scholarly journals Plasma Membrane Water Permeability of Cultured Cells and Epithelia Measured by Light Microscopy with Spatial Filtering

1997 ◽  
Vol 110 (3) ◽  
pp. 283-296 ◽  
Author(s):  
Javier Farinas ◽  
Malea Kneen ◽  
Megan Moore ◽  
A.S. Verkman
Keyword(s):  
Light Microscopy ◽  
Cell Volume ◽  
Water Permeability ◽  
Signal Intensity ◽  
Spatial Filtering ◽  
Water Channels ◽  
Molecular Water ◽  
Toad Urinary Bladder ◽  
Volume Changes ◽  
Cell Layers

A method was developed to measure the osmotic water permeability (Pf) of plasma membranes in cell layers and applied to cells and epithelia expressing molecular water channels. It was found that the integrated intensity of monochromatic light in a phase contrast or dark field microscope was dependent on relative cell volume. For cells of different size and shape (Sf9, MDCK, CHO, A549, tracheal epithelia, BHK), increased cell volume was associated with decreased signal intensity; generally the signal decreased 10–20% for a twofold increase in cell volume. A theory relating signal intensity to relative cell volume was developed based on spatial filtering and changes in optical path length associated with cell volume changes. Theory predictions were confirmed by signal measurements of cell layers bathed in solutions of various osmolarities and refractive indices. The excellent signal-to-noise ratio of the transmitted light detection permitted measurement of cell volume changes of <1%. The method was applied to characterize transfected cells and tissues that natively express water channels. Pf in control Chinese hamster ovary cells was low (0.0012 cm/s at 23°C) and increased more than fourfold upon stable transfection with aquaporins 1, 2, 4, or 5. Pf in apical and basolateral membranes in polarized epithelial cells grown on porous supports was measured. Pfbl and Pfap were 0.0011 and 0.0024 cm/s (MDCK cells), and 0.0039 and 0.0052 cm/s (human tracheal cells) at 23°C. In intact toad urinary bladder, basolateral Pf was 0.036 cm/s and apical membrane Pf after vasopressin stimulation was 0.025 cm/s at 23°C. The results establish light microscopy with spatial filtering as a technically simple and quantitative method to measure water permeability in cell layers and provide the first measurement of the apical and basolateral membrane permeabilities of several important epithelial cell types.

Progesterone inhibition of water permeability in Bufo arenarum oocytes and urinary bladder

1996 ◽  
Vol 270 (5) ◽  
pp. F880-F885 ◽  
Author(s):  
P. Ford ◽  
G. Amodeo ◽  
C. Capurro ◽  
C. Ibarra ◽  
R. Dorr ◽  
...  
Keyword(s):  
Urinary Bladder ◽  
Xenopus Laevis ◽  
Water Permeability ◽  
Water Channel ◽  
Water Channels ◽  
Toad Urinary Bladder ◽  
Osmotic Permeability ◽  
Bufo Arenarum ◽  
Mrna Extraction

The ovarian oocytes from Bufo arenarum (BAO) but not those from Xenopus laevis (XLO) would have water channels (WC). We now report that the injection of the mRNA from BAO into the oocytes from XLO increased their water osmotic permeability (Pi) (reduced by 0.3 mM HgCl2 and reversed by 5 mM beta-mercaptoethanol). A 30-min challenge with progesterone induced, 18 h later, a reduction of the mercury-sensitive fraction of Pf in the BAO (but not in XLO). The mRNA from BAO pretreated with progesterone lost its capacity to induce WC in the XLO, but the hormone did not affect the expression of the WC in XLO previously injected with the mRNA from BAO. Pf was also measured in urinary bladders of BAO. Eighteen hours after a challenge with progesterone, a reduction in the hydrosmotic response to oxytocin was observed. Finally, the mRNA from the urinary bladder of BAO was injected into XLO. An increase in Pf was observed. This was not the case if, before the mRNA extraction, the bladders were treated with progesterone. We conclude that the BAO WC share progesterone sensitivity with the oxytocin-regulated water channel present in the toad urinary bladder.

Apical membrane vesicles of ADH-stimulated toad bladder are highly water permeable

1990 ◽  
Vol 258 (2) ◽  
pp. F237-F243
Author(s):  
H. W. Harris ◽  
J. S. Handler ◽  
R. Blumenthal
Keyword(s):  
Water Permeability ◽  
Apical Membrane ◽  
Fluid Phase ◽  
Granular Cell ◽  
Water Channels ◽  
Apical Plasma Membrane ◽  
Toad Urinary Bladder ◽  
Erythrocyte Ghosts ◽  
Particle Aggregates ◽  
Particle Aggregate

Antidiuretic hormone (ADH) stimulation of the toad urinary bladder causes intracellular vesicles called aggrephores to fuse with the apical plasma membrane of granular cells. Aggrephore membranes contain particle aggregates. Particle aggregates are believed to be water channels that cause large increases in the water permeability (PF) of the granular cell apical membrane. Removal of ADH causes the retrieval of particle aggregate-containing apical membrane via endocytosis and a decline in PF. We have previously shown that fluid phase markers are sequestered in these particle aggregate-containing vesicles during retrieval of the apical membrane and that these vesicles can be recovered in cell homogenates. We have now loaded these vesicles with the self-quenching fluorophore carboxyfluorescein (CF) to measure and compare their PF with that of CF-loaded resealed human erythrocyte ghosts. The membranes of these retrieved vesicles have a very high water permeability. The minimum PF of 99% of these vesicles is 4.5 X 10(-2) cm/s. This PF is comparable with that of erythrocyte ghosts (5.4 X 10(-2) cm/s) measured under identical conditions. We conclude that these vesicles are highly permeable to water, and this is consistent with their postulated function of retrieving water channels that have been inserted into the apical membrane in response to ADH.

Mechanisms and regulation of water permeability in renal epithelia

1989 ◽  
Vol 257 (5) ◽  
pp. C837-C850 ◽  
Author(s):  
A. S. Verkman
Keyword(s):  
Urinary Bladder ◽  
Proximal Tubule ◽  
Water Permeability ◽  
Permeability Coefficient ◽  
Biological Membranes ◽  
Water Channels ◽  
Proton Pumps ◽  
Toad Urinary Bladder ◽  
Water Permeability Coefficient ◽  
Collecting Tubule

Water transport occurs in all biological membranes. A few selected membranes in the kidney, amphibian urinary bladder, and erythrocyte have very high water permeability and are thought to contain specialized water transporting units termed "water channels." The known biophysical properties of membranes containing water channels are a high osmotic water permeability coefficient (Pf), an osmotic-to-diffusional water permeability coefficient ratio (Pf/Pd) greater than unity, a low activation energy (Ea), and inhibition by mercurial compounds. The biochemical and molecular characteristics of water channel pathways are not known at present. Established and new methods to measure Pf and Pd in kidney tubules and in isolated membrane vesicles from kidney cells are reviewed and evaluated. In the mammalian proximal tubule, a high Pf results from transcellular movement of water across highly permeable apical and basolateral membranes containing water channels. It has been assumed that proximal tubule Pf is unregulated; however, recent results indicate that apical water channels are retrieved by endocytosis and that Pf is decreased fivefold with increasing transepithelial osmotic gradients. In the thin and thick ascending limbs, Pf is nearly the lowest of all biological membranes and is not subject to regulation. In contrast, collecting tubule Pf is subject to hormonal regulation by vasopressin. Vasopressin binding to receptors located at the basal membrane of principal cells initiates adenosine 3',5'-cyclic monophosphate production, which is thought ultimately to activate the exocytic insertion of intracellular vesicles containing water channels into the cell apical membrane. Vasopressin-induced endosomes from kidney collecting tubule and toad urinary bladder contain functional water channels but no proton pumps or urea transporters, supporting a membrane shuttle hypothesis that is selective for water channels. Future directions for the isolation and molecular cloning of kidney water channels are evaluated.

scholarly journals Antidiuretic hormone modulates membrane phosphoproteins in toad urinary bladder and retrieved water channel containing apical membrane vesicles.

1991 ◽  
Vol 1 (9) ◽  
pp. 1114-1122
Author(s):  
H W Harris
Keyword(s):  
Membrane Proteins ◽  
Urinary Bladder ◽  
Membrane Protein ◽  
Antidiuretic Hormone ◽  
Water Permeability ◽  
Apical Membrane ◽  
Water Channel ◽  
Water Channels ◽  
Toad Urinary Bladder ◽  
Granular Cells

Antidiuretic hormone (ADH) dramatically increases the water permeability of toad urinary bladder by insertion of unique highly selective water channels into the apical membranes of granular cells. Before ADH stimulation, water channels are stored in high concentrations in the limiting membranes of large cytoplasmic vesicles called aggrephores. ADH stimulation causes aggrephore fusion with the granular cell apical membrane and increases water permeability. Transepithelial osmotic water flow causes a rapid attenuation of the ADH-elicited increase in water permeability through a process called flux inhibition. Flux inhibition is due to retrieval of ADH water channels by apical membrane endocytosis. When phosphoproteins of intact bladders are labeled with (32P)orthophosphate, the 32P content of 34-, 28-, and 17-kDa proteins is increased by ADH stimulation. When flux inhibition occurs, the 32P-labelling of a 15.5-kDa protein is reduced to approximately one half its original value (Konieczkowski M, Rudolph SA, J Pharmacol Exp Ther 1985;234:515). These observations have been confirmed, and these studies have been extended, by using a combination of subcellular fractionation and membrane protein chemistry techniques. All four of these phosphoproteins are present in membrane fractions of granular cells. Analysis of membrane proteins by a combination of Triton X-114 partitioning and an alkaline stripping technique reveals that the 28- and 17-kDa species are integral membrane proteins of unknown function. In contrast, the 32P-labeled 15.5-kDa protein is a peripheral membrane protein. It is attached to the cytoplasmic (outer) surface of highly water-permeable vesicles retrieved during flux inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)

ADH-induced recycling of fluid-phase marker from endosomes to the mucosal surface in toad bladder

1994 ◽  
Vol 267 (1) ◽  
pp. C32-C38 ◽  
Author(s):  
R. A. Coleman ◽  
J. B. Wade
Keyword(s):  
Urinary Bladder ◽  
Horseradish Peroxidase ◽  
Antidiuretic Hormone ◽  
Water Permeability ◽  
Apical Membrane ◽  
Fluid Phase ◽  
Water Channels ◽  
Toad Urinary Bladder ◽  
Bathing Solution ◽  
Apical Surface

In the toad urinary bladder, the reversal of antidiuretic hormone (ADH) stimulation results in the endocytosis of apical membrane water channels, along with any fluid-phase marker present in the mucosal bathing solution. We have loaded vesicles with horseradish peroxidase (HRP), then restimulated the bladders and measured the reappearance of endocytosed HRP in the mucosal bath. HRP-loaded bladders that were restimulated showed HRP release that peaked sharply within 15 min after restimulation. Varying the interval between loading and restimulation did not vary HRP release significantly. Restimulation with forskolin gave HRP release values similar to ADH. The amount of HRP released correlated with the magnitude of water permeability induced. The demonstration that fluid-phase markers can be recycled from endosomes to the apical surface in a hormone-dependent fashion indicates that endocytosed membrane, containing water channels, is able to rapidly recycle back to the surface in response to hormone restimulation. In addition, marker release declined progressively with repeated restimulation, totaling < 30% of the retrieved amount. This result indicates that a relatively large proportion of the retrieved marker reaches a nonrecycling compartment.

Salt and water transport across alveolar and distal airway epithelia in the adult lung

1996 ◽  
Vol 270 (4) ◽  
pp. L487-L503 ◽  
Author(s):  
M. A. Matthay ◽  
H. G. Folkesson ◽  
A. S. Verkman
Keyword(s):  
Water Transport ◽  
Water Permeability ◽  
Fluid Transport ◽  
Water Channels ◽  
Molecular Water ◽  
Alveolar Epithelial ◽  
Distal Airway ◽  
Injured Lung

Substantial progress has been made in understanding the role of the distal airway and alveolar epithelial barriers in regulating lung fluid balance. Molecular, cellular, and whole animal studies have demonstrated that reabsorption of fluid from the distal air spaces of the lung is driven by active sodium transport. Several different in vivo, in situ, and isolated lung preparations have been used to study the mechanisms that regulate fluid transport in the normal and injured lung. Catecholamine-dependent and -independent regulatory mechanisms have been identified that modulate fluid transport, probably by acting on apical sodium channel uptake or the activity of the Na, K-ATPase pumps. Recently, a family of molecular water channels (aquaporins) has been identified that are small (approximately 30 kDa) integral membrane proteins expressed widely in fluid-transporting epithelia and endothelia. At present, four different water channels have been identified in trachea and lung. Measurements of osmotic water permeability in in situ perfused lung and isolated perfused airways suggest a significant contribution of these molecular water channels to measured water permeability. However, further studies are required to determine the role of these water channels in normal pulmonary physiology and disease. Recent studies have provided new insights into the role of the alveolar epithelial barrier in clinical and experimental acute lung injury. Unlike the lung endothelium, the alveolar epithelium is resistant to several clinically relevant types of injury, including endotoxemia and bacteremia as well as aspiration of hyperosmolar solutions. In addition, even when the alveolar barrier has been injured, its capacity to transport edema fluid from the distal air spaces of the lung recovers rapidly. Future studies need to integrate new insights into the molecular mechanisms of alveolar epithelial sodium and water transport with functional studies in the normal and injured lung.

scholarly journals A plate reader-based method for cell water permeability measurement

2010 ◽  
Vol 298 (1) ◽  
pp. F224-F230 ◽  
Author(s):  
R. A. Fenton ◽  
H. B. Moeller ◽  
S. Nielsen ◽  
B. L. de Groot ◽  
M. Rützler
Keyword(s):  
Cell Volume ◽  
Water Permeability ◽  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Phosphorylation Site ◽  
Type I ◽  
Volume Changes ◽  
Average Cell ◽  
Compound Libraries ◽  
Plate Reader

Cell volume and water permeability measurements in cultured mammalian cells are typically conducted under a light microscope. Many of the employed approaches are time consuming and not applicable to a study of confluent epithelial cell monolayers. We present here an adaptation of a calcein-quenching-based approach for a plate reader. A standard curve of fluorescence intensities at equilibrium has been recorded, following a shift from 285 mosmol/kgH2O to a series of altered extracellular osmolyte concentrations, ranging from final concentrations of 185 to 585 mosmol/kgH2O, by changing buffer d-mannitol concentrations. Similarly, according average cell volumes have been measured in suspension in a Coulter counter (particle-sizing device). Based on these measurements, we have derived an equation that facilitates the modeling of cell volume changes based on fluorescence intensity changes. We have utilized the method to study the role of a carboxyl-terminus aquaporin (AQP)-2 phosphorylation site, which is known to affect AQP2 membrane trafficking, in heterologous type I Madin-Darby canine kidney cells. We find that water permeability in cells expressing phosphorylation site mutants was in the following order: AQP2-S256D > AQP2 wild-type > AQP2-S256A. We propose that the method can be applied to study AQP function and more generally to study cell volume changes in adherent cell lines. Furthermore, it should be adaptable for AQP inhibitor screening in chemical compound libraries.

Confocal light microscopy and electrophysiology of neurons in culture

1991 ◽  
Vol 49 ◽  
pp. 400-401
Author(s):  
J. N. Turner ◽  
D. H. Szarowski ◽  
M. Fejtl ◽  
S. N. Ayrapetyan ◽  
D. O. Carpenter
Keyword(s):  
Electrical Properties ◽  
Light Microscopy ◽  
Cell Volume ◽  
Room Temperature ◽  
Good Method ◽  
Isolated Neurons ◽  
Volume Changes ◽  
Na Pump ◽  
Pump Activity ◽  
Physiologic Parameters

Cell volume changes and regulation are thought to be important in the physiology and pathology of neurons. Osmotic challenges alter a number of physiologic parameters including electrical properties, chemosensitivity, and Na pump activity. Thus, a good method for correlating images of the living cell's surface with electrophysiologic measures is needed, and we are exploring the use of confocal light microscopy for imaging neurons in culture.Isolated neurons from ganglia of Aplvsia California were plated on polylysine coated coverslips in L15 media with 20% hemolymph at room temperature. Cells stained with “Dil” were imaged with a Bio—Rad MRC—600 and an Olympus BH—2. Images were displayed using Bio—Rad's maximum projection procedure, or Vital Images’ Voxel View software run on an IBM RISC 6000 Powerstation 320. Electrophysiologic properties were assessed after imaging.

Liver cell hydration and integrin signaling

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Michele Bonus ◽  
Dieter Häussinger ◽  
Holger Gohlke
Keyword(s):  
Cell Volume ◽  
Liver Cell ◽  
Cell Function ◽  
The Other ◽  
Signaling Cascades ◽  
Integrin Signaling ◽  
Volume Changes ◽  
The One ◽  
And Oxidative Stress

Abstract Liver cell hydration (cell volume) is dynamic and can change within minutes under the influence of hormones, nutrients, and oxidative stress. Such volume changes were identified as a novel and important modulator of cell function. It provides an early example for the interaction between a physical parameter (cell volume) on the one hand and metabolism, transport, and gene expression on the other. Such events involve mechanotransduction (osmosensing) which triggers signaling cascades towards liver function (osmosignaling). This article reviews our own work on this topic with emphasis on the role of β1 integrins as (osmo-)mechanosensors in the liver, but also on their role in bile acid signaling.

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