Osmotic gradient-induced water permeation across the sarcolemma of rabbit ventricular myocytes

The mechanism of water permeation across the sarcolemma was characterized by examining the kinetics and temperature dependence of osmotic swelling and shrinkage of rabbit ventricular myocytes. The magnitude of swelling and the kinetics of swelling and shrinkage were temperature dependent, but the magnitude of shrinkage was very similar at 6 degrees, 22 degrees, and 37 degrees C. Membrane hydraulic conductivity, Lp, was approximately 1.2 x 10(-10) liter.N-1.s-1 at 22 degrees C, corresponding to an osmotic permeability coefficient, Pf, of 16 microns.s-1, and was independent of the direction of water flux, the magnitude of the imposed osmotic gradient (35-165 mosm/liter), and the initial cell volume. This value of Lp represents an upper limit because the membrane was assumed to be a smooth surface. Based on capacitive membrane area, Lp was 0.7 to 0.9 x 10(-10) liter.N-1.s-1. Nevertheless, estimates of Lp in ventricle are 15 to 25 times lower than those in human erythrocytes and are in the range of values reported for protein- free lipid bilayers and biological membranes without functioning water channels (aquaporin). Evaluation of the effect of unstirred layers showed that in the worst case they decrease Lp by < or = 2.3%. Analysis of the temperature dependence of Lp indicated that its apparent Arrhenius activation energy, Ea', was 11.7 +/- 0.9 kcal/mol between 6 degrees and 22 degrees C and 9.2 +/- 0.9 kcal/mol between 22 degrees and 37 degrees C. These values are significantly greater than that typically found for water flow through water-filled pores, approximately 4 kcal/mol, and are in the range reported for artificial and natural membranes without functioning water channels. Taken together, these data strongly argue that the vast majority of osmotic water flux in ventricular myocytes penetrates the lipid bilayer itself rather than passing through water-filled pores.     

The State of Water in Human and Dog Red Cell Membranes

The apparent activation energy for the water diffusion permeability coefficient, P_d, across the red cell membrane has been found to be 4.9 ± 0.3 kcal/mole in the dog and 6.0 ± 0.2 kcal/mole in the human being over the temperature range, 7° to 37°C. The apparent activation energy for the hydraulic conductivity, L_p, in dog red cells has been found to be 3.7 ± 0.4 kcal/mole and in human red cells, 3.3 ± 0.4 kcal/mole over the same temperature range. The product of L_p and the bulk viscosity of water, η, was independent of temperature for both dog and man which indicates that the geometry of the red cell membrane is not temperature-sensitive over our experimental temperature range in either species. In the case of the dog, the apparent activation energy for diffusion is the same as that for self-diffusion of water, 4.6–4.8 kcal/mole, which indicates that the process of water diffusion across the dog red cell membrane is the same as that in free solution. The slightly, but significantly, higher activation energy for water diffusion in human red cells is consonant with water-membrane interaction in the narrower equivalent pores characteristic of these cells. The observation that the apparent activation energy for hydraulic conductivity is less than that for water diffusion across the red cell membrane is characteristic of viscous flow and suggests that the flow of water across the membranes of these red cells under an osmotic pressure gradient is a viscous process.     

Cryopreservation and biophysical properties of articular cartilage chondrocytes

In order to successfully cryopreserve articular cartilage chondrocytes, it is important to characterize their osmotic response during the cryopreservation process, as the ice forms and the solutes concentrate. In this study, experimental work was undertaken to determine the osmotic parameters of articular cartilage chondrocytes. The osmotically inactive volume of articular cartilage chondrocytes was determined to be 44% of the isotonic volume. The membrane hydraulic conductivity parameters for water were determined by fitting a theoretical water transport model to the experimentally obtained volumetric shrinkage data; the membrane hydraulic conductivity parameter L(Pg) was found to be 0.0633 microm/min/atm, and the activation energy E, 8.23 kcal/mol. The simulated cooling process, using the osmotic parameters obtained in this study, suggests a cooling rate of 80 degrees C/min for the cryopreservation of the articular cartilage chondrocytes of hogs. The data obtained in this study could serve as a starting point for those interested in cryopreservation of chondrocytes from articular cartilage in other species in which there is clinical interest and there are no parameters for prediction of responses.     

Temperature Dependence of Vasopressin Action on the Toad Bladder

Toad bladders were challenged with vasopressin at one temperature, fixed on the mucosa with 1% glutaraldehyde, and then subjected to an osmotic gradient at another temperature. Thus, the temperature dependence of vasopressin action on membrane permeability was distinguished from the temperature dependence of osmotic water flux. As the temperature was raised from 20° to 38°C, there was a substantial increase in the velocity of vasopressin action, but osmotic flux was hardly affected. In this range of temperature the apparent energy of activation for net water movement across the bladder amounted to only 1.2 kcal/mole, a value well below the activation energy for bulk water viscosity. It is suggested that osmotic water flux takes place through narrow, nonpolar channels in the membrane. When the temperature was raised from 4° to 20°C, both vasopressin action as well as osmotic water flux were markedly enhanced. Activation energies for net water movement were now 8.5 kcal/mole (4°–9°C) and 4.1 kcal/mole (9°–20°C), indicating that the components of the aqueous channel undergo conformational changes as the temperature is lowered from 20°C. At 43°C bladder reactivity to vasopressin was lost, and irreversible changes in selective permeability were observed. The apparent energy of activation for net water movement across the denatured membrane was 6.6 kcal/mole. Approximately 1 µosmol of NaCl was exchanged for 1 µl of H₂O across the denatured membrane.     

Mercuric Chloride Effects on Root Water Transport in Aspen Seedlings

HgCl(2) (0.1 mM) reduced pressure-induced water flux and root hydraulic conductivity in the roots of 1-year-old aspen (Populus tremuloides Michx.) seedlings by about 50%. The inhibition was reversed with 50 mM mercaptoethanol. Mercurial treatment reduced the activation energy of water transport in the roots from 10.82 +/- 0.700 kcal mol(-1) to 6.67 +/- 0.193 kcal mol(-1) when measured over the 4 degrees C to 25 degrees C temperature range. An increase in rhodamine B concentration in the xylem sap of mercury-treated roots suggested a decrease in the symplastic transport of water. However, the apoplastic pathway in both control and mercury-treated roots constituted only a small fraction of the total root water transport. Electrical conductivity and osmotic potentials of the expressed xylem sap suggested that 0.1 mM HgCl(2) and temperature changes over the 4 degrees C to 25 degrees C range did not induce cell membrane leakage. The 0.1 mM HgCl(2) solution applied as a root drench severely reduced stomatal conductance in intact plants, and this reduction was partly reversed by 50 mM mercaptoethanol. In excised shoots, 0.1 mM HgCl(2) did not affect stomatal conductance, suggesting that the signal that triggered stomatal closure originated in the roots. We suggest that mercury-sensitive processes in aspen roots play a significant role in regulating plant water balance by their effects on root hydraulic conductivity.     

Computer Simulation of the Rapid Adaptation of Elongation Growth to Osmotic Stress in Segments of Cowpea Stem by Application of the Apoplast Canal Model

The transport of water during the adaptive rapid recovery ofelongation growth upon additional osmotic stress was examinedin the model stem segments of cowpea (Vigna unguiculata L.)by numerical solution of the extended canal equation, whichconsists of a set of time-dependent non-linear partial differentialequations. The calculated dynamic behaviour of the depletionof solute within the apoplast canal effectively explained thereported transient changes in water flow and, therefore, ingrowth during the adaptation to stress if the stress-inducedenhancement of net uptake of solute from the apoplast canalwas assumed. The extended canal model was also adequate forsimulation of the elastic shrinkage of hypocotyl segments uponexposure to osmotic stress which took place when the supplyof energy was limited. It appeared that the function of thecanal system in absorbing water is intrinsically stable againstperturbations of the water environment. (Received September 5, 1990; Accepted January 11, 1991)     

Ouabain-insensitive salt and water movements in duck red cells. I. Kinetics of cation transport under hypertonic conditions

Duck red cells in hypertonic media experience rapid osmotic shrinkage followed by gradual reswelling back toward their original volume. This uptake of salt and water is self limiting and demands a specific ionic composition of the external solution. Although ouabain (10(-4)M) alters the pattern of cation accumulation from predominantly potassium to sodium, it does not affect the rate of the reaction, or the total amount of salt or water taken up. To study the response without the complications of active Na-K transport, ouabain was added to most incubations. All water accumulated by the cells can be accounted for by net salt uptake. Specific external cation requirements for reswelling include: sufficient sodium (more than 23 mM), and elevated potassium (more than 7 mM). In the absence of external potassium cells lose potassium without gaining sodium and continue to shrink instead of reswelling. Adding rubidium to the potassium- free solution promotes an even greater loss of cell potassium, yet causes swelling due to a net uptake of sodium and rubidium followed by chloride. The diuretic furosemide (10(-3)M) inhibits net sodium uptake which depends on potassium (or rubidium), as well as inhibits net sodium uptake which depends on sodium. As a result, cell volume is stabilized in the presence of this drug by inhibition of shrinkage, at low, and of swelling at high external potassium. The response has a high apparent energy of activation (15-20 kcal/mol). We propose that net salt and water movements in hypertonic solutions containing ouabain are mediated by direct coupling or cis-interaction, between sodium and potassium so that the uphill movement of one is driven by the downhill movement of the other in the same direction.     

Permeability of human blood platelets to glycerol

The permeability of human platelets to glycerol was determined at 37 degrees C, 25 degrees C, and 0 degrees C from the rate of change of cell volume after abrupt addition of 0.5 mol/liter glycerol in phosphate-buffered saline. Intracellular water volume was measured employing both tritiated water and a photometric method. Intracellular glycerol was measured employing tritiated glycerol. The glycerol permeability coefficient derived from the tracer cell volume data was 4.0 +/- 0.7 X 10(-7) cm/s at 37 degrees C, and 1.1 +/- 0.4 X 10(-7) cm/s at 25 degrees C, and the photometric data gave a permeability coefficient of 5.4 +/- 0.4 X 10(-7) cm/s at 37 degrees C. The activation energy between 23 degrees C and 37 degrees C for glycerol permeation was 19.8 kcal/mol. The cells were virtually impermeable to glycerol at 0 degrees C. The minimum intracellular water volume attained after the addition of 0.5 mol/liter glycerol at 37 degrees C determined by the photometric method was 47.8% of normal water volume, whereas the minimum water volume calculated assuming that glycerol exerted its full osmotic effect (i.e., sigma = 1) was 45.6%. The reflexion coefficient was therefore assumed to be unity. Neither method of cell volume determination could be used with 1 or 2 mol/liter glycerol: adequate separation of the cells from the labeled medium could not be achieved in the tracer method; in the photometric method, it was apparent that transmittance (660 nm) was influenced by one or more variables in addition to cell volume.     

Dependence of water conductivity on pressure and temperature in plant stems

The effects of pressure and temperature on water conductivity were examined in okra stem segments. Segments were incubated in various concentrations of sorbitol solution at various temperatures. Water was found to pass mainly through the lateral side of the segments. The shrinkage rate was found to be proportional to the difference in the water potential between the inside of the cells and the ambient solution, while the rate was inversely proportional to the viscosity of water, which is a function of temperature. The nature of the media for water conductivity was found to be consistent with Darcy's law and with Hagen-Poiseuille's law with a rough approximation. An attempt was made to estimate the size of the water path. Okra stem segments were incubated with and without the plant hormone auxin before transference to sorbitol solution. Shrinkage rates of segments showed that auxin caused an increase in water conductivity and thus the size of the water path.     

Permeability of the Ehrlich Ascites Tumor Cell to Water

The osmotic permeability coefficient for water has been measured for the Ehrlich mouse ascites tumor cell. Measurements were made of the rate of cell shrinkage in hyperosmotic solutions of NaCI, a functionally impermeable solute. During the first 9 months of weekly serial transplantation the mean was 6.4 µ³/µ³/atm. ± 0.8 (S.E.). By the end of the 2nd year the permeability coefficient was much lower and averaged 1.6 ± 0.09. There were no significant differences in the volume of the tumor cells which could explain the discrepancy on the basis of a change in the volume to surface area ratio. Studies of the effect of temperature were done and Eyring's theory of absolute reaction rates was applied to the data. The apparent energy of activation was 9.6 kcal./mol and ΔS‡ was 39.1 entropy units. The thermodynamic data are twice as high as data reported by Wang for self-diffusion and viscous properties of water. Two alternate explanations have been advanced based on the pore hypothesis of membrane permeability. One explains the thermodynamic data from a change in the A'/Δx available for water movement; the other assumes A'/Δx constant and bases the results on the interaction of water dipoles with each other and the membrane.     

Effect of atrial natriuretic factor on the water permeability of endothelial cells.

Atrial natriuretic factor increases the water permeability of the whole endothelium. This study investigates how it would affect the transcellular osmotic water permeability of bovine artery endothelial cells. The cyclic-GMP production by the isolated cells was maximal for 10(-6)M atrial natriuretic factor within 30 minutes at 37 degrees C. The cyclic-GMP protein kinase cell concentration was 1.87 +/- 0.15 ng/mg protein. The control apparent water permeability of the cells measured by light scattering was 195 +/- 11 microns/sec (n = 5). Membrane folding revealed by light and scanning electron microscopy indicated that their true water permeability values would be close to 20-40 microns/sec, similar to the values for lipid membranes. The energy activation calculated from the temperature dependence of water permeability between 15 degrees C and 37 degrees C was 9.3 kcal/mol. This value suggests water movement through the lipid bilayer and not through water channels. Atrial natriuretic factor 10(-6)M did not significantly increase the water permeability of the cells. Hence, atrial natriuretic factor-stimulated increase in water permeability of the endothelium is more related to changes in paracellular water pathways than in transcellular water flux.     

Water permeability of human hematopoietic stem cells

It is currently impossible to isolate or identify human hematopoietic progenitor cells from the bone marrow, yet the biophysical properties of these cells are important for the development of techniques to isolate and preserve stem cells for transplantation. Osmotic permeability properties of human bone marrow stem cells were estimated from the kinetics of cell damage in a hypotonic solution measured using in vitro colony assays for multipotential (CFU-GEMM) and committed (BFU-E, CFU-GM) progenitor cells. Cells exposed to a hypotonic solution swell as a result of water influx, and the rate of change of volume is proportional to the hydraulic conductivity of the plasma membrane. Cell damage occurs when the cell volume exceeds the maximum tolerable volume, so the hydraulic conductivity can be estimated from the kinetics of cell damage. For all the progenitor cells studied, the mean value of the hydraulic conductivity was 0.283 micron3/micron2/min/atm at 20 degrees C, with an Arrhenius activation energy of 6.41 kcal/mole. No significant differences were observed in the osmotic properties of the various progenitor cells. These data were used to predict the osmotic responses of human bone marrow stem cells at subzero temperatures during freezing.     

Demonstration of Respiration-Dependent Water Uptake and Turgor Generation in Vigna Hypocotyl

Respiration-dependent water uptake and turgor change were observedby the xylem perfusion technique. Immediate and reversible shrinkagewith anoxia were repeatedly demonstrated under appropriate osmoticstress in elongating cow pea hypocotyl segments. Such shrinkageand re-elongation were always preceded by reversible inhibitionand re-activation of the electrogenic xylem pump, respectively.In mature zone segments where cell wall extensibility had beenshown to be practically null by means of the turgor jump method,anoxia and reaeration caused elastic shrinkage and expansion,respectively. The extent of respiration-dependent turgor wascalculated from the amplitudes of the elastic volume changeinduced by pressure jump and anoxia. In such segments, the directionof water flow across the xylem-symplast interface should bedetermined solely by the cell wall elasticity and the changein apoplasmic concentration of osmotica controlled by the xylempump activity, irrespective of any change in water conductivityor cell wall extensibility. (Received December 11, 1987; Accepted February 20, 1988)     

Measurements of the membrane water permeability (Lp) and its temperature dependence (activation energy) in human fresh and failed-to-fertilize oocytes and mouse oocyte.

The volumetric response of oocytes during rapid alterations of the extracellular osmotic environment were recorded using video microscopy. From these observations, the kinetics of water loss for human and mouse oocytes were determined over the temperature range 37 to 10 degrees C, including 37, 30, 20, and 10 degrees C. The changes in diameter of oocytes were measured over a 5-min period and a computer model was used to derive values for membrane water permeability (Lp) and inactive volume (Vb) and to compare the experimental data to the predicted values. The results for the mouse oocyte Lp were comparable to values determined by other methods. However the human data, for both failed-to-fertilize and fresh oocytes, have a wide range of values with large standard deviations. The Lp values at the various temperatures were used to calculate the Arrhenius activation energy (Ea). An Ea value of 9.48 kcal/mol was found for the fresh mouse oocyte, whereas the activation energy for human oocytes was extremely low, 3.73 kcal/mol for fresh oocytes and 1.93 kcal/mol for failed-to-fertilize oocytes.     

Effect of the gel to liquid crystalline phase transition on the osmotic behaviour of phosphatidylcholine liposomes.

Aspects of osmotic properties of liposomes, prepared from synthetic lecithin, above, at and below the gel to liquid crystalline phase transition temperature are described. The experiments show that liposomal membranes with their lipids in the gel state are still permeable to water. The rate of water permeation changes drastically on passing the transition temperature. The water permeation has activation energies of 9.5 +/- 1.28 and 26.4 +/- 0.9 kcal/mol above and below the transition temperature, respectively, indicating that the diffusion processes take place by different mechanisms. With respect to the barrier properties of the liposomes in the vicinity of the transition temperature, the following conclusions can be made. (1) Studying the osmotic shrinkage of liposomes at a fixed temperature near the transition point, the experiments indicate that dimyristoyl phosphatidylcholine membranes are highly permeable to glucose under these conditions, where liquid and solid domains co-exist. Under the same conditions the osmotic experiments did not indicate a strong increase in glucose permeability of dipalmitoyl phosphatidylcholine membranes as compared to the situation above and below the transition temperature. (2) On the other hand, perturbations of the phase equilibrium by temperature varations resulted in a marked increase of the glucose permeation through dipalmitoyl phosphatidylcholine bilayers. Once a new phase equilibrium of liquid and solid regions is established the permeation rate of glucose is much less.     

Roles of Aquaporin-3 Water Channels in Volume-Regulatory Water Flow in a Human Epithelial Cell Line

Membrane water transport is an essential event not only in the osmotic cell volume change but also in the subsequent cell volume regulation. Here we investigated the route of water transport involved in the regulatory volume decrease (RVD) that occurs after osmotic swelling in human epithelial Intestine 407 cells. The diffusion water permeability coefficient (Pd) measured by NMR under isotonic conditions was much smaller than the osmotic water permeability coefficient (Pf) measured under an osmotic gradient. Temperature dependence of Pf showed the Arrhenius activation energy (Ea) of a low value (1.6 kcal/mol). These results indicate an involvement of a facilitated diffusion mechanism in osmotic water transport. A mercurial water channel blocker (HgCl₂) diminished the Pf value. A non-mercurial sulfhydryl reagent (MMTS) was also effective. These blockers of water channels suppressed the RVD. RT-PCR and immunocytochemistry demonstrated predominant expression of AQP3 water channel in this cell line. Downregulation of AQP3 expression induced by treatment with antisense oligodeoxynucleotides was found to suppress the RVD response. Thus, it is concluded that AQP3 water channels serve as an essential pathway for volume-regulatory water transport in, human epithelial cells.     

Binding to tubulin of the colchicine analog 2-methoxy-5-(2', 3', 4'-trimethoxyphenyl)tropone. Thermodynamic and kinetic aspects

The thermodynamics and kinetics of the binding to tubulin of the colchicine analog 2-methoxy-5-(2', 3', 4'-trimethoxyphenyl) tropone (termed AC because it lacks the B-ring of colchicine) have been characterized by fluorescence techniques. The fluorescence of AC is weak in aqueous solution and is enhanced 250-fold upon binding to tubulin. The following thermodynamic values were obtained for the interaction at 37 degrees C: K = 3.5 X 10(5) M-1; delta G0 = -7.9 kcal/mol; delta H0 = -6.8 kcal/mol; delta S0 = 3.6 entropy units. The AC-tubulin complex is 1-2 kcal/mol less stable than the colchicine-tubulin complex. The change in fluorescence of AC was employed to measure the kinetics of the association process, and quenching of protein fluorescence was used to measure both association and dissociation. The association process, like that of colchicine, could be resolved into a major fast phase and a minor slow phase. The apparent second order rate constant for the fast phase was found to be 5.2 X 10(4) M-1 S-1 at 37 degrees C, and the activation energy was 13 kcal/mol. This activation energy is 7-11 kcal/mol less than that for the binding of colchicine to tubulin. The difference in activation energies can most easily be rationalized by a mechanism involving a tubulin-induced conformational change in the ligand ( Detrich , H. W., III, Williams, R. C., Jr., Macdonald, T. L., Wilson, L., and Puett , D. (1981) Biochemistry 20, 5999-6005). Such a change would be expected to have a small activation energy in AC because it possesses a freely rotating single bond in place of the B-ring of colchicine.     

Effect of para-chloromercuribenzenesulfonic acid and temperature on cell water osmotic permeability of proximal straight tubules

The apparent Arrhenius energy of activation (Ea) of the water osmotic permeability (Pcos) of the basolateral plasma cell membrane of isolated rabbit proximal straight tubules has been measured under control conditions and after addition of 2.5 mM of the sulfhydryl reagent, para-chloromercuribenzenesulfonic acid (pCMBS), of mersalyl and of dithiothreitol. Ea (kcal/mol) was 3.2 +/- 1.4 (controls) and 9.2 +/- 2.2 (pCMBS), while Pcos decreased with pCMBS to 0.26 +/- 0.17 of its control value. Mersalyl also decreased Pcos both in vitro and in vivo (using therapeutical doses). These actions of pCMBS and mersalyl were quickly reverted with 5 mM dithiothreitol and prevented by 0.1 M thiourea. Ea for free viscous flow is 4.2 and greater than 10 for non-pore-containing lipid membranes. By analogy with these membranes and with red blood cells, where similar effects of pCMBS on Pos are observed, it is concluded that cell membranes of the proximal tubule are pierced by aqueous pores which are reversibly shut by pCMBS. Part of the action of mercurial diuretics can be explained by their action on Pcos.     

Osmotic parameters of cells from a bioengineered human corneal equivalent and consequences for cryopreservation

A human corneal equivalent is under development with potential applications in pharmaceutical testing, biomedical research, and transplantation, but the ability to distribute this engineered tissue, depends on successful cryopreservation. Tissue recovery after exposure to conditions during cryopreservation depends on the response of its constituent cells to the changing environment as ice forms and solutes concentrate. This study defines the osmotic properties that define the rate of water movement across the plasma membrane of isolated human corneal endothelial, stroma, and epithelial cells. Cells were transferred from an isotonic (300 mosm/kg) to an anisotonic (150-1500 mosm/kg) solution at constant temperature, and cell volumes monitored using an electronic particle counter. Histograms describing cell volume changes over time after anisosmotic exposure allowed calculation of hydraulic conductivity (L(p)) and osmotically inactive volume fraction (V(b)). Experimental values for L(p) at 4, 13, 22, and 37 degrees C were used to determine the Arrhenius activation energy (E(a)). The L(p) for endothelial, stroma, and epithelial cells at 37 degrees C was 1.98+/-0.32,1.50+/-0.30, and 1.19+/-0.14 microm/min/atm, and the V(b) was 0.28, 0.27, and 0.41, respectively. The E(a) for endothelial, stroma, and epithelial cells was 14.8, 12.0, and 14.1 kcal/mol, respectively, suggesting the absence of aqueous pores. These osmotic parameters and temperature dependencies allow simulation of osmotic responses of human corneal cells to cryopreservation conditions, allowing amount of supercooling to be calculated to indicate the likelihood of intracellular freezing. Simulations show that differences in the osmotic parameters for the constituent cells in the bioengineered cornea result in significant implications for cryopreservation of the engineered corneal equivalent.     

Water Relations of Growing Maize Coleoptiles : Comparison between Mannitol and Polyethylene Glycol 6000 as External Osmotica for Adjusting Turgor Pressure

Water relations of growing segments of maize (Zea mays L.) coleoptiles were investigated with osmotic methods using either mannitol (MAN) or polyethylene glycol 6000 (PEG) as external osmotica. Segments were incubated in MAN or PEG solutions at 0 to - 15 bar water potential (Ψ_o) and the effects were compared on elongation growth, osmotic shrinkage, cell sap osmolality (OC), and osmotic pressure (π_i). The nonpenetrating osmoticum PEG affects π_i in agreement with Boyle-Mariotte's law, i.e. the segments behave in principle as ideal osmometers. There is no osmotic adjustment in the Ψ_o range permitting growth (0 to −5 bar) nor in the Ψ_o range inducing osmotic shrinkage (−5 to −10 bar). Promoting growth by auxin (IAA) has no effect on the osmotic behavior of the tissue toward PEG. In contrast to PEG, MAN produces an apparent increase in π_i accompanied by anomalous effects on segment elongation and shrinkage leading to a lower value for Ψ_o which establishes a growth rate of zero and to an apparent recovery from osmotic shrinkage after 2 hours of incubation. These effects can be quantitatively attributed to uptake of MAN into the tissue. MAN is taken up into the apoplastic space and the symplast as revealed by a large temperature-dependent component of MAN uptake. It is concluded that MAN, in contrast to PEG, is unsuitable as an extemal osmoticum for the quantitative determination of water relations of growing maize coleoptiles.