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 exper iments on membrane fractions were performed to characterize angiotensi n II (AII) formation in human isolated bladder smooth muscle. 2 A nove l human chymase inhibitor CH 5450 (Z-Ile-Glu-Pro-Phe-CO2Me) and a rece ntly developed human chymase substrate Pro(11)-,D-Ala(12))-angiotensin I, claimed to be resistant to angiotensin converting enzyme (ACE) and carboxypeptidase, were used. 3 Angiotensin I (AI) (0.3 mu M) induced a contractile response amounting to 58+/-5% (n=12) of the initial K+ ( 124 mh I)-induced contractions. This response was reduced to 36+/-3% ( n=8) by the ACE-inhibitor enalaprilat (10 mu M), while pretreatment wi th soybean trypsin inhibitor (STI 200 mu g ml(-1)) or CH 5450 (10 mu M ) had no effect. However, the combination of enalaprilat and STI reduc ed the AI-induced contractions to 19+/-5% (n=6), and the combination o f enalaprilat and CH 5450 caused an almost complete inhibition of the AI-induced contractions to 1+/-1% (n=6). 4 The substrate (Pro(11)-,D-A la(12))-AI (3 mu M) produced contractions which amounted to 57+/-4% (n =13) of the initial K+ (124 mM) contractions. These contractions were not affected by enalaprilat (10 mu M). On the other hand, STI (200 mu g ml(-1)) and CH 5450 (10 mu M) added separately, depressed the (Pro(1 1)-,D-Ala(12))-AI-induced contractions to 34+/-5% (n=6) and 24+/-4% (n =6), respectively. The combination of enalaprilat and STI or enalapril at and CH 5450 did not produce any further inhibition. 5 Experiments w ith detrusor membrane fractions incubated with AI (50 mu M) were perfo rmed. In the presence of enalaprilat (100 mu M), carboxypeptidase inhi bitor CPI (10 mu g ml(-1)) and aprotinin (15 mu M), CH 5450 (10 nM-1 m u M) caused a concentration-dependent inhibition of AII formation. 6 T he 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.