Water transporting properties of hepatocyte basolateral and canalicular plasma membrane domains

Previous work from our laboratory supports an important role for aquaporins (AQPs), a family of water channel proteins, in bile secretion by hepatocytes. To further define the pathways and molecular mechanisms for water movement across hepatocytes, we directly assessed osmotic water permeability (Pf) and activation energy (Ea) in highly purified, rat hepatocytes basolateral membrane vesicles (BLMV) and canalicular membrane (CMV) vesicles by measuring scattered light intensity using stopped-flow spectrophotometry. The time course of scattered light for BLMV and CMV fit well to a single-exponential function. In BLMV, Pf was 108 +/- 4 mum.s-1 (25 degrees C) with an Ea of 7.7 kcal/mol; in CMV, Pf was 86 +/- 5 mum.s-1 (25 degrees C) with an Ea of 8.0 kcal/mol. The AQP blocker, dimethyl sulfoxide, significantly inhibited the Pf of both basolateral (81 +/- 4 mum.s-1; -25%) and canalicular (59 +/- 4 mum.s-1; -30%) membrane vesicles. When CMV were isolated from hepatocytes treated with dibutyryl cAMP, a double-exponential fit was needed, implying two functionally different vesicle populations; one population had Pf and Ea values similar to those of CMV from untreated hepatocytes, but the other population had a very high Pf (655 +/- 135 mum.s-1, 25 degrees C) and very low Ea (2.8 kcal/mol). Dimethyl sulfoxide completely inhibited the high Pf value in this second vesicle population. In contrast, Pf and Ea of BLMV were unaltered by cAMP treatment of hepatocytes. Our results are consistent with the presence of both lipid- and AQP-mediated pathways for basolateral and canalicular water movement across the hepatocyte plasma membrane barrier. Our data also suggest that the hepatocyte canalicular membrane domain is rate-limiting for transcellular water transport and that this domain becomes more permeable to water when hepatocytes are exposed to a choleretic agonist, presumably by insertion of AQP molecules. These data suggest a molecular mechanism for the efficient coupling of osmotically active solutes and water transport during canalicular bile formation.     

Expression of mRNA coding for kidney and red cell water channels in Xenopus oocytes

The existence and identity of protein water transporters in biological membranes has been uncertain. Osmotic water permeability (Pf) was measured in defolliculated Xenopus oocytes microinjected with water or mRNA from kidney cortex, kidney papilla, reticulocyte, brain, and muscle. Pf was measured by quantitative image analysis from the time course of oocyte swelling in response to an osmotic gradient. When assayed at 10 degrees C, Pf in water-injected oocytes increased from (3.6 +/- 0.9) x 10(-4) cm/s (S.D., n = 16) to 74 x 10(-4) cm/s with addition of amphotericin B, showing absence of unstirred layers. At 48-72 h after injection of 50 ng of unfractionated mRNA, Pf (in cm/s x 10(-4] was: 4.0 +/- 1.5 (rabbit brain, n = 15), 4.2 +/- 1.8 (rabbit muscle, n = 10), 18.4 +/- 6.3 (rabbit reticulocyte, n = 20), 16.1 +/- 5.6 (rat renal papilla, n = 24), 12.9 +/- 6.3 (rat renal cortex, n = 20), 14.4 +/- 6.1 (rabbit renal papilla, n = 15), and 11.8 +/- 3.4 (rabbit renal cortex, n = 8). In oocytes injected with mRNA from rat renal papilla, Pf was inhibited reversibly by 0.3 mM HgCl2 (4.1 +/- 1.6, n = 10); expressed water channels from kidney and red cell had activation energies of less than 4 kcal/mol. These results show functional oocyte expression of water channels from red cell, kidney proximal tubule (cortex), and the vasopressin-sensitive kidney collecting tubule (papilla), indicating that water channels are proteins, and providing an approach for the expression cloning of water channels.     

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.     

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.     

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)     

High microvascular endothelial water permeability in mouse lung measured by a pleural surface fluorescence method.

Transport of water between the capillary and airspace compartments in lung encounters serial barriers: the alveolar epithelium, interstitium, and capillary endothelium. We previously reported a pleural surface fluorescence method to measure net capillary-to-airspace water transport. To measure the osmotic water permeability across the microvascular endothelial barrier in intact lung, the airspace was filled with a water-immiscible fluorocarbon. The capillaries were perfused via the pulmonary artery with solutions of specified osmolalites containing a high-molecular-weight fluorescent dextran. An increase in perfusate osmolality produced a prompt decrease in surface fluorescence due to dye dilution in the capillaries, followed by a slower return to initial fluorescence as capillary and lung interstitial osmolality equilibrate. A mathematical model was developed to determine the osmotic water permeability coefficient (Pf) of lung microvessels from the time course of pleural surface fluorescence. As predicted, the magnitude of the prompt change in surface fluorescence increased with decreased pulmonary artery perfusion rate and increased osmotic gradient size. With raffinose used to induce the osmotic gradient, Pf was 0.03 cm/s at 23 degrees C and was reduced 54% by 0.5 mM HgCl2. Temperature dependence measurements gave an Arrhenius activation energy (Ea) of 5.4 kcal/mol (12-37 degrees C). The apparent Pf induced by the smaller osmolytes mannitol and glycine was 0.021 and 0.011 cm/s (23 degrees C). Immunoblot analysis showed approximately 1.4 x 10(12) aquaporin-1 water channels/cm2 of capillary surface, which accounted quantitatively for the high Pf. These results establish a novel method for measuring osmotically driven water permeability across microvessels in intact lung. The high Pf, low Ea, and mercurial inhibition indicate the involvement of molecular water channels in water transport across the lung endothelium.     

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.     

Expression of multiple water channel activities in Xenopus oocytes injected with mRNA from rat kidney

To test the hypothesis that renal tissue contains multiple distinct water channels, mRNA prepared from either cortex, medulla, or papilla of rat kidney was injected into Xenopus oocytes. The osmotic water permeability (Pf) of oocytes injected with either 50 nl of water or 50 nl of renal mRNA (1 microgram/microliter) was measured 4 d after the injection. Pf was calculated from the rate of volume increase on exposure to hyposmotic medium. Injection of each renal mRNA preparation increased the oocyte Pf. This expressed water permeability was inhibited by p-chloromercuriphenylsulfonate and had a low energy of activation, consistent with the expression of water channels. The coinjection of an antisense oligonucleotide for CHIP28 protein, at an assumed > 100-fold molar excess, with either cortex, medulla, or papilla mRNA reduced the expression of the water permeability by approximately 70, 100, and 30%, respectively. Exposure of the oocyte to cAMP for 1 h resulted in a further increase in Pf only in oocytes injected with medulla mRNA. This cAMP activation was not altered by the CHIP28 antisense oligonucleotide. These results suggest that multiple distinct water channels were expressed in oocytes injected with mRNA obtained from sections of rat kidney: (a) CHIP28 water channels in cortex and medulla, (b) cAMP-activated water channels in medulla, and (c) cAMP-insensitive water channels in papilla.     

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.     

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.     

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.     

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)     

Cloning, functional analysis and cell localization of a kidney proximal tubule water transporter homologous to CHIP28

The localization and transporting properties of a kidney protein homologous to human erythrocyte protein CHIP28 was evaluated. The cDNA encoding rat kidney protein CHIP28k was isolated from a rat renal cortex cDNA library. A 2.8-kb cDNA was identified which contained an 807 bp open reading frame encoding a 28.8 kD protein with 94% amino acid identity to CHIP28. in vitro translation of CHIP28k cDNA in rabbit reticulocyte lysate generated a 28-kD protein; addition of ER-derived microsomes gave a 32-kD transmembrane glycoprotein. Translation of truncated RNA demonstrated glycosylation of residue Asn42 which is predicted to lie between the first and second transmembrane domains. Expression of in vitro transcribed mRNA encoding CHIP28k in Xenopus oocytes increased oocyte osmotic water permeability (Pf) from (4 +/- 1) x 10(-4) to (33 +/- 4) x 10(-4) cm/s at 10 degrees C; the increase in oocyte Pf was weakly temperature dependent and inhibited by HgCl2. Two- electrode voltage clamp measurements indicated that CHIP28k was not permeable to ions. Oocyte Pf also increased with expression of total mRNA from kidney cortex and papilla; the increase in Pf with mRNA from cortex, but not kidney papilla, was blocked by coinjection with excess antisense CHIP28k cRNA. In situ hybridization of a 150 base cRNA antisense probe to tissue sections from rat kidney showed selective CHIP28k localization to epithelial cells in proximal tubule and thin descending limb of Henle. Pf in purified apical membrane vesicles from rat and human proximal tubule, and in proteoliposomes reconstituted with purified protein, was very high and inhibited by HgCl2; stripping of apical vesicles with N-lauroylsarcosine enriched a 28-kD protein by 25-fold and yielded a vesicle population with high water, but low urea and proton permeabilities. CHIP28k identity was confirmed by NH2- terminus sequence analysis. These results indicate that CHIP28k is a major and highly selective water transporting protein in the kidney proximal tubule and thin descending limb of Henle, but not collecting duct.     

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.     

Water, proton, and urea transport in toad bladder endosomes that contain the vasopressin-sensitive water channel

Vasopressin (VP) increases the water permeability of the toad urinary bladder epithelium by inducing the cycling of vesicles containing water channels to and from the apical membrane of granular cells. In this study, we have measured several functional characteristics of the endosomal vesicles that participate in this biological response to hormonal stimulation. The water, proton, and urea permeabilities of endosomes labeled in the intact bladder with fluorescent fluid-phase markers were measured. The diameter of isolated endosomes labeled with horse-radish peroxidase was 90-120 nm. Osmotic water permeability (Pf) was measured by a stopped-flow fluorescence quenching assay (Shi, L.-B., and A. S. Verkman. 1989. J. Gen. Physiol. 94:1101-1115). The number of endosomes formed when bladders were labeled in the absence of a transepithelial osmotic gradient increased with serosal [VP] (0-50 mU/ml), and endosome Pf was very high and constant (0.08-0.10 cm/s, 18 degrees C). When bladders were labeled in the presence of serosal-to-mucosal osmotic gradient, the number of functional water channels per endosome decreased (at [VP] = 0.5 mU/ml, Pf = 0.09 cm/s, 0 osmotic gradient; Pf = 0.02 cm/s, 180 mosmol gradient). Passive proton permeability was measured from the rate of pH decrease in voltage-clamped endosomes in response to a 1 pH unit gradient (pHin = 7.5, pHout = 6.5). The proton permeability coefficient (PH) was 0.051 cm/s at 18 degrees C in endosomes containing the VP-sensitive water channel; PH was not different from that measured in vesicles not containing water channels. Measurement of urea transport by the fluorescence quenching assay gave a urea reflection coefficient of 0.97 and a permeability coefficient of less than 10(-6) cm/s. These results demonstrate: (a) VP-induced endosomes from toad urinary bladder have extremely high Pf. (b) In states of submaximal bladder Pf, the density of functional water channels in endosomes in constant in the absence of an osmotic gradient, but decreases in the presence of a serosal-to-mucosal gradient, suggesting that the gradient has a direct effect on the efficiency of packaging of water channels into endosomes. (c) The VP-sensitive water channel does not have a high proton permeability. (d) Endosomes that cycle the water channel do not contain urea transporters. These results establish a labeling procedure in which greater than 85% of labeled vesicles from toad urinary bladder are endosomes that contain the VP-sensitive water channel in a functional form.     

Alterations in phospholipid-dependent (Na+ +K+)-ATPase activity due to lipid fluidity. Effects of cholesterol and Mg2+.

The (Na+ +K+)-activated, Mg2+-dependent ATPase from rabbit kidney outer medulla was prepared in a partially inactivated, soluble form depleted of endogenous phospholipids, using deoxycholate. This preparation was reactivated 10 to 50-fold by sonicated liposomes of phosphatidylserine, but not by non-sonicated phosphatidylserine liposomes or sonicated phosphatidylcholine liposomes. The reconstituted enzyme resembled native membrane preparations of (Na+ +K+)-ATPase in its pH optimum being around 7.0, showing optimal activity at Mg2+:ATP mol ratios of approximately 1 and a Km value for ATP of 0.4 mM. Arrhenius plots of this reactivated activity at a constant pH of 7.0 and an Mg2+: ATP mol ratio of 1:1 showed a discontinuity (sharp change of slope) at 17 degrees C, with activation energy (Ea) values of 13-15 kcal/mol above this temperature and 30-35 kcal below it. A further discontinuity was also found at 8.0 degrees C and the Ea below this was very high (greater than 100 kcal/mol). Increased Mg2+ concentrations at Mg2+:ATP ratios in excess of 1:1 inhibited the (Na+ +K+)-ATPase activity and also abolished the discontinuities in the Arrhenius plots. The addition of cholesterol to phosphatidylserine at a 1:1 mol ratio partially inhibited (Na+ +K+)-ATPase reactivation. Arrhenius plots under these conditions showed a single discontinuity at 20 degrees C and Ea values of 22 and 68 kcal/mol above and below this temperature respectively. The ouabain-insensitive Mg2+-ATPase normally showed a linear Arrhenius plot with an Ea of 8 kcal/mol. The cholesterol-phosphatidylserine mixed liposomes stimulated the Mg2+-ATPase activity, which now also showed a discontinuity at 20 degrees C with, however, an increased value of 14 kcal/mol above this temperature and 6 kcal/mol below. Kinetic studies showed that cholesterol had no significant effect on the Km values for ATP. Since both cholesterol and Mg2+ are known to alter the effects of temperature on the fluidity of phospholipids, the above results are discussed in this context.     

Optical measurement of osmotic water transport in cultured cells. Role of glucose transporters

Methodology was developed to measure osmotic water permeability in monolayer cultured cells and applied to examine the proposed role of glucose transporters in the water pathway (1989. Proc. Natl. Acad. Sci. USA. 86:8397-8401). J774 macrophages were grown on glass coverslips and mounted in a channel-type perfusion chamber for rapid fluid exchange without cell detachment. Relative cell volume was measured by 45 degrees light scattering using an inverted microscope; measurement accuracy was validated by confocal imaging microscopy. The time required for greater than 90% fluid exchange was less than 1 s. In response to a decrease in perfusate osmolality from 300 to 210 mosM, cells swelled without lag at an initial rate of 4.5%/s, corresponding to a water permeability coefficient of (6.3 +/- 0.4) x 10(-3) cm/s (SE, n = 20, 23 degrees C), assuming a cell surface-to-volume ratio of 4,400 cm-1. The initial rate of cell swelling was proportional to osmotic gradient size, independent of perfusate viscosity, and increased by amphotericin B (25 micrograms/ml), and had an activation energy of 10.0 +/- 1 kcal/mol (12-39 degrees C). The compounds phloretin (20 microM) and cytochalasin B (2.5 micrograms/ml) inhibited glucose transport by greater than 85% but did not influence Pf in paired experiments in which Pf was measured before and after inhibitor addition. The mercurials HgCl2 (0.1 mM) and p-chloromercuribenzoate (1 mM) did not inhibit Pf. A stopped-flow light scattering technique was used to measure Pf independently in J774 macrophages grown in suspension culture. Pf in suspended cells was (4.4 +/- 0.3) x 10(-3) cm/s (assuming a surface-to-volume ratio of 8,800 cm-1), increased more than threefold by amphotericin B, and not inhibited by phloretin and cytochalasin B under conditions of strong inhibition of glucose transport. The glucose reflection coefficient was 0.98 +/- 0.03 as measured by induced osmosis, assuming a unity reflection coefficient for sucrose. These results establish a quantitative method for measurement of osmotic water transport in adherent cultured cells and provide evidence that glucose transporters are not involved in the water transporting pathway.     

Transalveolar osmotic and diffusional water permeability in intact mouse lung measured by a novel surface fluorescence method

A surface fluorescence method was developed to measure transalveolar transport of water, protons, and solutes in intact perfused lungs. Lungs from c57 mice were removed and perfused via the pulmonary artery (approximately 2 ml/min). The airspace was filled via the trachea with physiological saline containing a membrane-impermeant fluorescent indicator (FITC-dextran or aminonapthalene trisulfonic acid, ANTS). Because fluorescence is detected only near the lung surface due to light absorption by lung tissue, the surface fluorescence signal is directly proportional to indicator concentration. Confocal microscopy confirmed that the fluorescence signal arises from fluorophores in alveoli just beneath the pleural surface. Osmotic water permeability (Pf) was measured from the time course of intraalveolar FITC-dextran fluorescence in response to changes in perfusate osmolality. Transalveolar Pf was 0.017 +/- 0.001 cm/s at 23 degrees C, independent of the solute used to induce osmosis (sucrose, NaCl, urea), independent of osmotic gradient size and direction, weakly temperature dependent (Arrhenius activation energy 5.3 kcal/mol) and inhibited by HgCl2. Pf was not affected by cAMP activation but was decreased by 43% in lung exposed to hyperoxia for 5 d. Diffusional water permeability (Pd) and Pf were measured in the same lung from intraalveolar ANTS fluorescence, which increased by 1.8-fold upon addition of 50% D2O to the perfusate, Pd was 1.3 x 10(-5) cm/s at 23 degrees C. Transalveolar proton transport was measured from FITC-dextran fluorescence upon switching perfusate pH between 7.4 and 5.6; alveolar pH half-equilibrated in 1.9 and 1.0 min without and with HCO3-, respectively. These results indicate high transalveolar water permeability in mouse lung, implicating the involvement of molecular water channels, and establish a quantitative surface fluorescence method to measure water and solute permeabilities in intact lung.     

Rapid development of vasopressin-induced hydroosmosis in kidney collecting tubules measured by a new fluorescence technique.

The pre-steady-state kinetics of the vasopressin-induced increase in collecting tubule osmotic water permeability (Pf) has been measured by a new fluorescence technique. Isolated cortical collecting tubules (CCT) from rabbit kidney were perfused with physiological buffers containing the impermeant fluorophores fluorescein sulfonate (FS) and pyrenetetrasulfonic acid (PTSA). Tubules were subject to a 120 mOsm bath-to-lumen osmotic gradient in the presence and absence of 250 microU/ml vasopressin. The magnitude of transepithelial volume flow was determined from the self-quenching of FS, or from the ratio of PTSA/FS fluorescence, measured at 380 nm excitation and 420 +/- 10 nm (PTSA) and greater than 530 nm (FS) emission wavelengths. Pf was calculated from the magnitude of transepithelial volume flow, lumen and bath osmolarities, lumen perfusion rate, and tubule geometry. The instrument response time for a change in bath osmolality was less than 3 s. At 37 degrees C, CCT Pf was (in units of cm/s x 10(4] 13 +/- 2 (mean +/- SE, 16 tubules) before, and 227 +/- 10 after addition of vasopressin to the bath. CCT Pf began to increase in 23 +/- 3 s after vasopressin addition and was half-maximal after 186 +/- 20 s. At 23 degrees C, Pf was 9 +/- 1 (seven tubules) before, and 189 +/- 12 after vasopressin addition. Pf began to increase in 40 +/- 4 s and was half-maximal after 195 +/- 35 s. After vasopressin removal from the bath, Pf decreased to its baseline value with a half-time of 14 min.(ABSTRACT TRUNCATED AT 250 WORDS)