Chymase-dependent angiotensin II formation in human vascular tissue.
Chymase-dependent angiotensin II formation in human vascular tissue.
复制标题
人类血管组织中食糜酶依赖性血管紧张素 II 的形成。
作者：
S. Takai;D. Jin;M. Sakaguchi;M. Miyazaki
背景
一些报道表明，在体外，人心脏匀浆中的乳糜酶在天然蛋白酶抑制剂(如α-抗胰蛋白酶)存在的情况下，对血管紧张素II的形成几乎没有贡献。我们研究了与肝素结合的凝乳酶，类似于体内的形式，在天然蛋白酶抑制剂存在的情况下是否有助于血管紧张素II的形成。
方法和结果
在含有Ang I的人血管组织提取液(1 mg蛋白质/m L)中孵育30分钟后，Ang II的浓度为1.67+/-0.06 nmol/m L，我们认为这个量为100%。1 mg/L赖诺普利、100 mg/L糜抑素和0.1g/Lα-抗胰蛋白酶对血管紧张素Ⅱ的抑制作用分别为10%、95%和96%。将提取物应用于肝素亲和层析柱。用PBS洗脱柱后，洗脱的PBS含有微弱的Ang II形成活性，赖诺普利完全抑制该活性。洗脱后的PBS有较强的血管紧张素Ⅱ生成活性，但可被凝乳素和α-抗胰蛋白酶抑制。提取液上柱后，用PBS洗脱柱，然后在PBS中加入Ang I溶液。从孵育柱中洗脱出来的PBS中Ang II的形成随时间而增加。孵育30min后，PBS(1mL)中Ang II的浓度为2.56+/-0.28nmol/mL，我们认为这个量是100%。为了研究抑制剂的作用，提取液(1 mg蛋白质/m L)用于肝素亲和柱(1m L)，该柱与PBS(3 M L)预平衡，然后在PBS(1m L)中加入100m ol/L糜抑素或0.1g/Lα-抗胰蛋白酶(1m L)。色谱柱用3mLPBS洗涤后，加入1 mg/mLPBS的Ang I溶液，孵育30min。应用乳糜蛋白酶抑制从柱子洗脱出来的PBS中血管紧张素Ⅱ的生成，最高可抑制5%，但这不受α-抗胰蛋白酶的影响。
结论
这些发现表明，与肝素结合的人凝乳酶在天然蛋白酶抑制剂(如α-抗胰蛋白酶)存在的情况下，在Ang II的形成中起着重要作用。
BACKGROUND
Some reports have suggested that, in vitro, human heart chymase in homogenates contributes little to angiotensin (Ang) II formation in the presence of natural protease inhibitors such as alpha-antitrypsin. We studied whether chymase bound to heparin, resembling an in vivo form, could contribute to Ang II formation in the presence of natural protease inhibitors.
METHODS AND RESULTS
The Ang II formation was increased time-dependently after incubation in an extract (1 mg of protein/mL) of human vascular tissues containing Ang I. The concentration of Ang II in the extract after incubation for 30 minutes was 1.67+/-0.06 nmol/mL, and we regarded this quantity of Ang II as 100%. The Ang II formation was inhibited 10%, 95%, and 96% by 1 micromol/L lisinopril, 100 micromol/L chymostatin, and 0.1 g/L alpha-antitrypsin, respectively. The extract was applied to a heparin affinity column. After the column was washed with PBS, the eluted PBS contained a weak Ang II-forming activity, which was completely inhibited by lisinopril. The eluted PBS, to which >0.8 mol/L NaCl had been added, showed a strong Ang II-forming activity which was inhibited by chymostatin and alpha-antitrypsin. After the application of the extract, the column was washed with PBS and then an Ang I solution in PBS was applied to the column. The Ang II formation in the PBS eluted from the incubated column was increased time-dependently. The concentration of Ang II in the PBS (1 mL) eluted from the column after incubation for 30 minutes was 2.56+/-0.28 nmol/mL, and we regarded this quantity of Ang II as 100%. To study the effects of inhibitors, the extract (1 mg of protein/mL) was applied to a heparin affinity column (1 mL) which was preequilibrated with PBS (3 mL); 100 micromol/L chymostatin or 0.1 g/L alpha-antitrypsin in PBS (1 mL) was then applied to the column. After the column was washed with PBS (3 mL), Ang I solution (1 mg/mL) in PBS was applied to the column, and the column was incubated for 30 minutes. The Ang II formation in the PBS eluted from the column was suppressed up to 5% by application of chymostatin, although this was not affected by application of alpha-antitrypsin.
CONCLUSIONS
These findings suggest that human chymase bound to heparin plays a functional role in Ang II formation in the presence of natural protease inhibitors such as alpha-antitrypsin.
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