OSMOTIC PROPERTIES OF THE EGG CELLS OF THE OYSTER (OSTREA VIRGINICA)

Investigations of the osmotic properties of oyster eggs by a diffraction method for measuring volumes have led to the following conclusions: 1. The product of cell volume and osmotic pressure is approximately constant, if allowance is made for osmotically inactive cell contents (law of Boyle-van''t Hoff). The space occupied by osmotically inactive averages 44 per cent of cell volume. 2. Volume changes over a wide range of pressures are reversible, indicating that the semipermeability of the cell during such changes remains intact. 3. The kinetics of endosmosis and of exosmosis are described by the equation, See PDF for Equation, where dV is rate of volume change; S, surface area of cell, (P-P_e), the difference in osmotic pressure between cell interior and medium, and K, the permeability of the cell to water. 4. Permeability to water during endosmosis is 0.6µ³ of water per minute, per square micron of cell surface, per atmosphere of pressure. The value of permeability for exosmosis is closely the same; in this respect the egg cell of the oyster appears to be a more perfect osmometer than the other marine cells which have been studied. Permeability to water computed by the equation given above is in good agreement with computations by the entirely different method devised by Jacobs. 5. Permeability to diethylene glycol averages 27.2, and to glycerol 20.7. These values express the number of mols x 10^–15 which enter per minute through each square micron of cell surface at a concentration difference of 1 mol per liter and a temperature of 22.5°C. 6. Values for permeability to water and to the solutes tested are considerably higher for the oyster egg than for other forms of marine eggs previously examined. 7. The oyster egg because of its high degree of permeability is a natural osmometer particularly suitable for the study of the less readily penetrating solutes.     

FURTHER STUDIES ON THE KINETICS OF OSMOSIS IN LIVING CELLS

Using unfertilized eggs of Arbacia punctulata as natural osmometers an attempt has been made to account for the course of swelling and shrinking of these cells in anisotonic solutions by means of the laws governing osmosis and diffusion. The method employed has been to compute permeability of the cell to water, as measured by the rate of volume change per unit of cell surface per unit of osmotic pressure outstanding between the cell and its medium. Permeability to water as here defined and as somewhat differently defined by Northrop is approximately constant during swelling and shrinking, at least for the first several minutes of these processes. Permeability is found to be independent of the osmotic pressure of the solution in which cells are swelling. Water is found to leave cells more readily than it enters, that is, permeability is greater during exosmosis than during endosmosis.     

Analysis of transcellular water movement inNitella

Summary The mechanism of transcellular osmosis was analyzed on the assumption that the driving force, which is equal to the osmotic pressure of the mannitol solution given to the exosmosis side, is divided into two parts; one causing the inward water flow on the water side, the other causing the outward water flow on the solution side, when each force drives an equal amount of water. Based on this analysis a new procedure was developed to measure the endosmotic and exosmotic water permeabilities of the membranes independently. It involved measurement of volume of water transported transcellularly, change in turgor pressure, and water permeability of the cell wall alone.Experiments following the new procedure revealed that in aNitella internode positioned across a partition wall with equal length both the endosmotic and exosmotic water permeabilities remained constant during transcellular osmosis induced with 0.4M mannitol, at least for the first minute. It was found that the permeability coefficient for endosmosis (3.9 × 10^–5 cm sec^–1 atm^–1) was very much higher than that for exosmosis (1.4× 10^–5 cm sec^–1 atm^–1). Treatment of the endosmotic cell part with 5% ethanol conspicuously decreased the water permeability of the cell on this side down to 1/2.4 the value obtained without ethanol but never affected the permeability on the other side (exosmosis side).This work was supported partly by a Research Grant from the Ministry of Education of Japan.     

Nature of the water channels in the internodal cells ofNitellopsis

Summary The hydraulic resistance was measured on internodal cells ofNitellopsis obtusa using the method of transcellular osmosis. The hydraulic resistance was approximately 2.65 pm^–1 sec Pa, which corresponds to an osmotic permeability of 101.75 m sec^–1 (at 20°C).p-Chloromercuriphenyl sulfonic acid (pCMPS) (0.1–1mm, 60 min) reversibly increases the hydraulic resistance in a concentration-dependent manner.pCMPS does not have any effect on the cellular osmotic pressure.pCMPS increases the activation energy of water movement from 16.84 to 32.64 kJ mol^–1, indicating that it inhibits water movement by modifying a low resistance pathway.pCMPS specifically increases the hydraulic resistance to exosmosis, but does not influence endosmosis. By contrast, nonyltriethylammonium (C₉), a blocking agent of K⁺ channels, increases the hydraulic resistance to endosmosis, but does not affect that to exosmosis. These data support the hypothesis that water moves through membrane proteins in characean internodal cells and further that the polarity of water movement may be a consequence of the differential gating of membrane proteins on the endo- and exoosmotic ends.     

Hydraulic conductivity of tonoplast-freeChara cells

Summary This study is the first trial to measure the osmotic water permeability or the hydraulic conductivity of the plasmalemma alone of a plant cell. For this purpose tonoplast-free cells were prepared from intenodal cells ofChara australis and their hydraulic conductivities were measured by the transcellular osmosis method.The transcellular hydraulic conductivity did not change after removing the tonoplast. The transcellular hydraulic conductivity of the tonoplast-free cells was dependent on the internal osmotic pressure as is the case in the tonoplast-containing normal cells. The hydraulic conductivities for both endosmosis and exosmosis of the tonoplast-free cells were equal to respective values of the normal cells. Consequently the ratio between the inward and outward hydraulic conductivities did not change due to the loss of the tonoplast. The results indicate that the resistance of the tonoplast to water flow is negligibly small as compared with that of the plasmalemma and further that the tonoplast is not a factor responsible for the direction-dependency of hydraulic conductivity. The hydraulic conductivity of the plasmalemma is invariable for wide variations of K⁺ and Ca²⁺ in the cytoplasm.     

THE KINETICS OF OSMOTIC SWELLING IN LIVING CELLS

The rate of swelling of unfertilized sea urchin eggs in hypotonic sea water was investigated. Analysis of curves leads to the following conclusions. 1. The rate of swelling follows the equation, See PDF for Equation where V _eq., V ₀, and V_t stand for volume at equilibrium, at first instant, and at time t, respectively, the other symbols having their usual significance. This equation is found to hold over a wide range of temperatures and osmotic pressures. This relation is the one expected in a diffusion process. 2. The rate of swelling is found to have a high temperature coefficient (Q ₁₀ = 2 to 3, or µ = 13,000 to 19,000). This deviation from the usual effect of temperature on diffusion processes is thought to be associated with changes in cell permeability to water. The possible influence of changes in viscosity is discussed. 3. The lower the osmotic pressure of the solution, the longer it takes for swelling of the cell. Thus at 15° in 80 per cent sea water, the velocity constant has a value of 0.072, in 20 per cent sea water, of 0.006.     

THE EFFECT OF SALT CONCENTRATION OF THE MEDIUM ON THE RATE OF OSMOSIS OF WATER THROUGH THE MEMBRANE OF LIVING CELLS

1. Using the unfertilized egg of the sea urchin, Arbacia, as osmometer, it was found that the rate with which water enters or leaves the cell depends on the osmotic pressure of the medium: the velocity constant of the diffusion process is higher when the cell is in concentrated sea water, and lower when the sea water medium is diluted with distilled water. Differences of more than tenfold in the value of the velocity constant were obtained in this way. When velocity constants are plotted against concentration of medium, a sigmoid curve is obtained. 2. These results are believed to indicate that cells are more permeable to water when the osmotic pressure of the medium is high than when it is low. This relation would be accounted for if water should diffuse through pores in a partially hydrated gel, constituting the cell membrane. In a medium of high osmotic pressure, the gel is conceived to give up water, to shrink, and therefore to allow widening of its pores with more ready diffusion of water through them. Conversely, in solutions of lower osmotic pressure, the gel would take up water and its pores become narrow.     

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.     

SOME ASPECTS OF SECRETION : I. SECRETION OF WATER

If we increase the osmotic pressure at one end of a Nitella cell by applying a solution of sucrose and if we subsequently submerge the entire cell in water we find that water enters at the end where the osmotic pressure is higher and comes out of the cell at the other end. If similar inequalities of osmotic pressure should arise as the result of metabolism we can understand how a secreting cell might take up water at one spot on its surface and expel it in another spot and thus bring about the secretion of water. The Nitella cell can expel water from a region of the cell which is in contact with water, air, or mineral oil.     

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.     

STUDIES ON OSMOTIC EQUILIBRIUM AND ON THE KINETICS OF OSMOSIS IN LIVING CELLS BY A DIFFRACTION METHOD

1. Osmotic equilibrium and kinetics of osmosis of living cells (unfertilized eggs of Arbacia punctulata) have been studied by a diffraction method. This method consists of illuminating a suspension of cells by parallel monochromatic light and measuring, by means of telescope and scale, the angular dimensions of the resulting diffraction pattern from which the average volume of the cells may be computed. The method is far less laborious and possesses several advantages over direct measurement of individual cells. The average size of a large number of cells is obtained from a single measurement of the diffraction pattern and thus individual variability is averaged out. The observations can be made at intervals of a few seconds, permitting changes in volume to be followed satisfactorily. During the measurements the cells are in suspension and are constantly stirred. 2. Volumes of cells in equilibrium with solutions of different osmotic pressure have been determined. In agreement with our previous experiments, based upon direct microscope measurements, we have confirmed the applicability of the law of Boyle-van''t Hoff to these cells; that is to say, the product of volume and pressure has been found to be approximately constant if allowance be made for the volume of osmotically inactive material of the cell contents. The volume of osmotically inactive material was found to be, on the average, 12 per cent of the initial cell volume; in eggs from different animals this value ranged from 6 to 20 per cent. 3. Permeability to water of the Arbacia egg has been found to average, at 22°C., 0.106 cubic micra of water per square micron of cell surface, per minute, per atmosphere of difference in osmotic pressure. 4. Permeability to ethylene glycol has been found to average, at 24°C., 4.0 x 10^–15 mols, per square micron of cell surface, per minute, for a concentration difference of 1 mol per liter. This is in agreement with the values reported by Stewart and Jacobs.     

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.     

THE COMPOSITION OF THE CELL SAP OF THE PLANT IN RELATION TO THE ABSORPTION OF IONS

1. Chemical examination of the cell sap of Nitella showed that the concentrations of all the principal inorganic elements, K, SO₄, Ca, Mg, PO₄, Cl, and Na, were very much higher than in the water in which the plants were growing. 2. Conductivity measurements and other considerations lead to the conclusion that all or nearly all of the inorganic elements present in the cell sap exist in ionic state. 3. The insoluble or combined elements found in the cell wall or protoplasm included Ca, Mg, S, Si, Fe, and Al. No potassium was present in insoluble form. Calcium was predominant. 4. The hydrogen ion concentration of healthy cells was found to be approximately constant, at pH 5.2. This value was not changed even when the outside solution varied from pH 5.0 to 9.0. 5. The penetration of NO₃ ion into the cell sap from dilute solutions was definitely influenced by the hydrogen ion concentration of the solution. Penetration was much more rapid from a slightly acid solution than from an alkaline one. It is possible that the NO₃ forms a combination with some constituent of the cell wall or of the protoplasm. 6. The exosmosis of chlorine from Nitella cells was found to be a delicate test for injury or altered permeability. 7. Dilute solutions of ammonium salts caused the reaction of the cell sap to increase its pH value. This change was accompanied by injury and exosmosis of chlorine. 8. Apparently the penetration of ions into the cell may take place from a solution of low concentration into a solution of higher concentration. 9. Various comparisons with higher plants are drawn, with reference to buffer systems, solubility of potassium, removal of nitrate from solution, etc.     

Water balance in Collembola and its relation to habitat selection: Water content,haemolymph osmotic pressure and transpiration during an instar

In the two collembolan species Orchesella cincta and Tomocerus minor the water content, haemolymph osmotic pressure and transpiration rate fluctuate with the feeding rhythm during each instar. The changes in water content, however, are due to changes in dry weight, because the absolute water weight stays constant during the instar.The intake of food is probably the cause of the increase in haemolymph osmotic pressure. Increase of osmotically active substances in the blood and/or blood volume reduction may be responsible for the rise in osmotic pressure. This change in osmotic pressure in turn may affect the responsiveness of the animals to water as well as their feeding behaviour.Changes in the epicuticle and in epidermal cell membranes may cause changes in the rate of transpiration. The high rate observed during ecdysis and during the mid-instar may explain the behaviour of the animals in varied water conditions.Dehydration during the instar causes an equivalent rise in osmotic pressure for both Tomocerus minor and Orchesella cincta. The water loss appears to involve the haemolymph. The physiological state of the animal does not influence the rise in osmotic pressure. There are no signs of any osmoregulation in the two species.     

THE OSMOTIC PROPERTIES OF LIVING CELLS (EGGS OF ARBACIA PUNCTULATA)

We have attempted to answer the question: How nearly ideal, as an osmometer, is the unfertilized Arbacia egg? The following conclusion have been reached: 1. Volumes can be measured accurately over a wide range of pressures since the cell is in general spherical and does not suffer deformation from its own weight or other factors. 2. The product of volume and pressure is approximately constant, if allowance be made for osmotically inactive cell contents. It is computed that from 7 to 14 per cent of cell volume is occupied by osmotically inactive material. 3. Evidence is presented that no appreciable escape of cell contents occurs while the cell is in hypotonic sea water; that, therefore, the semipermeability of the membrane is approximately perfect, so long as injury to the cell is avoided. 4. In comparison with osmotic pressure the influence of other forces, such as elasticity or surface tension, on cell volume must in these experiments be slight.     

THE OSMOTIC EFFECTS OF ELECTRON MICROSCOPE FIXATIVES

The reflecting cells on the scales of sprat and herring contain ordered arrays of guanine crystals. The spacing of the crystals within these cells determines the wave bands of the light which they reflect, hence volume changes in the reflecting cells can be observed as color changes directly. This property of the scales is used to show that (a) fixation with osmium tetroxide solutions destroys osmotic activity; (b) fixation with aldehyde solutions does not destroy osmotic activity and does not cause volume changes if the aldehydes are made up in salt or sucrose solutions whose osmolarities, discounting the aldehyde, are about 60% of those to which the cells are in equilibrium in life, and (c) after aldehyde fixation the cells are osmotically active but come to a given volume in salt and sucrose solutions of concentrations only 60% of those which give their volume before fixation. Various possible mechanisms underlying the change of osmotic equilibrium caused by aldehyde fixation are discussed.     

Permeability of lipid membranes revised in relation to freeze-thaw processes.

Water and solute activity gradients created during freeze-thaw processes produce water and solute fluxes across the cell membrane resulting in volume changes. Under these conditions, osmotic and thermal stresses affect the curvature, the phase behavior, and the surface properties of the lipid bilayer. These structural changes are not considered by the classical formalisms describing permeability of lipid membranes to water and nonelectrolytes such as the Nernst-Planck equation, Eyring's absolute rate theory, and Kedem-Katchalsky's thermodynamic of irreversible processes approach. In this paper, the influence of such changes on the glycerol permeation kinetics are reported. The results indicate that osmotic and chemical effects of the cryoprotectant on the membrane properties affect the rate of volume swelling depending on whether the membrane is in the gel or in the liquid crystalline state.     

Membrane tension regulates water transport in yeast

Evidence that membrane surface tension regulates water fluxes in intact cells of a Saccharomyces cerevisiae strain overexpressing aquaporin AQY1 was obtained by assessing the osmotic water transport parameters in cells equilibrated in different osmolarities. The osmotic water permeability coefficients (P_f) obtained for yeast cells overexpressing AQY1 incubated in low osmolarity buffers were similar to those obtained for a double mutant aqy1aqy2 and approximately three times lower (with higher activation energy, E_a) than values obtained for cells incubated in higher osmolarities (with lower E_a). Moreover, the initial inner volumes attained a maximum value for cells equilibrated in lower osmolarities (below 0.75 M) suggesting a pre-swollen state with the membrane under tension, independent of aquaporin expression. In this situation, the impairment of water channel activity suggested by lower P_f and higher E_a could probably be the first available volume regulatory tool that, in cooperation with other osmosensitive solute transporters, aims to maintain cell volume. The results presented point to the regulation of yeast water channels by membrane tension, as previously described in other cell systems.     

THE CELL SAP OF HYDRODICTYON

Analysis of the cell sap of Hydrodictyon patenaeforme Pocock, from California indicates the usual marked accumulation of potassium, which is 4000 times as concentrated as in the surrounding pond water. Small amounts of sodium and calcium were found. Chloride makes up about three-fourths of the anions, with a very high sulfate, and much lower bicarbonate concentration accounting for most of the remainder. Electrical conductivity and osmotic studies indicate that the analyzed elements are ionized, and account for most of the sap''s osmotic pressure. pH is 5.5 to 6.0. The analytical procedure was designed to determine as many of the cations as possible on one small sample. Hydrodictyon is a large multinucleate cell belonging to an order (Chlorococcales) new to permeability and accumulation studies.     

The physico-chemical mechanism of mediated transport. II. Osmotic and isosmotic volume flow

The process of volume change of cells subject to osmotic shocks or isosmotic entrance of permeant solute is formulated on the basis of the accepted structure for the plasma membrane and a physico-chemical approach similar to that recently developed. The effect of relevant parameters is discussed and theoretical equilibrium values for the variables are calculated in connection with water and permeant solute permeability determinations. Although a sorption-diffusional mechanism for solute and/or water volume flow within the membrane is assumed in both cases, the kinetics of volume change is shown to be totally different between them. In the isosmotic process a fixed relationship, given by the total solute concentration, is shown to exist between the permeant solute and volume fluxes to the cell, thereby implying a definite value for the volume fraction of water in the migration pathway, higher than 90%. The bi-phase osmotic regulatory response caused by permeant solute is simulated on the basis of an osmotic and isosmotic processes in series, showing good agreement with general behavior. Finally, an explanation to the problem of volume flow and forces in connection with a diffusional mechanism in biological and artificial membranes, is presented.