Characterization of angiotensin II formation in human isolated bladder by selective inhibitors of ACE and human chymase: a functional and biochemical study.

1. Functional recordings of smooth muscle tension and biochemical experiments on membrane fractions were performed to characterize angiotensin II (AII) formation in human isolated bladder smooth muscle. 2. A novel human chymase inhibitor CH 5450 (Z-Ile-Glu-Pro-Phe-CO2Me) and a recently developed human chymase substrate Pro11-,D-Ala12)-angiotensin I, claimed to be resistant to angiotensin converting enzyme (ACE) and carboxypeptidase, were used. 3. Angiotensin I (AI) (0.3 microM) induced a contractile response amounting to 58 +/- 5% (n=12) of the initial K+ (124 mM)-induced contractions. This response was reduced to 36 +/- 3% (n=8) by the ACE-inhibitor enalaprilat (10 microM), while pretreatment with soybean trypsin inhibitor (STI 200 microg ml(-1)) or CH 5450 (10 microM) had no effect. However, the combination of enalaprilat and STI reduced the AI-induced contractions to 19 +/- 5% (n=6), and the combination of enalaprilat and CH 5450 caused an almost complete inhibition of the AI-induced contractions to 1+/-1% (n=6). 4. The substrate (Pro11-,D-Ala12)-AI (3 microM) produced contractions which amounted to 57 +/- 4% (n=13) of the initial K+ (124 mM) contractions. These contractions were not affected by enalaprilat (10 microM). On the other hand, STI (200 microg ml(-1)) and CH 5450 (10 microM) added separately, depressed the (Pro11-,D-Ala12)-AI-induced contractions to 34 +/- 5% (n=6) and 24 +/- 4% (n=6), respectively. The combination of enalaprilat and STI or enalaprilat and CH 5450 did not produce any further inhibition. 5. Experiments with detrusor membrane fractions incubated with AI (50 microM) were performed. In the presence of enalaprilat (100 microM), carboxypeptidase inhibitor CPI (10 microg ml(-1)) and aprotinin (15 microM), CH 5450 (10 nM-1 microM) caused a concentration-dependent inhibition of AII formation. 6. The results confirm that AII is a potent contractile agent in the human isolated detrusor muscle. They also indicate that the serine protease responsible for AII formation in the human bladder in vitro is human chymase or an enzyme similar to human chymase.