Functional water channels are present in clathrin-coated vesicles from bovine kidney but not from brain

Targeting of water channels in renal epithelia may involve trafficking of clathrin-coated vesicles. We have isolated and measured the osmotic water permeability (Pf) of purified clathrin-coated vesicles from bovine kidney cortex and inner medulla, and bovine brain, a tissue not expected to contain "water channels." Brain-coated vesicles had a diameter of 80 nm in negatively stained preparations. Pf was measured by a stopped-flow light scattering technique. In brain-coated vesicles, water transport was functionally homogeneous with a low Pf of 0.0016 +/- 0.0001 cm/s (seven preparations, 23 degrees C). Pf was independent of osmotic gradient size (25-300 mOsm), not inhibited by mercurials, and not altered by removal of the clathrin coat. The activation energy (Ea) for Pf was high (11 +/- 1 kcal/mol less than 34 degrees C, 17 +/- 2 kcal/mol greater than 34 degrees C). Therefore, water channels are absent from brain-coated vesicles. In contrast, there were two functional populations of vesicles in coated vesicle preparations from both kidney cortex and medulla. One population of vesicles had low water permeability and no water channels, whereas a second population had high Pf (0.02 cm/s, 21 degrees C) that was inhibited by HgCl2, and low Ea (2-3 kcal/mol). The fraction of vesicles with high Pf was 52 +/- 3% (S.D., n = 3, cortical vesicles) and 26 +/- 3% (medullary vesicles). These results provide evidence that functional water channels are not present in clathrin-coated vesicles from the brain, whereas they are found in a population of coated vesicles from kidney cortex and medulla, tissues in which water channels are recycled between the plasma membrane, and an intracellular compartment.     

Proton nuclear magnetic resonance measurement of diffusional water permeability in suspended renal proximal tubules.

Diffusional water permeability was measured in renal proximal tubule cell membranes by pulsed nuclear magnetic resonance using proton spin-lattice relaxation times (T1). A suspension of viable proximal tubules was prepared from rabbit renal cortex by Dounce homogenization and differential sieving. T1 measured in a tubule suspension (22% of exchangeable water in the intracellular compartment) containing 20 mM extracellular MnCl2 was biexponential with time constants 1.8 +/- 0.1 ms and 8.3 +/- 0.2 ms (mean +/- SD, n = 8, 37 degrees C, 10 MHz). The slower time constant, representing diffusional exchange of water between intracellular and extracellular compartments, increased to 11.6 +/- 0.6 ms (n = 6) after incubation of tubules with 5 mM parachloromercuribenzene sulfonate (pCMBS) for 60 min at 4 degrees C and was temperature dependent with activation energy Ea = 2.9 +/- 0.4 kcal/mol. To relate T1 data to cell membrane diffusional water permeabilities (Pd), a three-compartment exchange model was developed that included intrinsic decay of proton magnetization in each compartment and apical and basolateral membrane water transport. The model predicted that the slow T1 was relatively insensitive to apical membrane Pd because of low luminal/cell volume ratio. Based on this analysis, basolateral Pd (corrected for basolateral membrane surface convolutions) is 2.0 X 10(-3) cm/s, much lower than corresponding values for basolateral Pf (10-30 X 10(-3) cm/s) measured in the intact tubule and in isolated basolateral membrane vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Water permeabilities and salt reflection coefficients of luminal, basolateral and intracellular membrane vesicles isolated from rabbit kidney proximal tubule

The mechanisms of water transport across the rabbit renal proximal convoluted tubule were approached by measuring osmotic permeabilities and solute reflection coefficients of the brush-border and the basolateral membranes. Plasma and intracellular membrane vesicles were isolated from rabbit renal cortex by centrifugation on a Percoll gradient. Three major turbidity bands were obtained: a fraction of purified basolateral membranes (BLMV), the two others being brush-border (BBMV) and endoplasmic reticulum (ERMV) membrane vesicles. The osmotic permeability (Pf) of the three types of vesicle was measured using stop-flow techniques and their geometry was determined by quasi-elastic light scattering. Pf was equal to 123 +/- 8 microns/s (n = 10) for BBMV, 166 +/- 10 microns/s (n = 10) for BLMV and 156 +/- 9 microns/s (n = 4) for ERMV (T = 26 degrees C). A transcellular water permeability, per unit of apical surface area, of 71 microns/s was calculated considering that the luminal and the basolateral membranes act as two conductances in series. This value is in close agreement, after appropriate normalizations, with previously reported transepithelial water permeabilities obtained using in vitro microperfusion techniques thus supporting the hypothesis of a predominantly transcellular route for water flow across rabbit proximal convoluted tubule. The addition of 0.4 mM HgCl2, a sulfhydryl reagent, decreased Pf about 60% in three types of membrane providing evidence for the existence of proteic pathways. NaCl and KCl reflection coefficients were measured and found to be close to one for plasma and intracellular membranes suggesting that the water channels are not shared by salts.     

High channel-mediated water permeability in rabbit erythrocytes: characterization in native cells and expression in Xenopus oocytes.

Erythrocytes from several mammalian species contain mercurial-sensitive water transporters. By a stopped-flow light scattering technique, osmotic water permeability (Pf) was exceptionally high in rabbit erythrocytes (0.053 +/- 0.002 cm/s) and reversibly inhibited by 98% by p-(chloromercuri)benzenesulfonate (pCMBS). The activation energy (Ea) was 4.6 kcal/mol (15-37 degrees C). pCMBS inhibition was half-maximal at 0.1 mM (60-min incubation); at 1 mM pCMBS, half-maximal inhibition occurred in 8 min. Pf was also inhibited by HgCl2 and pCMB with greater than 90% inhibition in 5 min. There was no inhibition by high concentrations of phloretin, DNDS, cytochalasin B, amiloride, ouabain, furosemide, and several proteases. In defolliculated Xenopus oocytes microinjected with 50 nL of water or unfractionated mRNA (1 mg/mL) from rabbit reticulocytes, oocyte Pf assayed at 10 degrees C after 72-h incubation increased from (4 +/- 1) X 10(-4) cm/s (water injected) to (18 +/- 2) X 10(-4) cm/s (mRNA injected). Pf increased linearly with [mRNA] (0-75 ng/oocyte) and was inhibited slowly and reversibly by pCMBS and immediately by HgCl2 but not by cytochalasin B, phloretin, or DNDS. Ea was 9.6 kcal/mol (water injected) and 2.6 kcal/mol (mRNA injected). These results demonstrate that rabbit erythrocytes have the highest Pf and the greatest percentage inhibition of Pf by mercurials of any mammalian erythrocyte studied. The characteristics of the expressed and native water channels were similar, suggesting that the erythrocyte water channel is a membrane protein suitable for expression cloning.     

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     

Vasopressin-dependent water permeability of the basolateral membrane of the kidney outer medullary collecting duct in postnatal ontogenesis in rats

Kidneys of new-born animals are resistant to arginine vasopressin (AVP). The ability of the hormone to regulate water permeability of the collecting duct can be seen from weaning period, probably due to the maturation of the intracellular signaling pathway. The purpose of the present work was to investigate the effect of V2 receptor agonist dDAVP on the water permeability of OMCD basolateral membrane in 10-, 22- and 60-day old Wistar rats. We also estimated ontogenetic gene expression of AQP2, AQP3, AQP4 and V2 receptor. Osmotic water permeability (Pf) of the basolateral membrane of microdissected OMCD was measured under control conditions and after incubation with the agonist V2 receptor desmopressin (dDAVP; 10(-7) M). Water permeability in 10- and 22-day old rats under control conditions were significantly higher than in adults. Desmopressin stimulated significant increase of this parameter in 22-day old pups (Pf = = 125 +/- 4.85; Pf = 174 +/- 8.2 microns/s, p < 0.001) and adult rats (Pf = 100.5 +/- 7.38; Pf = 178.8 +/- 9.54 microns/s, p < 0.001). Osmotic water permeability of the OMCD basolateral membrane in 10-day old rats does not depend on dDAVP (Pf = 172.5 +/- 23.8; Pf = 164.8 +/- 34 microns/s). With the RT-PCR, we observed a gradual increase of AQP2 and V2 receptor genes expression during postnatal ontogenesis. The gene expression of AQP3 and AQP4 remained unchanged during postnatal ontogenesis. In general, the water permeability of the OMCD basolateral membrane of rats can be stimulated by AVP since the 22nd day of postnatal life. The water permeability of the OMCD basolateral membrane under control conditions gradually decreased during postnatal development, while gene expression of AQP3 and AQP4 was unchanged. The mechanism of this decrease remains to be established.     

Ontogeny of rabbit proximal tubule urea permeability

Urea transport in the proximal tubule is passive and is dependent on the epithelial permeability. The present study examined the maturation of urea permeability (P(urea)) in in vitro perfused proximal convoluted tubules (PCT) and basolateral membrane vesicles (BLMV) from rabbit renal cortex. Urea transport was lower in neonatal than adult PCT at both 37 and 25 degrees C. The PCT P(urea) was also lower in the neonates than the adults (37 degrees C: 45.4 +/- 10.8 vs. 88.5 +/- 15.2 x 10(-6) cm/s, P < 0.05; 25 degrees C: 28.5 +/- 6.9 vs. 55.3 +/- 10.4 x 10(-6) cm/s; P < 0.05). The activation energy for PCT P(urea) was not different between the neonatal and adult groups. BLMV P(urea) was determined by measuring vesicle shrinkage, due to efflux of urea, using a stop-flow instrument. Neonatal BLMV P(urea) was not different from adult BLMV P(urea) at 37 degrees C [1.14 +/- 0.05 x 10(-6) vs. 1.25 +/- 0.05 x 10(-6) cm/s; P = not significant (NS)] or 25 degrees C (0.94 +/- 0.06 vs. 1.05 +/- 0.10 x 10(-6) cm/s; P = NS). There was no effect of 250 microM phloretin, an inhibitor of the urea transporter, on P(urea) in either adult or neonatal BLMV. The activation energy for urea diffusion was also identical in the neonatal and adult BLMV. These findings in the BLMV are in contrast to the brush-border membrane vesicles (BBMV) where we have previously demonstrated that urea transport is lower in the neonate than the adult. Urea transport is lower in the neonatal proximal tubule than the adult. This is due to a lower rate of apical membrane urea transport, whereas basolateral urea transport is the same in neonates and adults. The lower P(urea) in neonatal proximal tubules may play a role in overall urea excretion and in developing and maintaining a high medullary urea concentration and thus in the ability to concentrate the urine during renal maturation.     

Histamine treatment induces rearrangements of orthogonal arrays of particles (OAPs) in human AQP4-expressing gastric cells

To test the involvement of the water channel aquaporin (AQP)-4 in gastric acid physiology, the human gastric cell line (HGT)-1 was stably transfected with rat AQP4. AQP4 was immunolocalized to the basolateral membrane of transfected HGT-1 cells, like in native parietal cells. Expression of AQP4 in transfected cells increased the osmotic water permeability coefficient (Pf) from 2.02 +/- 0.3 x 10-4 to 16.37 +/- 0.5 x 10-4 cm/s at 20 degrees C. Freeze-fracture EM showed distinct orthogonal arrays of particles (OAPs), the morphological signature of AQP4, on the plasma membrane of AQP4-expressing cells. Quantitative morphometry showed that the density of OAPs was 2.5 +/- 0.3% under basal condition and decreased by 50% to 1.2 +/- 0.3% after 20 min of histamine stimulation, mainly due to a significant decrease of the OAPs number. Concomitantly, Pf decreased by approximately 35% in 20-min histamine-stimulated cells. Both Pf and OAPs density were not modified after 10 min of histamine exposure, time at which the maximal hormonal response is observed. Cell surface biotinylation experiments confirmed that AQP4 is internalized after 20 min of histamine exposure, which may account for the downregulation of water transport. This is the first evidence for short term rearrangement of OAPs in an established AQP4-expressing cell line.     

Osmotic gradient-induced water permeation across the sarcolemma of rabbit ventricular myocytes

The mechanism of water permeation across the sarcolemma was characterized by examining the kinetics and temperature dependence of osmotic swelling and shrinkage of rabbit ventricular myocytes. The magnitude of swelling and the kinetics of swelling and shrinkage were temperature dependent, but the magnitude of shrinkage was very similar at 6 degrees, 22 degrees, and 37 degrees C. Membrane hydraulic conductivity, Lp, was approximately 1.2 x 10(-10) liter.N-1.s-1 at 22 degrees C, corresponding to an osmotic permeability coefficient, Pf, of 16 microns.s-1, and was independent of the direction of water flux, the magnitude of the imposed osmotic gradient (35-165 mosm/liter), and the initial cell volume. This value of Lp represents an upper limit because the membrane was assumed to be a smooth surface. Based on capacitive membrane area, Lp was 0.7 to 0.9 x 10(-10) liter.N-1.s-1. Nevertheless, estimates of Lp in ventricle are 15 to 25 times lower than those in human erythrocytes and are in the range of values reported for protein- free lipid bilayers and biological membranes without functioning water channels (aquaporin). Evaluation of the effect of unstirred layers showed that in the worst case they decrease Lp by < or = 2.3%. Analysis of the temperature dependence of Lp indicated that its apparent Arrhenius activation energy, Ea', was 11.7 +/- 0.9 kcal/mol between 6 degrees and 22 degrees C and 9.2 +/- 0.9 kcal/mol between 22 degrees and 37 degrees C. These values are significantly greater than that typically found for water flow through water-filled pores, approximately 4 kcal/mol, and are in the range reported for artificial and natural membranes without functioning water channels. Taken together, these data strongly argue that the vast majority of osmotic water flux in ventricular myocytes penetrates the lipid bilayer itself rather than passing through water-filled pores.     

Signals that regulate GLUT4 translocation

We have shown that there is a maturational increase in osmotic water permeability (Pf) of rabbit renal brush border membrane vesicles (BBMV). The purpose of the present study was to further investigate the changes in proximal tubule water transport that occur during postnatal development. Diffusional water permeability (PDW) has not been measured directly in adult or neonatal BBMV. We validated the method described by Ye and Verkman (Simultaneous optical measurement of osmotic and diffusional water permeability in cells and liposomes. Biochemistry 28:824-829, 1989) to measure PDW in red cell ghosts and liposomes, to examine the maturational changes in PDW in BBMV. This method utilizes the sensitivity of 8-aminonaphtalene-1,3,6-trisulfonic acid (ANTS) fluorescence to the D2O-H2O content of the solvent. ANTS-loaded neonatal (11 days old) and adult BBMV were rapidly mixed with two volumes of isoosmotic D2O solution using a stopped-flow apparatus at 5 degrees -37 degrees C. PDW was lower in neonatal than adult BBMV at 5 degrees (3.77 +/- 0.34 vs. 5.35 +/- 0.43 mm/sec, respectively, p<0.05) and 20 degrees C (7.03 +/- 0.40 vs. 9.04 +/- 0.25 mm/sec, respectively, p<0.001), but was not different at 30 degrees and 37 degrees C. The activation energy (Ea) was higher in neonatal than in adult BBMV (9.29 +/- 0.56 kcal/mol vs. 6.46 +/- 0.56 kcal/mol, p<0.001). In adult BBMV, PDW was inhibited by 0.5 mM HgCl2 by 46.6 +/- 3.6%, while it was not affected in neonatal BBMV (p<0.001). The results indicate that PDW can be measured in rabbit renal BBMV. There are significant changes in water transport across the apical membrane during postnatal development, consistent with a maturational increase in channel-mediated water transport.     

Lipid-phase structure in epithelial cell membranes: comparison of renal brush border and basolateral membranes

The lipid-phase structures of brush border membrane vesicles (BBMV) and basolateral membrane vesicles (BLMV) isolated from rabbit renal cortex were compared by steady-state and phase-modulation measurements of diphenylhexatriene (DPH) and trans- and cis-parinaric acid (tPnA and cPnA) fluorescence. A temperature-scanning system was used which gave reproducible temperature profiles of steady-state and dynamic fluorescence parameters with a resolution of 0.1 degrees C. Steady-state anisotropy of DPH showed a triphasic dependence on temperature with slope discontinuities at 22 +/- 4 and 47 +/- 3 degrees C (BBMV) and at 23 +/- 2 and 48 +/- 1 degrees C (BLMV). At all temperatures, DPH anisotropy in BBMV was greater than that in BLMV. Ground-state heterogeneity analysis of tPnA and cPnA fluorescence lifetime data demonstrated the presence of long (greater than 12 ns) and short (less than 5 ns) lifetime components, interpreted in terms of solid-phase and fluid-phase lipid domains. The fraction of solid-phase phospholipid decreased from 0.9 to 0.1 for BBMV and from 0.7 to 0.3 in BLMV with increasing temperature (10-50 degrees C). In both membranes, tryptophan-PnA fluorescence energy-transfer measurements showed that membrane proteins were surrounded by a fluidlike phospholipid phase. These results demonstrate the inadequacy of steady-state DPH anisotropy data in defining the structural characteristics of complex biological membranes. Results obtained with the phase-sensitive parinaric acid probes demonstrate major differences in the phase structure of the two opposing cell membranes in both the bulk lipid and the lipid microenvironment around membrane proteins.     

Very high water permeability in vasopressin-induced endocytic vesicles from toad urinary bladder

The regulation of transepithelial water permeability in toad urinary bladder is believed to involve a cycling of endocytic vesicles containing water transporters between an intracellular compartment and the cell luminal membrane. Endocytic vesicles arising from luminal membrane were labeled selectively in the intact toad bladder with the impermeant fluid-phase markers 6-carboxyfluorescein (6CF) or fluorescein-dextran. A microsomal preparation containing labeled endocytic vesicles was prepared by cell scraping, homogenization, and differential centrifugation. Osmotic water permeability was measured by a stopped-flow fluorescence technique in which microsomes containing 50 mM mannitol, 5 mM K phosphate, pH 8.5 were subject to a 60-mM inwardly directed gradient of sucrose; the time course of endosome volume, representing osmotic water transport, was inferred from the time course of fluorescence self-quenching. Endocytic vesicles were prepared from toad bladders with hypoosmotic lumen solution treated with (group A) or without (group B) serosal vasopressin at 23 degrees C, and bladders in which endocytosis was inhibited by treatment with vasopressin at 0-2 degrees C (group C), or with vasopressin plus sodium azide at 23 degrees C (group D). Stopped-flow results in all four groups showed a slow rate of 6CF fluorescence decrease (time constants 1.0-1.7 s for exponential fit) indicating a component of nonendocytic 6CF entrapment into sealed vesicles. However, in vesicles from group A only, there was a very rapid 6CF fluorescence decrease (time constant 9.6 +/- 0.2 ms, SEM, 18 separate preparations) with an osmotic water permeability coefficient (Pf) of greater than 0.1 cm/s (18 degrees C) and activation energy of 3.9 +/- 0.8 kcal/mol (16 kJ/mol). Pf was inhibited reversibly by greater than 60% by 1 mM HgCl2. The rapid fluorescence decrease was absent in vesicles in groups B, C, and D. These results demonstrate the presence of functional water transporters in vasopressin-induced endocytic vesicles from toad bladder, supporting the hypothesis that water channels are cycled to and from the luminal membrane and providing a functional marker for the vasopressin-sensitive water channel. The calculated Pf in the vasopressin-induced endocytic vesicles is the highest Pf reported for any biological or artificial membrane.     

Osmotic water permeability and regulatory volume decrease of rat thymocytes

Rat thymocytes displayed robust regulatory volume decrease (RVD) when suspended in NaCl-based hypotonic Ringer solutions. The RVD of thymocytes was completely abolished upon replacement of external Na+ ions with K+, indicating a role of coupled efflux of K+ and Cl- ions as a driving force of regulatory volume decrease. Osmotic water permeability (Pf) measured in KCl-based hypotonic solutions was (1.3 +/- 1.0 x 10(-4) cm/s at 25 degrees C and was temperature-dependent with low activation energy (Ea = 4.65 +/- 0.77 kcal/mol) characteristic to water transport through pores. HgCl2 and a sulfhydryl-blocking reagent, methyl methanethiosulphonate (MMTS), modulated the water permeability of thymocytes in a biphasic manner: inhibited at low dose (0.1-1 micromol/l) and restored or even enhanced at higher (10-100 micromol/l) concentrations. RVD paralleled the Pf: it was greatly suppressed at low dose of MMTS (sufficient to attenuate the water transport), but recovered at higher dose, when the water movement was restored. Therefore we suggest that thymocytes require the effective water transport for functional regulatory volume decrease.     

Functional colocalization of water channels and proton pumps in endosomes from kidney proximal tubule

The apical membrane of mammalian proximal tubule undergoes rapid membrane cycling by exocytosis and endocytosis. Osmotic water and ATP- driven proton transport were measured in endocytic vesicles from rabbit and rat proximal tubule apical membrane labeled in vivo with the fluid phase marker fluorescein-dextran. Osmotic water permeability (Pf) was determined from the time course of fluorescein-dextran fluorescence after exposure of endosomes to an inward osmotic gradient in a stopped- flow apparatus. Pf was 0.009 (rabbit) and 0.029 cm/s (rat) (23 degrees C) and independent of osmotic gradient size. Pf in rabbit endosomes was inhibited reversibly by HgCl2 (KI = 0.2 mM) and had an activation energy of 6.4 +/- 0.5 kcal/mol (15-35 degrees C). Endosomal proton ATPase activity was measured from the time course of internal pH, measured by fluorescein-dextran fluorescence, after the addition of external ATP. Endosomes contained an ATP-driven proton pump that was sensitive to N-ethylmaleimide and insensitive to vanadate and oligomycin. In response to saturating [ATP] the pump acidified the endosomal compartment at a rate of 0.17 (rat) and 0.029 pH unit/s (rabbit); at an external pH of 7.4, the steady-state pH was 6.4 (rat) and 6.5 (rabbit). To examine whether water channels and the proton ATPase were present in the same endosome, the time course of fluorescein-dextran fluorescence was measured in response to an osmotic gradient in the presence and absence of ATP. ATP did not alter endosome Pf, but decreased the amplitude of the fluorescence signal by 43 +/- 3% (rabbit) and 47 +/- 4% (rat).(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationship between lipid fluidity and water permeability of bovine tracheal epithelial cell apical membranes

Apical membrane vesicles were prepared from bovine tracheal epithelial cells. These membranes were enriched in alkaline phosphatase specific activity 35-fold compared to cellular homogenates. Steady-state fluorescence polarization studies of these membranes, using three fluorophores, demonstrated that they possessed a relatively low fluidity. Studies using the probe 1,6-diphenyl-1,3,5-hexatriene detected thermotropic transitions at 25.7 +/- 0.4 and 26.8 +/- 0.6 degrees C in these membranes and their liposomes, respectively. Analysis of the composition of these membranes revealed a fatty acyl saturation index of 0.59 +/- 0.02, a protein/lipid ratio (w/w) of 0.60 +/- 0.06, a cholesterol/phospholipid ratio (mol/mol) of 0.83 +/- 0.11, and a sphingomyelin/lecithin ratio (mol/mol) of 0.64 +/- 0.10. Membrane vesicles were osmotically active when studied by a stopped-flow nephelometric technique. Arrhenius plots of rates of osmotic water efflux demonstrated break points at approximately 28 and 18 degrees C, with activation energies of 16.7 +/- 0.2 kcal mol-1 from 35 to 28 degrees C, 8.3 +/- 0.5 kcal mol-1 from 28 to 18 degrees C, and approximately 3.0 kcal mol-1 below 18 degrees C. Treatment of membrane vesicles with benzyl alcohol, a known fluidizer, decreased lipid order (increased fluidity) and increased the rate of osmotic water efflux. The present results suggest that water crosses tracheal epithelial cell apical membranes by solubility-diffusion across the lipid domain and that increases in fluidity correlate with increases in the water permeability of these membranes.     

大鼠肝脏水通道蛋白7的表达

目的研究水通道蛋白7(AQP7)在大鼠肝脏中的表达和分布。方法选用成年健康SD大鼠,采用免疫组织化学的方法对肝脏中AQP7蛋白的表达进行定位检测。结果AQP7阳性免疫反应产物集中位于大鼠肝脏毛细胆管面的肝细胞质膜上,肝细胞的基膜面和血窦面未见阳性免疫反应产物。结论AQP7在肝脏中的表达及其空间上的分布提示其可能参与胆汁的分泌。     

Knockdown of hepatocyte aquaporin-8 by RNA interference induces defective bile canalicular water transport

Aquaporin-8 (AQP8) water channels, which are expressed in rat hepatocyte bile canalicular membranes, are involved in water transport during bile formation. Nevertheless, there is no conclusive evidence that AQP8 mediates water secretion into the bile canaliculus. In this study, we directly evaluated whether AQP8 gene silencing by RNA interference inhibits canalicular water secretion in the human hepatocyte-derived cell line, HepG2. By RT-PCR and immunoblotting we found that HepG2 cells express AQP8 and by confocal immunofluorescence microscopy that it is localized intracellularly and on the canalicular membrane, as described in rat hepatocytes. We also verified the expression of AQP8 in normal human liver. Forty-eight hours after transfection of HepG2 cells with RNA duplexes targeting two different regions of human AQP8 molecule, the levels of AQP8 protein specifically decreased by 60-70%. We found that AQP8 knockdown cells showed a significant decline in the canalicular volume of approximately 70% (P < 0.01), suggesting an impairment in the basal (nonstimulated) canalicular water movement. We also found that the decreased AQP8 expression inhibited the canalicular water transport in response either to an inward osmotic gradient (-65%, P < 0.05) or to the bile secretory agonist dibutyryl cAMP (-80%, P < 0.05). Our data suggest that AQP8 plays a major role in water transport across canalicular membrane of HepG2 cells and support the notion that defective expression of AQP8 causes bile secretory dysfunction in human hepatocytes.     

An ultrarapid filtration method adapted to the measurements of water and solute permeability of synthetic and biological vesicles.

An ultrarapid filtration method was adapted to the determination of water and solute permeability of membrane vesicles. This method consisted of measuring substance washout from vesicles first loaded with 3H2O or labeled solutes, placed on filters, and rinsed at high rates for short periods. The retention of the vesicles on the filters was analyzed and was found to be a function of the nature and porosity of the filters as well as of the vesicle origin. Washing buffer flow rate and washing duration did not affect vesicle retention. The diffusional water permeability of cholesterol-free liposomes was determined at 16 degrees C. Its value was reduced by a factor of 2.5 when the liposomes were prepared with 20% cholesterol and a threefold increase was noted when the liposomes were preincubated with gramicidin (6 mg/g lipid). Water permeability of liposomes was strongly temperature-dependent: Ea = 15.3 kcal/mol. Diffusional water permeability of pink ghosts was also measured: a value of (4.4 +/- 0.2) X 10(-3) cm/s (n = 3) was obtained at 13 degrees C. This permeability was reduced by 45.2% with 0.4 mM HgCl2. The urea permeability of intestinal and renal brush-border membrane vesicles was (1.15 +/- 0.18) X 10(-6) cm/s (n = 7) and (1.67 +/- 0.08) X 10(-6) cm/s (n = 9), respectively. The renal value was reduced by a factor of 4.4 by 100 mM thiourea. This ultrarapid filtration technique provides an accurate method of transport measurement in sealed membranes such as liposomes and plasma membrane vesicles.     

Structural and functional polarity of canalicular and basolateral plasma membrane vesicles isolated in high yield from rat liver

A method has been developed for routine high yield separation of canalicular (cLPM) from basolateral (blLPM) liver plasma membrane vesicles of rat liver. Using a combination of rate zonal floatation (TZ- 28 zonal rotor, Sorvall) and high speed centrifugation through discontinuous sucrose gradients, 9-16 mg of cLPM and 15-28 mg of blLPM protein can be isolated in 1 d. cLPM are free of the basolateral markers Na+/K+-ATPase and glucagon-stimulatable adenylate cyclase activities, but are highly enriched with respect to homogenate in the "canalicular marker" enzyme activities leucylnaphthylamidase (48-fold), gamma-glutamyl-transpeptidase (60-fold), 5'-nucleotidase (64-fold), alkaline phosphatase (71-fold), Mg++-ATPase (83-fold), and alkaline phosphodiesterase I (116-fold). In contrast, blLPM are 34-fold enriched in Na+/K+-ATPase activity, exhibit considerable glucagon-stimulatable adenylate cyclase activity, and demonstrate a 4- to 15-fold increase over homogenate in the various "canalicular markers." cLPM have a twofold higher content of sialic acids, cholesterol; and sphingomyelin compared with blLPM. At least three canalicular-(130,000, 100,000, and 58,000 mol wt) and several basolateral-specific protein bands have been detected after SDS PAGE of the two LPM subfractions. Specifically, the immunoglobin A-binding secretory component is restricted to blLPM as demonstrated by immunochemical techniques. These data indicate virtually complete separation of basolateral from canalicular LPM and demonstrate multiple functional and compositional polarity between the two surface domains of hepatocytes.