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Endogenous Digitalis-like Substance in Urine of DOCA-treated Hypertensive rats

DOCA治疗高血压大鼠尿液中内源性洋地黄样物质

基本信息

批准号：
62570132
负责人：
KATSUYA Nagai M.D.
金额：
$ 1.34万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1987
资助国家：
日本
起止时间：
1987 至 1988
项目状态：
已结题

项目摘要

Recently, possible involvement of the endogenous digitalis-like substance in the mechanism of essential hypertension has been suggested. In this work the purification and identification of this digitalis-like substance in the urine of DOCA (deoxycorticosterone acetate) treated hypertensive rats were attempted. This substance was purified using methods of an ultrafiltration (Amicon YM-10), a CM Sepharose column chromatography, a Sephadex G-10 column chromatography, an anti-digoxin antibody affinity column chromatography, a reversed phase HPLC and a molecular sieve HPLC. The purified material was recognized as a single substance by a TLC chromatography. This substance dose-dependently inhibited Na^+, K^+-STPase and binding of ^<125>I-digoxin to anti-digoxin antibody, and elevated the blood pressure with intravenous injection. This substance lost the inhibitory activity of Na^+, K^+-ATPase by the acid hydrolysis (6N HCl, 110 C, 20 h) and treatments with proteolytic enzymes such as pronase, prolidase, aminopeptidase P and lysine endopeptidase. The amino acid analysis of this substance gave 14 species of amino acids. Now, the amino acid sequence of this substance is under the investigation by protein sequencer. Existence of only a minute amount of this substance in the urine and a blockade of the N-terminal of this peptide might prevent rapid identification of this substance. A similar substance was observed in rat brain extract.

最近，有人提出内源性洋地黄样物质可能参与原发性高血压的发病机制。本工作试图从DOCA（醋酸脱氧皮质酮）处理的高血压大鼠尿中纯化和鉴定这种洋地黄样物质。使用超滤（Amicon YM-10）、CM Sepharose柱层析、Sephadex G-10柱层析、抗地高辛抗体亲和柱层析、反相HPLC和分子筛HPLC的方法纯化该物质。通过TLC色谱法将纯化的物质识别为单一物质。该物质呈剂量依赖性地抑制Na^+，K^+-ST段抬高和抑制<125>地高辛与地高辛抗体的结合，并使静脉注射的血压升高。该物质经酸水解（6 N HCl，110 ℃，20 h）和蛋白水解酶如链霉蛋白酶、脯氨酰二肽酶、氨肽酶P和赖氨酸内肽酶处理后，丧失了Na^+，K^+-ATP酶的抑制活性。氨基酸分析表明，该物质含有14种氨基酸。目前，该物质的氨基酸序列正在通过蛋白质测序仪进行研究。尿液中仅存在微量的该物质以及该肽的N-末端的阻断可能会阻止该物质的快速鉴定。在大鼠脑提取物中观察到类似物质。