NMR studies of glycerol permeability in lipid vesicles, erythrocytes and the alga Dunaliella

Glycerol diffusional permeabilities through the cytoplasmic cell membrane of Dunaliella salina, the cell envelope of pig erythrocyte and egg phosphattidylcholine vesicles were measured by NMR spectroscopy employing the spin-echo method and nuclear T₁ relaxation. The following permeability coefficients (P) and corresponding enthalpies of activation (ΔH^‡) were determined for glycerol at 25°C: for phosphatidylcholine vesicles 5·10⁻⁶ cm/s and 11±2 kcal/mol; for pig erythrocytes 7·10⁻⁸ cm/s and 18±3 kcal/mol, respectively; for the cytoplasmic membrane of D. salina the permeability at 17°C was found to be exceptionally low and only a lower limit (P<5·10⁻¹¹cm/s) could be calculated. At temperatures above 50°C a change in membrane permeability occurred leading to rapid leakage of glycerol accompanied by cell death. The data reinforce the notion that the cytoplasmic membrane of Dunaliella represents a genuine anomaly in its exceptional low permeability to glycerol.     

Conformational change of core particles studied by quasielastic light scattering

The diffusion translational coefficient D_T of core particles in monodisperse solutions has been measured by the quasielastic light scattering method in a large scale of salinities over the range 6.10⁻⁴ to 2M Na⁺ or K⁺. The observed values of D_T are independent of particle concentration in the range 0.1–2 mg/ml and do not vary with the scattering vector q corresponding to scattering angles between 40°–120°. When the salinity is progressively raised an increase of D_T from 1.9.10⁻⁷ cm²s⁻¹ to 3.2.10⁻⁷ cm²s⁻¹ was observed at about 2.10⁻³ M NaCl followed by a decrease of D_T beyond 0.6 M NaCl.The various possible causes of the changes of D_T such as interactions between particles or between particles and salt ions are discussed. We show that the single low ionic strength change is due to a conformational transition of the core particles, while the second variation of D_T accompanies the disorganization of the core particles.     

Osmotic permeabilities across corneal endothelium and antidiuretic hormone-stimulated toad urinary bladder structures

Osmotic permeabilities of several epithelial structures have been determined with novel optical procedures based on specular microscopy. The osmotic permeabilities of several tissue layers were determined by continuously monitoring the position of the apical tissue borders while an osmotic flow was imposed across those layers. The values found were (in μm/s; mean ± SE): corneal epithelium, 137 ± 30 (n = 5); antidiuretic hormone stimulated toad bladder, 429 ± 64 (n = 6); and corneal endothelium, 711 ± 34 (n = 7). In addition, the osmotically-induced transient change in thickness of the corneal endothelial cells was determined with the help of a computer, and the apparent osmotic permeability measured for the apical membrane was 1420 ± 160 μm/s (n = 5). It is concluded that the osmotic permeability across the endothelial layer is sizably larger than had been previously detected and that osmotic flows across such layer largely traverse the cellular membranes. With osmotic permeability values (per unit of cell membrane area) as large as presently reported, isotonic fluid transport by epithelia can be explained simply on the basis of local osmotic gradients.     

Permeability of bilayer lipid membranes for superoxide (O2) radicals

Egg yolk phosphatidylcholine monolamellar liposomes (1000 Å in diameter) loaded with cytochrome c were placed into an external solution, in which superoxide radicals, O₂⁻, were generated by a xanthine-xanthine oxidase system. The penetration of the superoxide radicals across the liposomal membrane was detected by cytochrome c reduction in the inner liposome compartment. The effects of modifiers and temperature on this process were studied. The permeability of liposomal membrane for O₂⁻(P′_O2⁻ = (7.6 ± 0.3) · 10^-8 cm/s), or HO₂^• (P′_HO2⁻ = 4.9 · 10^-4 cm/s) were determined. The effect of the transmembrane electric potential (K⁺ concentration gradient, valinomycin) on the permeability of liposomal membranes for O₂⁻ were investigated. It was found that O₂⁻ can penetrate across liposomal membrane in an uncharged form. The feasibility of penetration of superoxide radicals through liposomal membrane, predominantly via anionic channels, was demonstrated by the use of an intramolecular cholesterol-amphotericin B complex.     

The diffusional water permeability in the halotolerant alga Dunaliella as measured by nuclear magnetic resonance

T₁ nuclear relaxation measurements of ¹H and ¹⁷O of water have been applied to study the kinetics of the diffusional transport of water across the cytoplasmic cell membrane of Dunaliella salina and Dunaliella bardawil. The water permeability coefficients at 25°C were found to be 1.5·10⁻³ cm/s and 1.8·10⁻³ cm/s, respectively, with an activation energy of 3.7 kcal/mol. The results indicate that the cell membrane of Dunaliella exhibits high diffusional permeability to water, similar in magnitude to that found for other cells and model membranes, and a relatively low activation energy. This regularity is in contrast to the exceptionally low glycerol permeability of the membrane (Brown, F.F., Sussman, I., Avron, M. and Degani, H. (1982) Biochim. Biophys. Acta 690, 165–173).     

Effect of renoguanylin on hydrogen/bicarbonate ion transport in rat renal tubules

Renoguanylin (REN) is a recently described member of the guanylin family, which was first isolated from eels and is expressed in intestinal and specially kidney tissues. In the present work we evaluate the effects of REN on the mechanisms of hydrogen transport in rat renal tubules by the stationary microperfusion method. We evaluated the effect of 1 μM and 10 μM of renoguanylin (REN) on the reabsorption of bicarbonate in proximal and distal segments and found that there was a significant reduction in bicarbonate reabsorption. In proximal segments, REN promoted a significant effect at both 1 and 10 μM concentrations. Comparing control and REN concentration of 1 μM, JHCO₃⁻, nmol cm^− 2 s^− 1 − 1,76 ± 0,11_control × 1,29 ± 0,08_{REN 10 μM}; P < 0.05, was obtained. In distal segments the effect of both concentrations of REN was also effective, being significant e.g. at a concentration of 1 μM (JHCO₃⁻, nmol cm^− 2 s⁻ 1 − 0.80 ± 0.07_control × 0.60 ± 0.06_{REN 1 μM}; P < 0.05), although at a lower level than in the proximal tubule. Our results suggest that the action of REN on hydrogen transport involves the inhibition of Na⁺/H⁺exchanger and H⁺-ATPase in the luminal membrane of the perfused tubules by a PKG dependent pathway.     

Development of a microfluidic device for determination of cell osmotic behavior and membrane transport properties

An understanding of cell osmotic behavior and membrane transport properties is indispensable for cryobiology research and development of cell-type-specific, optimal cryopreservation conditions. A microfluidic perfusion system is developed here to measure the kinetic changes of cell volume under various extracellular conditions, in order to determine cell osmotic behavior and membrane transport properties. The system is fabricated using soft lithography and is comprised of microfluidic channels and a perfusion chamber for trapping cells. During experiments, rat basophilic leukemia (RBL-1 line) cells were injected into the inlet of the device, allowed to flow downstream, and were trapped within a perfusion chamber. The fluid continues to flow to the outlet due to suction produced by a Hamilton Syringe. Two sets of experiments have been performed: the cells were perfused by (1) hypertonic solutions with different concentrations of non-permeating solutes and (2) solutions containing a permeating cryoprotective agent (CPA), dimethylsulfoxide (Me₂SO), plus non-permeating solute (sodium chloride (NaCl)), respectively. From experiment (1), cell osmotically inactive volume (V_b) and the permeability coefficient of water (L_p) for RBL cells are determined to be 41% [n = 18, correlation coefficient (r²) of 0.903] of original/isotonic volume, and 0.32 ± 0.05 μm/min/atm (n = 8, r² > 0.963), respectively, for room temperature (22 °C). From experiment (2), the permeability coefficient of water (L_p) and of Me₂SO (P_s) for RBL cells are 0.38 ± 0.09 μm/min/atm and (0.49 ± 0.13) × 10⁻³ cm/min (n = 5, r² > 0.86), respectively. We conclude that this device enables us to: (1) readily monitor the changes of extracellular conditions by perfusing single or a group of cells with prepared media; (2) confine cells (or a cell) within a monolayer chamber, which prevents imaging ambiguity, such as cells overlapping or moving out of the focus plane; (3) study individual cell osmotic response and determine cell membrane transport properties; and (4) reduce labor requirements for its disposability and ensure low manufacturing costs.     

Determination of oocyte membrane permeability coefficients and their application to cryopreservation in a rabbit model

Having an effective means to cryopreserve human oocytes would offer more flexibility in healthcare services for infertility patients, and obviate cryopreservation of preimplantation embryos. It is essential to establish good animal models for human oocyte cryopreservation and the rabbit is a good candidate. Attempts to improve oocyte cryopreservation are often empirical, with results often being irreproducible. Cryopreservation protocols may be optimized by modeling the changes in oocyte volume and the associated damages incurred during the addition and dilution of cryoprotective agents (CPA). The objectives of the current study were to determine cryobiological properties of rabbit oocytes, including the isotonic volume, osmotically inactive cell fraction (V_b), hydraulic conductivity (L_p), permeability (P_s) to dimethylsulfoxide (Me₂SO), ethylene glycol (EG), and glycerol (GLY) and to examine the correlation between cell volume excursions and viability. This has led to the development of the accumulative osmotic damage (AOD) model associated with the processes of CPA addition/dilution. Mature rabbit oocytes were perfused with 15% (V/V) CPA medium (dissolved in 1× PBS). The osmotic responses of the oocytes were videotaped. A two-parameter model was fit to the experimental data to determine the values of L_p and P_s. Oocyte volumes reached upon equilibration with 285, 600, 900, and 1200 mOsm (milliosmolal) solutions of non-permeating compounds were plotted in a Boyle van’t Hoff plot. The average radius of rabbit oocytes in an isotonic solution was determined to be 55.7 ± 1.2 μm (n = 16). The rabbit oocyte exhibited an “ideal” osmotic response in the range from iso-osmolity to 1200 mOsm. The V_b was determined to be 20% of the isotonic value with r² = 0.97. The values of L_p were determined to be 0.79 ± 0.26, 0.82 ± 0.22, and 0.64 ± 0.16 μm min⁻¹ atm⁻¹ and the P_s values were determined to be 2.9 ± 1.3, 2.7 ± 1.3, and 0.27 ± 0.18 × 10⁻³ cm min⁻¹ for Me₂SO, EG and GLY, respectively. There were no significant differences (p > 0.05) between values for L_p and P_S in the presence of the Me₂SO and EG. However, these values were significantly different from the values in presence of GLY. We calculated the AOD values of those oocytes that experienced the process of CPA additions/dilutions and found that these values were highly correlated to the development rates of these oocytes after parthenogenetic activation (r = −0.98).     

Water transport in maize roots : measurement of hydraulic conductivity, solute permeability, and of reflection coefficients of excised roots using the root pressure probe

A root pressure probe has been used to measure the root pressure (P_r) exerted by excised main roots of young maize plants (Zea Mays L.). Defined gradients of hydrostatic and osmotic pressure could be set up between root xylem and medium to induce radial water flows across the root cylinder in both directions. The hydraulic conductivity of the root (Lp_r) was evaluated from root pressure relaxations. When permeating solutes were added to the medium, biphasic root pressure relaxations were observed with water and solute phases and root pressure minima (maxima) which allowed the estimation of permeability (P_Sr) and reflection coefficients (σ_sr) of roots. Reflection coefficients were: ethanol, 0.27; mannitol, 0.74; sucrose, 0.54; PEG 1000, 0.82; NaCl, 0.64; KNO₃, 0.67, and permeability coefficients (in 10⁻⁸ meters per second): ethanol, 4.7; sucrose, 1.6; and NaCl, 5.7. Lp_r was very different for osmotic and hydrostatic gradients. For hydrostatic gradients Lp_r was 1·10⁻⁷ meters per second per megapascal, whereas in osmotic experiments the hydraulic conductivity was found to be an order of magnitude lower. For hydrostatic gradients, the exosmotic Lp_r was about 15% larger than the endosmotic, whereas in osmotic experiments the polarity in the water movement was reversed. These results either suggest effects of unstirred layers at the osmotic barrier in the root, an asymmetrical barrier, and/or mechanical effects. Measurements of the hydraulic conductivity of individual root cortex cells revealed an Lp similar to Lp_r (hydrostatic). It is concluded that, in the presence of external hydrostatic gradients, water moves primarily in the apoplast, whereas in the presence of osmotic gradients this component is much smaller in relation to the cell-to-cell component (symplasmic plus transcellular transport).     

Bottlenecks to water transport in Quercus robur L.: the abscission zone and its physiological consequences

Cladoptosis, the abscission of twigs, is the main mechanism of changes in crown structure in senescing pedunculate oak (Quercus robur L.). We tested the hypotheses that abscission zones in nodes of old pedunculate oak trees reduce leaf-specific hydraulic conductance of shoots and thereby limit the stomatal conductance and assimilation.Hydraulic conductance and leaf-specific hydraulic conductance, measured with a high pressure flowmeter in 0.5–1.5 m long shoots, were significantly lower in shoots of low vigour compared to vigorous growing shoots in a 165-years-old stand in the southeast of Germany. Two types of bottlenecks to water transport could be identified in shoots of old oak trees, namely nodes and abscission zones. In young twigs, vessel diameter and vessel density in nodes with abscission zones were significantly reduced compared with internodes. In nodes without abscission zones, vessel density was significantly reduced. The reduction of hydraulic conductance was especially severe in the smallest and youngest shoots with diameters less than 2 mm. Internodes of 1–5 mm sapwood diameter had an average hydraulic conductance of 7.13×10⁻⁶±0.2×10⁻⁶ kg s⁻¹ m⁻¹ MPa⁻¹, compared to 4.54×10⁻⁶±0.3×10⁻⁶ kg s⁻¹ m⁻¹ MPa⁻¹ in those with nodes.Maximum stomatal conductance and maximum net assimilation rate increased significantly with hydraulic conductance and leaf-specific hydraulic conductance. Maximum rate of net photosynthesis A_max of the most vigorous shoots (VC0) (7.34±0.55 μmol m⁻² s⁻¹) was significantly higher (P<0.001) than in shoots of other vigour classes (5.97±0.28 μmol m⁻² s⁻¹). Our data support the hypothesis that the changes in shoot and consequently crown architecture that are observed in ageing and declining trees can limit photosynthesis by reducing shoot hydraulic conductance. Abscission zones increase the hydraulic disadvantage of less vigorous compared to vigorously growing twigs. Cladoptosis might serve as a mechanism of selection between twigs of different efficiency.     

Pressure jump relaxation kinetics of frog skin open circuit voltage and short circuit current

The relaxation kinetics of frog skin open circuit voltage, V_oc, and short circuit current I_sc, was studied by analyzing the effects of subjecting the tissue to sudden increments of hydrostatic pressure. Both V_oc and I_sc are perturbed by the pressure jump. Changes in V_oc can be resolved into three components: a rapid decrease (phase I), a second, additional decrease with time constant 2.2 s (phase II), and finally a very slow increase found only in some preparations. The amplitudes of phases I and II are linear in the range of pressures studied (<350 atm) and have respective pressure coefficients of −1.2 · 10⁻⁴atm⁻¹ and −3.7 · 10⁻⁴atm⁻¹.Under short circuit conditions phases I and II persist. The pressure coefficients of the amplitudes of phase I and II, −4.3 · 10⁻⁴atm⁻¹ and −5.0 · 10⁻⁴atm⁻¹, respectively, are larger than those of V_oc, but the time constant of phase II, 2.2 s, is the same. The sum of the amplitudes of phases I and II is directly proportional to I_sc when it is inhibited with ouabain. It is argued that in both electrical states pressure perturbs the same transport mechanism giving rise to phases I and II of V_oc and I_sc.The magnitude of the pressure coefficients of these processes implies that they arise from chemical reactions, rather than from simple, physical solution properties. Comparison of the pressure jump kinetics with the previous spectral analysis of the electrical fluctuations of frog skin suggests a common origin for both sets of phenomena.     

Nonelectrolyte diffusion through lecithin-water lamellar phases and red-cell membranes

The longitudinal diffusion of a homologous series of monoamides through lecithin-water lamellar phases with aqueous channel widths of 16–27 Å has been studied. The diffusion coefficients relative to water of the hydrophilic amides, formamide and acetamide, depend logarithmically on solute molar volume, as previously demonstrated in human red cells. Aqueous diffusion of amides in red-cell membranes is similar to that in a lecithin-water phase of aqueous channel width less than 16 Å, the smallest channel width used. Partition coefficients of the lipophilic amides, valeramide and isovaleramide, between lecithin vesicles and water are 1.64 and 1.15 at 20 °C. These data enabled us to compute a valeramide diffusion coefficient of 6.5 · 10⁻⁷cm² · s⁻¹ at 20 °C in the lipid region of a lamellar phase containing 30% water about one order of magnitude greater than the diffusion coefficient of spin-labelled analogs of phosphatidylcholine. The discrimination between the permeability coefficients of valeramide and isovaleramide is more than twice as great in the human red cell as between lipid diffusion coefficients in a phase containing 8% water. This suggests that the lipid region of the human red cell is more highly organized than lipid in the lecithin-water lamellar phase.     

Hydrodynamic parameters and isolation of mitochondria, microperoxisomes and microsomes of rat heart

1. Analytical differential centrifugation of rat heart homogenates revealed a single population of mitochondria and microperoxisomes. Using cytochorme c oxidase, malate dehydrogenase and amine oxidase as mitochondrial marker enzymes, the s̄-value of mitochondria was estimated to s̄ = 10326 ± 406 S (average for the three marker enzymes). The −s-value of microperoxisomes was found to be −s = 1381 ± 40 S using catalase as the marker enzyme. The −s-value for the two orgenelles did not change significantly when the isoosmotic sucrose medium was substituted by an isoosmotic mannitol medium. 2. Analytical differential centrifugation revealed a polydispercity of the microsomal fraction using glucose-6-phosphatase and NADPH-cytochrome c reductase as the marker enzymes. The s̄-values were found to be −s_H1 = 1569 ± 412 S (NADPH-cytochrome c reductase), (glucose-6-phosphatase) and (NADPH-cytochrome c reductase and glucose-6-phosphatase). The recovery of marker enzymes in the isolated subcellular fractions was in the range of 84–94%. 3. When the mitochondrial and microperoxisomal fractions were subjected to isopycnic gradient centrifugation, using a self-generating gradient of polyvinylpyrrolidone-coated colloidal silica particles (Percoll) in 0.25 M sucrose medium, buoyant densities of 1.10 g/cm³ (main fraction of mitochondria) and 1.06 g/cm³ (main fraction of microperixosomes) were obtained. The density gradient centrifugation separated microperoxisomes from contaminating lysosomes of high specific activity in acid phosphatase. A value 1.04 g/cm³ was foung for the density of the microsomal fraction. 4. Based on the estimated -values, an optimal procedure is described for the isolattion of mitochondrial and microperoxisomal fractions from rat heart muscle.     

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     

Interaction of anthelmintic benzimidazoles and benzimidazole derivatives with bovine brain tubulin

The binding and inhibitory properties of 11 benzimidazoles for bovine brain tubulin were investigated. The effects of the benzimidazoles on the initial rates of microtubule polymerization were determined by a turbidimetric assay. The median inhibitory concentrations (I₅₀) for nocodazole, oxibendazole, parbendazole, mebendazole and fenbendazole ranged from 1.97 · 10⁻⁶ to 6.32 · 10⁻⁶ M. Benomyl, cambendazole and carbendazim had I₅₀ values from 5.83 · 10⁻⁵ to 9.01 · 10⁻⁵ M. Thiabendazole had an I₅₀ value of 5.49 · 10⁻⁴ M. Inhibitor constants (K_i) were determined by the colchicine binding assay. Oxibendazole, fenbendazole, and cambendazole had K_i values of 3.20 · 10⁻⁵, 1.73 · 10⁻⁵ and 1.10 · 10⁻⁴ M, respectively. Oxibendazole and fenbendazole were competitive inhibitors of colchicine. In contrast, cambendazole was a noncompetitive inhibitor of colchicine. The ability of these benzimidazoles to inhibit microtubule polymerization and the mode of action for the anthelmintic benzimidazoles is discussed.     

High-performance affinity capture-removal of bacterial pyrogen from solutions

Synthetic peptide S3Δ has high affinity for bacterial endotoxin or lipopolysaccharide (LPS). Under tested conditions of pH 5–9 and 0–0.4 M NaCl, the affinity constant, K_D ranged from 2·10⁻⁶ to 2·10⁻⁹ M⁻¹. A novel affinity matrix based on peptide S3Δ was developed for removal of LPS from solutions such as: water; buffers with a wide range of ionic strength and pH; medium for cell culture; and protein solutions under optimized conditions. At a starting LPS of ≈100 EU/ml, a post-purification level below 0.005 EU/ml was achieved.     

Dopamine-stimulated cyclic AMP and binding of [H]dopamine in acini from dog pancreas

Dispersed acini from dog pancreas were used to examine the ability of dopamine to increase cyclic AMP cellular content and the binding of [³H]dopamine. Cyclic AMP accumulation caused by dopamine was detected at 1·10⁻⁸ M and was half-maximal at 7.9±3.4·10⁻⁷M. The increase at 1·10⁻⁵ M, (7.5-fold) was equal to the half-maximal increase caused by secretin at 1·10⁻⁹ M. Haloperidol, a dopaminergic receptor antagonist inhibited cyclic AMP accumulation caused by dopamine. The IC₅₀ value for haloperidol, calculated from the inhibition of cyclic AMP increase caused by 1·10⁻⁵ M dopamine was 2.3±0.9·10⁻⁶M. Haloperidol did not alter basal or secretin-stimulated cyclic AMP content. [³H]Dopamine binding was studied on the same batch of cells as cyclic AMP accumulation. At 37°C, it was rapid, reversible, saturable and stereospecific. The K_d value for high affinity binding sites was 0.43±0.1·10⁻⁷M and 4.7±1.6·10⁻⁷M for low affinity binding sites. The concentration of drugs necessary to inhibit specific binding of dopamine by 50% was 1.2±0.4·10^/t-7M noradrenaline, 2·10^/t-7 M epinine, 4.1±1.8·10^/t-6M fluphenazine, 8.0±1.6·10^/t-6M haloperidol, 4.2±1.2·10⁻⁶Mcis-flupenthixol, 2.7±0.4·10⁻⁵Mtrans-flupenthixol, >1·10⁻⁵M apomorphine, sulpiride, naloxone and isoproterenol.     

Kinetic analyses for bindings of concanavalin A to dispersed and condensed mannose surfaces on a quartz crystal microbalance

A biotinylated mannotriose (Man3-bio) was dispersively immobilized in the matrix of biotinylated lactose (Gal-Glc-bio) on a streptavidin-covered, 27-MHz quartz crystal microbalance (QCM), and binding kinetics of concanavalin A (Con A) to Man3-bio in the Gal-Glc-bio matrix could be obtained from frequency decreases (mass increases) of the QCM. Association constants (K_a) and binding and dissociation rate constants (k_on and k_off) could be determined separately as the 1:1 and 1:2 bindings of Con A to Man3-bio on the surface. When Man3-bio was immobilized with content of 1 to 5 mol% in the matrix, the 1:1 binding of Con A to Man3-bio was obtained as K_a = (4 ± 1) × 10⁶ M⁻¹, k_on = (4 ± 1) × 10⁴ M⁻¹ s⁻¹, and k_off = (12 ± 2) × 10^–3 s⁻¹. On the contrary, when Man3-bio was immobilized with content of 20 to 100 mol% in the matrix, the 1:2 binding of Con A to Man3-bio was obtained as K_a = (14 ± 2) × 10⁶ M⁻¹, k_on = (14 ± 2) × 10⁴ M⁻¹ s⁻¹, and k_off = (7 ± 2) × 10^–3 s⁻¹. Thus, K_a for the 1:2 binding was 10 times larger than that for the 1:1 binding, with a three times larger binding rate constant (k_on) and a three times smaller dissociation rate constant (k_off). This is the first example to obtain separate kinetic parameters for the 1:1 and 1:2 bindings of lectins to carbohydrates on the surface.     

Active NaCl absorption across split lamellae of posterior gills of Chinese crabs (Eriocheir sinensis) adapted to different salinities

Split lamellae of posterior gills of Eriocheir sinensis adapted to fresh water, brackish waters (9 or 18‰) or seawater (36‰) were mounted in Ussing chambers, and transepithelial short-circuit currents and conductances were measured with salines, containing approximately in vivo-like NaCl concentrations. Active sodium and chloride absorption (I_Na and I_Cl), the transcellular conductances and the leak conductance were identified with external amiloride and/or DIDS. Split gill lamellae of crabs adapted to fresh water displayed similar magnitudes of I_Na and I_Cl with 10 mmol l⁻¹ NaCl in the external medium (internally haemolymph-like NaCl saline). Augmenting external NaCl (50 mmol l⁻¹) resulted in an increase of I_Cl, whereas I_Na decreased. Split gill lamellae of crabs adapted to brackish waters (external NaCl of 125 and 225 mmol l⁻¹, respectively) showed lower currents than preparations of freshwater crabs (50 mmol l⁻¹ external NaCl). With split gill lamellae of seawater crabs no currents were detected (450 mmol l⁻¹ NaCl on both sides). The transcellular conductances showed similar changes as the currents. The leak conductance of split gill lamellae of crabs adapted to fresh or brackish waters was low (0.3–0.8 mS cm⁻²), whereas it was much higher (7 mS cm⁻²) with preparations of seawater crabs.     

Thyroid hormone: Aldosterone antagonism in cultured epithelial cells

Thyroid hormone (T₃) has been demonstrated to inhibit the action of aldosterone on sodium transport in toad urinary bladder and rat kidney. We have exammined the effect of T₃ on aldosterone action and specific nuclear binding in cultured epithelial cells derived from toad urinary bladder. In cell line TB6-C, addition of 5·10⁻⁸ M T₃ to culture media for up to 3 days results in no change in short-circuit current or transepithelial resistance. This concentration of T₃ completely inhibits the maximal increase in short-circuit current in response to 1·10⁻⁷ M aldosterone. The inhibition can be demonstrated with 18 h preincubation or with simultaneous addition of T₃ and aldosterone. The half-maximal concentration for the inhibition of the aldosterone effect is approx. 5·10⁻⁹ M T₃. T₃ has no effect on cyclic AMP-stimulated short-circuit current in these cells. The effect of T₃ on nuclear binding of [³H]aldosterone was examined using a filtration assay with data analysis by at least-squares curve-fitting program. Best fit was obtained with a model for two binding sites. The dissociation constants for the binding were K′_d1 = (0.82 ± 0.36)·10⁻¹⁰ M and K′_d2 = (3.2±0.60)·10⁻⁸ M.The half-maximal concentration for aldosterone-stimulated sodium transport in these cells is approx. 1·10⁻⁸ M. Analysis of nuclear aldosterone binding in cells preincubated for 18 h with 5·10⁻⁸ M T₃ showed a K′_d1 = (0.15 ± 0.10)·10⁻¹⁰ M and K′_d2 = (3.5 ± 0.10)·10⁻⁸ M. We conclude that T₃ i action of aldosterone on sodium transport at a site after receptor binding in the nucleus.