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  • 1
    Electronic Resource
    Electronic Resource
    The sodium concentration of the lateral intercellular spaces of MDCK cells: A microspectrofluorimetric study (1995)
    Springer
    The journal of membrane biology 144 (1995), S. 11-19 
    Details
    ISSN: 1432-1424
    Keywords: Epithelia ; Fluid transport ; Fluorescence microscopy ; Tissue culture ; Na+ transport
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract MDCK cell monolayers grown on glass coverslips were used to examine the Na+ concentration in individual lateral intercellular spaces (LIS) by video fluorescence microscopy. The LIS was filled with the Na+-sensitive fluorescent dye SBFO by incubation of the monolayers for 75–90 min with 250 μm of the membrane impermeant form of the dye. After dye loading, the monolayers were perfused at 37°C with solutions buffered with HEPES or bicarbonate/CO2 containing 142 mm Na+. Ratios of the fluorescence images after sequential excitation with 340 nm and 380 nm light were performed and in situ calibration of LIS Na+ was accomplished after blocking the Na+ pump with 5 × 10−4 m ouabain. Measurements of Na+ along the basolateral-to-apical axis of the LIS at 1.0 or 1.5 μm intervals did not reveal a Na+ gradient when the perfusate was either HEPES or bicarbonate/CO2 solutions. In bicarbonate solutions, the mean Na+ concentration (mm) was 157.2 ± 2.3, ∼15 mm higher than the bath Na+ concentration. In HEPES solutions, however, the Na+ concentration was not different from the bath concentration (142.7 ± 3.1 mm). The time course of Na+ changes in LIS was investigated by rapidly switching the perfusate from 142 to 80 mm Na+ and measuring the Na+ changes at one focal plane.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00238412
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  • 2
    Electronic Resource
    Electronic Resource
    Acidic pH of the lateral intercellular spaces of MDCK cells cultured on permeable supports (1994)
    Springer
    The journal of membrane biology 140 (1994), S. 89-99 
    Details
    ISSN: 1432-1424
    Keywords: Epithelia ; Fluid transport ; Fluorescence microscopy ; Tissue culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract The pH of the lateral intercellular space (LIS) of Madin-Darby canine kidney (MDCK) cell monolayers grown on permeable supports was investigated by microspectrofluorimetry using BCECF (2′,7′-bis(carboxyethyl)-5,6-carboxyfluorescein). The permeability of the support was selectively reduced by growing ZnAl-silicate crystals inside its pores. The diffusion of BCECF across the filter was sufficiently retarded to allow measurements of fluorescence in the LIS. The LIS pH and intracellular pH of the cells surrounding them were determined in HEPES-buffered solutions. When the perfusate pH was 7.4, the LIS pH was more acidic (7.06±0.02) and equaled the cytoplasmic pH (7.08±0.05). When perfusate was changed to pH 7.0 or 7.8, the LIS changed linearly by about half the magnitude of the perfusate pH. Intracellular pH followed LIS pH variations between perfusate pH 7.0 and 7.4 but was significantly higher when perfusate pH was 7.8. Tight junctional H+ permeability was undetectably low. The low steady-state pH in the LIS was not altered by inhibitors of acid transport or low temperature. Rapid perturbations of pH in the LIS showed that protons were not immobilized in the LIS. The acidic microenvironment within the LIS may be the result of buffering by the cell surface proteins.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00232897
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  • 3
    Electronic Resource
    Electronic Resource
    The chloride concentration in the lateral intercellular spaces of MDCK cell monolayers (1995)
    Springer
    The journal of membrane biology 144 (1995), S. 21-30 
    Details
    ISSN: 1432-1424
    Keywords: Epithelia ; Fluid transport ; Cl transport ; ABQ ; Fluorescence microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract We measured the Cl concentration of the lateral intercellular spaces (LIS) of MDCK cell monolayers, grown on glass coverslips, by video fluorescence microscopy. Monolayers were perfused at 37°C either with HEPES-buffered solutions containing 137 mm Cl or bicarbonate/CO2-buffered solutions containing 127 mm Cl. A mixture of two fluorescent dyes conjugated to dextrans (MW 10,000) was microinjected into domes and allowed to diffuse into the nearby LIS. The Cl sensitive dye, ABQ-dextran, was selected because of its responsiveness at high Cl concentrations; a Clinsensitive dye, Cl-NERF-dextran, was used as a reference. Both dyes were excited at 325 nm, and ratios of the fluorescence intensity at spectrally distinct emission wavelengths were obtained from two intensified CCD cameras, one for ABQ-dextran the other for Cl-NERFdextran. LIS Cl concentration was calibrated in situ by treating the monolayer with digitonin or ouabain and varying the perfusate Cl between 0 and 137 mm (HEPES buffer) or between 0 and 127 mm (bicarbonate/CO2 buffer). LIS Cl in HEPES-buffered solutions averaged 176 ± 19 mm (n = 12), calibrated with digitonin, and 170 ± 9 mm (n = 12), calibrated with ouabain. LIS Cl in bicarbonate/CO2-buffered solutions averaged 174 ± 10 mm (n = 7) using the ouabain calibration. The Cl concentration of MDCK cell domes, measured with Clsensitive microelectrodes and by microspectrofluorimetry, did not differ significantly. Images of the LIS at 3 focal planes, near the tight junction, midway and basal, failed to reveal any gradients in Cl concentration along the LIS. LIS Cl changed rapidly in response to perfusate Cl with characteristic times of 0.8 ± 0.1 min (n = 21) for Cl decrease and 0.3 ± 0.04 min (n=21) for Cl increase. In conclusion, (i) Cl concentration is higher in the LIS than in the bathing medium, (ii) no gradients of Cl along the depth of LIS are detectable, (iii) junctional Cl permeability is high.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00238413
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  • 4
    Electronic Resource
    Electronic Resource
    Chloride movement across the basolateral membrane of proximal tubule cells (1981)
    Springer
    The journal of membrane biology 58 (1981), S. 35-42 
    Details
    ISSN: 1432-1424
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary Electrophysiologic and tracer experiments have shown that Cl− entersNecturus proximal tubule cells from the tubule lumen by a process coupled to the flow of Na+, and that Cl− entry is electrically silent. The mechanism of Cl− exit from the cell across the basolateral membrane has not been directly studied. To evaluate the importance of the movement of Cl− ions across the basolateral membrane, the relative conductance of Cl− to K+ was determined by a new method. Single-barrel ion-selective microelectrodes were used to measure intracellular Cl− and K+ as a function of basolateral membrane PD as it varied normally from tubule to tubule. Basolateral membrane Cl− conductance was about 10% of K+ conductance by this method. A second approach was to voltage clamp the basolateral PD to 20 mV above and below the spontaneous PD, while sensing intracellular Cl− activity with the second barrel of a double-barrel microelectrode. An axial wire electrode in the tubule lumen was used to pass current across the tubular wall and thereby vary the basolateral membrane PD. Cell Cl− activity was virtually unaffected by the PD changes. We conclude that Cl− leavesNecturus proximal tubule cells by a neutral mechanism, possibly coupled to the efflux of Na+ or K+.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01871032
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  • 5
    Electronic Resource
    Electronic Resource
    Determination of the Na permeability of the tight junctions of MDCK cells by fluorescence microscopy (1995)
    Springer
    The journal of membrane biology 148 (1995), S. 223-232 
    Details
    ISSN: 1432-1424
    Keywords: Na ; cAMP ; SBFO ; Protein kinase A ; Transference number
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract The kinetics of Na movement across the tight junctions of MDCK cells, grown on coverslips and perfused with HEPES or bicarbonate Ringer at 37°C, were investigated after filling the lateral intercellular spaces (LIS) of the epithelium with SBFO, an Na-sensitive fluorescent dye. Dilution and bi-ionic potential measurements showed that MDCK cell tight junctions, although cation-selective, were poorly permeable to N-methyl-D-glucamine Cl (NMDG) but freely permeable to Li. In previous experiments in which Na was replaced by NMDG, a very slow decrease in LIS Na concentration (time constant = 4.8 min) resulted. In the present study, reduction of perfusate Na from 142 to 14 or 24 mm with Na replaced by Li caused LIS Na concentration to decrease with a time constant of 0.43 min. The time constant for Na increase of the LIS was 0.28 min, significantly shorter than that for Na decrease because of the additional component of transcellular Na influx. Ouabain eliminated the transcellular component and equalized the time constants for Na influx and efflux. These results were incorporated into a mathematical model which enabled calculation of the transcellular and paracellular Na fluxes during fluid reabsorption. Regulation of the Na permeability of individual tight junctions by protein kinase A (PKA) was evaluated by treating the monolayers with the Sp-cAMPS, a cAMP substitute, or Rp-cAMPS, a specific inhibitor of PKA. Stimulation of PKA strikingly increased tight junctional permeability while PKA inhibition diminished junctional Na permeability.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00235040
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  • 6
    Electronic Resource
    Electronic Resource
    Chloride transport activation by plasma osmolarity during rapid adaptation to high salinity of Fundulus heteroclitus (1995)
    Springer
    The journal of membrane biology 143 (1995), S. 207-217 
    Details
    ISSN: 1432-1424
    Keywords: Gill chloride cell ; Cl− secretion ; Fundulus heteroclitus ; Cell volume regulation ; Na+/H+ exchanger ; Cl−/HCO 3 − exchanger
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract Transition from low salt water to sea water of the euryhaline fish, Fundulus heteroclitus, involves a rapid signal that induces salt secretion by the gill chloride cells. An increase of 65 mOsm in plasma osmolarity was found during the transition. The isolated, chloridecell-rich opercular epithelium of sea-water-adapted Fundulus exposed to 50 mOsm mannitol on the basolateral side showed a 100% increase in chloride secretion, which was inhibited by bumetanide 10−4 m and 10−4 m DPC (N-Phenylanthranilic acid). No effect of these drugs was found on apical side exposure. A Na+/H+ exchanger, demonstrated by NH4Cl exposure, was inhibited by amiloride and its analogues and stimulated by IBMX, phorbol esters, and epithelial growth factor (EGF). Inhibition of the Na+/H+ exchanger blocks the chloride secretion increase due to basolateral hypertonicity. A Cl−/HCO 3 − exchanger was also found in the chloride cells, inhibited by 10−4 m DIDS but not involved in the hyperosmotic response. Ca2+ concentration in the medium was critical for the stimulation of Cl− secretion to occur. Chloride cell volume shrinks in response to hypertonicity of the basolateral side in sea-water-adapted operculi; no effect was found on the apical side. Freshwater-adapted fish chloride cells show increased water permeability of the apical side. It is concluded that the rapid signal for adaptation to higher salinities is an increased tonicity of the plasma that induces chloride cell shrinkage, increased chloride secretion with activation of the Na+K+2Cl− cotransporter, the Na+/H+ exchanger and opening of Cl− channels.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00233449
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  • 7
    Electronic Resource
    Electronic Resource
    The volume of mitochondria-rich cells of frog skin epithelium (1986)
    Springer
    The journal of membrane biology 92 (1986), S. 21-26 
    Details
    ISSN: 1432-1424
    Keywords: cell volume ; chloride transport ; quantitative light microscopy ; volume regulation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary The pathway for movement of chloride ions across frog skin is not well understood. Mitochondria-rich (MR) cells have been proposed as the route for chloride across the skin. To test this hypothesis we studied the MR cells of the skin of the frog,Rana pipiens, by quantitative light microscopic determination of cell volume. MR cell volume was influenced by changes in the chloride concentration or osmolality of the outside bathing solution. MR cells shrank about 23% when all chloride was removed from the outside (mucosal) bathing solution. MR cells were also shown to be responsive to changes in the osmolality of either the mucosal or serosal bath. Osmotically-induced swelling caused by dilution of the serosal bath resulted in volume regulatory decrease. These results are consistent with the hypothesis that MR cells constitute the pathway for chloride movement across frog skin.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01869012
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  • 8
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    Uptake of fluorescent dyes associated with the functional expression of the cystic fibrosis transmembrane conductance regulator in epithelial cells. (1996)
    National Academy of Sciences
    Details
    Publication Date: 1996-02-06
    Print ISSN: 0027-8424
    Electronic ISSN: 1091-6490
    Topics: Biology , Medicine , Natural Sciences in General
    Published by National Academy of Sciences
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  • 9
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    Ketoconazole activates Cl- conductance and blocks Cl- and fluid absorption by cultured cystic fibrosis (CFPAC-1) cells. (1993)
    National Academy of Sciences
    Details
    Publication Date: 1993-05-01
    Print ISSN: 0027-8424
    Electronic ISSN: 1091-6490
    Topics: Biology , Medicine , Natural Sciences in General
    Published by National Academy of Sciences
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  • 10
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    Water does not flow across the tight junctions of MDCK cell epithelium (1998)
    National Academy of Sciences
    Details
    Publication Date: 1998-05-26
    Print ISSN: 0027-8424
    Electronic ISSN: 1091-6490
    Topics: Biology , Medicine , Natural Sciences in General
    Published by National Academy of Sciences
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