Réaux A, de Mota N, Zini S, Cadel S, Fournié-Zaluski M C, Roques B P, Corvol P, Llorens-Cortès C
Institut National de la Santé et de la Recherche Médicale, Unité 36, Collège de France, Paris, France.
Neuroendocrinology. 1999 May;69(5):370-6. doi: 10.1159/000054439.
Angiotensin III (AngIII), which is metabolized in vivo by aminopeptidase N (APN), was previously shown to be one of the main effector peptides of the brain renin-angiotensin system (RAS) in the control of vasopressin release. Recently, a potent APN inhibitor, PC18 (2-amino-4-methylsulfonyl butane thiol, methionine thiol), has been developed. In this study, we first checked the in vitro selectivity of PC18 towards APN, aminopeptidase A (APA) and aminopeptidase B (APB), three zinc metalloproteases with significant identity between their amino acid sequences. The Ki values of this compound on APN were found to be in the nanomolar range (Ki = 8.0 +/- 1.7 nM) but it was 2,150 and 125 times less active on APA and APB, respectively. Secondly, we evaluated in vivo the effect of brain APN inhibition with PC18 on the inactivation of brain AngIII and on vasopressin secretion in mice. For this purpose, mice received [3H]AngII intracerebroventricularly in the presence or absence of the APN inhibitor PC18 (30 microg). At different times after the injection, [3H]AngIII levels were evaluated from hypothalamus homogenates after separation by cation-exchange chromatography. PC18 induced an accumulation of [3H]AngIII, increasing its half-life 3.9 times as compared with control values. In addition, the effect of PC18 on vasopressin release was studied in mice. PC18 (10-100 microgram) was injected intracerebroventricularly, and plasma vasopressin levels were estimated by radioimmunoassay. PC18 increased vasopressin levels in a dose-dependent manner. The maximal increase in vasopressin release (+220%) is observed for a dose of PC18 of 100 microgram and was inhibited 75% by the coadministration of the AngII receptor antagonist (Sar1-Ala8)-AngII (0.5 microgram). These results indicate that in vivo, in the mouse brain, APN inhibition by PC18 increases the half-life of endogenous AngIII, resulting in an enhanced vasopressin release.
血管紧张素III（AngIII）在体内由氨肽酶N（APN）代谢，先前已证明它是脑肾素-血管紧张素系统（RAS）中控制血管加压素释放的主要效应肽之一。最近，一种有效的APN抑制剂PC18（2-氨基-4-甲基磺酰基丁烷硫醇，蛋氨酸硫醇）已被开发出来。在本研究中，我们首先检测了PC18对APN、氨肽酶A（APA）和氨肽酶B（APB）这三种氨基酸序列具有显著同源性的锌金属蛋白酶的体外选择性。发现该化合物对APN的Ki值处于纳摩尔范围（Ki = 8.0 +/- 1.7 nM），但对APA和APB的活性分别低2150倍和125倍。其次，我们在体内评估了用PC18抑制脑APN对小鼠脑内AngIII失活和血管加压素分泌的影响。为此，小鼠在有或无APN抑制剂PC18（30微克）的情况下脑室内注射[3H]AngII。注射后不同时间，通过阳离子交换色谱分离后，从下丘脑匀浆中评估[3H]AngIII水平。PC18诱导[3H]AngIII积累，其半衰期比对照值增加3.9倍。此外，在小鼠中研究了PC18对血管加压素释放的影响。脑室内注射PC18（10 - 100微克），通过放射免疫测定法估计血浆血管加压素水平。PC18以剂量依赖性方式增加血管加压素水平。在PC18剂量为100微克时观察到血管加压素释放的最大增加（+220%），并且通过共同给予血管紧张素II受体拮抗剂（Sar1-Ala8）-AngII（0.5微克）可抑制75%。这些结果表明，在体内，在小鼠脑中，PC18抑制APN可增加内源性AngIII的半衰期，导致血管加压素释放增强。
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