Ion transport and barrier function in a telomerase-immortalized human nondysplastic, Barrett's cell line (BAR-T)

• Published in: Diseases of the esophagus 2009-08, Vol.22 (5), p.386-395
• Main Authors: Jovov, Biljana, Orlando, Geraldine S., Tobey, Nelia A., Brown, Karen L., Djukic, Zorka, Carson, John L., Brighton, Louisa E., Orlando, Roy C.
• Format: Article
• Language: English
• Subjects: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid - pharmacology; Autoantigens - analysis; Barrett Esophagus - metabolism; Barrett Esophagus - pathology; Barrett Esophagus - physiopathology; Buffers; Cell Culture Techniques; Cell Differentiation; Cell Line; Cell Proliferation; claudin; Claudin-1; Claudin-4; Claudins; Culture Media; Electric Conductivity; Electric Impedance; Enzyme Inhibitors - pharmacology; Epithelial Cells - drug effects; Epithelial Cells - metabolism; Epithelial Cells - pathology; HEPES - pharmacology; HET-1A; Humans; ion transport; Ion Transport - drug effects; Ion Transport - physiology; Membrane Potentials - physiology; Membrane Proteins - analysis; Nerve Tissue Proteins - analysis; Ouabain - pharmacology; Phosphoproteins - analysis; resistance; Sodium-Potassium-Exchanging ATPase - antagonists & inhibitors; tight junction; Tight Junctions - physiology; ZO1; Zonula Occludens-1 Protein
• ISSN: 1120-8694; 1442-2050
• DOI: 10.1111/j.1442-2050.2008.00907.x

SUMMARY Barrett's specialized columnar epithelium (SCE) replaces reflux‐damaged squamous epithelium. The benefits of SCE lie in its superior protection of the esophagus against further reflux damage. It was shown that this protection is dependent on ion transport and barrier function of SCE. The risks of SCE lie in its higher predisposition to malignant transformation. An understanding of underlying mechanisms of both processes would benefit considerably from greater knowledge of the structure and function of native SCE – the latter recently advanced by the availability of a telomerase‐immortalized, nonneoplastic, human Barrett's cell line (BAR‐T). Some of BAR‐T characteristics for growth and differentiation have been described recently, but not its capacity to serve as a model for ion transport and barrier function of SCE. To determine the latter, BAR‐T cells were grown in enriched media, seeded on permeable supports, and subjected to electrical, biochemical, and morphologic study. HET‐1A (esophageal epithelial cell line), a nonneoplastic, human esophageal squamous cell line, was also studied for comparison. BAR‐T, but not HET‐1A cells in HEPES Ringer solution behaved as polarized monolayers with the capacity for ion transport and barrier function. This was evident electrically with a volt–ohm meter (EVOM), which recorded in BAR‐T a resting potential difference of 2.0 ± 0.2 mV, Isc of 17.4 ± 3.3 µAmps/cm2 and resistance of 103 ± 12 ohms·cm2. Further, Isc in BAR‐T was inhibitable by exposure to Na‐free solution, serosal ouabain, and luminal 4‐acetamido4′‐isothiocyano‐2,2′‐stilbenedisulfonic acid. Expression of tight junction genes were determined in BAR‐T and HET‐1A cells using quantitative reverse transcriptase‐polymerase chain reaction, with expression of zonula occludens‐1 (ZO‐1) set at 1 as reference. Claudins 1, 4, and 12 were prominently expressed in BAR‐T (0.2–0.6 of ZO‐1), while claudins 1, 11, and 12 were prominently expressed in HET‐1A (0.1–0.8 of ZO‐1). BAR‐T, but not HET‐1A, expressed claudins 4, 8, 16, 18, and 23, and HET‐1A, but not BAR‐T, expressed claudins 11, 15, and 20. Protein expression of prominently expressed claudins in BAR‐T correlated with mRNA expression. Immunofluorescence and confocal microscopy localized claudins 1 and 4 in BAR‐T to cell membranes and claudin 18, specifically to the tight junction. Membrane polarization was also documented in BAR‐T by immunolocalization of NaK,ATPase to the basolateral membrane. BAR‐T, but not