Permeability of rabbit polymorphonuclear leukocyte membranes to urea and other nonelectrolytes

The diffusional permeability coefficients of rabbit polymorphonuclear leukocyte membranes to urea, methylurea and thiourea have been measured. It was found that the permeability coefficient of these membranes to urea is very low and that thiourea was more permeable than methylurea which was, in turn, more permeable than urea. These results suggest that there is no need to postulate a carrier-mediated mechanism for urea transport across biological membranes and that the concept of “aqueous pores” is not a general property of biological membranes but restricted only to certain cases.     

Comparative transport efficiencies of urea analogues through urea transporter UT-B

Expression of urea transporter UT-B confers high urea permeability to mammalian erythrocytes. Erythrocyte membranes also permeate various urea analogues, suggesting common transport pathways for urea and structurally similar solutes. In this study, we examined UT-B-facilitated passage of urea analogues and other neutral small solutes by comparing transport properties of wildtype to UT-B-deficient mouse erythrocytes. Stopped-flow light-scattering measurements indicated high UT-B permeability to urea and chemical analogues formamide, acetamide, methylurea, methylformamide, ammonium carbamate, and acrylamide, each with P(s)>5.0 x 10(-6) cm/s at 10 degrees C. UT-B genetic knockout and phloretin treatment of wildtype erythrocytes similarly reduced urea analogue permeabilities. Strong temperature dependencies of formamide, acetamide, acrylamide and butyramide transport across UT-B-null membranes (E(a)>10 kcal/mol) suggested efficient diffusion of these amides across lipid bilayers. Urea analogues dimethylurea, acryalmide, methylurea, thiourea and methylformamide inhibited UT-B-mediated urea transport by >60% in the absence of transmembrane analogue gradients, supporting a pore-blocking mechanism of UT-B inhibition. Differential transport efficiencies of urea and its analogues through UT-B provide insight into chemical interactions between neutral solutes and the UT-B pore.     

Comparative transport efficiencies of urea analogues through urea transporter UT-B

Expression of urea transporter UT-B confers high urea permeability to mammalian erythrocytes. Erythrocyte membranes also permeate various urea analogues, suggesting common transport pathways for urea and structurally similar solutes. In this study, we examined UT-B-facilitated passage of urea analogues and other neutral small solutes by comparing transport properties of wildtype to UT-B-deficient mouse erythrocytes. Stopped-flow light-scattering measurements indicated high UT-B permeability to urea and chemical analogues formamide, acetamide, methylurea, methylformamide, ammonium carbamate, and acrylamide, each with P_s > 5.0 × 10^− 6 cm/s at 10 °C. UT-B genetic knockout and phloretin treatment of wildtype erythrocytes similarly reduced urea analogue permeabilities. Strong temperature dependencies of formamide, acetamide, acrylamide and butyramide transport across UT-B-null membranes (E_a > 10 kcal/mol) suggested efficient diffusion of these amides across lipid bilayers. Urea analogues dimethylurea, acryalmide, methylurea, thiourea and methylformamide inhibited UT-B-mediated urea transport by > 60% in the absence of transmembrane analogue gradients, supporting a pore-blocking mechanism of UT-B inhibition. Differential transport efficiencies of urea and its analogues through UT-B provide insight into chemical interactions between neutral solutes and the UT-B pore.     

Changes in cell membrane permeability and lipid content of wheat root cortex cells induced by NaCl

Wheat seedlings were grown hydroponically in absence and presence of 100 mM NaCl for 7 d. Cell membrane permeability to nonelectrolytes and water was determined by the plasmometric method for individual intact cells. NaCl increased membrane permeability to urea, methylurea and ethylurea and decreased permeability to water. Membrane lipid partiality was decreased by NaCl. The effects of NaCl on cell permeability parallel changes in the lipid composition of the plasma membranes induced by NaCl stress suggesting that nonelectrolyte permeability is a useful tool to probe alterations in the lipid matrix of the membrane.     

Urea and water permeability in dogfish (Squalus acanthias) gills

We used a perfused gill preparation from dogfish to investigate the origin of low branchial permeability to urea. Urea permeability (14C-urea) was measured simultaneously with diffusional water permeability (3H2O). Permeability coefficients for urea and ammonia in the perfused preparation were almost identical to in vivo values. The permeability coefficient of urea was 0.032 x 10(-6) cm/sec and of 3H2O 6.55 x 10(-6) cm/sec. Adrenalin (1 x 10(-6) M) increased water and ammonia effluxes by a factor of 1.5 and urea efflux by a factor of 3.1. Urea efflux was almost independent of the urea concentration in the perfusion medium. The urea analogue thiourea in the perfusate had no effect on urea efflux, whereas the non-competitive inhibitor of urea transport, phloretin, increased efflux markedly. The basolateral membrane is approximately 14 times more permeable to urea than the apical membrane. We conclude that the dogfish apical membrane is extremely tight to urea, but the low apparent branchial permeability may also relate to the presence of an active urea transporter on the basolateral membrane that returns urea to the blood and hence reduces the apical urea gradient.     

Inhibiting nitrification and increasing yield of barley by band placement of thiourea with fall-applied urea

Incubation and field experiments were conducted on the influence of thiourea in inhibiting nitrification of urea N, and subsequently on reducing over-winter losses of fallapplied N. Under incubation, most of the added urea placed in bands was nitritified within five or six weeks. However, thiourea when pelleted with urea (21 urea to thiourea by weight) reduced the amount of nitrification to less than one-half during the same period.In two uncropped field experiments in an early dry fall, the application of pelleted urea+thiourea (21) in bands resulted in almost complete inhibition of nitrification of urea for four weeks. In two other uncropped field experiments begun in June with the same fertilizer in bands, half or less of applied N appeared as nitrate after eight weeks. In 10 cropped field experiments with 56 kg N ha^–1, urea+thiourea placed in bands depressed nitrification of fall-applied urea over the winter. By early May, the urea mixed into the soil in the previous fall was nearly all nitrified, while only one-half of the banded urea+thiourea was nitrified. The loss of mineral N by early May was 38% with urea mixed into the soil, but only 18% with bands of urea+thiourea.The 10 sites were cropped to spring barley. The increase in yield of grain or the increase in %N uptake from fertilier N was approximately only one-half as much with fall-applied urea mixed into the soil as compared to spring-applied urea added in the same way. Specifically, fall-applied mixed urea produced 930 kg ha^–1 less grain yield and 32% less N uptake from fertilizer N than did mixed urea in spring. On fall-application there was some benefit from banding of urea or with mixing urea+thiourea pellets into the soil, but the banding of urea+thiourea pellets gave more benefit. Among the fall applications, banded urea+thiourea pellets produced 670 kg ha^–1 more grain yield and 26% more N uptake in grain from fertilizer N than did urea mixed into the soil.     

Development of a biosensor for urea assay based on amidase inhibition,using an ion-selective electrode

Abstract

A biosensor for urea has been developed based on the observation that urea is a powerful active-site inhibitor of amidase, which catalyzes the hydrolysis of amides such as acetamide to produce ammonia and the corresponding organic acid. Cell-free extract from Pseudomonas aeruginosa was the source of amidase (acylamide hydrolase, EC 3.5.1.4) which was immobilized on a polyethersulfone membrane in the presence of glutaraldehyde; an ion-selective electrode for ammonium ions was used for biosensor development. Analysis of variance was used for optimization of the biosensor response and showed that 30 μL of cell-free extract containing 7.47 mg protein mL^?1, 2 μL of glutaraldehyde (5%, v/v) and 10 μL of gelatin (15%, w/v) exhibited the highest response. Optimization of other parameters showed that pH 7.2 and 30 min incubation time were optimum for incubation of membranes in urea. The biosensor exhibited a linear response in the range of 4.0–10.0 μM urea, a detection limit of 2.0 μM for urea, a response time of 20 s, a sensitivity of 58.245 % per μM urea and a storage stability of over 4 months. It was successfully used for quantification of urea in samples such as wine and milk; recovery experiments were carried out which revealed an average substrate recovery of 94.9%. The urea analogs hydroxyurea, methylurea and thiourea inhibited amidase activity by about 90%, 10% and 0%, respectively, compared with urea inhibition.     

Permeability of lipid bilayers to water and ionic solutes

The lipid bilayer moiety of biological membranes is considered to be the primary barrier to free diffusion of water and solutes. This conclusion arises from observations of lipid bilayer model membrane systems, which are generally less permeable than biological membranes. However, the nature of the permeability barrier remains unclear, particularly with respect to ionic solutes. For instance, anion permeability is significantly greater than cation permeability, and permeability to proton-hydroxide is orders of magnitude greater than other monovalent inorganic ions. In this review, we first consider bilayer permeability to water and discuss proposed permeation mechanisms which involve transient defects arising from thermal fluctuations. We next consider whether such defects can account for ion permeation, including proton-hydroxide flux. We conclude that at least two varieties of transient defects are required to explain permeation of water and ionic solutes.     

Urea permeability of human red cells

The rate of unidirectional [14C]urea efflux from human red cells was determined in the self-exchange and net efflux modes with the continuous flow tube method. Self-exchange flux was saturable and followed simple Michaelis-Menten kinetics. At 38 degrees C the maximal self-exchange flux was 1.3 X 10(-7) mol cm-2 s-1, and the urea concentration for half-maximal flux, K1/2, was 396 mM. At 25 degrees C the maximal self-exchange flux decreased to 8.2 X 10(-8) mol cm-2 s-1, and K1/2 to 334 mM. The concentration-dependent urea permeability coefficient was 3 X 10(-4) cm s-1 at 1 mM and 8 X 10(-5) cm s-1 at 800 mM (25 degrees C). The latter value is consonant with previous volumetric determinations of urea permeability. Urea transport was inhibited competitively by thiourea; the half-inhibition constant, Ki, was 17 mM at 38 degrees C and 13 mM at 25 degrees C. Treatment with 1 mM p-chloromercuribenzosulfonate inhibited urea permeability by 92%. Phloretin reduced urea permeability further (greater than 97%) to a "ground" permeability of approximately 10(-6) cm s-1 (25 degrees C). This residual permeability is probably due to urea permeating the hydrophobic core of the membrane by simple diffusion. The apparent activation energy, EA, of urea transport after maximal inhibition was 59 kJ mol-1, whereas in control cells EA was 34 kJ mol-1 at 1 M and 12 kJ mol-1 at 1 mM urea. In net efflux experiments with no extracellular urea, the permeability coefficient remained constantly high, independent of a variation of intracellular urea between 1 and 500 mM, which indicates that the urea transport system is asymmetric. It is concluded that urea permeability above the ground permeability is due to facilitate diffusion and not to diffusion through nonspecific leak pathways as suggested previously.     

Some studies on lipid peroxidation in monomolecular and bimolecular lipid films

Summary Hydrogen peroxide generated from dissolved oxygen through the alloxandialuric acid cycle affected both the permeability and the stability of lipid bilayer membranes. The permeability of the artificial membranes varied directly with the hydrogen peroxide concentration. Membrane stability varied inversely with the hydrogen peroxide concentration. Bilayers formed from solutions containing both phospholipid and the antioxidant vitamin E were less permeable and more stable in the presence of hydrogen peroxide than bilayers generated from solutions containing phospholipid alone. Peroxidation of phospholipid monolayers caused first an expansion of the films presumably through the introduction of peroxide groups. Further oxidation of phospholipid monolayers led to contraction of the films presumably through the formation of water-soluble products. The results of the monolayer studies and a consideration of the possible kinetics for the peroxidation reaction sequence have been used to explain the changes in the permeability and the stability of lipid bilayer membranes. Our data suggest that oxidation of lipid in biological membranes may first increase membrane permeability and then decrease membrane stability.     

Selective fixation with glutaraldehyde of ADH-induced urea permeability sites in toad bladder

Toad bladders exposed to vasopressin (ADH) and then fixed on the mucosal surface with 1% glutaraldehyde were highly permeable to water and to urea compared to control bladders fixed in the absence of hormone. When identical conditions of fixation were were used, but the concentration of glutaraldehyde was decreased to 0.25%, the ADH-induced increase in membrane permeability to urea was preserved whereas water permeability was not. About 74% of the hormone-induced urea permeability sites were preserved by glutaraldehyde and were stable to changes in temperature as suggested by a constant value for the activation energy of urea movement of 5.4 kcal/mole (4-33 degrees C). In other studies bladders were exposed at low temperatures to 0.17% glutaraldehyde applied either to the serosal or the mucosal surface. The ADH-induced increase in membrane permeability to urea, bulk water, and tritiated water was well preserved with serosal fixation, but not with mucosal fixation. The observation that the urea pathway can be selectively preserved with 0.25% glutaraldehyde applied to the mucosa indicates that this structure is more accessible and (or) more sensitive to low-dose glutaraldehyde than is the ADH-induced water pathway. The observation that glutaraldehyde is more effective in stabilizing the ADH-induced urea channels from the serosal than from the mucosal surface indicates that these channels are not fixed at the extracellular surface of the apical plasma membrane. It appears, rather, that glutaraldehyde exerts its effects from an intracellular position, where it cross-links components of the urea channels at the cytoplasmic surface of the apical membrane and (or) inactivates the intracellular machinery responsible for the removal or dispersal of the ADH-induced urea permeability sites.     

Organ specificity and lactate-dehydrogenase activity. Differential inhibition by urea and related compounds

1. The effect of urea on the lactate-dehydrogenase activities of human-heart and -liver tissue extracts and on crystalline ox-heart and rabbit-muscle enzyme have been determined. Similar studies on electrophoretically separated isoenzyme fractions have shown an inverse relationship between sensitivity to urea inhibition and electrophoretic mobility. 2. With pyruvate as substrate a sharp change in the nature of the inhibition of tissue lactate dehydrogenase with increasing concentrations of urea occurs at 1 m or 4 m with the electrophoretically slow and fast isoenzymes respectively. 3. At concentrations of urea less than 1 m, inhibition of the purified enzymes is competitive with respect to pyruvate and 2-oxobutyrate. 4. Similar studies have been carried out with methylurea and hydantoic acid, both of which are more potent inhibitors than urea.     

Selective permeabilization of lipid membranes by photodynamic action via formation of hydrophobic defects or pre-pores

To gain insight into mechanisms of photodynamic modification of biological membranes, we studied an impact of visible light in combination with a photosensitizer on translocation of various substances across artificial (vesicular and planar) bilayer lipid membranes (BLMs). Along with induction of carboxyfluorescein leakage from liposomes, pronounced stimulation of lipid flip-flop between the two monolayers was found after photosensitization, both processes being prevented by the singlet oxygen quencher sodium azide. On the contrary, no enhancement of potassium chloride efflux from liposomes was detected by conductometry under these conditions. Illumination of planar BLMs in the presence of a photosensitizer led to a marked increase in membrane permeability to amphiphilic 2-n-octylmalonic acid, but practically no change in the permeability to ammonia, which agreed with selective character of the photosensitized leakage of fluorescent dyes from liposomes (Pashkovskaya et al., Langmuir, 2010). Thus, the effect on transbilayer movement of molecules elicited by the photodynamic treatment substantially depended on the kind of translocated species, in particular, on their lipophilicity. Based on similarity with results of previous electroporation studies, we hypothesized about photodynamic induction of "pre-pores" or "hydrophobic defects" permeable to amphiphilic compounds and less permeable to hydrophilic substances and inorganic ions.     

A dielectric dispersion technique for measuring the ionic permeability of internal membranes of isolated chloroplasts

Summary A method has been developed in which a chloroplast suspension is placed between electrodes to which a variable AC potential is applied. The dielectric constant of the suspension varies inversely as the square root of frequency within the range 0.5 to 50 MHz. Results are consistent with the view that this dielectric dispersion is due to ion movement across the chloroplast internal membranes, under the influence of the applied potential. The slope of the dispersion depends on the permeability of the membranes, and thus enables the mobility of externally added ions to be calculated. Results imply that the internal membranes of sugar cane chloroplasts are more permeable to K⁺ than Na⁺, and more permeable to Cl^– than CH₃COO^–. Results also confirm the view that Triton X-100 increases the ionic permeability of membranes.     

Urea and ethylene glycol-facilitated transport systems in the human red cell membrane. Saturation, competition, and asymmetry

The equilibrium exchange of [14C]urea and ethylene glycol was measured using a new type of fast flow system. Approximately equal volumes of saline and air were mixed to form a segmented fluid stream into which 14C-loaded red cells are injected. The stream flows through three filter chambers which allow sampling of the 14C in the extracellular fluid at three time points. The chambers are designed so that they do not disrupt the segmented bubble pattern. The alternating air and saline segments prevent laminar dispersion in the flowing stream and ensure good mixing at the injection and sampling sites. The equilibrium exchange of both urea and ethylene glycol showed saturation kinetics. The maximum permeability (Po) measured in the limit of zero solute concentration is 1.6 X 10(-3) cm/s for urea and 4.8 X 10(-4) cm/s for ethylene glycol (T = 23 degrees C). The apparent dissociation constant (Km) was 218 mM for urea and 175 mM for ethylene glycol. The Po for thiourea is 2.3 X 10(-6) cm/s and the Km is 19 mM. Urea and thiourea inhibit the transport of each other and the inhibition constant (KI) is approximately equal to the Km for both compounds. 53 other analogues of urea were screened for their inhibition of urea or thiourea transport. Several analogues [e.g., 1-(3,4-dichloro-phenyl)-2-thiourea] had a KI in the range of 0.03 mM. The affinity of the inhibitor increased as it was made more hydrophobic. The urea analogues did not significantly inhibit the ethylene glycol or osmotic permeability. Glycerol inhibited ethylene glycol permeability with a KI of 1,200 mM.     

Permeability of Sugar-cane Chloroplasts to Sucrose

The Sucrose permeability of chloroplast-bounding membranes wasmeasured by three different methods. These gave similar valuesand suggested that the chloroplast membrane was fairly rapidlypermeable to sucrose. The chloroplast membranes were found tobe about 10⁴-fold more permeable to sucrose efflux than carrotcallus cells.     

Rate of hydrolysis of urea as influenced by thiourea and pellet size

Summary Two incubation experiments and a number of field experiments were conducted to determine the effect of soil moisture tension, pellet size and addition of thiourea to urea on the rate of urea hydrolysis. In the incubation experiments at 20°C, the rate of hydrolysis of urea increased from 15 bar to 1/3 bar soil moisture tension, with the largest change (doubling) occurring from 15 bar to 7 bar moisture tension. Increasing pellet size reduced the rate of urea hydrolysis by about 12% with urea pellets weighing 0.21 g as compared to 0.01 g urea pellets after 114h. When thiourea (a metabolic inhibitor) was pelleted with urea in a ratio of two parts urea and one part thiourea, the rate of hydrolysis was halved.In a field experiment, the addition of thiourea to urea and increasing pellet size suppressed the rate of urea hydrolysis considerably for 8 days. The amount of urea hydrolyzed with urea+thiourea (21) pellets weighing 2.51 g was one-fourth of the amount of urea hydrolyzed with 0.01 g pellets of urea alone. In the other six field experiments which were set out in October, only 22% to 39% of urea +thiourea (21) was hydrolyzed at two weeks after application, while almost all of the urea was hydrolyzed when it was mixed into the soil without an inhibitor.Unter our field conditions, we would estimate that the hydrolysis of urea can be inhibited for at least one week. The inhibition of urea hydrolysis appears to be great enough that the problems encountered from the rapid hydrolysis of urea, wherever these occur, may be reduced by combined use of thiourea and either increased pellet size or band placement.     

Plasmolysis shape in relation to freeze-hardening of cabbage plants and to the effect of penetrating solutes*

Abstract In conformity with earlier results, low-temperature hardening of cabbage seedlings lowered the osmotic potential and increased the permeability to thiourea of the petiole cells. It also decreased the time required for rounding-up of the protoplasts in cells plasmolysed in 1. 5 × isotonic (or higher) glucose or CaCl₂ solutions. Solutions of dimethylsulphoxide (DMSO), thiourea, urea, and glycerol each accelerated the rate of rounding-up of protoplasts in plasmolysed cells, compared to the rate in glucose solution of the same hypertonicity. Each also penetrated the cell membranes as indicated by deplasmolysis. Only in the case of DMSO, in which there was very rapid deplasmolysis (5–6 min), was this rounding-up due to protoplast expansion. In the case of thiourea (deplasmolysis within 30–60 min) rounding-up occurred almost immediately (less than 2 min), before protoplast expansion was sufficient to induce it. It was concluded that the accelerated rounding-up was due to a rapid osmotic adjustment in the protoplasm by the penetrating solution, which increased its water content and decreased its viscosity.     

The permeability of liposomes to nonelectrolytes. I. Activation energies for permeation.

The effect of temperature on the permeability of nonelectrolytes across liposomal membranes above and below their transition temperature has been studied by using an osmotic method. Below their transition temperature, liposomes are osmotically insensitive structures but, on addition of gramicidin A, the water permeability so increased that the permeability of solutes could be studied. The measured activation energies for permeation of a variety of nonelectrolytes has been found to increase when a) there is an increase in the capability of the solutes to form hydrogen bonds in water, b) the cholesterol concentration in the membranes increases and c) the membranes pass from a liquid-crystalline to a solid-crystalline state. The change in the activation energy for permeation per hydrogen bond is about 1.8 kcal/mole for all the different liposome systems investigated; the only solute tested that deviated from this correlation was urea, whose activation energy for permeation across a gramicidin-containing system was much lower than expected from its hydrogen-bonding capacity. This finding suggests that urea is permeating across the gramicidin pore. Although the literature contains only incomplete data relating the activation energies for permeation of nonelectrolytes across biological membranes to their hydrogen-bonding capacity, the available evidence suggests that there is a similar correlation to that found in liposomes. Thus, the average increase in the activation energy per hydrogen bond for permeation across ox red cell membranes (Jacobs, Glassman & Parpart, J. Cell. Comp. Physiol. 7:197, 1935) is 2.2 plus or minus 0.4 kcal/mole, a value that is similar to that obtained in liposomes. However, the activation energies for water and urea are - in such a system - very much lower than expected, suggesting that they, too, are permeating by some parallel route such as an aqueous pore.     

Membrane Transport in Primitive Cells

Although model protocellular membranes consisting of monoacyl lipids are similar to membranes composed of contemporary diacyl lipids, they differ in at least one important aspect. Model protocellular membranes allow for the passage of polar solutes and thus can potentially support cell-to functions without the aid of transport machinery. The ability to transport polar molecules likely stems from increased lipid dynamics. Selectively permeable vesicle membranes composed of monoacyl lipids allow for many lifelike processes to emerge from a remarkably small set of molecules.Lipid bilayer membranes are an integral component of living cells, providing a permeability barrier that is essential for nutrient transport and energy production. It is reasonable to assume that a similar boundary structure would be required for the origin of cellular life (Szostak et al. 2001). Even though bilayer membranes are a cellular necessity, they also pose a significant obstacle to early cellular functions, the most obvious being that the permeability barrier would inhibit chemical exchange with the environment. Such an exchange is important not only for acquiring nutrient substrates for primitive metabolic processes, but also for the release of inhibitory side-products.Contemporary cells circumvent the permeability problem by incorporating complex transmembrane protein machinery that provides specific transport capabilities. It is unlikely that Earth’s first cells assembled bilayer membranes together with specific membrane protein transporters. Rather, intermediate evolutionary steps must have existed in which simple lipid molecules provided many of the characteristics of contemporary membranes without relying on advanced protein machinery. What seems to have been necessary was the appearance of a simple membrane system capable of retaining and releasing specific molecules. In short, a protocell needed to be selectively permeable.