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1 Cystic Fibrosis Research Laboratory, Stanford University, Stanford, CA, USA
* To whom correspondence should be addressed. E-mail: wine{at}stanford.edu.
We developed a new apparatus, the virtual gland (VG), for measuring the rate of fluid secretion (Jv), 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 priming 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 Jv and collected for analysis. Calu-3 cells were mounted in the VG with a basolateral bath consisting of Krebs-HCO3- buffer at 37° C. Basal Jv was 2.7 ± 0.1 µl/cm2/hr (n=42), and TEP was -9.2 ± 0.6 mV (n=33); both measures were reduced to zero by ouabain (n=6). Jv/TEP were stimulated 64/59% by 5 µM forskolin (n=10), 173/101 % by 1 mM 1-EBIO (n=5), 213/122 % by 333 nM thapsigargin (n=5), and 520/240 % by combined forskolin and thapsigargin (n=6). Basal Jv/TEP were inhibited to 82/63 % with 10 µM bumetanide (n=5), 71/82% with 100 µM acetazolamide (n=5), and 47/56% with 600 µM glibenclamide (n=4). Basal Jv/TEP were 52/89 % of control values after HCO3- replacement with HEPES (n=16). The net [HCO3-] of the secreted fluid was close to 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 Jv in Calu-3 cells.
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