Grupp C, Stelmach R, Troche I, Müller G A
Abteilung Nephrologie und Rheumatologie, Georg August-Universität Göttingen, Germany.
Pflugers Arch. 1999 Dec;439(1-2):174-85. doi: 10.1007/s004249900147.
To better characterize loop diuretic-sensitive ion fluxes in the inner medullary collecting duct (IMCD) we examined them in IMCD cells grown as a primary culture on permeable supports. A polarization of the cells with their basolateral side to the support was confirmed morphologically by electron microscopy and functionally by flux studies with ouabain. Within 7 days cells developed a transepithelial resistance of 974+/-52 omega per cm2 and a low transepithelial potential difference (-0.7+/-0.8 mV). Measurements of intracellular ion content by electron probe microanalysis in IMCD depleted of intracellular ions by preincubation in a Na(+)-K(+)-Cl(-)-free medium revealed, compared to the control receiving solvent, significant reductions in intracellular Na+ content (-17.6% within 10 min) and intracellular Cl- content (-43.8% within 30 min) by the addition of bumetanide (10(-4) mol/l) to the apical but not basolateral incubation medium. In 22Na+ and 86Rb+ isotope uptake studies, fluxes from the apical side were significantly inhibited at bumetanide concentrations of 100 micromol/l by 0.27+/-0.10 and 0.21+/-0.04 nmol/cm2 in 10 min, respectively, whereas basolateral fluxes of 86Rb+ but not 22Na+ were significantly reduced by this substance. Removal of Cl- had a similar but not additional effect. mRNA encoding the apical isoform of the Na+2Cl(-)K+ cotransporter could be specifically amplified by reverse transcriptase polymerase chain reaction from the inner medulla and highly purified IMCD cells. Northern blot of mRNA isolated from the inner medulla with a riboprobe of the apical isoform revealed a transcript of approximately 4.9 kb. This probe localized under "low-stringency" conditions to the IMCD in in situ hybridization studies. These results suggest the presence of an apically localized isoform of bumetanide-sensitive Na+2Cl(-)K+ cotransport in at least a subfraction of IMCD cells. This transport may be involved in the ultimate adjustment of urinary electrolyte concentration by this final segment of the tubular system.
为了更好地表征髓质内层集合管（IMCD）中对袢利尿剂敏感的离子通量，我们在可渗透支持物上以原代培养方式生长的IMCD细胞中对其进行了研究。通过电子显微镜在形态学上以及通过用哇巴因进行通量研究在功能上证实了细胞的极化，其基底外侧面向支持物。在7天内，细胞形成了每平方厘米974±52Ω的跨上皮电阻和低跨上皮电位差（-0.7±0.8 mV）。通过电子探针微分析测量在无Na(+)-K(+)-Cl(-)培养基中预孵育以耗尽细胞内离子的IMCD中的细胞内离子含量，与接受溶剂的对照相比，通过向顶端而非基底外侧孵育培养基中添加布美他尼（10(-4)mol/l），细胞内Na+含量显著降低（10分钟内降低17.6%），细胞内Cl-含量显著降低（30分钟内降低43.8%）。在22Na+和86Rb+同位素摄取研究中，在布美他尼浓度为100μmol/l时，顶端侧的通量在10分钟内分别被显著抑制0.27±0.10和0.21±0.04 nmol/cm2，而该物质显著降低了86Rb+的基底外侧通量，但未降低22Na+的基底外侧通量。去除Cl-有类似但无额外的作用。编码Na+2Cl(-)K+共转运体顶端异构体的mRNA可通过逆转录聚合酶链反应从髓质内层和高度纯化的IMCD细胞中特异性扩增。用顶端异构体的核糖探针从髓质内层分离的mRNA的Northern印迹显示约4.9 kb的转录本。在原位杂交研究中，该探针在“低严格度”条件下定位到IMCD。这些结果表明，至少在IMCD细胞的一个亚组分中存在顶端定位的对布美他尼敏感的Na+2Cl(-)K+共转运异构体。这种转运可能参与了肾小管系统这一终末节段对尿液电解质浓度的最终调节。
