Molecular cloning of cDNA coding for kidney aldose reductase. Regulation of specific mRNA accumulation by NaCl-mediated osmotic stress.
Molecular cloning of cDNA coding for kidney aldose reductase. Regulation of specific mRNA accumulation by NaCl-mediated osmotic stress.
复制标题
DOI：
--
复制
发表时间：
1989-10
期刊：
影响因子：
--
通讯作者：
A. García-Pérez;B. Martin;H. Murphy;S. Uchida;H. Murer;B. Cowley;J. Handler;M. Burg
中科院分区：
文献类型：
--
作者：
A. García-Pérez;B. Martin;H. Murphy;S. Uchida;H. Murer;B. Cowley;J. Handler;M. Burg
细胞通常通过积累非扰动有机渗透剂来应对长期高渗应激。与细菌不同的是，细菌中涉及渗透调节剂积累增加的分子机制已被确定，而多细胞生物中的分子机制实际上是未知的。在哺乳动物中，在抗利尿期间，肾内髓质细胞暴露于高且变化的细胞外 NaCl。在这些条件下，细胞含有高水平的山梨醇和其他渗透剂，有助于平衡高细胞外渗透压。 PAP-HT25 是源自兔肾内髓质的连续细胞系。当通过提高 NaCl 浓度来增加培养基渗透压时，这些细胞会积累山梨醇。山梨醇是在醛糖还原酶催化的反应中由葡萄糖合成的。当培养基处于高渗状态时，由于酶量增加较多，醛糖还原酶活性增加。这种增加是由于醛糖还原酶蛋白的合成速率加快而产生的。本研究的目的是通过测量醛糖还原酶 mRNA 的相对丰度来检查醛糖还原酶蛋白增加的机制。分离出编码兔肾醛糖还原酶的cDNA克隆。从该克隆转录的反义 RNA 探针在 Northern 印迹中与 1.5-1.6 kb mRNA 特异性杂交。在高渗培养基中长期生长的细胞具有相对丰度的这种特定mRNA，是在等渗培养基中生长的细胞的六倍。当在等渗培养基中生长的细胞切换至高渗培养基时，醛糖还原酶 mRNA 水平在 18-24 小时达到峰值（18 倍）。渗透压对醛糖还原酶 mRNA 的诱导是可逆的。我们发现直接响应高渗应激的特定 mRNA 丰度增加，这是关于动物中这种效应的首次报告。
Cells generally respond to long-term hyperosmotic stress by accumulating nonperturbing organic osmolytes. Unlike bacteria, in which molecular mechanisms involved in the increased accumulation of osmolytes have been identified, those in multicellular organisms are virtually unknown. In mammals, during antidiuresis, cells of the renal inner medulla are exposed to high and variable extracellular NaCl. Under these conditions, the cells contain a high level of sorbitol and other osmolytes which help balance the high extracellular osmolality. PAP-HT25 is a continuous line of cells derived from rabbit renal inner medulla. When medium osmolality is increased by raising the NaCl concentration, these cells accumulate sorbitol. The sorbitol is synthesized from glucose in a reaction catalyzed by aldose reductase. When the medium is made hyperosmotic, aldose reductase activity increases because of a larger increase in the amount of enzyme. This increase is produced by the accelerated rate of synthesis of aldose reductase protein. The purpose of the present studies was to examine the mechanism of this increase in aldose reductase protein by measuring the relative abundance of aldose reductase mRNA. A cDNA clone coding for rabbit kidney aldose reductase was isolated. Antisense RNA probes transcribed from this clone hybridized specifically with a 1.5-1.6 kilobase mRNA in Northern blots. Cells grown chronically in hyperosmotic medium had a relative abundance of this specific mRNA which was six times that of cells grown in isoosmotic medium. When cells grown in isoosmotic medium were switched to hyperosmotic medium, the level of aldose reductase mRNA peaked (18-fold) at 18-24 h. The induction of aldose reductase mRNA by osmotic stress was reversible. Our finding of increased abundance of a specific mRNA in direct response to hyperosmotic stress represents the first report of such an effect in animals.