A virtual gland method for quantifying epithelial fluid secretion

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-mul 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 degreesC. Basal J(v) was 2.7 +/- 0.1 mul . 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 muM 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 muM bumetanide ( n = 5), 71 and 82% with 100 muM acetazolamide ( n = 5), and 47 and 56% with 600 muM 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 (similar to 80 mM). These results validate the use of the VG apparatus and provide the first direct measures of J(v) in Calu-3 cells.