ウサギ肺アンジオテンシンI変換酵素の生化学的性質とその生理的役割

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It has become apparent that the lung has many functions other than respiration within recent years. Among them, the metabolism of vasoactive substances in pulmonary circulation has been interested in by many investigators because of its influence upon the pathophysiology of the systemic circulation. Angiotensin I-converting enzyme (ACE) converts inactive angiotensin I(AI) to angiotensin II(AII), a potent vasopressor octapeptide, by releasing the C-terminal dipeptide of AI. Recently the enzyme is also thought to be capable of inactivating bradykinin (BK) and to be identical with kininase II. The lung has been pointed out as an organ which is rich of this enzyme by many investigators. So, if the enzyme acts on both renin-angiotensin and kallikrein-kinin system in the pulmonary circulation, the change of this enzymic activity in the pathological lung is considered to have very important influence on the metabolism of those both systems, resulting in the change of systemic circulation. To elucidate the physiological roles of ACE in lung, the following three experiments were performed. 1) Purification of ACE from rabbits lung and determination of properties and characteristics of the purified enzyme. 2) Estimation of ACE activity in pulmonary tissue and in plasma of normal rabbits and rabbits with chemically induced pneumonitis. 3) Perfusion experiment with isolated lung of normal rabbits and of rabbits with chemically induced pneumonitis for determining metabolic activity of AI and BK. Materials and Methods: Purification and determination of properties of ACE from rabbit lung: The membrane fraction of the lung was precipitated from lung homogenate by acid treatment (pH 5.2). ACE was solubilized from the membrane fraction using trypsin treatment and purified using columns of DE-52 cellulose, hydroxylapatite and Sephadex G-200. The analytical disc gel electrophoresis was performed. The activity of ACE was assayed by the spectrophotometric method using Hip-His-Leu as the substrate. Biological assay of the enzymic activity was also performed in the isolated rat uterus using AI, AII and BK as the substrate. The products of the reaction were identified by high voltage paper electrophoresis. The molecular weight of the enzyme was determined by gel filtration of Sephadex G-200. Km value, optimal pH, Cl^- dependency and inhibition by Arg-Pro-Pro, SQ 20,881 and SQ 14,225 were also examined. Production of chemically induced pneumonitis: Rabbits were treated with intravenous injection of complete Frund's adjuvant (FA) for the acute experiment or N-nitroso-N-methylurethane (NMU) for the chronic experiment. Measurement of ACE activity in pulmonary tissue and in plasma: Lungs were homogenized and sonicated by Polytron^R. ACE activity was measured spectrophotometrically and expressed as nmol/mg prot./min for the lung and nmol/ml/min for the plasma. Perfusion of isolated lung: Lungs were perfused with constant volume of Tyrode's solution from a cannula placed in the pulmonary artery without taking out of the thorax, AI, AII or BK were injected via the canula and the perfusate was collected respectively from left ventricle. AI and AII were assayed radioimmunologically. Rate of conversion of AI and of inactivation of BK were calculated. Mesurment of systemic blood pressure: A catheter was placed in femoral artery of unanesthetized rabbits and connected to mercurial manometer.
1979-12-20

ウサギ肺アンジオテンシンI変換酵素の生化学的性質とその生理的役割

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