Purification of New Peptidal Hypertensive Substances and Their Role in the Control of Blood Pressure

近年来，内源性洋地黄类物质与抗地高辛抗体发生交叉反应，被认为可能是引起高血压的原因之一，因为据报道，这种物质在该病患者的血和尿中增加。这种物质增强钠尿，抑制Na~+，K~+-ATPase，被认为有升高血压的作用。因此，以大鼠脑组织Na+，K+-ATPase抑制活性为指标，从高血压大鼠一侧肾切除后的尿液中提纯该物质，注射脱氧皮质酮醋酸酯，饮用1%的氯化钠溶液作为饮用水。在盐酸存在下收集尿液并煮沸后，用CM-Sepharose柱层析、Sephadex G-10柱层析和反相高效液相色谱法进行纯化。分离纯化了3种对Na~+，K~+-ATPase具有抑制活性的物质。抑制活性呈剂量依赖关系，激活…更多的IES不受胰酶消化的影响。酸解后，3种物质的上述活性消失，3种物质中有2种物质的活性在脯氨酸酶消化后消失。这些物质对抗地高辛抗体也表现出剂量依赖的交叉反应，这种交叉反应与Na~+，K~+-ATPase的抑制活性平行。其中一种物质在侧脑室注射时也表现出升压作用。这些发现表明，这些物质是多肽或蛋白质。对这些物质的氨基酸分析得到了氨基酸，但使用氨基酸测序仪进行的分析没有显示任何有关序列的信息。由于如果多肽的N-端被封闭，就不可能确定氨基酸序列，所以我们试图解除封锁。然而，我们无法获得序列。因此，我们认为这些物质在尿液中的含量很低，无法获得足够的物质来分析氨基酸序列。因此，现在我们正试图获得足够的物质来分析序列。在成功鉴定序列后，我们将检查这些物质的生理作用。较少

Recently, the endogenous digitalis like substance, which shows cross-reactivity with anti-digoxin antibody, is thought a possible cause of the essential hypertension, since this substance is reported to increase in the blood and urine of the patients of this disease. This substance enhances natriuresis, inhibits Na^+, K^+-ATPase and is considered to elevate blood pressure. Thus, using the inhibitory activity of rat brain Na^+, K^+ATPase as a marker, this substance was purified from urine of hypertensive rats which were removed of unilateral kidney, were injected wit deoxy-corti-costerone acetate and were given 1 % NaCl solution as drinking water. After collecting urine in the presence of HCL and boiling the ruine, the purification was done using CM-sepharose column chromatography, Sephadex G-10 column chromatography and reversed phase HPLC. Three substances which had inhibitory activity of the Na^+, K^+ATPase were purified. The inhibitory activities were dose-dependent, and the activit … More ies were not affected by the trypsin-digestion. however, these activities of the three substance were disappeared after the acid hydrolysis, and these of 2 substances out of the 3 were disappeared after the prolidase digestion. These substances also showed cross-reactivity to anti-digoxin antibody in dose-dependent manners and the cross-reactivity was parallel with the inhibitory activity of the Na^+, K^+-ATPase. One of the 3 substances also showed hypertensive action when it was injected into the lateral cerebral ventricle. These findings suggest that these substances are peptides or proteins. Amino acid analysis of these substances gave amino acids, but analysis using amino acid sequencer did not show any information about the sequences. Since it is impossible to determine amino acid sequence if the N-terminal of the peptide is blocked, we tried to remove the blockade. However, we could not to get the sequences. Therefore, we think contents of these substances in the urine were very low and we could not get enough substances to analyze the amino acid sequence. Thus, now we are trying to get enough substances to analyze the sequence. After succeeding to identify the sequences, we are going to examine the physiological roles of these substances. Less