Electrophysiological studies on primary cultures of proximal tubule cells.
Electrophysiological studies on primary cultures of proximal tubule cells.
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近曲小管细胞原代培养物的电生理学研究。
DOI：
10.1152/ajprenal.1986.251.3.f490
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发表时间：
1986
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
原汇合单层生长从近端小管片段的兔肾。通过梯度离心获得片段，并将其接种在底部为可渗透且透明的胶原膜的特设培养皿上。培养基是50%Ham’s F-12和50%Dulbecco’s改良Eagle’s培养基的混合物，补充有胰岛素、转铁蛋白、乙醇胺、亚硒酸钠和氨基酸。在接种后6-14天研究单层。透射电子显微镜显示立方细胞高8.5-10.5微米，顶端刷状缘1.5 - 2.5微米，丰富的线粒体、空泡、溶酶体和不规则的基底交错突起。环磷酸腺苷合成被甲状旁腺素刺激，对加压素和异丙肾上腺素不敏感。使用相同的生理盐溶液对两侧进行的电生理研究显示，跨上皮电压为-2.6 +/- 0.6 mV（n = 10），基底外侧膜电压为-51.0 +/- 4.5 mV（n = 13），均指基础溶液。跨上皮电阻为7 +/- 2 Ω X cm 2。顶端膜去极化的葡萄糖添加到顶端侧和超极化的葡萄糖去除。加入不同葡萄糖浓度（[Na] = 135 mM，37 ℃）时顶端膜电压的变化证明存在葡萄糖转运系统，表观Km为3.54 +/- 0.54，Vmax为7.2 +/- 0.4 mV。因此，该制剂具有近端小管细胞的形态学和电生理学特征;这些研究证明了使用细胞内微电极技术研究培养上皮细胞的运输特性的可行性。
Primary confluent monolayers were grown from proximal tubule fragments of rabbit kidneys. The fragments were obtained by gradient centrifugation and seeded on an ad hoc dish whose bottom was a permeable and transparent collagen membrane. The culture medium was a mixture of 50% Ham's F-12 and 50% Dulbecco's modified Eagle's medium supplemented with insulin, transferrin, ethanolamine, sodium selenite, and amino acids. The monolayers were studied at 6-14 days after seeding. Transmission electron microscopy revealed cuboidal cells 8.5-10.5 microns high, with a 1.5 to 2.5-microns apical brush border, abundant mitochondria, vacuoles, lysosomes, and irregular basal interdigitating processes. Cyclic AMP synthesis was stimulated by parathyroid hormone and was insensitive to vasopressin and isoproterenol. Electrophysiological studies performed with the same physiological salt solution on both sides revealed a transepithelial voltage of -2.6 +/- 0.6 mV (n = 10) and a basolateral membrane voltage of -51.0 +/- 4.5 mV (n = 13), both referred to the basal solution. The transepithelial electrical resistance was 7 +/- 2 omega X cm2. The apical membrane depolarized on addition of glucose to the apical side and hyperpolarized on removal of glucose. Changes in apical membrane voltage on addition of varying glucose concentrations (at [Na] = 135 mM, 37 degrees C) demonstrate the presence of a glucose transport system with an apparent Km of 3.54 +/- 0.54 and a Vmax of 7.2 +/- 0.4 mV. Thus this preparation exhibits morphological and electrophysiological characteristics of proximal tubule cells; these studies demonstrate the feasibility of the use of intracellular microelectrode techniques to study the transport properties of cultured epithelia.