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     

Microscopical determination of the filtration permeability of the mucosal surface of the goldfish intestinal epithelium

Summary The rate of shrinkage of the mucosal folds of goldfish intestine in response to mucosal hypertonicity was measured by microscopic means. Because of the geometry of the intestinal folds the rate of shrinkage could be directly related to the loss of volume from the fold through the brush border membranes and tight junctions. Experimentally a wide range of velocities was observed, reflecting the difficulty of rapidly estabilishing a uniform osmotic gradient at the preparation's mucosal surface. The initial velocity of volume loss provided a measure of the filtration permeability (P _f ) of the mucosal surface. From the highest velocities observed the filtration permeability was estimated to be approximately 14×10^–3 cm/sec related to the folded mucosal surface and 65×10^–3 cm/sec related to the straight serosal surface. Consideration of the experimental errors and unstirred layer effects make it probable that the latter value is still an underestimate of the trueP _f . The series barriers of the epithelium cause the total tissueP _f to be less than theP _f of the mucosal surface alone. In addition theP _f measured in the presence of an osmotic gradient may differ substantially from the tissue filtration permeability which exists in the absence of a change in osmolarity.     

The role of the lateral intercellular spaces and solute polarization effects in the passive flow of water across the rabbit gallbladder

Summary Osmotic water flows were measured acrossin vitro preparations of the rabbit gallbladder by a gravimetric technique. The bladders exhibited asymmetrical osmotic behavior, in which theL _p (hydraulic conductivity) for water flow from mucosa to serosa was up to four times greater than theL _p for water flow in the opposite direction. This result is similar to the effects of osmotic gradients on ion and nonelectrolyte permeability reported in the first paper. As in the case of solute permeability, these changes inL _p are accounted for by changes in the dimensions of the lateral intercellular spaces of the epithelium. These spaces are thus a final common pathway for the movement of both solutes and water across the epithelium. We also observed osmotic flow transients in which the initialL _p was about an order of magnitude greater than the steady stateL _p . These transients are largely explained by solute polarization in the unstirred layers adjacent to the epithelial membranes. A comparison between streaming potentials and water flows showed that streaming potentials are directly proportional to the rate of flow only over a limited range. These observations are readily explained on the basis of structural changes and solute polarization effects. Finally, the routes of water flow across epithelia are discussed in the light of our observations.     

Filtration Coefficient of the Axon Membrane As Measured with Hydrostatic and Osmotic Methods

The hydraulic conductivity of the membranes surrounding the giant axon of the squid, Dosidicus gigas, was measured. In some axons the axoplasm was partially removed by suction. Perfusion was then established by insertion of a second pipette. In other axons the axoplasm was left intact and only one pipette was inserted. In both groups hydrostatic pressure was applied by means of a water column in a capillary manometer. Displacement of the meniscus in time gave the rate of fluid flowing across the axon sheath. In both groups osmotic differences across the membrane were established by the addition of a test molecule to the external medium which was seawater. The hydraulic conductivity determined by application of hydrostatic pressure was 10.6 ± 0.8.10^-8 cm/sec cm H₂O in perfused axons and 3.2 ± 0.6.10^-8 cm/sec cm H₂O in intact axons. When the driving force was an osmotic pressure gradient the conductivity was 4.5 ± 0.6 x 10^-10 cm/sec cm H₂O and 4.8 ± 0.9 x 10^-10 cm/sec cm H₂O in perfused and intact axons, respectively. A comparable result was found when the internal solution was made hyperosmotic. The fluid flow was a linear function of the hydrostatic pressure up to 70 cm of water. Glycerol outflux and membrane conductance were increased 1.6 and 1.1 times by the application of hydrostatic pressure. These increments do not give an explanation of the difference between the filtration coefficients. Other possible explanations are suggested and discussed.     

Osmotic water permeabilities of human placental microvillous and basal membranes

Summary Literature data suggest that water accumulation by the human fetus is driven by osmotic gradients of small solutes. However, the existence of such gradients has not been supported by prior measurements. Attempts to estimate the size of the gradient necessary to drive net water movement have been seriously hampered by the lack of permeability data for the syncytiotrophoblast membranes. Stopped-flow light scattering techniques were employed to measure the osmotic water permeability (P _f )of microvillous (MVM) and basal membrane (BM) vesicles isolated from human term placenta. At 37°C, the P _f was determined to be 1.9±0.06 × 10^+–3 cm/sec for MVM and 3.1±0.20 × 10^+–3 cm/sec for BM (mean ±SD, n = 6). At 23°C, P _f was reduced to 0.7±0.04 × 10^+–3 cm/sec in MVM and 1.6±0.05 × 10^+–3 cm/sec in BM. These P _f values are comparable to those observed in membranes where water has been shown to permeate via a lipid diffusive mechanism. Arrhenius plots of P _f over the range 20–40°C were linear, with activation energies of 13.6 ± 0.6 kcal/mol for MVM and 12.9±1.0 kcal/mol for BM. Water permeation was not affected by mercurial sulfhydryl agents and glucose transport inhibitors. These data clearly suggest that water movement across human syncytiotrophoblast membranes occurs by a lipid diffusion pathway. As noted in several other epithelial tissues, the basal membrane has a higher water permeability than the microvillous membrane. It is speculated that water accumulation by the human fetus could be driven by a solute gradient small enough to be within the error of osmolarity measurements.We thank the staff of the labor and delivery ward at University of San Francisco Medical Center for help in obtaining placental tissue. This work was supported by NIH grant HD 26392. Dr. Jansson was supported by the Sweden-America Foundation, The Swedish Society of Medicine, The Swedish Society for Medical Research, and the Swedish Medical Research Council.     

Apoplastic transport across young maize roots: effect of the exodermis

The uptake of water and of the fluorescent apoplastic dye PTS (trisodium 3-hydroxy-5,8,10-pyrenetrisulfonate) by root systems of young maize (Zea mays L.) seedlings (age: 11–21 d) has been studied with plants which either developed an exodermis (Casparian band in the hypodermis) or were lacking it. Steady-state techniques were used to measure water uptake across excised roots. Either hydrostatic or osmotic pressure gradients were applied to induce water flows. Roots without an exodermis were obtained from plants grown in hydroponic culture. Roots which developed an exodermis were obtained using an aeroponic (=mist) cultivation method. When the osmotic concentration of the medium was varied, the hydraulic conductivity of the root (Lp _r in m³ · m⁻² · MPa⁻¹ · s⁻¹) depended on the osmotic pressure gradient applied between root xylem and medium. Increasing the gradient (i.e. decreasing the osmotic concentration of the medium; range: zero to 40 mM of mannitol), increased the osmotic Lp _r. In the presence of hydrostatic pressure gradients applied by a pressure chamber, root Lp _r was constant over the entire range of pressures (0–0.4 MPa). The presence of an exodermis reduced root Lp _r in hydrostatic experiments by a factor of 3.6. When the osmotic pressure of the medium was low (i.e. in the presence of a strong osmotic gradient between xylem sap and medium), the presence of an exodermis caused the same reduction of root Lp _r in osmotic experiments as in hydrostatic ones. However, when the osmotic concentration of the medium was increased (i.e. the presence of low gradients of osmotic pressure), no marked effect of growth conditions on osmotic root Lp _r was found. Under these conditions, the absolute value of osmotic root Lp _r was lower by factors of 22 (hydroponic culture) and 9.7 (aeroponic culture) than in the corresponding experiments at low osmotic concentration. Apoplastic flow of PTS was low. In hydrostatic experiments, xylem exudate contained only 0.3% of the PTS concentration of the bathing medium. In the presence of osmotic pressure gradients, the apoplastic flow of PTS was further reduced by one order of magnitude. In both types of experiments, the development of an exodermis did not affect PTS flow. In osmotic experiments, the effect of the absolute value of the driving force cannot be explained in terms of a simple dilution effect (Fiscus model). The results indicate that the radial apoplastic flows of water and PTS across the root were affected differently by apoplastic barriers (Casparian bands) in the exodermis. It is concluded that, unlike water, the apoplastic flow of PTS is rate-limited at the endodermis rather than at the exodermis. The use of PTS as a tracer for apoplastic water should be abandoned. Received: 9 October 1997 / Accepted: 5 February 1998     

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).     

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.     

Radial and axial water transport in the sugar beet storage root

To evaluate the contribution of transcellular, apoplastic and symplastic pathways to water movements, horizontal (axial pathway) and vertical (radial pathway) sugar beet root (Beta vulgaris L.) slices were studied. Volume flows (Jv) were measured under hydrostatic and/or osmotic gradients, using a computer-based data-acquisition system. When tissues were tested under hydrostatic gradients (0.3 MPa m^-1) a much more important permeability was observed in the axial pathway, as compared with the radial one. Negative pressure gradients (tensions) were as effective as positive ones in inducing a net water movement. After the establishment of a concentration gradient in the radial pathway (obtained by adding 300 M m^-3 mannitol to the employed solution) an osmotic flux, sensitive to HgCI2, was observed. The inhibitory effect of mercurial compounds was reversed by -mercaptoethanol while [¹⁴C] mannitol unidirectional fluxes were not affected by mercurial agents. In the axial pathway, the presence of a mannitol gradient did not develop a sustained osmotic flux. After an initial Jv in the expected direction, the Jv reversed and moved in the opposite way. It is concluded that, in the sugar beet root, water channels play a significant role in water transfers in the radial pathway. On the other side, water and solutes are transported by a hydrostatic gradient in the xylem vessels. In general, these results extend and adapt to a storage root the 'composite transport model' first proposed by Steudle et al.     

Osmotic water permeabilities of brush border and basolateral membrane vesicles from rat renal cortex and small intestine

Summary The osmotic water permeabilityP _f of brush border (BBM) and basolateral (BLM) membrane vesicles from rat small intestine and renal cortex was studied by means of stopped-flow spectrophotometry. Scattered light intensity was used to follow vesicular volume changes upon osmotic perturbation with hypertonic mannitol solutions. A theoretical analysis of the relationship of scattered light intensity and vesicular volume justified a simple exponential approximation of the change in scattered light intensity. The rate constants extracted from fits to an exponential function were proportional to the final medium osmolarity as predicted by theory. For intestinal membranes, computer analysis of optical responses fitted well with a single-exponential treatment. For renal membranes a double-exponential treatment was needed, implying two distinct vesicle populations.P _f values for BBM and BLM preparations of small intestine were equal and amount to 60 m/sec. For renal preparations,P _f values amount to 600 m/sec for the fast component, BBM as well as BLM, and to 50 (BBM) and 99 (BLM) m/sec for the slow component. The apparent activation energy for water permeation in intestinal membranes was 13.3±0.6 and in renal membranes, 1.0±0.3 kCal/mole, between 25 and 35°C. The mercurial sulfhydryl reagentpCMBS inhibited completely and reversibly the highP _f value in renal brush border preparations. These observations suggest that in intestinal membranes water moves through the lipid matrix but that in renal plasma membranes water channels may be involved. From the highP _f values of renal membrane vesicles a transcellular water permeability for proximal tubules can be calculated which amounts to 1 cm/sec. This value allows for an entirely transcellular route for water flow during volume reabsorption.     

Osmosis in Cortical Collecting Tubules : A Theoretical and Experimental Analysis of the Osmotic Transient Phenomenon

This paper reports a theoretical analysis of osmotic transients and an experimental evaluation both of rapid time resolution of lumen to bath osmosis and of bidirectional steady-state osmosis in isolated rabbit cortical collecting tubules exposed to antidiuretic hormone (ADH). For the case of a membrane in series with unstirred layers, there may be considerable differences between initial and steady-state osmotic flows (i.e., the osmotic transient phenomenon), because the solute concentrations at the interfaces between membrane and unstirred layers may vary with time. A numerical solution of the equation of continuity provided a means for computing these time-dependent values, and, accordingly, the variation of osmotic flow with time for a given set of parameters including: P_f (cm s^–1), the osmotic water permeability coefficient, the bulk phase solute concentrations, the unstirred layer thickness on either side of the membrane, and the fractional areas available for volume flow in the unstirred layers. The analyses provide a quantitative frame of reference for evaluating osmotic transients observed in epithelia in series with asymmetrical unstirred layers and indicate that, for such epithelia, P_f determinations from steady-state osmotic flows may result in gross underestimates of osmotic water permeability. In earlier studies, we suggested that the discrepancy between the ADH-dependent values of P_f and P_DDw (cm s^–1, diffusional water permeability coefficient) was the consequence of cellular constraints to diffusion. In the present experiments, no transients were detectable 20–30 s after initiating ADH-dependent lumen to bath osmosis; and steady-state ADH-dependent osmotic flows from bath to lumen and lumen to bath were linear and symmetrical. An evaluation of these data in terms of the analytical model indicates: First, cellular constraints to diffusion in cortical collecting tubules could be rationalized in terms of a 25-fold reduction in the area of the cell layer available for water transport, possibly due in part to transcellular shunting of osmotic flow; and second, such cellular constraints resulted in relatively small, approximately 15%, underestimates of P_f.     

Osmotic responses of maize roots

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

Water permeability ofNecturus gallbladder epithelial cell membranes measured by nuclear magnetic resonance

Summary In order to assess the contribution of transcellular water flow to isosmotic fluid transport acrossNecturus gallbladder epithelium, we have measured the water permeability of the epithelial cell membranes using a nuclear magnetic resonance method. Spin-lattice (T ₁) relaxation of water protons in samples of gallbladder tissue where the extracellular fluid contained 10 to 20mm Mn²⁺ showed two exponential components. The fraction of the total water population responsible for the slower of the two was 24±2%. Both the size of the slow component, and the fact that it disappeared when the epithelial layer was removed from the tissue, suggest that it was due to water efflux from the epithelial cells. The rate constant of efflux was estimated to be 15.6±1.0 sec¹ which would be consistent with a diffusive membrane water permeabilityP _d of 1.6×10³ cm sec¹ and an osmotic permeabilityP _os of between 0.3×10⁴ and 1.4×10⁴ cm sec¹ osmolar¹. Using these data and a modified version of the standing-gradient model, we have reassessed the adequacy of a fluid transport theory based purely on transcellular osmotic water flow. We find that the model accounts satisfactorily for near-isosmotic fluid transport by the unilateral gallbladder preparation, but a substantial serosal diffusion barrier has to be included in order to account for the transport of fluid against opposing osmotic gradients.     

Kinetics of Water Transport in Eel Intestinal Vesicles

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

Water and ion permeability of a native collagen membrane

With the help of a ribonucleoprotein it is possible to precipitate collagen in a layer of fibers with a 700 Å period. As collagen is a constituent of many membrane systems in the body, it seemed interesting to investigate the permeability of ions and water through a native collagen membrane.The experiments were carried out with the help of an acryl glass apparatus, where an osmotic pressure, a hydrostatic pressure difference or both can be maintained between the two bulk phases separated by the membrane. The diffusion coefficients for NaCl and KCl were found to be comparable with those in other biological membranes (D_s = 9 · 10⁻⁷cm² · s⁻¹) whereas there is difference of more than three orders of magnitude in the hydraulic permeability (L_p = 6 cm⁴ · J⁻¹ · s⁻¹).Volume flow measurements caused by an osmotic gradient indicated that the reflection coefficient for NaCl and KCl is very small. In hydrostatic pressure experiments, the membrane shows a preferred direction for volume flows which seems to have something to do with the mode of preparation of the membrane.     

Epithelial water transport in a balanced gradient system.

The relationship between epithelial fluid transport, standing osmotic gradients, and standing hydrostatic pressure gradients has been investigated using a perturbation expansion of the governing equations. The assumptions used in the expansion are: (a) the volume of lateral intercellular space per unit volume of epithelium is small; (b) the membrane osmotic permeability is much larger than the solute permeability. We find that the rate of fluid reabsorption is set by the rate of active solute transport across lateral membranes. The fluid that crosses the lateral membranes and enters the intercellular cleft is driven longitudinally by small gradients in hydrostatic pressure. The small hydrostatic pressure in the intercellular space is capable of causing significant transmembrane fluid movement, however, the transmembrane effect is countered by the presence of a small standing osmotic gradient. Longitudinal hydrostatic and osmotic gradients balance such that their combined effect on transmembrane fluid flow is zero, whereas longitudinal flow is driven by the hydrostatic gradient. Because of this balance, standing gradients within intercellular clefts are effectively uncoupled from the rate of fluid reabsorption, which is driven by small, localized osmotic gradients within the cells. Water enters the cells across apical membranes and leaves across the lateral intercellular membranes. Fluid that enters the intercellular clefts can, in principle, exit either the basal end or be secreted from the apical end through tight junctions. Fluid flow through tight junctions is shown to depend on a dimensionless parameter, which scales the resistance to solute flow of the entire cleft relative to that of the junction. Estimates of the value of this parameter suggest that an electrically leaky epithelium may be effectively a tight epithelium in regard to fluid flow.     

Root hydraulic properties of spruce measured with the pressure probe

The root pressure probe was used for the first time to measure the hydraulic properties of entire root systems of youngPicea abies. Hydraulic conductance was measured by osmotic and hydrostatic pressure relaxation techniques. Osmotic experiments were conducted by changing the nutrient solution and hydrostatic experiments by causing flow across the root with the pressure probe and with external pressure applied to the root system or to the cut stem of the excised root system. Usually,Picea abies root systems did not develop appreciable root pressure (< 0.02 MPA) and could be induced to reach a root pressure of 0.07 MPa by treating with KNO₃. In general, hydraulic conductance of the root system was large, but it was much smaller in the osmotic than in the hydrostatic experiments. Both hydrostatic techniques gave similar results. The results were explainable by a composite transport model of the root.     

Maturational Changes in Rabbit Renal Basolateral Membrane Vesicle Osmotic Water Permeability

We have recently demonstrated that while the osmotic water permeability (P _f ) of neonatal proximal tubules is higher than that of adult tubules, the P _f of brush-border membrane vesicles from neonatal rabbits is lower than that of adults. The present study examined developmental changes in the water transport characteristics of proximal tubule basolateral membranes by determining aquaporin 1 (AQP1) protein abundance and the P _f in neonatal (10–14 days old) and adult rabbit renal basolateral membrane vesicles (BLMV). At 25°C the P _f of neonatal BLMV was significantly lower than the adult BLMV at osmotic gradients ranging from 40 to 160 mOsm/kg water. The activation energies for osmotic water movement were identical in the neonatal and adult BLMV (8.65 ± 0.47 vs. 8.86 ± 1.35 kcal · deg⁻¹· mol⁻¹). Reflection coefficients for sodium chloride and sodium bicarbonate were identical in both the neonatal and adult BLMV and were not different from one. Mercury chloride (0.5 mm) reduced osmotic water movement by 31.3 ± 5.5% in the adult BLMV, but by only 4.0 ± 4.0% in neonatal vesicles (P < 0.01). Adult BLMV AQP1 abundance was higher than that in the neonate. These data demonstrate that neonatal BLMV have a lower P _f and AQP1 protein abundance than adults and that a significantly greater fraction of water traverses the basolateral membrane lipid bilayer and not water channels in neonates compared to adults. The lower P _f of the neonatal BLMV indicates that the basolateral membrane is not responsible for the higher transepithelial P _f in the neonatal proximal tubule. Received: 8 July 1999/Revised: 9 November 1999     

Evidence for water channels in renal proximal tubule cell membranes

Summary Water transport mechanisms in rabbit proximal convoluted cell membranes were examined by measurement of: (1) osmotic (P _f ) and diffusional (P _d ) water permeabilities, (2) inhibition ofP _f by mercurials, and (3) activation energies (E _a ) forP _f .P _f was measured in PCT brush border (BBMV) and basolateral membrane (BLMV) vesicles, and in viable PCT cells by stopped-flow light scattering;P _d was measured in PCT cells by proton NMR T_i relaxation times using Mn as a paramagnetic quencher. In BLMV,P _f (0.019 cm/sec, 23°C) was inhibited 65% by 5mm pCMBS and 75% by 300 m HgCl₂ (K _l =42 m);E _a increased from 3.6 to 7.6 kcal/mole (15–40°C) with 300 m HgCl₂. In BBMV,P _f (0.073 cm/sec, 23°C,E _a =2.8 kcal/mole, <33°C and 13.7 kcal/mole, >33°C) was inhibited 65% with HgCl₂ withE _a =9.4 kcal/mole (15–45°C). Mercurial inhibition in BLMV and BBMV was reversed with 10 m mercaptoethanol. Viable PCT cells were isolated from renal cortex by Dounce homogenization and differential seiving. Impedence sizing studies show that PCT cells are perfect osmometers (100–1000 mOsm). Assuming a cell surface-to-volume ratio of 25,000 cm^–1,P _f was 0.010±0.002 cm/sec (37°C) andP _d was 0.0032 cm/sec.P _f was independent of osmotic gradient size (25–1000 mOsm) withE _a 2.5 kcal/mole (<27°C) and 12.7 kcal/mole (>27°C). CellP _f was inhibited 53% by 300 m HgCl₂ (23°C) withE _a 6.2 kcal/mole. These findings indicate that cellP _f is not restricted by extracellular or cytoplasmic unstirred layers and that cellP _f is not flow-dependent. The high BLMV and BBMVP _f , inhibition by HgCl₂, lowE _a which increases with inhibition, and the measuredP _f /P _d >1 in cells in the absence of unstirred layers provide strong evidence for the existence of water channels in proximal tubule brush border and basolateral membranes. These channels are similar to those found in erythrocytes and are likely required for rapid PCT transcellular water flow.     

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