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A "virtual gland" method for quantifying epithelial fluid secretion

Cystic Fibrosis Research Laboratory, Stanford University, Stanford, California 94305-2130

Submitted 5 April 2004 ; accepted in final form 22 May 2004

We developed a new apparatus, the virtual gland (VG), for measuring the rate of fluid secretion (J_v), its composition, and the transepithelial potential (TEP) in cultured epithelial cells under open circuit. The VG creates a 10-µl chamber above the apical surface of epithelial cells on a Costar filter with a small hole leading to an oil-filled reservoir. After the chamber is primed with a fluid of choice, secreted fluid is forced through the hole into the oil, where it forms a bubble that is monitored optically to determine J_v and collected for analysis. Calu-3 cells were mounted in the VG with a basolateral bath consisting of Krebs-Ringer bicarbonate buffer at 37°C. Basal J_v was 2.7 ± 0.1 µl·cm^–2·h^–1 (n = 42), and TEP was –9.2 ± 0.6 mV (n = 33); both measures were reduced to zero by ouabain (n = 6). J_v and TEP were stimulated 64 and 59%, respectively, by 5 µM forskolin (n = 10), 173 and 101% by 1 mM 1-ethyl-2-benzimidazolinone (n = 5), 213 and 122% by 333 nM thapsigargin (n = 5), and 520 and 240% by forskolin + thapsigargin (n = 6). Basal J_v and TEP were inhibited to 82 and 63%, respectively, with 10 µM bumetanide (n = 5), 71 and 82% with 100 µM acetazolamide (n = 5), and 47 and 56% with 600 µM glibenclamide (n = 4). Basal J_v and TEP were 52 and 89% of control values, respectively, after HCO₃^– replacement with HEPES (n = 16). The net HCO₃^– concentration of the secreted fluid was close to that of the bath (25 mM), except when stimulated with forskolin or VIP, when it increased (80 mM). These results validate the use of the VG apparatus and provide the first direct measures of J_v in Calu-3 cells.

cystic fibrosis; submucosal gland; serous cell; mucus; airway

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