Imaging of filter-grown epithelial cells: MDCK Na+-H+ exchanger is basolateral

S. O. Rosenberg, P. A. Berkowitz, L. Li, V. L. Schuster

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Abstract

We report a simple method for growing epithelial cells on permeable supports and for imaging the cells from the apical side using an inverted microscope. Madin-Darby canine kidney (MDCK) cells were either seeded onto the conventional side of Millipore-CM filters or onto ''inverted'' filters. The peak transepithelial resistance of confluent monolayers was the same with either system. Cells on inverted filters that were stained with various dyes and imaged by epifluorescence exhibited more distinct intercellular spaces, cell margins, nuclei, and subapical vesicles. We also perfused both sides of inverted filters with HCO3/CO2-free saline and measured intracellular pH (pH(i)) using 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF) and digital imaging. The intrinsic buffer capacity of MDCK cells increased exponentially as pH(i) decreased. After an NH4Cl load, the H+ extrusion rate (J(H+)) in control saline was 2.42 ± 0.62 mM/min. J(H+) was completely blocked by 1 mM basolateral amiloride. In contrast, 1 mM apical amiloride had no effect. We conclude that 1) growth of epithelial cells on an inverted filter system is useful for the microspectrofluorimetric determination of pH(i) in single cells and for the imaging of apical/subapical structures, and 2) the Na+-H+ exchanger of MDCK cells is functionally polarized to the basolateral membrane.

Original languageEnglish (US)
Pages (from-to)C868-C876
JournalAmerican Journal of Physiology - Cell Physiology
Volume260
Issue number4 29-4
StatePublished - May 28 1991

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Keywords

  • 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein
  • Digital imaging
  • Epithelial transport
  • Fluorescence microscopy
  • Image analysis
  • Intracellular pH

ASJC Scopus subject areas

  • Physiology
  • Cell Biology

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