A "virtual gland" method for quantifying epithelial fluid secretion
A "virtual gland" method for quantifying epithelial fluid secretion
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DOI：
10.1152/ajplung.00124.2004
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发表时间：
2004-10-01
影响因子：
4.9
通讯作者：
Wine, JJ
中科院分区：
文献类型：
--
作者：
Irokawa, T;Krouse, ME;Wine, JJ
我们研制了一种新的仪器，虚拟腺体(VG)，用于测量开放电路下培养的上皮细胞的液体分泌率(J(V))、液体成分和跨上皮电位(TEP)。VG在Costar过滤器上的上皮细胞顶端表面上方创建一个10-mul的小室，其中有一个小孔通向充满油的储油池。在腔内注入精选的液体后，分泌的液体通过孔进入油中，在那里形成一个气泡，通过光学监测确定J(V)并收集进行分析。CALU-3细胞在37℃的Krebs-Ringer碳酸氢盐缓冲液中固定在VG中。基础J(V)为2.7+/-0.1mul。Cm(-2)。H(-1)(n=42)，TEP为-9.2+/-0.6 mV(n=33)；哇巴因使两项指标均降至零(n=6)。1 mM 1-乙基-2-苯并咪唑啉酮(n=5)对J(V)和TEP分别有173和101%的刺激作用，333 nM的thapsigargin(n=5)对J(V)和TEP的刺激分别为213%和122%，Forsklin+thapsigargin(n=6)的刺激分别为520%和240%。布美他尼组(n=5)对基础J(V)和TEP的抑制分别为82%和63%，100 mU乙酰唑胺(n=5)组分别为71%和82%，格列本脲600 mU组(n=4)分别为47%和56%。用HEPES替代HCO3后，基础J(V)和TEP分别为对照组的52%和89%(n=16)。分泌液的HCO3-净浓度与浴液接近(25 MM)，但用Forsklin或VIP刺激时增加(接近80 mM)。这些结果验证了VG装置的使用，并首次提供了CALU-3细胞中J(V)的直接测量。
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.