Expression of colonic H-K-ATPase mRNA in cortical collecting duct: regulation by acid/base balance.
Expression of colonic H-K-ATPase mRNA in cortical collecting duct: regulation by acid/base balance.
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皮质集合管中结肠 H-K-ATPase mRNA 的表达：酸/碱平衡的调节。
DOI：
10.1152/ajprenal.1995.269.4.f551
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Naray-Fejes-Toth,A
中科院分区：
文献类型：
--
作者：
Fejes-Toth,G;Rusvai,E;Longo,KA;Naray-Fejes-Toth,A
除了H-K-ATP酶的胃亚型外，结肠亚型也在肾脏中表达，但其在肾内的定位和确切功能尚不清楚。本研究的目的是确定结肠H-K-ATP酶是否在兔皮质集合管（CCD）中表达，以及它是否受酸碱平衡变化的调节。用定量逆转录聚合酶链反应（RT-PCR）与RNA分离的免疫兔CCD细胞和简并寡核苷酸引物，PCR产物的预测大小（约430 bp）进行了扩增。通过巢式PCR和测序进一步表征扩增的DNA。对434 bp的PCR产物进行序列测定，结果表明该基因与大鼠结肠H-K-ATP酶基因在核苷酸水平上的同源性为83%，在推导的氨基酸水平上的同源性为80.4%。用相同的引物和源自兔远端结肠的cDNA，扩增出与源自CCD细胞的DNA片段大小和核苷酸序列相同的DNA片段。此外，使用PCR筛选，我们从兔CCD文库中分离并测序了1.5 kb的cDNA克隆。该cDNA编码的蛋白质的氨基酸序列与豚鼠和大鼠结肠H-K-ATP酶相应区域的同源性分别为85%和82%，与最近从海蟾蜍中克隆的H-K-ATP酶的同源性为70%，而与大鼠Na-K-ATP酶α 1亚基和大鼠胃H-K-ATP酶的同源性分别为45%和42%。（250字处删节）
In addition to the gastric isoform of H-K-ATPase, the colonic isoform is also expressed in the kidney, but its intrarenal localization and exact function are not known. The goal of this study was to determine whether the colonic H-K-ATPase is expressed in the rabbit cortical collecting duct (CCD) and whether it is regulated by changes in acid/base balance. With quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) with RNA isolated from immunodissected rabbit CCD cells and degenerate oligonucleotide primers, a PCR product of the predicted size (approximately 430 bp) was amplified. The amplified DNA was further characterized by nested PCR and sequencing. Direct sequencing of the 434-bp PCR product revealed 83% identity at the nucleotide level and an 80.4% identity at the deduced amino acid level to the rat colonic H-K-ATPase. With the same primers and cDNA originating from rabbit distal colon, a DNA fragment with a size and nucleotide sequence identical to that originating from CCD cells was amplified. Furthermore, using PCR screening, we isolated and sequenced a 1.5-kb cDNA clone from a rabbit CCD library. The predicted amino acid sequence of the protein encoded by this cDNA is 85 and 82% identical to the corresponding regions of the guinea pig and rat colonic H-K-ATPase, respectively, and 70% identical to the H-K-ATPase recently cloned from Bufo marinus, whereas it shows only 45 and 42% homology to the rat Na-K-ATPase alpha 1-subunit and the rat gastric H-K-ATPase, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)