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

最近，内源性洋地黄样物质可能参与了高血压的发病机制。本工作对去氧皮质酮醋酸酯(DOCA)治疗的高血压大鼠尿液中的洋地黄类物质进行了纯化和鉴定。采用超滤(Amcon YM-10)、CM Sepharose柱层析、Sephadex G-10柱层析、抗地高辛抗体亲和柱层析、反相高效液相色谱法和分子筛层析法对该物质进行纯化。经薄层层析鉴定为单一物质。该物质剂量依赖性地抑制Na~+，K~+-STPase和~(125)&gt；i-地高辛与抗地高辛抗体的结合，静脉注射使血压升高。该物质在酸性条件下(6NHCl，110℃，20h)和蛋白水解酶如链霉蛋白酶、脯氨酸酶、氨基肽酶P和赖氨酸内肽酶处理，失去了对Na~+，K~+-ATPase的抑制活性。对该物质进行氨基酸分析，得到14种氨基酸。目前，该物质的氨基酸序列正在用蛋白质测序仪进行研究。尿液中只有极少量的这种物质存在，这种多肽的N-末端被阻断，可能会阻碍这种物质的快速鉴定。在大鼠脑提取液中也观察到了类似的物质。

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.