Aldosterone-dependent regulation of Na-K-ATPase subunit mRNA in the rat CCD: competitive PCR analysis.
Aldosterone-dependent regulation of Na-K-ATPase subunit mRNA in the rat CCD: competitive PCR analysis.
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大鼠 CCD 中 Na-K-ATP 酶亚基 mRNA 的醛固酮依赖性调节：竞争性 PCR 分析。
DOI：
10.1152/ajprenal.1996.271.1.f7
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Welling,PA
中科院分区：
文献类型：
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作者：
Tsuchiya,K;Giebisch,G;Welling,PA
在皮质集合管（CCD）中，醛固酮通过涉及Na-K-ATP酶α 1和β 1亚基蛋白丰度的亚型特异性增加的机制增加功能活性Na-K-腺苷三磷酸酶（Na-K-ATP酶）分子的数量。然而，反应的分子基础，特别是在哺乳动物CCD在体内，仍然不清楚。为了解决这个问题，采用逆转录（RT）和竞争性聚合酶链反应（PCR）来研究盐皮质激素依赖性对大鼠CCD中α 1和β 1亚基mRNA的调节。用Na-K-ATP酶亚基特异性寡核苷酸引物进行PCR扩增，从单个显微切割的CCD中扩增逆转录亚基mRNA（RT-mRNA）。构建对照模板（大鼠Na-K-ATP酶α 1-亚基cDNA的84-bp缺失突变和β 1-亚基cDNA的70-bp缺失），连续稀释，并与来自单个CCD的野生型Na-K-ATP酶亚基RT-mRNA共扩增。通过溴化乙锭染色观察预测大小的PCR产物。用内部亚基特异性寡核苷酸进行Southern印迹证实了Na-K-ATP酶α 1-和β 1-亚基的同一性。发现扩增的野生型与突变体PCR产物的比率在测试的输入对照cDNA的范围内是线性的，使得可以确定亚基mRNA的量。双侧肾上腺切除术（7天）导致皮质类固醇水平长期降低，α 1亚基转录物的表观水平降低了54.0 +/- 6.3%，但β 1亚基没有降低。给予醛固酮至生理水平足以在6小时内将CCD α 1亚基mRNA丰度恢复至对照水平。我们得出以下结论：1）体内CCD的Na-K-ATP酶的调节至少部分归因于Na-K-ATP酶α 1亚基mRNA丰度的盐皮质激素依赖性控制; 2）竞争性PCR可以提供一种灵敏的定量工具来确定肾单位节段中mRNA丰度的盐皮质激素依赖性调节。
In the cortical collecting duct (CCD), aldosterone increases the number of functionally active Na-K-adenosin-etriphosphatase (Na-K-ATPase) molecules by a mechanism involving an isoform-specific increase in the abundance of the Na-K-ATPase alpha 1- and beta 1-subunit protein. However, the molecular basis for the response, particularly in the mammalian CCD in vivo, has remained unclear. To resolve this issue, reverse transcription (RT) and a competitive polymerase chain reaction (PCR) were employed to study mineralocorticoid-dependent regulation of alpha 1- and beta 1-subunit mRNA in the rat CCD. Na-K-ATPase subunit-specific oligonucleotides primers were used in the PCR to amplify reverse-transcribed subunit mRNA (RT-mRNA) from single microdissected CCD. Control templates were constructed (84-bp deletion mutation of the rat Na-K-ATPase alpha 1-subunit cDNA and 70-bp deletion of the beta 1-subunit cDNA), serially diluted, and coamplified with the wild-type Na-K-ATPase subunit RT-mRNA from single CCD. PCR products of predicted size were observed by ethidium bromide staining. Southern blots with an internal subunit-specific oligonucleotide confirmed Na-K-ATPase alpha 1- and beta 1-subunit identity. The ratio of the amplified wild-type to mutant PCR products was found to be linear over the range of input control cDNA tested so that the amount of subunit mRNA could be determined. A chronic reduction in corticosteroid levels by bilateral adrenalectomy (7 days) reduced the apparent level of alpha 1-subunit transcript by 54.0 +/- 6.3% but not the beta 1-subunit. Administering aldosterone to physiological levels is sufficient to restore CCD alpha 1-subunit mRNA abundance toward control levels within 6 h. We conclude the following: 1) regulation of Na-K-ATPase of CCD in vivo can be attributed, at least in part, to mineralocorticoid-dependent control of Na-K-ATPase alpha 1-subunit mRNA abundance; and 2) competitive PCR may provide a sensitive and quantitative tool for determining hormone-dependent regulation of mRNA abundance in nephron segments.