Hydraulic Properties of MDCK Cell Epithelium

Timbs, Melanie M.; Spring, Kenneth R.

doi:10.1007/s002329900104

Cited by 30 publications

(17 citation statements)

References 23 publications

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“…No immunopositive bands were detected in parental cell membranes using either anti-AQP-8 CT (lane 4) or anti-AQP-8 NT (lane 6). The lack of AQP5 and AQP8 expression in MDCK-II cells is consistent with results showing that the water permeabilities of the apical and basolateral membranes of low-resistance MDCK cells are similar to those of an unmodified lipid bilayer (26).…”

Section: Resultssupporting

confidence: 90%

Polarized Sorting of Aquaporins 5 and 8 in Stable MDCK-II Transfectants

Wellner

Baum

2001

Biochemical and Biophysical Research Communications

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Section: Resultssupporting

confidence: 90%

Polarized Sorting of Aquaporins 5 and 8 in Stable MDCK-II Transfectants

Wellner

Baum

2001

Biochemical and Biophysical Research Communications

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“…Ϫ5 m/s, depending on the experimental system and most likely also on precise cell culture conditions (3,5,16,26,31). In light of these results, our values for P f appear somewhat high (6 ϫ10 Ϫ5 m/s) but agree well with other values suggested for water permeability through a lipid bilayer (28,35).…”

supporting

confidence: 78%

A plate reader-based method for cell water permeability measurement

Fenton

Moeller

Nielsén

et al. 2010

American Journal of Physiology-Renal Physiology

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Fenton RA, Moeller HB, Nielsen S, de Groot BL, Rü tzler M. A plate reader-based method for cell water permeability measurement. Am J Physiol Renal Physiol 298: F224 -F230, 2010. First published November 4, 2009 doi:10.1152/ajprenal.00463.2009.-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 Dmannitol concentrations. Similarly, according average cell volumes have been measured in suspension in a Coulter counter (particlesizing 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 Ͼ AQP2 wild-type Ͼ 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.aquaporin-2; calcein, Madin-Darby canine kidney cells WATER HOMEOSTASIS IN HIGHER organisms requires precise regulation of water and solute reabsorption and excretion by the kidney. To date, numerous transporters and channels necessary for these processes have been characterized (7). These include several aquaporin (AQP) water channels, expressed in the proximal tubule and thin descending limb (AQP1) and the principal cells of the collecting duct (AQP2, -3, and -4), as well as the facilitative urea transporters expressed in the inner medullary collecting duct (UT-A1/3), descending thin limb of Henle's loop (UT-A2), and descending vasa recta (UT-B) (8). Water flux through AQPs is typically measured as the rate of volume change in cells or vesicles following osmotic challenge with a nonpermeable solute. Several experimental setups have been utilized to date (for a comprehensive review, see Ref. 35).AQP channel function can be studied in proteoliposomes utilizing the stopped-flow, light-scattering technique (37), where vesicles are exposed to a rapid change in buffer osmolality and scattered light intensity is recorded to monitor vesicle volume change. Similarly, vesicles may be loaded with high concentrations of a fluorescent dye. Upon a decrease in vesicle volume, dye self-quenching occurs by a process that...

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“…We calculated a value of 52.62 μm 2 from the measurements of (Wang et al, 1990). A value of 57.2 μm 2 is given by Timbs and Spring (1996) for the average smooth surface area. We assume the cell apical surface area to remain constant and take this measurement to represent the surface area when the cell cytoskeleton is relaxed.…”

Section: Parameter Valuesmentioning

confidence: 99%