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.

CHANGES IN RESTING POTENTIAL DUE TO A SHIFT OF ELECTROLYTES IN THE CELL PRODUCED BY NON-ELECTROLYTES

Experiments on Nitella indicate that the resting potential is due chiefly to the outwardly directed diffusion potential of electrolytes which is set up at the inner, non-aqueous, protoplasmic surface surrounding the vacuole. We might therefore expect that any change in the concentration of these electrolytes would affect the resting potential. The experiments described here indicate that this expectation is justified. When a sucrose solution is applied at one end of the cell and water is placed at another spot, water enters at the latter, passes along inside the cell, and escapes into the sucrose solution, but the electrolytes are unable to escape into the sucrose solution (except very slowly) so that the concentration of electrolytes increases in the region in contact with the sucrose solution. Hence the potential at this spot increases. At the other spot where the water enters, the concentration of electrolytes decreases and the potential at this spot falls off. The changes can be carried out reversibly without injury to the cell.

The Effects of High Hydrostatic Pressures on a Suctorian

1. The suctorian Discophrya piriformis Guilcher has been subjected to pressures ranging from 1000 to 15,000 lb./sq.in. (68-1020 atm.). 2. Pressures of 2000 lb./sq.in. and over cause a creasing of the body surface. Except at the higher pressures this occurs some seconds after the application of pressure. 3. Creasing is accompanied by an expansion of the pellicle. At the lower pressures, there is also an expansion of the protoplasmic surface. A comparison may be made with the expansion of the body surface which occurs during feeding. 4. In many cases, and particularly at the higher pressures, the protoplasm later separates from the expanded pellicle. With prolonged treatment it sometimes rounds up, and there is evidence of a loss of volume of the protoplasm. 5. On release of pressure the protoplasm spreads back to the pellicle, usually within a few minutes. The wrinkled and expanded pellicle is then slowly reorganized to its normal shape and size, over a period of many hours.

THE MECHANISM OF ACCUMULATION IN LIVING CELLS

When a compound enters a living cell until its activity becomes greater inside than outside, it may be said to accumulate. Since it moves from a region where its activity is relatively low to a region where its activity is relatively high, it is evident that work must be done to bring this about. The following explanation is suggested to account for accumulation. The protoplasmic surface is covered with a non-aqueous layer which is permeable to molecules but almost impermeable to ions. Hence free ions cannot enter except in very small numbers. The experiments indicate that ions combine at the outer surface with organic molecules (carrier molecules) and are thus able to enter freely. If upon reaching the aqueous protoplasm these molecules are decomposed or altered so as to set the ions free, the ions must be trapped since they cannot pass out except in very small numbers. If we adopt this point of view we can suggest answers to some important questions. Among these are the following: 1. Why accumulation is confined to electrolytes. This is evident since only ions will be trapped. 2. Why ions appear to penetrate against a gradient. Actually there is no such penetration since the ions enter in combination with molecules. The energy needed to raise the activity of entering compounds is furnished by the reactions involved in the process of accumulation. 3. Why, in absence of injury, ions do not come out when the cell is placed in distilled water. Presumably the outgoing ions will combine at the outer surface with carrier molecules and then move inward in the same way as ions coming from without. 4. Why the relative rate of penetration falls off as the external concentration increases. This is because the entrance of ions is limited by the number of carrier molecules but no such limitation exists when ions move outward since they can do so without combining with carrier molecules. 5. Why accumulation is promoted by constructive metabolism which is needed to build up the organic molecules and by destructive metabolism which brings about their decomposition. 6. Why measuring the mobilities of ions in the outer protoplasmic surface does not enable us to predict the relative rate of entrance of ions. We find for example in Nitella that K+ has a much higher mobility than Na+ but the accumulation of these ions does not differ greatly. This is to be expected if they enter by combining with molecules at the surface. Only if K+ is able to combine preferentially will it accumulate preferentially. 7. Why ions may come out in anoxia and at low temperatures. If these conditions depress the formation of carrier molecules and their decomposition in the protoplasm, the balance between intake and outgo of ions will be disturbed and relatively more may come out. 8. Why the excess of internal over external osmotic pressure is less in sea water than in fresh water. As the external concentration of ions increases the rate of intake does not increase in direct proportion since the number of carrier molecules does not increase and this slows down the relative rate of intake of ions. But it does not slow down the rate of exit of ions since they need not combine with carrier molecules in order to pass out. Hence the excess of ions inside will be relatively less as the concentration of external ions increases. 9. How water is pumped from solutions of higher to solutions of lower osmotic pressure. If metabolism and consequently accumulation is higher at one end of a cell than at the other, the internal osmotic pressure will be higher at the more active end and this makes it possible for the cell to pump water from solutions of higher osmotic pressure at the more active end to solutions of lower osmotic pressure at the less active, as shown experimentally for Nitella. This might help to explain the action of kidney cells and the production of root pressure in plants.

HIGHER PERMEABILITY FOR WATER THAN FOR ETHYL ALCOHOL IN NITELLA

If we apply water at one end of a Nitella cell, A, and place at the other end, B, a solution of a substance which does not penetrate, such as sucrose, water enters the cell at A, passes along inside the cell, and escapes at B. But if in place of sucrose we use a substance which penetrates such as ethyl alcohol the flow of water is lessened and this fact makes it possible to measure the amount of alcohol which enters. (An increase in the size of cells placed in solutions of alcohol does not necessarily indicate that the number of mols of alcohol entering is greater than the number of mols of water leaving the cell.) The permeability for water is more than 18 times as great as for ethyl alcohol. The behavior of the 2 substances was compared in the same individual cell with a driving force which at the start was the same for both substances. The number of mols entering per second per cm.2 of surface with a driving force of 1 atmosphere at 25°C. is 0.772 (10–6) for water and 0.042 (10–6) for ethyl alcohol. The experiments indicate that the non-aqueous substance at the surface of the protoplasm has a higher partition coefficient for water than for ethyl alcohol, although the protoplasmic surface is composed of materials not miscible with water.

ABNORMAL PROTOPLASMIC PATTERNS AND DEATH IN SLIGHTLY HYPERTONIC SOLUTIONS

Some interesting properties of protoplasm are revealed when slightly hypertonic solutions of sugars or of electrolytes are applied to Nitella. The chloroplasts contract and the space between them increases and forms a characteristic pattern consisting of clear areas extending lengthwise along the cell and tapering off at both ends. The development of these areas is irreversible from the start. If the cell is returned to water after plasmolysis begins these areas continue to enlarge in much the same fashion as when no change is made in the external solution. The cell soon dies whether returned to water or left in the plasmolyzing solution. Similar results are obtained with other sugars, with NaCl, CaCl2, and sea water. Similar reactions are also brought about by strong ingoing or outgoing currents of water. This suggests that mechanical action may be chiefly responsible for the result and this idea is in harmony with other facts. It seems possible that the retraction of the protoplasm from the cellulose wall may disturb the delicate non-aqueous film which covers the outer surface of the protoplasm and thus produce injury. Such an effect might take place even without visible retraction if the injury occurred in protoplasmic projections extending into the cellulose wall. A study of this behavior may throw light on the nature of the protoplasmic surface and on the properties of protoplasmic gels as well as on the process of death. An understanding of the mechanism involved may help to explain the action of hypertonic solutions in other cases as, for example, in the artificial parthenogenesis of marine eggs.

EFFECTS OF HYDROXYL ON NEGATIVE AND POSITIVE CELLS OF NITELLA

Remarkable changes are brought about by KOH in transforming negative cells of Nitella (showing dilute solution negative with KOH) to positive cells (showing dilute solution positive with KOH). NaOH is less effective as a transforming agent. This might be explained on the ground that the protoplasm contains an acid (possibly a fatty acid) which makes the cell negative and which is dissolved out more rapidly by KOH than by NaOH, as happens with the fatty acids in ordinary soaps. Part of a negative cell can be changed to positive by exposure to KOH while the untreated portion remains negative. After exposure to KOH the potential the protoplasm has when in contact with NaCl may increase. At the same time there may be an increase in the potassium effect; i.e., in the change of P.D. in a positive direction observed when 0.01 M KCl is replaced by 0.01 M NaCl. In some cases the order of ionic mobilities is uK > vOH > uNa. This shows that the protoplasmic surface cannot be a pore system: for in such a system all cations must have greater mobilities than all anions or vice versa.

DIFFERING RATES OF DEATH AT INNER AND OUTER SURFACES OF THE PROTOPLASM

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.

DIFFERING RATES OF DEATH AT INNER AND OUTER SURFACES OF THE PROTOPLASM

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.