in darkness.     

THE GEOTROPIC REACTION OF RODLESS MICE IN LIGHT AND IN DARKNESS

"Black—short ears—kinky tail—rodless" mice, controlled by "pink eyed—dilute—brown" mice, were tested on an inclined plane in order to determine if they are photically sensitive, and, if so, to get a quantitative expression for their visual receptivity. Rodless and control animals were tested in the dark to obtain an expression for normal geotropic orientation. Light was then introduced to modify these reactions if possible. Under light, the controls failed to orient, whereas the rodless gave reactions almost identical with those in the dark. This test has failed in this experiment to suggest sight in the rodless mouse.     

LEAF DEVELOPMENT OF PHASEOLUS VULGARIS L. IN LIGHT AND IN DARKNESS

Development of the primary bean leaf in the dark and under continuous white light was studied during 14 days after sowing. The increase in surface area of the blade is the result of a number of sequential processes. Both in the darkness and under illumination, leaf growth is characterized by an initial cell enlargement followed by intensive cell division. Cell division in etiolated leaves continues for one day longer than in illuminated ones, but it proceeds at a slower rate. Mature leaves grown under white light undergo a phase of cell enlargement after cell division has stopped. This increases their surface area up to 800 times when compared with the blade area of the embryo. This enlargement phase is almost absent in dark-grown seedlings. Consequently the blade area of etiolated leaves is only 50 times that of the embryonic state. Thus light appears to have a dual effect on leaf development: it activates cell division and induces cell expansion.     

THE KINETICS OF PENETRATION : XIV. THE PENETRATION OF IODIDE INTO VALONIA

When 0.1 M NaI is added to the sea water surrounding Valonia iodide appears in the sap, presumably entering as NaI, KI, and HI. As the rate of entrance is not affected by changes in the external pH we conclude that the rate of entrance of HI is negligible in comparison with that of NaI, whose concentration is about 10⁷ times that of HI (the entrance of KI may be neglected for reasons stated). This is in marked contrast with the behavior of sulfide which enters chiefly as H₂S. It would seem that permeability to H₂S is enormously greater than to Na₂S. Similar considerations apply to CO₂. In this respect the situation differs greatly from that found with iodide. NaI enters because its activity is greater outside than inside so that no energy need be supplied by the cell. The rate of entrance (i.e. the amount of iodide entering the sap in a given time) is proportional to the external concentration of iodide, or to the external product [N⁺]_o [I^-l_o, after a certain external concentration of iodide has been reached. At lower concentrations the rate is relatively rapid. The reasons for this are discussed. The rate of passage of NaI through protoplasm is about a million times slower than through water. As the protoplasm is mostly water we may suppose that the delay is due chiefly to the non-aqueous protoplasmic surface layers. It would seem that these must be more than one molecule thick to bring this about. There is no great difference between the rate of entrance in the dark and in the light.     

THE KINETICS OF PENETRATION : VI. SOME FACTORS AFFECTING PENETRATION

Some of the factors affecting penetration in living cells may be advantageously studied in models in which the organic salts KG and NaG diffuse from an aqueous solution A, through a non-aqueous layer B (representing the protoplasmic surface) into an aqueous solution C (representing the sap and hence called artificial sap) where they react with CO₂ to form KHCO₃ and NaHCO₃. Their relative proportions in C depend chiefly on the partition coefficients and on the diffusion constants in the non-aqueous layer. But the ratio is also affected by other variables, among which are the following: 1. Temperature, affecting diffusion constants and partition coefficients and altering the thickness of the unstirred layers by changing viscosity. 2. Viscosity (especially in the non-aqueous layers) which depends on temperature and the presence of solutes. 3. Rate of stirring, which affects the thickness of the unstirred layers and the transport of electrolyte in those that are stirred. 4. Shape and surface area of the non-aqueous layer. 5. Surface forces. 6. Reactions occurring at the outer surface such as loss of water by the electrolyte or its molecular association in the non-aqueous phase. The reverse processes will occur at the inner surface and here also combinations with acids or other substances in the "artificial sap" may occur. 7. Outward diffusion from the artificial sap. The outward movement of KHCO₃ and NaHCO₃ is small compared with the inward movement of KG and NaG when the concentrations are equal. This is because the partition coefficients³ of the bicarbonates are very low as compared with those of NaG and KG. Since CO₂ and HCO₃ ^- diffuse into A and combine with KG and NaG the inward movement of potassium and sodium falls off in proportion as the concentration of KG and NaG is lessened. 8. Movement of water into the non-aqueous phase and into the artificial sap. This may have a higher temperature coefficient than the penetration of electrolytes. 9. Variation of the partition coefficients with concentration and pH. Many of these variables may occur in living cells. (It happens that the range of variation in the ratio of potassium to sodium in the models resembles that found in Valonia.)     

THE KINETICS OF PENETRATION : XII. HYDROGEN SULFIDE

The rate of entrance of H₂S into cells of Valonia macrophysa has been studied and it has been shown that at any given time up to 5 minutes the rate of entrance of total sulfide (H₂S + S^-) into the sap is proportional to the concentration of molecular H₂S in the external solution. This is in marked contrast with the entrance of ammonia, where Osterhout has shown that the rate of entrance of total ammonia (NH₃ + NR₄ ⁺) does not increase in a linear way with the increase in the external concentration of NH₃, but falls off. The strong base guanidine also acts thus. It has been shown that the rate of entrance of H₂S is best explained by assuming that it enters by diffusion of molecular H₂S through the non-aqueous protoplasmic surface. It has been pointed out that the simple diffusion requires that the rate of entrance might be expected to be monomolecular. Possible causes of the failure of H₂S to follow this relationship have been discussed.     

GROWTH PATTERNS OF PEA SEEDLINGS IN DARKNESS AND IN RED AND WHITE LIGHT

Thomson , Betty F., and Pauline Monz Miller . (Connecticut Coll., New London.) Growth patterns of pea seedlings in darkness and in red and white light. Amer. Jour. Bot. 48(3): 256–261. Illus. 1961.—Seedlings of peas were grown in vermiculite at 22°C. and exposed 16 hr. daily to red or white light or kept in darkness. Others were grown in soil in the greenhouse. Samples harvested daily to 16 days were dissected, the length of each internode and leaf measured and the total number of leaves and leaf primordia counted. The form of the stem apex and youngest primordia and interrodes is the same in light as in darkness. Leaf production is accelerated very slightly and the growth of leaves and internodes is decidedly accelerated by light. Leaf-leaf, leaf-internode and internode-internode correlations indicate that the morphogenetic effect of light is limited to later stages of organ growth. Dry weight is consumed more rapidly in light than in darkness, probably because of more rapid growth and slightly greater amounts of respiring tissue in light-grown plants.     

THE KINETICS OF PENETRATION : XV. THE RESTRICTION OF THE CELLULOSE WALL

When Valonia cells are impaled on capillaries, it is in some ways equivalent to removing the comparatively inelastic cellulose wall. Under these conditions sap can migrate into a free space and it is found that on the average the rate of increase of volume of the sap is 15 times what it is in intact cells kept under comparable conditions. The rate of increase of volume is a little faster during the first few hours of the experiment, but it soon becomes approximately linear and remains so as long as the experiment is continued. The slightly faster rate at first may mean that the osmotic pressure of the sap is approaching that of the sea water (in the intact cell the sap osmotic pressure is always slightly above that of the sea water). This might result from a more rapid entrance of water than of electrolyte, as would be expected when the restriction of the cellulose wall was removed. During the linear part of the curve the osmotic concentration and the composition of the sap suffer no change, so that entrance of electrolyte must be 15 times as fast in the impaled cells as it is in the intact cells. The explanation which best accords with the facts is that in the intact cell the entrance of electrolyte tends to increase the osmotic pressure. As a consequence the protoplasm is partially dehydrated temporarily and it cannot take up more water until the cellulose wall grows so that it can enclose more volume. The dehydration of the protoplasm may have the effect of making the non-aqueous protoplasm less permeable to electrolytes by reducing the diffusion and partition coefficients on which the rate of entrance depends. In this way the cell is protected against great fluctuations in the osmotic concentration of the sap.     

THE KINETICS OF PENETRATION : IX. MODELS OF MATURE CELLS

To imitate cells which have ceased to grow we have made models in which artificial sap is separated from the external solution by a non-aqueous layer (representing the protoplasm). A stream of CO₂ is bubbled through the artificial sap to imitate its production by the living cell. Potassium passes from the external solution through the non-aqueous layer into the artificial sap and there reacts with CO₂ to form KHCO₃: its rate of entrance depends on the supply of CO₂. Hence the increase of volume depends on the supply of CO₂ (as is probably true of the living cell). By regulating the supply of CO₂ and the osmotic pressure we are able to keep the volume and composition of the artificial sap approximately constant while maintaining a higher concentration of potassium than in the external solution. In these respects the model resembles certain mature cells which have ceased to grow.     

THE KINETICS OF PENETRATION : XIX. ENTRANCE OF ELECTROLYTES AND OF WATER INTO IMPALED HALICYSTIS

When cells of Halicystis are impaled on a capillary so that space is provided into which the sap can migrate, the rate of entrance of water and of electrolyte is increased about 10-fold. In impaled Valonia cells the rate is increased about 15-fold. After a relatively rapid non-linear rate of increase of sap volume immediately after impalement (which may possibly represent the partial dissipation of the difference of the osmotic energy between intact and impaled cells) the volume increases at a linear rate, apparently indefinitely. Since the halide concentration of the sap at the end of the experiment is (within the limits of natural variation) the same as in the intact cell, we conclude that electrolyte also enters the sap about 10 times as fast as in the intact cell. As in the case of Valonia we conclude that there is a mechanism whereby in the intact cell the osmotic concentration of the sap is prevented from greatly exceeding that of the sea water. This may be associated with the state of hydration of the non-aqueous protoplasmic surfaces.     

THE KINETICS OF PENETRATION : V. THE KINETICS OF A MODEL AS RELATED TO THE STEADY STATE

An organic potassium salt, KG, passes from an aqueous phase, A, through a non-aqueous layer, B, into a watery solution, C. In C it reacts with CO₂ to form KHCO₃. The ionic activity product (K) (G) in C is thus kept at such a low level that KG continues to diffuse into C after the concentration of potassium becomes greater in C than in A. Hence potassium accumulates in C, the osmotic pressure rises, and water goes in. A steady state is eventually reached in which potassium and water enter C in a constant ratio. The rate of entrance of potassium (with no water penetrating into C) may fall off in a manner approximately exponential. But water enters and may produce an exponential decrease in concentration. This suggests that the kinetics may be treated like that of two consecutive monomolecular reactions. Calculations made on this basis agree very well with the observed values. The rate of penetration appears to be proportional to the concentration gradient of KG in the non-aqueous layer and in consequence depends upon the partition coefficients which determine this gradient. Exchange of ions (passing as such through the non-aqueous layer) does not seem to play an important rôle in the entrance of potassium. The kinetics of the model may be similar to that of living cells.     

THE KINETICS OF PENETRATION : I. EQUATIONS FOR THE ENTRANCE OF ELECTROLYTES

When the only solute present is a weak acid, HA, which penetrates as molecules only into a living cell according to a curve of the first order and eventually reaches a true equilibrium we may regard the rate of increase of molecules inside as See PDF for Equation where P_M is the permeability of the protoplasm to molecules, M_o, denotes the external and M_i the internal concentration of molecules, A_i denotes the internal concentration of the anion A^- and See PDF for Equation (It is assumed that the activity coefficients equal 1.) Putting P_MF_M = V_M, the apparent velocity constant of the process, we have See PDF for Equation where e denotes the concentration at equilibrium. Then See PDF for Equation where t is time. The corresponding equation when ions alone enter is See PDF for Equation. where K is the dissociation constant of HA, P_A is the permeability of the protoplasm to the ion pair H⁺ + A^-, and A_ie denotes the internal concentration of A_i at equilibrium. Putting P_AKF_M = V_A, the apparent velocity constant of the process, we have See PDF for Equation and See PDF for Equation When both ions and molecules of HA enter together we have See PDF for Equation where S_i = M_i + A_i and S_ie is the value of S_i at equilibrium. Then See PDF for Equation V_M, V_A, and V_MA depend on F_M and hence on the internal pH value but are independent of the external pH value except as it affects the internal pH value. When the ion pair Na⁺ + A^- penetrates and Na_i = BA_i, we have See PDF for Equation and See PDF for Equation where P _NaA is the permeability of the protoplasm to the ion pair Na⁺ + A^-, Na_o and Na_i are the external and internal concentrations of Na⁺, See PDF for Equation, and V _Na is the apparent velocity constant of the process. Equations are also given for the penetration of: (1) molecules of HA and the ion pair Na⁺ + A^-, (2) the ion pairs H⁺ + A^- and Na⁺ + A^-, (3) molecules of HA and the ion pairs Na⁺ + A^- and H⁺ + A^-. (4) The penetration of molecules of HA together with those of a weak base ZOH. (5) Exchange of ions of the same sign. When a weak electrolyte HA is the only solute present we cannot decide whether molecules alone or molecules and ions enter by comparing the velocity constants at different pH values, since in both cases they will behave alike, remaining constant if F_M is constant and falling off with increase of external pH value if F_M falls off. But if a salt (e.g., NaA) is the only substance penetrating the velocity constant will increase with increase of external pH value: if molecules of HA and the ions of a salt NaA. penetrate together the velocity constant may increase or decrease while the internal pH value rises. The initial rate See PDF for Equation (i.e., the rate when M_i = 0 and A_i = 0) falls off with increase of external pH value if HA alone is present and penetrates as molecules or as ions (or in both forms). But if a salt (e.g., NaA) penetrates the initial rate may in some cases decrease and then increase as the external pH value increases. At equilibrium the value of M_i equals that of M_o (no matter whether molecules alone penetrate, or ions alone, or both together). If the total external concentration (S_o = M_o + A_o) be kept constant a decrease in the external pH value will increase the value of M_o and make a corresponding increase in the rate of entrance and in the value at equilibrium no matter whether molecules alone penetrate, or ions alone, or both together. What is here said of weak acids holds with suitable modifications for weak bases and for amphoteric electrolytes and may also be applied to strong electrolytes.     

THE PENETRATION OF AMMONIA INTO FROG MUSCLE

1. A study was made of the electrolyte changes which occur when frog muscles are immersed in a Ringer solution with 1/5 of the Na replaced by NH₄Cl. Analyses were made in the solution and in the muscles for K and NH₃, and the muscles were also analyzed for Cl, HCO₃, and Na. Control muscles were immersed in normal Ringer''s solution and similarly analyzed. 2. The amount of ammonia taken up was about equal to the K and Na lost. There was also a small increase in chloride content. The bicarbonate content was the same in both experimental and control muscles, indicating no change in the muscle pH due to the NH₃ which penetrated. An increased loss of K due to the penetration of NH₃ was also demonstrated by the use of radioactive K. 3. After 5 hours, the concentration of ammonia per gram of muscle is about the same as the concentration in the solution. After 4 or 5 days, the concentration in the muscle is about 1.5 times that in the solution. The inside to outside NH₃ ratio is about equal to the corresponding H ion ratio, but is much less than the K ratio. 4. The rate of penetration of the NH₃ is increased by a rise of temperature, by stirring the solution, and by decrease in the concentration of Na, K, Ca, or Mg in the solution; it is decreased by increasing the size of the muscles or by killing them with chloroform or boiling. 5. Liver, smooth muscle, skin, and kidney, in a few experiments, behaved much like muscle except that there was a formation of urea in the case of liver. 6. The injection of NH₄Cl into anesthetized cats causes an increase in the level of K in the blood plasma.     

THE KINETICS OF PENETRATION : III. EQUATIONS FOR THE EXCHANGE OF IONS

Equations are given for a variety of cases when ions of the same sign are exchanged between the interior of a living cell and the external solution.