Dog Red Blood Cells : Adjustment of salt and water content in vitro

Dog red blood cells (RBC) lack a ouabain-sensitive sodium pump, and yet they are capable of volume regulation in vivo. The present study was designed to find in vitro conditions under which dog RBC could transport sodium outward, against an electrochemical gradient. Cells were first loaded with sodium chloride and water by preincubation in hypertonic saline. They were then incubated at 37°C in media containing physiologic concentrations of sodium, potassium, chloride, bicarbonate, glucose, and calcium. The cells returned to a normal salt and water content in 16–20 h. Without calcium in the medium the cells continued slowly to accumulate sodium. Removal of glucose caused rapid swelling and lysis, whether or not calcium was present. The net efflux of sodium showed a close relationship to medium calcium over a concentration range from 0 to 5 mM. Extrusion of salt and water was also demonstrated in fresh RBC (no hypertonic preincubation) when calcium levels in the media were sufficiently raised. The ion and water movements in these experiments were not influenced by ouabain or by removal of extracellular potassium. Magnesium could not substitute for calcium. It is concluded that dog RBC have an energy-dependent mechanism for extruding sodium chloride which requires external calcium and is quite distinct from the sodium-potassium exchange pump.     

Volume Control by Muscle Fibers of the Blue Crab : Volume readjustment in hypotonic salines

Single isolated muscle fibers from the walking legs of the blue crab, Callinectes sapidus act as Boyle-van't Hoff osmometers with an osmotically inactive volume of 33 %. Fibers in hypotonic salines undergo a spontaneous volume readjustment toward the initial volumes of the cells found in isotonic salines. The volume readjustment is initiated by the increase in cell volume in hypotonic salines and appears to be dependent on the duration of exposure of the fiber to external sodium, the sodium concentration, and the pH of the external medium. The volume-readjusted cells continue to behave as osmometers, but with an increased relative osmotically inactive volume and a decreased internal resistivity. The decreases in cell volumes appear to be, in large part, due to losses of osmotically active nonelectrolytes from the cells.     

Cell volume regulation in frog urinary bladder

We have studied the problem of cell volume homeostasis in toad and frog urinary bladder by using electrophysiological measurements and an optical measure of cell volume. After osmotically induced swelling, urinary bladder cells spontaneously regulate their volume through a net loss of potassium, chloride, and water. During inhibition of sodium transport by amiloride the cells swell to the same extent as controls, but the volume-regulatory process is blocked. Electrophysiological results under isosmotic conditions indicate that basolateral membrane resistance increases simultaneously with the amiloride-induced rise in apical membrane resistance during transport inhibition. These independent observations indicate that inhibition of apical membrane sodium entry results in a secondary decrease in basolateral membrane potassium permeability. When cells are exposed to calcium-free, hyposmotic Ringer's solution, cell volume regulation is blocked; subsequent addition of the calcium ionophore A23187 is ineffective in restoring the regulatory process. The ionophore does induce volume regulation, however, in amiloride-inhibited, osmotically swollen cells in the presence of external calcium. Calcium thus seems to control basolateral membrane potassium permeability and may be the intracellular mediator of apical and basolateral membrane interactions.     

Further studies of the volume-regulatory response of Amphiuma red cells in hypertonic media. Evidence for amiloride-sensitive Na/H exchange

When Amphiuma red cells are shrunken in hypertonic media, they return toward their original volume by gaining Na through an amiloride-sensitive pathway. As cells recover their volume during this volume-regulatory increase (VRI) response, acid is extruded into the medium. Medium acidification is correlated with cell Na uptake. Both medium acidification and cell Na uptake are blocked by 10(-3) M amiloride or by replacing medium Na with K or choline. Perturbations that increase cell Na uptake (such as increasing medium osmolality) also increase medium acidification. As the medium becomes more acidic, the cells become more alkaline. These changes in cell and medium pH are increased if pH equilibration across the cell membrane is prevented by inhibiting the anion exchanger with SITS (4-acetamido-4'-isothiocyano-2,2'-stilbene disulfonic acid). The quantity of acid extruded by SITS-treated cells is the same as the quantity of Na gained, which strongly suggests 1:1 exchange of Na for H. Cell enlargement in SITS-treated cells results from the exchange of osmotically active Na ions for H ions that are not osmotically active when combined with cellular buffers. Previous evidence indicates that the normal VRI response involves an increase in the cellular content of Cl as well as Na. We show that SITS completely blocks net Cl uptake, which suggests that Cl enters via the anion exchanger. SITS also slows Na entry, presumably as a result of the above-mentioned increase in cell pH caused by SITS. We suggest that the initial event in the VRI response is net Na uptake via a Na/H exchanger, and that net Cl uptake results from secondary Cl/HCO3 exchange via the anion exchanger.     

Growth, mineral uptake and skeletal calcium deposition in brown trout, Salmo trutta L., yolk-sac fry exposed to aluminium and manganese in soft acid water

In 30-day exposures in artificial soft water medium, survival of brown trout alevins was not affected by low pH (4.5,4.8, 5.4), by low calcium concentration (10.25 μmol l⁻¹) or by manganese (≤20 μmol l⁻¹), but was impaired by aluminium (6–8 μmol l⁻¹) at low calcium concentration (10 μmol l⁻¹) irrespective of pH (4.5 or 5.4). Manganese (6.6, 20 μmol l⁻¹) impaired net calcium uptake and calcium deposition in the skeleton at low calcium concentration (25 μmol l⁻¹) irrespective of pH. Aluminium (2–8 μmol l⁻¹) impaired gross development, net uptake of calcium, potassium and sodium, and calcium deposition in the skeleton, and slightly increased the net loss of magnesium, some of these effects being more severe at calcium concentration 10 μmol l⁻¹ than 50 μmol l⁻¹, and some more severe at pH 5.4 than pH 4.5. Net uptake of calcium and sodium were impaired at low pH (4.5, 4.8), and skeletal calcium deposition was impaired at low calcium concentration (10 μmol l⁻¹), but these effects of low pH and low calcium concentration were slight compared with those of the trace metals. The possible role of trace metals in reports of the deleterious effects on fish of low pH levels is discussed.     

Electrolyte and non-electrolyte distribution in the ehrlich ascites tumor cells during the cell cycle

In a previous study, evidence was presented for changes in the state of water and osmotically active solutes during the cell cycle. Total water was constant at 82% (w/w), while the fraction of water that was osmotically active decreased from a maximum during S to a minimum at mitosis. Total Na⁺, K⁺, and C1^? in milliequivalents per liter of cell water remained constant. Therefore, electrolytes are sequestered in the osmotically inactive water. Evidence is now presented that Na⁺ exists primarily as one compartment, with a second, slower compartment appearing during S and disappearing during G2. Na⁺ is completely exchangeable during the entire cell cycle. The distribution of other penetrating solutes was also investigated. When placed in hyperosmotic ethylene glycol solutions, cells first shrink, then swell to their original volumes. ¹⁴C-ethylene glycol distributes in 89% of cell water throughout the cell cycle. However, ¹⁴C-urea distributes in anywhere from 86–100% of the cell water, depending on the stage in the cell cycle. Both solutes are at chemical equilibrium in water in which they are distributed, but they differ in their effects on cell volume. The final volume at which cells equilibrate in urea varies with the concentration of urea in the environment and with time into the cell cycle. Results suggest a loss of osmotically active particles or decreased osmotic activity of urea.     

Calcium transport in intact human erthrocytes

Intact human erythrocytes can be readily loaded with calcium by incubation in hypersomotic media at alkaline pH. Erythrocyte calcium content increases from 15-20 to 120-150 nmol/g hemoglobin after incubation for 2 h at 20 degree C in a 400 mosmol/kg, pH 7.8 solution containing 100 mM sodium chloride, 90 mM tetramethylammonium chloride, 1 mM potassium chloride, and 10 mM calcium chloride. Calcium uptake is a time-dependent process that is associated with an augmented efflux of potassium. The ATP content in these cells remains at more than 60% of normal and is not affected by calcium. Calcium uptake is influenced by the cationic composition of the external media. The response to potassium is diphasic. With increasing potassium concentrations, the net accumulation of calcium initially increases, becoming maximal at 1 mM potassium, then diminishes, falling below basal levels at concentrations above 3 mM potassium. Ouabain inhibits the stimulatory effect of low concentrations of potassium. The inhibitory effects of higher concentrations of potassium are ouabain insensitive and independent of the external calcium concentration. Sodium also inhibits calcium uptake but this inhibition can be modified by altering the external concentration of calcium. The effux of calcium from loaded erythrocytes is not significantly altered by changes in osmolality, medium ion composition, or ouabain. It is concluded that hypertonicity increases the net uptake of calcium by increasing the influx of calcium and that some part of the sodium potassium transport system is involved in this influx process.     

Determination of the permeability of human lymphocytes with a microscope diffusion chamber

A diffusion chamber similar to that proposed by J.J. McGrath (J. Microsc., in press) was constructed which allows microscopic observation of osmotically induced volume changes of individual cells in small (microliter) sample volumes. The cells are kept fixed in position in the upper compartment of the chamber by means of a highly permeable membrane and exposed to a step-like change in concentration generated in the lower compartment. An electrical conductivity probe in the upper compartment was used to monitor the temporal change of salt concentration as experienced by the cells. The rise from isotonic to hypertonic can be approximated by an exponential function. Its time constant of tau = 2.08 sec seems to be mainly determined by the change in flushing solution as tau = 1.48 sec was measured with no membrane installed. With human lymphocytes, no loss of cell volume was detected before 5 sec, i.e., when 95% of the final concentration was reached extracellularly. A step change can hence be assumed when modeling exosmosis for determining the lymphocyte membrane permeability. The equations for coupled transport of water and salt were solved numerically and fitted to the experimental data. The results were also compared to various other transport models described in the literature. Human lymphocytes are almost ideally semipermeable with a hydraulic reference permeability of Lp = 4.23 X 10(-4) cm/sec (3.13 X 10(-3) micron X atm-1 X sec-1) at T = 23 degrees C. The temperature and concentration dependence are described by an activation energy Ea = 14.3 kJ/mol and a concentration coefficient alpha 2 = 0.261 osmol/kg. An osmotically inactive volume fraction of 36.9% was determined from the final cell volumes reached asymptotically after shrinkage.     

Volume regulation by Amphiuma red blood cells. The membrane potential and its implications regarding the nature of the ion-flux pathways

After osmotic perturbation, the red blood cells of Amphiuma exhibited a volume-regulatory response that returned cell volume back to or toward control values. After osmotic swelling, cell-volume regulation (regulatory volume decrease; RVD) resulted from net cellular loss of K, Cl, and osmotically obliged H2O. In contrast, the volume-regulatory response to osmotic shrinkage (regulatory volume increase; RVI) was characterized by net cellular uptake of Na, Cl, and H2O. The net K and Na fluxes characteristic of RVD and RVI are increased by 1-2 orders of magnitude above those observed in studies of volume-static control cells. The cell membrane potential of volume-regulating and volume-static cells was measured by impalement with glass microelectrodes. The information gained from the electrical and ion-flux studies led to the conclusion that the ion fluxes responsible for cell-volume regulation proceed via electrically silent pathways. Furthermore, it was observed that Na fluxes during RVI were profoundly sensitive to medium [HCO3] and that during RVI the medium becomes more acid, whereas alkaline shifts in the suspension medium accompany RVD. The experimental observations are explained by a model featuring obligatorily coupled alkali metal-H and Cl-HCO3 exchangers. The anion- and cation-exchange pathways are separate and distinct yet functionally coupled via the net flux of H. As a result of the operation of such pathways, net alkali metal, Cl, and H2O fluxes proceed in the same direction, whereas H and HCO3 fluxes are cyclic. Data also are presented that suggest that the ion-flux pathways responsible for cell-volume regulation are not activated by changes in cell volume per se but by some event associated with osmotic perturbation, such as changes in intracellular pH.     

Absorption of short-chain fatty acids,sodium and water from the forestomach of camels

In camelids the ventral parts of compartments 1 and 2 (C1/C2) and the total surface of compartment 3 of the forestomach are lined with tubular glands, whereas in ruminants the surface of the forestomach is composed entirely of stratified, squamous epithelium. Thus, differences in absorption rates between these foregut fermenters can be expected. In five camels C1/C2 was temporarily isolated, washed and filled with buffer solutions. Absorption of short-chain fatty acids (SCFA) and net absorption of sodium and water were estimated relative to Cr-ethylenediaminetetraacetic acid as a fluid marker. SCFA were extensively absorbed in the forestomach; clearance rates of SCFA with different chain lengths were equal. After lowering the pH of solutions SCFA absorption rates increased, but much less than the increase of the non-ionized fraction. Absorption of propionate was lower when acetate had been added. Findings suggest that most of the SCFA in camels are transported in the ionized form, most likely via an anion exchange mechanism. Net water absorption is closely related to net sodium absorption. Apparently water absorption results from an iso-osmotic process. Differences between absorption mechanisms of SCFA from the forestomach of camelids and ruminants are discussed.     

Osmotic Properties of Mitochondria

The osmotic behavior of rat liver mitochondria has been studied in a sucrose medium. The mitochondria behave like a two compartment system. One compartment is permeable to sucrose and has a volume of 1.22 µl/(mg mitochondrial dry weight) in a 272 milliosmol sucrose medium; the second, inaccessible to sucrose, has a volume of 0.555 µl/mg dry weight) under the same conditions. Part of the water in the sucrose inaccessible space is apparently not free to participate in osmotic phenomena. This volume is 0.272 µl/(mg dry weight) under the same conditions. It is suggested that the osmotically inactive water corresponds to the water of hydration of the mitochondrial macromolecules. The volume of the remainder of the water in the sucrose inaccessible space depends inversely on the osmolality of the medium, as is to be expected. The volume of water in the sucrose accessible space is constant, independent of the osmolality of the medium, as is the volume of the mitochondrial framework plus the nonvolatile solutes.     

Determinants of epithelial cell volume

Epithelial cell volume is determined by the concentration of intracellular, osmotically active solutes. The high water permeability of the cell membrane of most epithelia prevents the establishment of large osmotic gradients between the cell and the bathing solutions. Steady-state cell volume is determined by the relative rates of solute entry and exit across the cell membranes. Inhibition of solute exit leads to cell swelling because solute entry continues; inhibition of solute entry leads to cell shrinkage because solute exit continues. Cell volume is then a measure of the rate and direction of net solute movements. Epithelial cells are also capable of regulation of the rate of solute entry and exit to maintain intracellular composition. Feedback control of NaCl entry into Necturus gallbladder epithelial cells is demonstrable after inhibition of the Na,K-ATPase or reduction in the NaCl concentration of the serosal bath. Necturus gallbladder cells respond to a change in the osmolality of the perfusion solution by rapidly regulating their volume to control values. This regulatory behavior depends on the transient activation of quiescent transport systems. These transport systems are responsible for the rapid readjustments of cell volume that follow osmotic perturbation. These powerful transporters may also play a role in steady-state volume regulation as well as in the control of cell pH.     

The effect of monovalent cations on calcium efflux in yeasts

The properties of the calcium efflux system in the yeast Saccharomyces cerevisiae were investigated. After growing the cells overnight in medium containing ⁴⁵Ca, the cells were transferred to medium containing glucose, Hepes buffer (pH 5.2) and monovalent cations. The presence of potassium or sodium in the medium induced efflux of calcium from the cells. The magnitude of the efflux was dependent on the concentration of these cations in the medium. The time course of calcium efflux was analyzed, and two types of exchangeable calcium pools, which turned over at different rates, were detected: ‘Fast turnover’ and ‘slow turnover’. Increase in the concentration of monovalent cations in the medium caused an increase in the fraction of cellular calcium which turned over at a fast rate, and activation of calcium efflux from the ‘slow turnover’ calcium pool. The specific changes in the parameters of calcium efflux induced by monovalent cations were different from those reported previously to be induced by divalent cations. Both processes, i.e. activation of calcium efflux by monovalent and by divalent cations, were found to be additive, indicating that they operate via different mechanisms. Experiments using the respiratory inhibitor Antimycin A, showed that stimulation of calcium efflux by monovalent cations is energy dependent. Lanthanum ions which are known to inhibit calcium influx into yeast cells, inhibitted the activation of calcium efflux by both divalent and monovalent cations. Determination of the cationic composition of the cells indicated that the stimulation of calcium efflux was accompanied by influx of potassium or sodium into the cells.     

Rapid electrogenic sulfate-chloride exchange mediated by chemically modified band 3 in human erythrocytes

One of the modes of action of the red blood cell anion transport protein is the electrically silent net exchange of 1 Cl- for 1 SO4= and 1 H+. Net SO4(=)-Cl- exchange is accelerated by low pH or by conversion of the side chain of glutamate 681 into an alcohol by treatment of intact cells with Woodward's reagent K (WRK) and BH4-. The studies described here were performed to characterize the electrical properties of net SO4(=)-Cl- exchange in cells modified with WRK/BH4-. The SO4= conductance measured in 100 mM SO4= medium is smaller in modified cells than in control cells. However, the efflux of [35S] SO4= into a 150-mM KCl medium is 80-fold larger in modified cells than in control cells and is inhibited 99% by 10 microM H2DIDS. No detectable H+ flux is associated with SO4(=)-Cl- exchange in modified cells. In the presence of gramicidin to increase the cation permeability, the stoichiometry of SO4(=)-Cl- exchange is not distinguishable from 1:1. In modified cells loaded with SO4=, the valinomycin-mediated efflux of 86Rb+ into an Na- gluconate medium is immediately stimulated by the addition of 5 mM extracellular Cl-. Therefore, SO4(=)-Cl- exchange in modified cells causes an outward movement of negative charge, as expected for an obligatory 1:1 SO4(=)-Cl- exchange. This is the first example of an obligatory, electrogenic exchange process in band 3 and demonstrates that the coupling between influx and efflux does not require that the overall exchange be electrically neutral. The effects of membrane potential on SO4(=)-SO4= exchange and SO4(=)-Cl- exchange in modified cells are consistent with a model in which nearly a full net positive charge moves inward through the transmembrane field during the inward Cl- translocation event, and a small net negative charge moves with SO4= during the SO4= translocation event. This result suggests that, in normal cells, the negative charge on Glu 681 traverses most of the transmembrane electric field, accompanied by Cl- and the equivalent of two protein-bound positive charges.     

Accumulation of weak bases in relation to intralysosomal pH in cultured human skin fibroblasts

The volume of the lysosomal compartment in cultured human skin fibroblasts was estimated from the distribution between the cells and the medium of tracer amounts of labelled methylamine and chloroquine, which accumulate in the lysosomes, 2,2-dimethyloxazolidine-2,4-dione, which accumulates in the soluble cytoplasmic compartment relative to the lysosomes, and sucrose, which is excluded by the cells. In a foetal fibroblast line, the fractional volume of the lysosomal compartment was $\text{0.044 ± 0.007}$ ($\text{n}\text{= 8}$). In fibroblasts from a patient with the I-cell disease, the fractional volume was 0.15.The fractional volume of the lysosomal compartment was used to calculate the intralysosomal pH from the accumulation of the weak bases in the cells. The mean value obtained was $\text{5.29 ± 0.04}$ ($\text{n}\text{= 8}$).In fibroblasts incubated with various concentrations of chloroquine, the fractional volume of the lysosomal compartment and the accumulation of chloroquine in the cells were used to calculate the concentration of chloroquine in the lysosomes. The intralysosomal concentration increased from 3 to 114 mM as the extracellular concentration increased from 1 to 100 μM. Concomitantly, the intralysosomal pH increased from 5.3 in the absence of chloroquine to 5.9 in the presence of 100 μM chloroquine. A similar increase in intralysosomal pH could be calculated in fibroblasts incubated with different concentrations of ammonia.     

Ouabain-insensitive salt and water movements in duck red cells. II. The role of chloride in the volume response

This paper describes the effect of external chloride on the typical swelling response induced in duck red cells by hypertonicity or norepinephrine. Lowering chloride inhibits swelling and produces concomitant changes in net movements of sodium and potassium in ouabain-treated cells, which resemble the effect of lowering external sodium or potassium. Inhibition is the same whether chloride is replaced with gluconate or with an osmotic equivalent of sucrose. Since changes in external chloride also cause predictable changes in cell chloride, pH, and water, these variables were systematically investigated by varying external pH along with chloride. Lowering pH to 6.60 does not abolish the response if external chloride levels are normal, although the cells are initially swollen due to the increased acidity. Cells deliberately preswollen in hypotonic solutions with appropriate ionic composition can also respond to norepinephrine by further swelling. These results rule out initial values of cell water, chloride, and pH as significant variables affecting the response. Initial values of the chloride equilibrium potential do have marked effect on the direction and rate of net water movement. If chloride is lowered by replacement with the permeant anion, acetate, E(Cl) is unchanged and a normal response to norepinephrine, which is inhibited by furosemide, is observed. Increasing internal sodium by the nystatin technique also inhibits the response. A theory is developed which depicts that the cotransport carrier proposed in the previous paper (W.F. Schmidt and T.J. McManus. 1977b. J. Gen. Physiol. 70:81-97) moves in response to the net electrochemical potential difference driving sodium and potassium across the membrane. Predictions of this theory fit the data for both cations and anions.     

Different patterns of cell volume regulation in hyposmotic media between attached and suspended HeLa cells.

Both attached and suspended HeLa cells swelled in a medium of a hypotonic osmolality of 235 mosmol/kg H2O. When the osmolality was further decreased to 166 mosmol/kg H2O, attached cells instantly swelled and then rapidly lost water and K+, followed by slow gains of them. Suspended cells instantly swelled and then K+ loss and regulatory volume decrease (RVD) occurred. Neither 0.1 mM ouabain nor 10 mM TEA changed the water loss of attached cells, whereas ouabain inhibited RVD of suspended cells. Quinine (1 mM) inhibited water losses from both cells and comparison of the losses implies stronger activation of K+ channel in attached cells than in suspended cells. Omission of medium Ca2+ or addition of 10 mM BaCl2 inhibited RVD in part. These results suggest that hyposmotic stress induces net water loss from attached cells, associated with K+ release through the Ca(2+)-dependent K+ channel. Suspended cells osmotically swell, followed by RVD with K+ and Na+ releases through the K+ channel and Na(+)-pump, respectively. The different patterns of volume changes may relate to the difference of activity or time of activation of the K+ channel between both cells.     

THE SIGNIFICANCE OF IONIC CONCENTRATIONS IN THE INTERNAL MEDIA OF ANIMALS

1. The electrical and ionic gradients across a cell membrane depend on its permeability properties, on the concentration and net valency of the organic constituents of the cytoplasm and on the critical energy barrier to the extrusion of sodium. Such considerations do not, however, explain the small extent to which the concentration of potassium varies in myoplasm which may, instead, be related to the effects of potassium on particular enzymes. 2. The fact that the apparent optimum level of potassium cannot usually be maintained in animals in which the extracellular level of sodium is below about 140 mM may explain why so many non-marine animals have internal media of about that concentration, for more concentrated body fluids would require more work for their regulation. 3. In axoplasm, the concentration of potassium is more nearly proportional to the concentration of sodium in the internal medium and this may partly explain the general correlation between the extracellular levels of sodium and potassium. 4. The relation between pH and temperature in poikilothermic vertebrates is such as to suggest that the prime function of acid-base regulation is to control the ionization of imidazole groups. 5. High tensions of carbon dioxide cannot be maintained in water-breathing animals because of the high solubility of this gas in water as compared with oxygen. Bicarbonate levels are correspondingly low to give a suitable pH. Higher tensions are possible in air-breathing animals, and also necessary if water and heat are to be conserved, but an uncertain upper limit is set by the need for oxygen. The associated higher levels of bicarbonate confer the advantage of better buffering. 6. Calcium and bicarbonate levels are not obviously limited by the solubility of calcium carbonate and a more general limitation on the composition of body fluids seems to arise from the low solubilities of calcium phosphates. 7. The pattern of ionic balance in vertebrate plasma, reflected in a nearly constant value to the molar ratio ([Ca] + 5 × 10^--⁴)/([K] +0.034 [Na]), may be explained in terms of the maintenance of a constant electrical gradient across certain areas of cell membrane, between the inner and outer double layers. 8. The patterns of cation balance in the haemolymphs of molluscs, crustacea and insects are also reviewed, with emphasis on the correlations existing between the concentrations of different cations. An attempt is made to relate the correlations in the mollusca to the concentrations of cations at the surfaces of cells.     

On the osmotically unresponsive water compartment in cells

Differences in colligative properties (freezing point, boiling point, vapor pressure and osmotic behavior) between water in living cells and pure bulk water were investigated by re-evaluating reports of the osmotic behavior of mammalian cells. In five different animal cells, osmotically unresponsive water (OUW) values ranged from 1.1 to 2.2 g per g dry mass. Detailed analysis of human red blood cell (RBC) data indicates a major role for hemoglobin OUW-values, aggregation and packing in cell volume regulation that can be explained for the first time in relevant molecular terms.     

Growth,cell volume,and fine structure of Porphyra umbilicalis in relation to osmotic tolerance

Porphyra umbilicalis, a marine red alga occurring in the intertidal zone of the cold North Sea, tolerates a wide range of osmotic conditions from 0.2 x to 6 x artificial seawater medium ASP₁₂. In cells osmotically adapted for two weeks, photosynthesis and respiration are progressively inhibited in media more concentrated than 2 x. In both hypo- and hyperosmotic stress ranges, the most striking fine structural change is the development of vacuoles. In comparison to 1 x medium, where vacuoles are virtually lacking, the vacuolar part of the protoplasm increases 6-fold in 0.2 x and 10-fold in 3.5 x medium, respectively. However, at extreme hyperosmotic stress (6 x medium) the vacuolar part is extremely small. The largest cell volumes are found in 0.2 x and 3.5 x media, the smallest one in 6 x medium. In the osmotically regulated range (0.2–3.5 x medium), the regulated parameter is the volume of the protoplasm without the vacuolar system. It is suggested that at hyperosmotic stress the vacuoles may serve as osmotically active compartment, probably by accumulation of inorganic ions. The intracellular content of Floridean starch granules decreases with increasing osmotic pressure, possibly indicating the significance of soluble organic constituents as osmotically active solutes.Member of the Arbeitsgemeinschaft für Elektronenmikroskople un der Ticrärztlichen Hochschule Hannover