Electrophysiological studies of primary cultures of rabbit distal tubule cells.
Electrophysiological studies of primary cultures of rabbit distal tubule cells.
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兔远端肾小管细胞原代培养物的电生理学研究。
DOI：
10.1152/ajprenal.1987.252.5.f899
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发表时间：
1987
期刊：
影响因子：
--
通讯作者：
Weber,MR
中科院分区：
文献类型：
--
作者：
Bello-Reuss,E;Weber,MR
接种后6-14天，在融合后，研究了从Percoll梯度离心的F1带生长的原代单层（“远端”单层）。透射和扫描电子显微镜显示，两种类型的细胞，类似于主细胞的兔皮质集合管（CCT）和插入细胞的CCT或连接小管，构成约96%的单层。当用荧光花生凝集素处理时，大约三分之二的嵌入细胞发出荧光。间接特异性免疫细胞荧光染色显示96%的细胞有荧光，证实单层细胞来源于CCT或连接小管细胞。单细胞层暴露于加压素或异丙肾上腺素可增加细胞和浸浴介质中的环磷酸腺苷（cAMP）含量，而甲状旁腺激素则无效。电生理学研究显示跨上皮电压（VT）为-11 +/- 2 mV，基底外侧膜电压（Vb）为-77 +/- 5 mV（n = 20）。跨上皮电阻（RT）为1，870 +/- 250 Ω X cm 2（n = 13）。在6个单层细胞中的3个中，将阿米洛利（10（-5）M）施加于顶侧，导致顶膜电压（Va）从-71 +/- 1增加到-89 +/- 9 mV），VT降低（从-10 +/- 1到-2 +/- 1 mV）。RT在阿米洛利暴露期间没有变化。顶膜暴露于140 mM K+-去极化Va从-67 +/- 7至-39 +/- 11 mV（P小于0.002），超极化VT从-7 +/- 2至-15 +/- 3 mV（P小于0.005）。从基底外侧暴露于高K+使Vb从-76 +/- 11去极化至-43 +/- 10 mV（P小于0.001），使VT从-9 +/-去极化至8 +/- 5 mV（P小于0.001）。该制剂适用于通过电生理学方法和其他技术研究上皮转运的基本方面。这一发现与通过离体灌流小管技术研究的家兔皮质集合小管的几个已知特性一致。
Primary monolayers grown from F1 band of a Percoll gradient centrifugation ("distal" monolayers) were studied, after confluency, 6-14 days after seeding. Transmission and scanning electron microscopy revealed that two cell types, resembling principal cells of the rabbit cortical collecting tubule (CCT) and intercalated cells of either CCT or connecting tubule, constitute approximately 96% of the monolayer. About two-thirds of the intercalated cells fluoresced when treated with fluorescent peanut lectin. Indirect specific immunocytofluorescent staining revealed fluorescence in 96% of the cells, confirming that the monolayers were derived from CCT or connecting tubule cells. Exposure of monolayers to vasopressin or isoproterenol increases adenosine 3',5'-cyclic monophosphate (cAMP) content in the cells and bathing medium, whereas parathyroid hormone was ineffective. Electrophysiological studies revealed a transepithelial voltage (VT) of -11 +/- 2 mV, and basolateral membrane voltage (Vb) of -77 +/- 5 mV (n = 20). The transepithelial electrical resistance (RT) was 1,870 +/- 250 omega X cm2 (n = 13). In three out of six monolayers, amiloride (10(-5) M) applied to the apical side produced an increase in apical membrane voltage (Va) from -71 +/- 1 to -89 +/- 9 mV) and a decrease in VT (from -10 +/- 1 to -2 +/- 1 mV). The RT did not change during amiloride exposure. Exposure of the apical membrane to 140 mM K+-depolarized Va from -67 +/- 7 to -39 +/- 11 mV (P less than 0.002) and hyperpolarized VT from -7 +/- 2 to -15 +/- 3 mV (P less than 0.005). Exposure to high K+ from the basolateral side depolarized Vb from -76 +/- 11 to -43 +/- 10 mV (P less than 0.001) and depolarized VT from -9 +/- to 8 +/- 5 mV (P less than 0.001). This preparation is suitable to study basic aspects of epithelial transport by electrophysiological methods and other techniques. The findings are consistent with several of the known properties of cortical collecting tubules from rabbits studied by the isolated perfused tubule technique.