Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (P_f) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the ‘PfFit’), to yield P_f.     

Osmotic Water Permeability of Vacuolar and Plasma Membranes Isolated from Maize Roots

The method of stopped flow was used to follow the changes in light scattering by the vesicles of plasmalemma and tonoplast isolated from maize (Zea maysL.) roots and treated by osmotic pressure. In both membrane preparations, the rate of the process depended on the osmotic gradient and was described with the simple exponential function. The rate constants derived from these functions were the following: the coefficient of water permeability in the tonoplast (P= 165 ± 7 m/s) exceeded by an order of magnitude the corresponding index for plasmalemma (11 ± 2 m/s). The presence of HgCl₂(1.6 nmol/g membrane protein) decreased the tonoplast water permeability by 80%. Microviscosity studies of the hydrocarbon zone in the isolated membranes by using a fluorescent diphenylhexatriene probe demonstrated that the two membranes do not differ in the phase state of their lipid bilayer. The authors conclude that the observed difference in water permeability does not depend on the state of the lipid phase and probably reflects the dissimilar functional activity of plasmalemma and tonoplast aquaporins.     

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.     

土壤缓慢脱水对开花期小麦根系及叶片渗透调节及渗透调节物质的影响

以抗旱性不同的小麦品种为材料,在小麦的水分临界期开花期进行缓慢脱水处理,分别在脱水的不同阶段取样测定叶片及根系的渗透调节能力及渗透调节物质。结果表明：随着土壤含水量的降低,叶片与根系的饱和渗透势同步下降,表现出叶片与根系对水分胁迫反应的一到场生,但根系的渗透调节能力低于叶片。根系与叶片的渗透调节物质,一方面在物质总含量方面,表现出与渗透调节能力的一致性,另一方面各种物质的相对含量又有一定差异,叶片中可溶性糖与K＋含量及增加量都高于根系,而根系中的游离氨基酸与Ca^2 的相对增加量则大于叶片。     

Aquaporins: Another piece in the osmotic puzzle

Osmolarity not only plays a key role in cellular homeostasis but also challenges cell survival. The molecular understanding of osmosis has not yet been completely achieved, and the discovery of aquaporins as molecular entities involved in water transport has caused osmosis to again become a focus of research. The main questions that need to be answered are the mechanism underlying the osmotic permeability coefficients and the extent to which aquaporins change our understanding of osmosis. Here, attempts to answer these questions are discussed. Critical aspects of the state of the state of knowledge on osmosis, a topic that has been studied since 19th century, are reviewed and integrated with the available information provided by in vivo, in vitro and in silico approaches.     

Plasma Membrane Water Permeability of Cultured Cells and Epithelia Measured by Light Microscopy with Spatial Filtering

A method was developed to measure the osmotic water permeability (P_f) of plasma membranes in cell layers and applied to cells and epithelia expressing molecular water channels. It was found that the integrated intensity of monochromatic light in a phase contrast or dark field microscope was dependent on relative cell volume. For cells of different size and shape (Sf9, MDCK, CHO, A549, tracheal epithelia, BHK), increased cell volume was associated with decreased signal intensity; generally the signal decreased 10–20% for a twofold increase in cell volume. A theory relating signal intensity to relative cell volume was developed based on spatial filtering and changes in optical path length associated with cell volume changes. Theory predictions were confirmed by signal measurements of cell layers bathed in solutions of various osmolarities and refractive indices. The excellent signal-to-noise ratio of the transmitted light detection permitted measurement of cell volume changes of <1%. The method was applied to characterize transfected cells and tissues that natively express water channels. P_f in control Chinese hamster ovary cells was low (0.0012 cm/s at 23°C) and increased more than fourfold upon stable transfection with aquaporins 1, 2, 4, or 5. P_f in apical and basolateral membranes in polarized epithelial cells grown on porous supports was measured. P_f ^bl and P_f ^ap were 0.0011 and 0.0024 cm/s (MDCK cells), and 0.0039 and 0.0052 cm/s (human tracheal cells) at 23°C. In intact toad urinary bladder, basolateral P_f was 0.036 cm/s and apical membrane P_f after vasopressin stimulation was 0.025 cm/s at 23°C. The results establish light microscopy with spatial filtering as a technically simple and quantitative method to measure water permeability in cell layers and provide the first measurement of the apical and basolateral membrane permeabilities of several important epithelial cell types.     

On the Protection by Inorganic Phosphate of Calcium-Induced Membrane Permeability Transition

The role of inorganic phosphate as inhibitor of mitochondrial membrane permeability transition was studied. It is shown that in mitochondria containing a high phosphate concentration, i.e., 68 nmol/mg, Ca²⁺ did not activate the pore opening. Conversely, at lower levels of matrix phosphate, i.e., 38 nmol/mg, Ca²⁺ was able to induce subsequent pore opening. The inhibitory effect of phosphate was apparent in sucrose-based media, but it was not achieved in KCl media. The matrix free Ca²⁺ concentration and matrix pH were lowered by phosphate, but they were always higher in K⁺-media. In the absence of ADP, phosphate strengthened the inhibitory effect of cyclosporin A on carboxyatractyloside-induced Ca²⁺ efflux. Acetate was unable to replace phosphate in the induction of the aforementioned effects. It is concluded that phosphate preserves selective membrane permeability by diminishing the matrix free Ca²⁺ concentration.     

Kinetics of Water Transport in Eel Intestinal Vesicles

Brush border membrane vesicles, BBMV, from eel intestinal cells or kidney proximal tubule cells were prepared in a low osmolarity cellobiose buffer. The osmotic water permeability coefficient P _f for eel vesicles was not affected by pCMBS and was measured at 1.6 × 10⁻³ cm sec⁻¹ at 23°C, a value lower than 3.6 × 10⁻³ cm sec⁻¹ exhibited by the kidney vesicles and similar to published values for lipid bilayers. An activation energy E _a of 14.7 Kcal mol⁻¹ for water transport was obtained for eel intestine, contrasting with 4.8 Kcal mol⁻¹ determined for rabbit kidney proximal tubule vesicles using the same method of analysis. The high value of E _a , as well as the low P _f for the eel intestine is compatible with the absence of water channels in these membrane vesicles and is consistent with the view that water permeates by dissolution and diffusion in the membrane. Further, the initial transient observed in the osmotic response of kidney vesicles, which is presumed to reflect the inhibition of water channels by membrane stress, could not be observed in the eel intestinal vesicles. The P _f dependence on the tonicity of the osmotic shock, described for kidney vesicles and related to the dissipation of pressure and stress at low tonicity shocks, was not seen with eel vesicles. These results indicate that the membranes from two volume transporter epithelia have different mechanisms of water permeation. Presumably the functional water channels observed in kidney vesicles are not present in eel intestine vesicles. The elastic modulus of the membrane was estimated by analysis of swelling kinetics of eel vesicles following hypotonic shock. The value obtained, 0.79 × 10⁻³ N cm⁻¹, compares favorably with the corresponding value, 0.87 × 10⁻³ N cm⁻¹, estimated from measurements at osmotic equilibrium. Received: 28 January 1999/Revised: 15 June 1999     

Water Permeability of Brush Border Membrane Vesicles from Kidney Proximal Tubule

Brush border membrane vesicles (BBMV) maintain an initial hydrostatic pressure difference between the intra- and extravesicular medium, which causes membrane strain and surface area expansion (Soveral, Macey & Moura, 1997). This has not been taken into account in prior osmotic water permeability P _f evaluations. In this paper, we find further evidence for the pressure in the variation of stopped-flow light scattering traces with different vesicle preparations. Response to osmotic shock is used to estimate water permeability in BBMV prepared with buffers of different osmolarities (18 and 85 mosM). Data analysis includes the dissipation of both osmotic and hydrostatic pressure gradients. P _f values were of the order of 4 × 10⁻³ cm sec⁻¹ independent of the osmolarity of the preparation buffer. Arrhenius plots of P _f vs. 1/T were linear, showing a single activation energy of 4.6 kcal mol⁻¹. The initial osmotic response which is significantly retarded is correlated with the period of elevated hydrostatic pressure. We interpret this as an inhibition of P _f caused by membrane strain and suggest how this inhibition may play a role in cell volume regulation in the proximal tubule. Received: 8 August 1996/Revised: 4 March 1997     

Botulinum Toxins Inhibit the Antidiuretic Hormone (ADH)-Stimulated Increase in Rabbit Cortical Collecting-Tubule Water Permeability

The mammalian renal collecting duct increases its water permeability in response to antidiuretic hormone (ADH). ADH causes cytoplasmic endosomes containing the water channel, aquaporin 2 (AQP2), to fuse with the apical membrane so that the water permeability of the tubule increases many times above baseline. SNARE proteins are involved in the docking and fusion of vesicles with the cell membrane in neuron synapses. Whether these proteins are involved in the fusion of vesicles to the cell membrane in other tissues is not entirely clear. In the present study, we examined the role of SNARE proteins in the insertion of water channels in the collecting-duct response to ADH by using botulinum toxins A, B and C. Toxins isolated from clostridium botulinum are specific proteases that cleave different SNARE proteins and inactivate them. Tubules were perfused in vitro with botulinum toxin in the perfusate (50 nM for A and B and 15 nM for C). ADH (200 pM) was then added to the bath after baseline measurements of osmotic water permeability (P_f) and the change in P_f was followed for one hour. Botulinum toxins significantly inhibited the maximum P_f by approximately 50%. Botulinum toxins A and C also decreased the rate of rise of P_f. Thus, SNARE proteins are involved in the insertion of the water channels in the collecting duct.     

Effects of Decreasing Mitochondrial Volume on the Regulation of the Permeability Transition Pore

The permeability transition pore (PTP) is a Ca²⁺-sensitive mitochondrial inner membrane channel involved in several models of cell death. Because the matrix concentration of PTP regulatory factors depends on matrix volume, we have investigated the role of the mitochondrial volume in PTP regulation. By incubating rat liver mitochondria in media of different osmolarity, we found that the Ca²⁺ threshold required for PTP opening dramatically increased when mitochondrial volume decreased relative to the standard condition. This shrinkage-induced PTP inhibition was not related to the observed changes in protonmotive force, or pyridine nucleotide redox state and persisted when mitochondria were depleted of adenine nucleotides. On the other hand, mitochondrial volume did not affect PTP regulation when mitochondria were depleted of Mg²⁺. By studying the effects of Mg²⁺, cyclosporin A (CsA) and ubiquinone 0 (Ub₀) on PTP regulation, we found that mitochondrial shrinkage increased the efficacy of Mg²⁺ and Ub₀ at PTP inhibition, whereas it decreased that of CsA. The ability of mitochondrial volume to alter the activity of several PTP regulators represents a hitherto unrecognized characteristic of the pore that might lead to a new approach for its pharmacological modulation.     

Analysis of the source of heterogeneity in the osmotic response of plant membrane vesicles

Plasma membrane vesicles have been widely employed to understand the biophysics of water movements, especially when active aquaporins are present. In general, water permeability coefficients in these preparations outcome from the analysis of the osmotic response of the vesicles by means of light scattering. As from now, this is possible by following a theoretical approach that assumes that scattered light follows a single exponential function and that this behavior is the consequence of vesicle volume changes due to an osmotic challenge. However, some experimental data do not necessarily fit to single exponentials but to double ones. It is argued that the observed double exponential behavior has two possible causes: different vesicle population in terms of permeability or in terms of size distribution. As classical models cannot identify this source of heterogeneity, a mathematical modeling approach was developed based on phenomenological equations of water transport. In the three comparative models presented here, it was assumed that water moves according to an osmotic mechanism across the vesicles, and there is no solute movement across them. Interestingly, when tested in a well described plasma membrane vesicle preparation, the application of these models indicates that the source of heterogeneity in the osmotic response is vesicles having different permeability, clearly discarding the variable size effect. In conclusion, the mathematical approach presented here allows to identify the source of heterogeneity; this information being of particular interest, especially when studying gating mechanisms triggered in water channel activity.     

Effect of Water Deficit and Membrane Destruction on Water Diffusion in the Tissues of Maize Seedlings

We investigated diffusion of water in maize seedlings (Zea mays L. cv. Dnepropetrovskaya) following addition of polyethylene glycol (PEG) 6000 (osmotic potential –0.1 and –0.3 MPa) to the root medium by NMR method with pulsed gradient of magnetic field. Diffusion coefficients of different water phases in plant tissues (water of apoplast and vacuoles, water transported through the membranes) have been estimated from multicomponent decays of echo amplitude. Different signs of changes of water diffusion coefficients of fast and slow components of diffusional echo decay in roots and leaves under the influence of PEG-induced water deficits were shown. It has been supposed that under water deficit a sharing of water flows takes places through the different pathways (apoplastic, symplastic and transmembrane). In roots, 1-h water deficit increased the rate of fast diffusing water (water of apoplasm, vacuoles and, perhaps, water contained in intercellular endoplasm system), and decreased the rate of slowly diffusing water (water passing across the membranes). A long-term water deficit increased to a small extent the rate of water transmembrane transfer in root tissue. Leaf response to water stress was in the intensification of rate of transmembrane water transport that could be connected with the expression of water channels, and in the decrease of apoplastic water flow and flow along endoplasm. The possibility of estimation of plant tissue (membrane) integrity on the basis of diffusional data has been demonstrated.     

Water permeability properties of the ovarian oocytes from Bufo arenarum and Xenopus laevis: A comparative study

The water permeability properties of ovarian oocytes from Xenopus laevis and Bufo arenarum, a toad species found in the Buenos Aires region, were studied. We report that: (i) the water osmotic permeability (P _f, cm/sec × 10^–4) was significantly higher in Bufo (6°C=12.3±2.4; 18°C = 20.8±4.8) than in Xenopus oocytes (6°C=5.3±0.3; 18°C=6.2±1.6). The corresponding water diffusion permeability values (P _d, cm/sec × 10^–4) were: Xenopus = 2.3±0.3 (6°C) and 4.8±0.7 (18°C); Bufo=2.7±0.4 (6°C) and 6.0 ±0.5 (18°C). (ii) Amphotericin B increased the P _f and P _d values. The observed P _fP _d ratio was not significantly different from the expected results (n=3), after amphotericin B incorporation in both species. This means that the influence of unstirred layers and other potential artifactual compounds did not significantly affect our experimental results, (iii) Preincubation with gramicidin during 12 hr induced a clear increase in the oocyte volume. After that, a hypotonic shock only slightly increased the oocyte volume. Conversely, a hypertonic challenge induced a volume change significantly higher than the one observed in control conditions, (iv) Mercury ions did not affect the osmotic permeability in Xenopus oocytes but clearly inhibited, in a reversible way, the osmotic permeability in oocytes from B. arenarum. (v) Mercury ions did not reduce P _d values in either species, (vi) The P _fP _d values calculated from the differences observed in these parameters between both species were 11.9±5.1 at 18°C and 15.5±2.4 at 6°C. These numbers are similar to those previously reported in the case of membranes having water channels. From these results, we propose that water channels are present in the ovarian oocyte from B. arenarum but not in the ovarian oocyte from X. laevis.This work was supported by Fundación Antorchas, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, Argentina) and Universidad de Buenos Aires (UBA). It was developed in the frame of an INSERM (France)-CONICET cooperative program.     

The role of aquaporin water channels in fluid secretion by the exocrine pancreas

The mammalian exocrine pancreas secretes a near-isosmotic fluid over a wide osmolarity range. The role of aquaporin (AQP) water channels in this process is now becoming clearer. AQP8 water channels, which were initially cloned from rat pancreas, are expressed at the apical membrane of pancreatic acinar cells and contribute to their osmotic permeability. However, the acinar cells secrete relatively little fluid and there is no obvious defect in pancreatic function in AQP8 knockout mice. Most of the fluid secreted by the pancreas is generated by ductal epithelial cells, which comprise only a small fraction of the gland mass. In the human pancreas, secretion occurs mainly in the intercalated ducts, where the epithelial cells express abundant AQP1 and AQP5 at the apical membrane and AQP1 alone at the basolateral membrane. In the rat and mouse, fluid secretion occurs mainly in the interlobular ducts where AQP1 and AQP5 are again co-localized at the apical membrane but appear to be expressed at relatively low levels. Nonetheless, the transepithelial osmotic permeability of rat interlobular ducts is sufficient to support near-isosmotic fluid secretion at observed rates. Furthermore, apical, but not basolateral, application of Hg²⁺ significantly reduces the transepithelial osmotic permeability, suggesting that apical AQP1 and AQP5 may contribute significantly to fluid secretion. The apparently normal fluid output of the pancreas in AQP1 knockout mice may reflect the presence of AQP5 at the apical membrane.     

干旱胁迫对霸王水分生理特征及细胞膜透性的影响

以2年生霸王为试验材料,用盆栽人工控制土壤水分,研究不同梯度土壤水分条件对霸王水分生理特征、丙二醛含量和细胞膜相对透性的影响.结果表明,与充足供水对照(土壤重量含水量为23%)相比,在严重干旱胁迫(土壤含水量为3%)20 d和45 d时,霸王叶水势和叶片相对含水量分别显著降低70.87%和45.54%、26.81%和12.66%;相应的叶片MDA含量和细胞膜相对透性分别显著增加33.81%和21.78%、17.51%和14.98%;严重干旱胁迫30 d使得霸王叶片保水力显著下降;同时随着土壤干旱胁迫程度的增强,霸王日蒸腾耗水量持续下降,且严重干旱胁迫处理的蒸腾耗水量日变化为水平直线型,其余处理均为单峰型.研究发现,严重干旱胁迫下随着胁迫时间的延长,霸王叶水势和叶片相对含水量有增加趋势,而叶片MDA含量和细胞膜相对透性则有减小趋势,它能够通过自身的调节机制,逐渐改善叶片水分状况,降低细胞膜伤害程度,从而适应严重干旱环境.     

Osmotic responses of maize roots

Water and solute relations of excised seminal roots of young maize (Zea mays L) plants, have been measured using the root pressure probe. Upon addition of osmotic solutes to the root medium, biphasic root pressure relaxations were obtained as theoretically expected. The relaxations yielded the hydraulic conductivity Lp _r) the permeability coefficient (P _sr), and the reflection coefficient (σ _sr) of the root. Values of Lp _r in these experiments were by nearly an order of magnitude smaller than Lp _r values obtained from experiments where hydrostatic pressure gradients were used to induce water flows. The value of P _sr was determined for nine different osmotica (electrolytes and nonelectrolytes) which resulted in rather variable values (0.1·10^-8–1.7·10^-8m·s^-1). The reflection coefficient σ _sr of the same solutes ranged between 0.3 and 0.6, i.e. σ _sr was low even for solutes for which cell membranes exhibit a σ _s≈1. Deviations from the theoretically expected biphasic responses occured which may have reflected changes of either P _sr or of active pumping induced by the osmotic change. The absolute values of Lp _r, P _sr, and σ _sr have been critically examined for an underestimation by unstirred layer effecs. The data indicate a considerable apoplasmic component for radial movement of water in the presence of hydrostatic gradients and also some solute flow byppassing root protoplasts. In the presence of osmotic gradients, however, there was a substantial cell-to-cell transport of water. Cutting experiments demonstrated that the hydraulic resistance for the longitudinal movement of water was much smaller than for radial transport except for the apical ends of the segments (length=5 to 20 mm). The differences in Lp _r as well as the low σ _sr values suggest that the simple osmometer model of the root with a single osmotic barrier exhibiting nearly semipermeable properties should be extended for a composite membrane model with hydraulic and osmotic barriers arranged in series and in parallel.     

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.     

On the Role of the Respiratory Complex I on Membrane Permeability Transition

In this work we studied permeability transition by incubating mitochondria in the presence of 50 M Ca²⁺ and malate/glutamate as substrates. This condition, besides inducing the release of pyridine nucleotides, promotes the generation of reactive oxygen-derived species by the complex I of the respiratory chain. The latter leads to the opening of the mitochondrial permeability transition pore. Ca²⁺ release, mitochondrial swelling and collapse of the transmembrane electric potential, were analyzed to assess this process. We propose that the mechanism for pore opening, in addition to the oxidative stress, involves the uncoupling effect of fatty acids providing activation of phospholipase A2, lipid peroxidation, and the oxidation of membrane thiols. This proposal emerges from the data indicating the protective effect of bovine serum albumin and N-ethylmaleimide. The key role of reactive oxygen species was implied based on the fact that the scavenger -phenyl-tert-butyl nitrone inhibited pore opening.     

Influence of Membrane Physical State on the Lysosomal Proton Permeability

Influence of membrane physical state on the proton permeability of isolated lysosomes was assessed by measuring the membrane potential with 3,3′-dipropylthiadicarbocyanine iodide and monitoring their proton leakage with p-nitrophenol. Changes in the membrane order were examined by the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene. Both the membrane potential and proton leakage increased with fluidizing the lysosomal membranes by benzyl alcohol and decreased with rigidifying the membranes by cholesteryl hemisuccinate. The proton permeability increased to the maximum of 42% by the benzyl alcohol treatment and decreased to the minimum of 38.1% by the cholesteryl hemisuccinate treatment. Treating the lysosomes with protonophore CCCP increased the proton permeability by 58%. The effects of the membrane fluidization and rigidification can be reversed by rigidifying the fluidized membranes and fluidizing the rigidified membranes, respectively. The results indicate that the proton permeability of lysosomes increased and decreased with increasing and decreasing their membrane fluidity, respectively. Moreover, the lysosomal proton permeability did not alter further if the changes, either an increase or a decrease, in the fluidity exceeded some amount. The results suggest that the proton permeability of lysosomes can be modulated finitely by the alterations in their membrane physical state. Received: 27 September 1999 / Revised: 27 December 1999