Influence of the growth at high osmolality on the lipid composition, water permeability and osmotic response of Lactobacillus bulgaricus

Changes in water permeability and membrane packing were measured in cells of Lactobacillus bulgaricus and in vesicles prepared with lipids extracted from them. The osmotic response of whole cells and vesicles is compared with the one of bacteria grown in a high osmolal medium. Both bacteria and vesicles, behave as osmometers. This means that the volume decrease is promoted by the outflow of water, driven by the NaCl concentration difference, arguing that neither Na+ nor Cl- permeates the cell or the lipid membrane in these conditions. Therefore, the volume changes can be correlated with the rate of water permeation across the cell or the vesicle membranes. The permeation of water was analyzed as a function of the lipid species by measuring the volume changes and the saturation ratio of the lipids. To put into relevance the membrane processes, the permeation properties of lipid vesicles prepared with lipids extracted from bacteria grown in normal and high osmolality conditions were also analyzed. The permeation response was correlated with the physical properties of the membrane of whole cells and vesicles, by means of fluorescence anisotropy of diphenyl hexatriene (DPH). The modifications in membrane properties are related with the changes in the membrane composition triggered by the growth in a high osmolal medium. The changes appear related to an increase in the sugar content of the whole pool of lipids and in the saturated fatty acid residues.     

Changes in osmotic water permeability of the eel gills during seawater and freshwater adaptation

Summary Changes in osmotic water permeability of the isolated gills of the Japanese eel,Anguilla japonica were studied during transfer to seawater or to fresh water. The water permeability increased gradually during the course of seawater transfer and attained a maximal level after 2 weeks. The water permeability of the freshwater eel gills was reduced when calcium ions were added to the incubation medium at a concentration of 1 mM, where-as no effect of the ion was observed on the gills of the seawater-adapted eel even at a higher concentration (10 mM). In contrast to seawater transfer, the water permeability decreased to a low level almost immediately (3 h) after transfer from seawater to fresh water. The acute reduction of the water permeability was also seen in the gills of the hypophysectomized eel after transfer to fresh water.The gradual increase in the gill water permeability during seawater transfer is correlated with an increase in the number of chloride cells. In scanning electron microscopy, chloride cells of seawater-adapted eel gills exhibit a pit-like structure, which was larger than in the freshwater eel. On transfer from seawater to fresh water, the pit diameter became smaller within 6 h. Hypophysectomy did not affect the change in gill surface structures during transfer to fresh water. The junctions between the chloride cells of seawater eel gills are reported to be of the leaky type. The parallel change in osmotic water permeability and in pit size of the chloride cells during seawater or freshwater transfer or after hypophysectomy suggests that these cells could provide a major route of water as well as ion movement.This paper is a portion of a thesis presented to Hokkaido University by t. Ogasawara in partial fulfilment of the requirements for Doctor of Fisheries     

The possible influence of osmotic poration on cell membrane water permeability

It has been hypothesized that pores in the plasma membrane form under conditions of rapid water efflux, allowing extracellular ice to grow into the cytoplasm under conditions of rapid freezing. When cells with intracellular ice are thawed slowly, the transmembrane ice crystal expands through recrystallization causing the cell to lyse. One of the implications of this hypothesis is that osmotic pores will provide an alternative route for water movement under conditions of osmotically induced flow. We show that the plasma membrane water permeability of a fibroblast cell changes as a function of the osmotic pressure gradient that is used to drive water movement. It is further shown that cell volume is more important than the magnitude of water flux in causing this departure from a uniform water permeability. We suggest that these data provide evidence of a transient route for water movement across cell membranes.     

Urinary bladder membrane permeability differentially induced by membrane lipid composition

The permeability barrier of the urothelium (covering the mammalian urinary tract) has stimulated interest in the role of the luminal membrane in the barrier function. To know how membrane lipids may affect the permeability barrier we prepare endocytic vesicles of different lipid composition entrapping a fluorescent dye (HPTS) and its quencher (DPX) using a dietary strategy (rats fed with commercial, oleic acid- or linoleic acid-enriched diets) followed by endocytosis induction. Vesicular leakage was measured by a fluorescence requenching technique. The results showed (1) endocytosed vesicles can release their content; (2) a linoleic acid-rich diet did not change either the mechanism of leakage or the amount of released material relative to the control; and (3) a oleic acid-rich diet greatly affected the mechanism of release. Thus, the dietary fatty acids can modify the urothelial cell physiology altering the pathway of endocytosed urinary fluid.     

Dynamic changes in the osmotic water permeability of protoplast plasma membrane

The osmotic water permeability coefficient (P(f)) of plasma membrane of maize (Zea mays) Black Mexican Sweet protoplasts changed dynamically during a hypoosmotic challenge, as revealed using a model-based computational approach. The best-fitting model had three free parameters: initial P(f), P(f) rate-of-change (slope(P(f))), and a delay, which were hypothesized to reflect changes in the number and/or activity of aquaporins in the plasma membrane. Remarkably, the swelling response was delayed 2 to 11 s after start of the noninstantaneous (but accounted for) bath flush. The P(f) during the delay was < or =1 microm s(-1). During the swelling period following the delay, P(f) changed dynamically: within the first 15 s P(f) either (1) increased gradually to approximately 8 microm s(-1) (in the majority population of low-initial-P(f) cells) or (2) increased abruptly to 10 to 20 microm s(-1) and then decreased gradually to 3 to 6 microm s(-1) (in the minority population of high-initial-P(f) cells). We affirmed the validity of our computational approach by the ability to reproduce previously reported initial P(f) values (including the absence of delay) in control experiments on Xenopus oocytes expressing the maize aquaporin ZmPIP2;5. Although mercury did not affect the P(f) in swelling Black Mexican Sweet cells, phloretin, another aquaporin inhibitor, inhibited swelling in a predicted manner, prolonging the delay and slowing P(f) increase, thereby confirming the hypothesis that P(f) dynamics, delay included, reflected the varying activity of aquaporins.     

Structural determinants of water permeability through the lipid membrane

Despite intense study over many years, the mechanisms by which water and small nonelectrolytes cross lipid bilayers remain unclear. While prior studies of permeability through membranes have focused on solute characteristics, such as size, polarity, and partition coefficient in hydrophobic solvent, we focus here on water permeability in seven single component bilayers composed of different lipids, five with phosphatidylcholine headgroups and different chain lengths and unsaturation, one with a phosphatidylserine headgroup, and one with a phosphatidylethanolamine headgroup. We find that water permeability correlates most strongly with the area/lipid and is poorly correlated with bilayer thickness and other previously determined structural and mechanical properties of these single component bilayers. These results suggest a new model for permeability that is developed in the accompanying theoretical paper in which the area occupied by the lipid is the major determinant and the hydrocarbon thickness is a secondary determinant. Cholesterol was also incorporated into DOPC bilayers and X-ray diffuse scattering was used to determine quantitative structure with the result that the area occupied by DOPC in the membrane decreases while bilayer thickness increases in a correlated way because lipid volume does not change. The water permeability decreases with added cholesterol and it correlates in a different way from pure lipids with area per lipid, bilayer thickness, and also with area compressibility.     

Determination of cells' water membrane permeability: unexpected high osmotic permeability of Saccharomyces cerevisiae

Water permeability (Lp), calculated from the volume variations of cells subjected to an osmotic shock, is classically used to characterize cell membrane properties. In this work, we have shown the importance of the kind of mixing reactor used to measure the Lp parameter. A mathematical model including the mixing time constant has been proposed allowing an accurate Lp estimation even though the mixing time constant is higher than the cell time constant obtained in response to a perfect shock. The estimated Lp values of human leukemia K562 cells were found to be the same whatever the mixing time constant. The Lp value of Saccharomyces cerevisiae could not be exactly estimated. However, S. cerevisiae has unexpectedly high water permeability, implying that this yeast may contain water channels in the membrane. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 62-70, 1997.     

Canine RBC osmotic tolerance and membrane permeability

The objective of this study was to determine the cryobiological characteristics of canine red blood cells (RBC). These included the hydraulic conductivity (L(p)), the permeability coefficients (P(s)) of common cryoprotectant agents (CPAs), the associated reflection coefficient (sigma), the activation energies (E(a)) of L(p) and P(s) and the osmotic tolerance limits. By using a stopped-flow apparatus, the changes of fluorescence intensity emitted by intracellularly entrapped 5-carboxyfluorescein diacetate (CFDA) were recorded when cells were experiencing osmotic volume changes. After the determination of the relationship between fluorescence intensity and cell volume, cell volume changes were calculated. These volume changes were used in three-parameter fitting calculations to determine the values of L(p), P(s), and sigma for common CPAs. These volume measurements and data analyses were repeated at three different temperatures (22, 14, 7 degrees C). Using the Arrhenius equation, the activation energies of L(p) and P(s) in the presence of CPAs were determined. The osmotic tolerance limits for canine RBC were determined by measuring the percentage of free hemoglobin in NaCl solutions with various osmolalities compared to that released by RBC incubated in double distilled water. The upper and lower osmotic tolerance limits were found to be 150mOsm (1.67V(iso)) and 1200mOsm (0.45V(iso)), respectively. These parameters were then used to calculate the amount of non-permeating solute needed to keep cell volume excursions within the osmotic tolerance limits during CPA addition and removal.     

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.     

Very low osmotic water permeability and membrane fluidity in isolated toad bladder granules

Summary Osmotic water permeability of the apical membrane of toad urinary epithelium is increased greatly by vasopressin (VP) and is associated with exocytic addition of granules and aggrephores at the apical surface. To determine the physiological role of granule exocytosis, we measured the osmotic water permeability and membrane fluidity of isolated granules, surface membranes and microsomes prepared from toad bladder in the presence and absence of VP.P _f was measured by stopped-flow light scattering and membrane fluidity was examined by diphenylhexatriene (DPH) fluorescence anisotropy. In response to a 75mm inward sucrose gradient, granule size decreased with a single exponential time constant of 2.3±0.1 sec (sem, seven preparations, 23°C), corresponding to aP _f of 5×10^–4 cm/sec; the activation energy (E _a ) forP _f was 17.6±0.8 kcal/mole. Under the same conditions, the volume of surface membrane vesicles decreased biexponentially with time constants of 0.13 and 1.9 sec; the fast component comprised 70% of the signal. Granule, surface membrane and microsome time constants were unaffected by VP. However, in surface membranes, there was a small decrease (6±2%) in the fraction of surface membranes with fast time constant. DPH anisotropies were 0.253 (granules), 0.224 (surface membrane fluidity is remarkably lower than that of surface and microsomal membranes, and (4) rapid water transport occurs in surface membrane vesicles. The unique physical properties of the granule suggests that apical exocytic addition of granule membrane may be responsible for the low water permeability of the unstimulated apical membrane.     

Cell volume and plasma membrane osmotic water permeability in epithelial cell layers measured by interferometry.

The development of strategies to measure plasma membrane osmotic water permeability (Pf) in epithelial cells has been motivated by the identification of a family of molecular water channels. A general approach utilizing interferometry to measure cell shape and volume was developed and applied to measure Pf in cell layers. The method is based on the cell volume dependence of optical path length (OPL) for a light beam passing through the cell. The small changes in OPL were measured by interferometry. A mathematical model was developed to relate the interference signal to cell volume changes for cells of arbitrary shape and size. To validate the model, a Mach-Zehnder interference microscope was used to image OPL in an Madin Darby Canine Kidney (MDCK) cell layer and to reconstruct the three-dimensional cell shape (OPL resolution < lambda/25). As predicted by the model, a doubling of cell volume resulted in a change in OPL that was proportional to the difference in refractive indices between water and the extracellular medium. The time course of relative cell volume in response to an osmotic gradient was computed from serial interference images. To measure cell volume without microscopy and image analysis, a Mach-Zehnder interferometer was constructed in which one of two interfering laser beams passed through a flow chamber containing the cell layer. The interference signal in response to an osmotic gradient was analyzed to quantify the time course of relative cell volume. The calculated MDCK cell plasma membrane Pf of 6.1 x 10(-4) cm/s at 24 degrees C agreed with that obtained by interference microscopy and by a total internal reflection fluorescence method. Interferometry was also applied to measure the apical plasma membrane water permeability of intact toad urinary bladder; Pf increased fivefold after forskolin stimulation to 0.04 cm/s at 23 degrees C. These results establish and validate the application of interferometry to quantify cell volume and osmotic water permeability in cell layers.     

Influence of growth temperature on cryotolerance and lipid composition of Lactobacillus acidophilus

In order to correlate the lipid composition of the membrane of Lactobacillus acidophilus CRL 640 with the freeze-thaw behaviour of the cultures grown at different temperatures, fatty acid methyl esters (FAMEs) from extracts grown at 25, 30, 37 and 40 degrees C were obtained and compared. Cultures grown at 25 degrees C (M25) exhibited more resistance to the freeze-thaw process probably because of an increase in C18:2 and C16:0 fatty acids. This culture also exhibited a lesser amount of phospholipids as shown by the sugar: phosphorus ratio. In all cases, the presence of the uncommon 10-hydroxyoctadecanoic acid was determined. From the extracts of the M25 and M37 cultures, diacylphosphatidylglycerol, cardiolipin, diglycosyldiglycerides, triglycosyldiglycerides and neutral lipids were isolated and identified. The structural elucidation was carried out by FAMEs and sugar analysis and by mass spectrometry using fast atom bombardment ionization. The changes in lipid composition due to different growth temperatures could be indicative of the resistance of the bacteria to freeze-thaw processes.     

Effect of streptolysin S on liposomes. Influence of membrane lipid composition on toxin action

The effect of the bacterial cytolytic toxin, streptolysin S, on liposomes composed of various phospholipids was investigated. Large unilamellar vesicles containing [14C]sucrose were prepared by reverse-phase evaporation, and membrane damage produced by the toxin was measured by following the release of labeled marker. The net charge of the liposomes had little or no effect on their susceptibility to steptolysin S and the toxin was about equally effective on liposomes composed of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylglycerol. Experiments with liposomes composed of synthetic phospholipids showed that the ability of the toxin to produce membrane damage depended on the degree of unsaturation of the fatty acyl chains. The order of sensitivity was C18 : 2 phosphatidylcholine greater than C18: I phosphatidylcholine greater than C18 : 0 phosphatidylcholine = C16 : 0 phosphatidylcholine. Liposomes containing the latter two phospholipids were virtually unaffected by streptolysin S, and experiments with C18 : 0 phosphatidylcholine suggested that toxin activity does not bind to liposomes composed of phospholipids with saturated fatty acyl chains. The inclusion of 40 mol% cholesterol in C16 : 0 phosphatidylcholine and C18 : 0 phosphatidylcholine liposomes made these vesicles sensitive to streptolysin S. Egg phosphatidylcholine liposomes, which were unaffected at 0 degrees C and 4 degrees C became susceptible to the toxin at these temperatures when cholesterol was included. Liposomes composed of C14 : 0 phosphatidylcholine were unaffected by streptolysin S at temperatures below the chain-melting transition temperature (23 degrees C) of this phospholipid, but became increasingly susceptible above this temperature. The results suggest that the fluidity of the phospholipid hydrocarbon chains in the membrane is important in streptolysin S action.     

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

The apparent Arrhenius energy of activation ($\text{E}{}_{\mathrm{<mtext>a</mtext>}}$) of the water osmotic permeability ($\text{P}{}_{\mathrm{<mtext>os</mtext>}}{}^{\mathrm{<mtext>c</mtext>}}$) 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. $\text{E}{}_{\mathrm{<mtext>a</mtext>}}$ (kcal/mol) was $\text{3.2 ± 1.4}$ (controls) and $\text{9.2 ± 2.2}$ (pCMBS), while $\text{P}{}_{\mathrm{<mtext>os</mtext>}}{}^{\mathrm{<mtext>c</mtext>}}$ decreased with pCMBS to $\text{0.26 ± 0.17}$ of its control value. Mersalyl also decreased $\text{P}{}_{\mathrm{<mtext>os</mtext>}}{}^{\mathrm{<mtext>c</mtext>}}$ 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. $\text{E}{}_{\mathrm{<mtext>a</mtext>}}$ 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 $\text{P}{}_{\mathrm{<mtext>os</mtext>}}$ 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 $\text{P}{}_{\mathrm{<mtext>os</mtext>}}{}^{\mathrm{<mtext>c</mtext>}}$.     

Role of lipids in the Neurospora crassa membrane. I. Influence of fatty acid composition on membrane lipid phase transitions.

The relationship between lipid composition and phase transition was investigated by differential scanning calorimetry for intact and membrane phospholipid extracts of wild-type (w/t) and the cel-(Tw 40) mutant of Neurospora crassa. The cel-(Tw 40) mutant (grown on minimal, sucrose medium supplemented with Tween 40 at approximately 34 degrees C) had approximately twice the saturated fatty acid content of w/t organisms grown at approximately 22 degrees C. The gel-liquid crystal phase transitions of ergosterol-free extracts derived from w/t and cel-(Tw 40) occur at -31 and -11 degrees C, respectively. The heats of transition (delta H) of these extracts were 1 and 13 cal/g, respectively. The addition of ergosterol (the predominant sterol in Neurospora) to the phospholipid extracts decreased the observed heats of transition, but did not alter the transition temperature. Intact Neurospora, whether w/t or cal-(Tw 40) did not manifest similar gel-liquid crystal phase transitions in the differential scanning calorimeter. However, an endothermic peak at approximately 30 degrees C was observed in intact cells and extracted phospholipids of both w/t and cel-(Tw 40) organisms. This peak was insensitive to the addition of ergosterol, had a low heat content (delta H congruent to 1 cal/g), and was reversible.     

Water permeability and lipid composition of toad urinary bladder: The influence of temperature

Summary The water diffusional permeability, its activation energy and the lipid composition were studied in urinary bladders from toads adapted to different temperatures. It was observed that the unidirectional water flux greatly depends on the temperature at which the experiments are performed. This dependence is greater in the animals adapted to higher temperatures. Toads adapted to cold show strong reduction in the activation energy for water diffusion permeability (from 11.4±1.9 kcal·mol^–1 to 4.4±1.1 kcal·mol^–1) and an increase of 30% in the amount of total lipids from bladder epithelial cells. There were no significant changes in the phospholipid/cholesterol ratio, composition of the paraffinic chains or protein concentration between toads adapted to both temperatures. The possibility that water translocates through the mucosal border of the toad bladder by partitioning in the polar zone and diffusioning between the hydrocarbon chains of the membrane lipids and that cold adaptation would induce a stronger packing of lipids in the membrane is discussed.     

Role of lipids in theNeurospora crassa membrane. I. Influence of fatty acid composition on membrane lipid phase transitions

Summary The relationship between lipid composition and phase transition was investigated by differential scanning calorimetry for intact and membrane phospholipid extracts of wild-type (w/t) and thecel ^– (Tw 40) mutant ofNeurospora crassa. Thecel ^– (Tw 40) mutant (grown on minimal, sucrose medium supplemented with Tween 40 at 34 °C) had approximately twice the saturated fatty acid content ofw/t organisms grown at 22 °C. The gel-liquid crystal phase transitions of ergosterol-free extracts derived fromw/t andcel ^– (Tw 40) occur at –31 and –11 °C, respectively. The heats of transition (H) of these extracts were 1 and 13 cal/g, respectively. The addition of ergosterol (the predominant sterol inNeurospora) to the phospholipid extracts decreased the observed heats of transition, but did not alter the transition temperature. IntactNeurospora, whetherw/t orcel ^– (Tw 40) did not manifest similar gel-liquid crystal phase transitions in the differential scanning calorimeter. However, an endothermic peak at approximately 30 °C was observed in intact cells and extracted phospholipids of bothw/t andcel ^– (Tw 40) organisms. This peak was insensitive to the addition of ergosterol, had a low heat content (H1 cal/g), and was reversible.     

Regulation of membrane fatty acid composition by temperature in mutants of Arabidopsis with alterations in membrane lipid composition

Background  

A wide range of cellular responses occur when plants are exposed to elevated temperature, including adjustments in the unsaturation level of membrane fatty acids. Although membrane bound desaturase enzymes mediate these adjustments, it is unknown how they are regulated to achieve these specific membrane compositions. Furthermore, the precise roles that different membrane fatty acid compositions play in photosynthesis are only beginning to be understood. To explore the regulation of the membrane composition and photosynthetic function in response to temperature, we examined the effect of temperature in a collection of mutants with altered membrane lipid fatty acid composition.     

Influence of enzymatic phospholipid cleavage on the permeability of the erythrocyte membrane. I. Transport of non-electrolytes via the lipid domain.

In order to further elucidate the influence of membrane lipids on transport via the lipid domain of the erythrocyte membrane, simple non-electrolyte diffusion was investigated by tracer flux measurements in whole cells after cleavage of up to 65% of phosphatidylcholine or sphingomyelin by phospholipase A2 from Naja naja, or by sphingomyelinase. A new type of labelled model non-electrolyte was used in this study, readily available by reacting a non-labelled thiol with a labelled alkylating SH-reagent. In spite of the marked enzymatic alterations of the membrane, which lead to the occurrence of large quantities of lysophosphatidylcholine and long chain fatty acids, or of ceramide, the permeability of the lipid domain remained unaffected. This finding is very surprising, since the physical properties of the lipid phase (microviscosity, structure of the membrane interface) are likely to be perturbed in the enzyme-treated membranes. Sphingomyelinase-treated cells undergo stomatocytic shape changes followed by deep invaginations of the membrane and finally endocytosis, while phospholipase A2-treated cells essentially maintain their normal shape.