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.     

Deposition of BaSO4 in the tight junctions of amphibian epithelia causes their opening; apical Ca2+ reverses this effect

Selective deposition of BaSO₄ in the tight junctions (TJs) of frog skins led to profound and reversible functional alterations of these structures, as revealed by changes of tissue conductance (G), clamping current (I), and fluxes of extracellular markers (sulfate (J^SO ₄) and sucrose (J^SUC)). Experiments were performed with nominally Ca²⁺ -free simple salt solutions on the apical side (usually KCl) and Na₂SO₄-Ringer on the inner side of skins. The deposition of BaSO₄ in the TJs was obtained by diffusion and/or migration through the paracellular path of Ba²⁺ from the apical solution and SO ₄ ^2– from the inner solution. A brief presence (2 to 6 min) of apical Ba²⁺ (Ba²⁺ pulse) is followed (i.e., when Ba²⁺ is removed from the apical fluid) by a large increase of G, I, J^SO ₄ and J^SUC, above pre-Ba²⁺ levels. These attain a steady state within 15 to 30 min (overshoot phase), characterizing a conspicuous increase of the paracellular permeability. During the overshoot phase, a second Ba²⁺ pulse blocks the paracellular route while apical Ba²⁺ is present, leading to a new and larger overshoot when the Ba²⁺ pulse is terminated. Addition of apical Ca²⁺ triggers the resealing of the TJs, resulting in a full recovery of G, I, J^SO ₄ and J^SUC. This Ca²⁺ -induced recovery persists when apical Ca²⁺ is removed. The presence of a normal Ca²⁺ concentration in the inner bathing Ringer does not induce the recovery process. Tissues remain viable after being submitted to the Ba²⁺ treatment and the subsequent overshoot. Experiments performed in the urinary bladder of Rana catesbeiana and skins and urinary bladders of Bufo marinus indicate that Ba²⁺ effect can also be elicited in these tissues. The above results seem to report general properties of the TJs. Incidentally, they warn about the use of Ba²⁺ as an ion channel blocker in epithelial membranes in association with SO ₄ ^2– -containing solutions on the contralateral side.This project was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (91/0293-7 to F.L.V., and 90/1788-1 to A.S.), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (410068/90-0 and 303633-85/BF to F.L.V.). J.A.C. received a doctoral fellowship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior/Fundação Universidade do Rio Grande. We thank Dr. Alice T. Ferreira for help in the measurements of free Ca²⁺ concentration.     

Chloride-activated water permeability in the frog corneal epithelium

We have previously reported that the isolated frog corneal epithelium (a Cl^–-secreting epithelium) has a large diffusional water permeability (P_dw 1.8×10^–4 cm/s). We now report that the presence of Cl^– in the apical-side bathing solution increases the diffusional water flux, J_dw (in both directions) by 63% from 11.3 to 18.4 l min^–1 · cm^–2 with 60 mm [Cl] exerting the maximum effect. The presence of Cl^– in the basolateral-side bathing solution had no effect on the water flux. In Cl^–-free solutions amphotericin B increased J_dw by 29% but only by 3% in Cl^–-rich apical-side bathing solution, suggesting that in Cl^–-rich apical side bathing solution, the apical barrier is no longer rate limiting. Apical Br^– (75 mm) also increased J_dw by 68%. The effect of Cl^– on J_dw was observed within 1 min after its addition to the apicalside bathing solution. HgCl₂ (0.5 mm) reduced the Cl^–-increased P_dw by 31%. The osmotic permeability (P_f) was also measured under an osmotic gradient yielding values of 0.34 and 2.88 (x 10^–3 cm/s) in Cl^–-free and Cl^–-rich apical-side bathing solutions respectively. It seems that apical Cl^–, or Cl^– secretion into the apical bath could activate normally present but inactive water channels. In the absence of Cl^–, water permeability of the apical membrane seems to be limited to the permeability of the lipid bilayer.This work was supported by National Eye Institute grants EY-00160 and EY-01867.     

Pre-steady-state analysis of the turn-on and turn-off of water permeability in the kidney collecting tubule

Summary Water transport across the mammalian collecting tubule is regulated by vasopressin-dependent water channel insertion into and retrieval from the cell apical membrane. The time course of osmotic water permeability (P _f ) following addition and removal of vasopressin (VP) and 8-Br-cAMP was measured continuously by quantitative fluorescence microscopy using an impermeant fluorophore perfused in the lumen. Cortical collecting tubules were subjected to a 120 mOsm bath-to-lumen osmotic gradient at 37°C with 10–15 nl/min lumen perfusion and 10–20 ml/min bath exchange rate. With addition of VP (250 U/ml), there was a 23±3 sec (sem,n=16) lag in whichP _f did not change, followed by a rise inP _f (initial rate 1.4±0.2×10^–4 cm/sec²) to a maximum of 265±10×10^–4 cm/sec. With addition of 8-Br-cAMP (0.01–1mm) there was an 11±2 sec lag. For [8-Br-cAMP]=0.01, 0.1 and 1mm, the initial rate ofP _f increase following the lag was (units 10^–4 cm/sec²): 1.1±0.1, 1.2±0.1 and 1.7±0.3. MaximumP _f was (units 10^–4 cm/sec): 64±4, 199±9 and 285±11. With removal of VP,P _f decreased to baseline (12×10^–4 cm/sec) with aT _1/2 of 18 min; removal of 0.1 and 1mm 8-Br-cAMP gaveT _1/2 of 4 and 8.5 min. These results demonstrate (i) a brief lag in theP _f response, longer for stimulation by VP than by 8-Br-cAMP, representing the transient build-up of biochemical intermediates proximal to the water channel insertion step, (ii) similar initialdP _f /dt (water channel insertion) over a wide range of [8-Br-cAMP] and steady-stateP _f values, and (iii) more rapidP _f decrease with removal of 8-Br-cAMP than with VP. These pre-steady-state results define the detailed kinetics of the turn-on and turn-off of tubuleP _f and provide kinetic evidence that the rate-limiting step for turn-on ofP _f is not the step at which VP regulates steady-stateP _f . If water channel insertion is assumed to be the rate-limiting step in the turn-on ofP _f , these results raise the possibility that water channels must be activated following insertion into the apical membrane.     

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.     

Electrolytes control flows of water across the apical barrier in toad skin: The hydrosmotic salt effect

Summary The reversible dependence of skin osmotic water permeability (L _PD ) upon the ionic concentration of the outer bathing solution — which we have called hydrosmotic salt effect (HSE) — was studied in the isolated skin of the toadBufo marinus ictericus. The skin osmotic water flow (J _V ) was measured as a function of outer bathing solution osmolality (O _e ).L _PD , calculated as (J _v /) _P=0 (where and P are the osmotic and hydrostatic pressure differences across the skin, respectively) was constant whenO _e was altered with sucrose, a nonelectrolyte. In contrast,L _PD increased continuously in the hypotonic range asO _e was raised from zero (distilled water) with NaCl or KCl. The HSE could also be evoked in the condition of reversed osmotic volume flow, with the outer bathing medium made hypertonic with sucrose.Diffusional¹⁴C-sucrose permeability, measured in theJ _v =0 condition to prevent solvent drag of sucrose in the paracellular pathways, indicate that the hydrosmotic salt effect cannot be explained by assuming a paracellular permeability increase, due to tight junction opening, but might be interpreted as due to changes in the osmotic water permeability of the apical membranes of the most superficial cells of the epithelium.The hydrosmotic salt effect can be elicited in control skins and in vasopressin-stimulated skins, on top of the hormonal response.The time course of the hydrosmotic salt effect is substantially different from that of the hydrosmotic response to vasopressin. Its half-time is 4 to 5 times faster than that of vasopressin action, with individual values as short as 1.5 min.The time courses of the hydrosmotic salt-effect onset and reversibility are exponential, clearly contrasting with the typical sigmoidal shape of vasopressin onset and washout time courses.Based on time course data and on speed of response we postulate that the mechanism underlying the hydrosmotic salt effect is due to modifications of existing water pathways in the apical membrane, rather than to incorporation and removal of water permeability units in this structure.     

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.     

Maturational Changes in Rabbit Renal Brush Border Membrane Vesicle Osmotic Water Permeability

We have recently shown that the osmotic water permeability (P _f ) of proximal tubules from neonatal rabbits is higher than that of adults (AJP 271:F871-F876, 1996). The developmental change in P _f could be due to differences in one or more of the components in the path for transepithelial water transport. The present study examined developmental changes in water transport characteristics of the proximal tubule apical membrane by determining P _f and aquaporin 1 (AQP1) expression in neonatal (10–14 days old) and adult rabbit renal brush border membrane vesicles (BBMV). AQP1 abundance in the adult BBMV was higher than the neonatal BBMV. At 25°C the P _f of neonatal BBMV was found to be significantly lower than the adult BBMV at osmotic gradients from 50 to 250 mOsm/kg water. The activation energy for osmotic water movement was higher in the neonatal BBMV than the adult BBMV (9.19 ± 0.37 vs. 5.09 ± 0.57 kcal · deg⁻¹· mol⁻¹, P < 0.005). Osmotic water movement in neonatal BBMV was inhibited 17.9 ± 1.3% by 1 mm HgCl₂ compared to 34.3 ± 3.8% in the adult BBMV (P < 0.005). These data are consistent with a significantly greater fraction of water traversing the apical membrane lipid bilayer in proximal tubules of neonates than adults. The lower P _f of the neonatal BBMV indicates that the apical membrane is not responsible for the higher transepithelial P _f in the neonatal proximal tubule. Received: 18 December 1997/Revised: 3 April 1998     

Osmotic water permeability of plasma and vacuolar membranes in protoplasts I. High osmotic water permeability in radish ( Raphanus sativus ) root cells as measured by a new method

Intra- and transcellular water movements in plants are regulated by the water permeability of the plasma membrane (PM) and vacuolar membrane (VM) in plant cells. In the present study, we investigated the osmotic water permeability of both PM (P _f1) and VM (P _f2), as well as the bulk osmotic water permeability of a protoplast (P _f(bulk)) isolated from radish (Raphanus sativus) roots. The values of P _f(bulk) and P _f2 were determined from the swelling/shrinking rate of protoplasts and isolated vacuoles under hypo- or hypertonic conditions. In order to minimize the effect of unstirred layer, we monitored dropping or rising protoplasts (vacuoles) in sorbitol solutions as they swelled or shrunk. P _f1 was calculated from P _f(bulk) and P _f2 by using the ‘three-compartment model’, which describes the theoretical relationship between P _f1, P _f2 and P _f(bulk) (Kuwagata and Murai-Hatano in J Plant Res, 2007). The time-dependent changes in the volume of protoplasts and isolated vacuoles fitted well to the theoretical curves, and solute permeation of PM and VM was able to be neglected for measuring the osmotic water permeability. High osmotic water permeability of more than 500 μm s⁻¹, indicating high activity of aquaporins (water channels), was observed in both PM and VM in radish root cells. This method has the advantage that P _f1 and P _f2 can be measured accurately in individual higher plant cells. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users. It includes four appendices, four tables and two figures. Mari Murai-Hatano and Tsuneo Kuwagata contributed equally to the paper. An erratum to this article is available at .     

Effect of dDAVP on basolateral cell surface water permeability in the outer medullary collecting duct

We report a novel approach for assessing the volume of living cells which allows quantitative, high-resolution characterization of dynamic changes in cell volume while retaining the cell functionality. The aim of this study was to evaluate the short-term effect of vasopressin on basolateral cell surface water permeability in the outer medullary collecting duct (OMCD). The permeability of the basolateral cell membrane was determined in the tubules where the apical membrane was blocked with oil injected into the lumen. The apparent coefficient of water permeability (P _f) was evaluated by measuring the cell swelling after the step from hypertonic to isotonic medium (600 mosm to 300 mosm). Desmopressin (dDAVP) induced an increase of the basolateral P _f from 113.7±8.5 μm/s in control cells to 186.6±11.4 μm/s in micro-dissected fragments of the OMCD incubated in vitro (10⁻⁷ M dDAVP, 30 min at 37 °C) (P<0.05). Mercury caused pronounced inhibition of basolateral water permeability (26.0±6.9 μm/s; P<0.05). The effect of mercury (1.0 mM HgCl₂) was reversible: after washing the fragments with PBS for 20 min, P _f values were restored to the control levels (125.0±9.5 μm/s). The results of the study indicate the existence of a mechanism controlling the osmotic water permeability of the basolateral cell membrane in the OMCD epithelium.     

Direct and Indirect Effects of Mutations at the Outer Mouth of the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Pore

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channel pore is thought to contain multiple binding sites for permeant and impermeant anions. Here, we investigate the effects of mutation of different positively charged residues in the pore on current inhibition by impermeant Pt(NO₂)₄²⁻ and suramin anions. We show that mutations that remove positive charges (K95, R303) influence interactions with intracellular, but not extracellular, Pt(NO₂)₄²⁻ ions, consistent with these residues being situated within the pore inner vestibule. In contrast, mutation of R334, supposedly located in the outer vestibule of the pore, affects block by both extracellular and intracellular Pt(NO₂)₄²⁻. Inhibition by extracellular Pt(NO₂)₄²⁻ requires a positive charge at position 334, consistent with a direct electrostatic interaction resulting in either open channel block or surface charge screening. In contrast, inhibition by intracellular Pt(NO₂)₄²⁻ is weakened in all R334-mutant forms of the channel studied, inconsistent with a direct interaction. Furthermore, mutation of R334 had similar effects on block by intracellular suramin, a large organic molecule that is apparently unable to enter deeply into the channel pore. Mutation of R334 altered interactions between intracellular Pt(NO₂)₄²⁻ and extracellular Cl⁻ but not those between intracellular Pt(NO₂)₄²⁻ and extracellular Pt(NO₂)₄²⁻. We propose that while the positive charge of R334 interacts directly with extracellular anions, mutation of this residue also alters interactions with intracellular anions by an indirect mechanism, due to mutation-induced conformational changes in the protein that are propagated some distance from the site of the mutation in the outer mouth of the pore.     

Effects of PCMBS on the water and small solute permeabilities in frog urinary bladder

Summary It has been reported that PCMBS (p-chloromercuribenzene sulfonate) blocks the water permeability of red cells and of the tubular kidney membranes. In this study we compare the effects of this mercurial compound on the permeability of water and other small solutes in the frog urinary bladder.We observed that: (i) 5mm PCMBS applied at pH 5.0 to the mucosal side inhibited the net and unidirectional water fluxes induced by oxytocin without changing the P _f/P _d ratio. (ii) The oxytocin-induced urea and Na⁺ influxes were also inhibited by PCMBS. (iii) The unidirectional Cl^– movement was first reduced and then increased during the course of PCMBS treatment. (iv) The short-circuit measured at low mucosal Na⁺ concentration (10mm), diminished continuously, whereas the transepithelial resistance first increased and then diminished. (v) Mannitol, raffinose, -methyl-glucose, antipyrine, caffeine and Rb⁺ movements were not changed significantly during the first 26 min of the water permeability inhibition. In conclusion: (i) The ADH-sensitive water, urea and Na⁺ transport systems were inhibited by PCMBS, (ii) PCMBS did not induce a nonspecific and general effect on the permeability of the membrane during the development of the water permeability inhibition, and (iii) in terms of water channels, the inhibition of water transport with the maintenance of a highP _f/P _d ratio suggests that PCMBS closes the water channels in an all or none manner, reducing their operative number in the apical border of frog bladder.     

Control of sodium permeability of the outer barrier in toad skin

Summary The²⁴Na efflux (J _eff ^Na ) (i.e., the rate of appearance of²⁴Na in the outer compartment) in the isolated short-circuited toad skin bathed by NaCl-Ringer's solution on both sides is composed of para- and transcellular components of almost equal magnitudes. This relies on the assumption that amiloride acts on the transcellular component only and could block it completely.Ouabain induces a large transient increase of the transcellular component. This increase, which starts within a few minutes after the addition of ouabain, is due to electrical depolarization of the outer barrier, rather than a consequence of blocking Na recirculation across the inner barrier. The subsequent decline ofJ _eff ^Na , which takes place after the ouabain-inducedJ _eff ^Na peak, is due to a progressive block of outer barrier Na channels with time, which can eventually be complete, depending on the duration of action of ouabain. As the external Na concentration was always kept high and constant in these experiments, the results indicate that a rise in cell Na concentration, and not in the outer bathing solution, is the signal that triggers the reduction of outer barrier Na permeability (P ₀ ^Na ).Ouabain has no effect uponJ _eff ^Na with Na-free solution bathing the outer and NaCl-Ringer's solution the inner skin surface, showing the importance of Na penetration across the outer barrier, and not across the inner barrier due to its low Na permeability, in the process of closing the Na channels of this structure.Step changes from Na 115mm to Na-free external solution, or vice-versa, may affect both the outer barrier electrical potential difference (PD₀) and cell Na concentration (Na) _c . Therefore, the behavior ofJ _eff ^Na depends on which variable (if PD₀ or (Na) _c regulated outer barrier Na permeability) is most affected by step changes in outer bathing solution Na concentration.Amiloride in the control condition blocks the transcellular component ofJ _eff ^Na . However, in the condition of approximate short-circuiting of the outer barrier and high cellular Na concentration induced by long term effects of ouabain, when the Na channels of the outer barrier are already blocked by elevated cell Na concentration, amiloride may induce the opposite effect, increasing Na permeability of the outer barrier.With outer barrier Na channels completely blocked by high cell Na concentration, PCMB in the outer bathing medium induces a large increase ofJ _eff ^Na , rendering these channels again amiloride sensitive.The results are consistent with the notion that Na efflux from cell compartment to the outer bathing solution goes through the amiloride-sensitive Na channels of the apical border of the superficial cell layer of toad skin, with an apparent Na permeability modulated by cell ionic environment, most probably the cell Na concentration.The ensemble of the present results are consistent with Na permeability regulation taking place at the outer barrier level. However, this precise location could only be made unambiguously by measurements across the individual outer cell membranes.     

Osmotic water permeability of plasma and vacuolar membranes in protoplasts II. Theoretical basis

Water permeability of the plasma membrane (PM) and the vacuolar membrane (VM) is important for intracellular and transcellular water movement in plants, because mature plant cells have large central vacuoles. We have developed a new method for measuring the osmotic water permeability of the PM and VM (P _f1 and P _f2, respectively) in individual plant cells. Here, the theoretical basis and procedure of the method are discussed. Protoplasts isolated from higher plant tissues are used to measure P _f1 and P _f2. Because of the semi-permeability (selective permeability) of cellular membranes, protoplasts swell or shrink under hypotonic or hypertonic conditions. A theoretical three-compartment model is presented for simulating time-dependent volume changes in the vacuolar and cytoplasmic spaces in a protoplast during osmotic excursions. The model describes the theoretical relationships between P _f1, P _f2 and the bulk osmotic water permeability of protoplasts (P _f(bulk)). The procedure for measuring the osmotic water permeability is: (1) P _f(bulk) is calculated from the time when half of the total change in protoplast volume is completed, by assuming that the protoplast has a single barrier to water movement across it (two-compartment model); (2) P _f2 of vacuoles isolated from protoplasts is obtained in the same manner; and (3) P _f1 is determined from P _f(bulk) and P _f2 according to the three-compartment model. The theoretical relationship between P _fl (m s⁻¹) and L _Pl (hydraulic conductivity, l=1, 2) (m s⁻¹ Pa⁻¹) is also discussed. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorised users. Tsuneo Kuwagata and Mari Murai-Hatano contributed equally to the paper.     

Ion selectivity of the apical membrane Na channel in the toad urinary bladder

The ion selectivity of the apical membrane Na channel in the toad urinary bladder was investigated. The electrical potential difference and resistance across the basal-lateral membrane were reduced using high concentrations of KCl in the serosal bathing medium, and gradients for various ions were imposed across the apical membrane by altering the composition of the mucosal bathing medium. Ion fluxes through the channel were measured as the transepithelial current inhibited by amiloride, a specific blocker of the channel's Na conductance. The selectivity sequence for alkali metal cations was H greater than Li greater than Na much greater than K. K permeability was barely detectable; the selectivity for Na over K was about 1000:1. Ammonium, hydroxyl ammonium and hydrazinium ions were, like K, virtually impermeant. The results suggest that the size of the unhydrated ion is an important factor in determining permeability in this channel.     

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.     

Serial permeability barriers to water transport in human placental vesicles

Summary Microvillous vesicles were prepared from term human placenta by shearing, differential centrifugation and Mg²⁺ precipitation. Vesicles were purified further on a sucrose density gradient producing two bands with densities of 1.16 to 1.18 g/ml (C1) and 1.13 to 1.15 g/ml (C2). The C2 fraction, which had a 24-fold enrichment of alkaline phosphatase and a three-fold reduction in Na⁺, K⁺-ATPase activity compared to homogenates, was used to measure osmotic water (P _f ) permeability.P _f was measured from the time course of scattered light intensity following exposure of vesicles to specified gradients of impermeant solutes.P _f decreased from 3.0×10^–3 to 0.6×10^–3 cm/sec with increasing gradient size (65 to 730mm; 23°C). Four possible causes of this behavior were examined theoretically and experimentally: an unstirred layer, saturation of water transport, large changes in the vesicle surface area with changes in volume and a structural restriction to vesicle volume change. The measured dependence ofP _f on gradient size and the effect of the channel-forming ionophore gramicidin onP _f fit best to the theoretical dependences predicted by a structural restriction mechanism. This finding was supported by experiments involving the effects onP _f of increased solution viscosity, initial vesicle volume, the magnitude of transmembrane volume flow, and the effects of gradient size on activation energy (E _a) forP _f . The decreasedP _f resulting from a structural restriction limiting vesicle volume change was modeled mathematically as a second barrier in series with the vesicle membrane.E _a measured using a 250-mm inwardly directed sucrose gradient was 5.4±0.6 kcal/mol (T>27°C) and 10.0±0.6 kcal/mol (T<27°C).E _a above 27°C is in the range normally associated with transmembrane passage of water via aqueous channels. Water transport was not inhibited byp-chloromercuribenzenesulfonate.     

Osmosis in Cortical Collecting Tubules : ADH-Independent Osmotic Flow Rectification

The present experiments were designed to evaluate the effects of varying the osmolality of luminal solutions on the antidiuretic hormone (ADH)-independent water and solute permeability properties of isolated rabbit cortical collecting tubules. In the absence of ADH, the osmotic water permeability coefficient (cm s^–1) P_f^l→b, computed from volume flows from hypotonic lumen to isotonic bath, was 20 ± 4 x 10^–4 (SEM); the value of P_f^b→l in the absence of ADH, computed from volume flows from isotonic bath to hypertonic lumen, was 88 ± 15 x 10^–4 cm s^–1. We also measured apparent urea permeability coefficients (cm s^–1) from ¹⁴C-urea fluxes from lumen to bath (P_DDurea^l→b) and from bath to lumen (P_DDurea^b→l). For hypotonic luminal solutions and isotonic bathing solutions, P_DDurea^l→b was 0.045 ± 0.004 x 10^–4 and was unaffected by ADH. The ADH-independent values of P_DDurea^l→b and P_urea^b→l were, respectively, 0.216 ± 0.022 x 10^–4 cm s^–1 and 0.033 ± 0.002 x 10^–4 cm s^–1 for isotonic bathing solutions and luminal solutions made hypertonic with urea, i.e., there was an absolute increase in urea permeability and asymmetry of urea fluxes. Significantly, P_DDurea^l→b did not rise when luminal hypertonicity was produced by sucrose; and, bathing fluid hypertonicity did not alter tubular permeability to water or to urea. We interpret these data to indicate that luminal hypertonicity increased the leakiness of tight junctions to water and urea but not sucrose. Since the value of P_f^b→l in the absence of ADH, when tight junctions were open to urea, was approximately half of the value of P_f^l→b in the presence of ADH, when tight junctions were closed to urea, we conclude that tight junctions are negligible paracellular shunts for lumen to bath osmosis with ADH. These findings, together with those in the preceding paper, are discussed in terms of a solubility-diffusion model for water permeation in which ADH increases water solubility in luminal plasma membranes.     

Effects of external sodium and cell membrane potential on intracellular chloride activity in gallbladder epithelium

Summary Conventional and Cl-selective liquid ion-exchanger intracellular microelectrodes were employed to study the effects of extracellular ionic substitutions on intracellular Cl activity (aCl _i ) inNecturus gallbladder epithelium. As shown previously (Reuss, L., Weinman, S.A., 1979;J. Membrane Biol. 49:345), when the tissue was exposed to NaCl-Ringer on both sidesaCl _i was about 30mm, i.e., much higher than the activity predicted from equilibrium distribution (aCl_eq) across either membrane (5–9mm). Removal of Cl from the apical side caused a reversible decrease ofaCl _i towards the equilibrium value across the basolateral membrane. A new steady-stateaCl _i was reached in about 10 min. Removal of Na from the mucosal medium or from both media also caused reversible decreases ofaCl _i when Li, choline, tetramethylammonium or N-methyl-d-glucamine (NMDG) were employed to replace Na. During bilateral Na substitutions with choline the cells depolarized significantly. However, no change of cell potential was observed when NMDG was employed as Na substitute. Na replacements with choline or NMDG on the serosal side only did not changeaCl _i . When K substituted for mucosal Na, the cells depolarized andaCl _i rose significantly. Combinations of K for Na and Cl for SO₄ substitutions showed that net Cl entry during cell depolarization can take place across either membrane. The increase ofaCl _i in depolarized cells exposed to K₂SO₄-Ringer on the mucosal side indicates that the basolateral membrane Cl permeability, (P _Cl) increased. These results support the hypothesis that NaCl entry at the apical membrane occurs by an electroneutral mechanism, driven by the Na electrochemical gradient. In addition, we suggest that Cl entry during cell depolarization is downhill and involves an increase of basolateral membraneP _Cl.     

Aldosterone control of the density of sodium channels in the toad urinary bladder

Summary Near-instantaneous current-voltage relationships and shot-noise analysis of amiloride-induced current fluctuations were used to estimate apical membrane permeability to Na (P _Na), intraepithelial Na activity (Na _c ), single-channel Na currents (i) and the number of open (conducting) apical Na channels (N₀), in the urinary bladder of the toad (Bufo marinus). To facilitate voltageclamping of the apical membrane, the serosal plasma membranes were depolarized by substitution of a high KCl (85mm) sucrose (50mm) medium for the conventional Na-Ringer's solution on the serosal side.Aldosterone (5×10^–7 m, serosal side only) elicited proportionate increases in the Na-specific current (I _Na and inP _Na, with no significant change in the dependence ofP _Na on mucosal Na (Na _o ).P _Na and the control ofP _Na by aldosterone were substrate-dependent: In substrate-depleted bladders, pretreatment with aldosterone markedly augmented the response to pyruvate (7.5×10^–3 m) which evoked coordinate and equivalent increases inI _Na andP _Na.The aldosterone-dependent increase inP _Na was a result of an equivalent increase in the area density of conducting apical Na channels. The computed single-channel current did not change. We propose that, following aldosterone-induced protein synthesis, there is a reversible metabolically-dependent recruitment of preexisting Na channels from a reservoir of electrically undetectable channels. The results do not exclude the possibility of a complementary induction of Na-channel synthesis.